A Damaged Oxidative Phosphorylation Mechanism Is Involved in the Antifungal Activity of Citral against Penicillium digitatum

PubMed Central

OuYang, Qiuli; Tao, Nengguo; Zhang, Miaoling

2018-01-01

Citral exhibits strong antifungal activity against Penicillium digitatum. In this study, 41 over-expressed and 84 repressed proteins in P. digitatum after 1.0 μL/mL of citral exposure for 30 min were identified by the iTRAQ technique. The proteins were closely related with oxidative phosphorylation, the TCA cycle and RNA transport. The mitochondrial complex I, complex II, complex III, complex IV and complex V, which are involved in oxidative phosphorylation were drastically affected. Among of them, the activities of mitochondrial complex I and complex IV were apparently suppressed, whereas those of mitochondrial complex II, complex III and complex V were significantly induced. Meanwhile, citral apparently triggered a reduction in the intracellular ATP, the mitochondrial membrane potential (MMP) and glutathione content, in contrast to an increase in the glutathione S-transferase activity and the accumulation of reactive oxygen species (ROS). Addition of exogenous cysteine decreased the antifungal activity. In addition, cysteine maintained the basal ROS level, deferred the decrease of MMP and the membrane damage. These results indicate that citral inhibited the growth of P. digitatum by damaging oxidative phosphorylation and cell membranes through the massive accumulation of ROS. PMID:29503638

A Damaged Oxidative Phosphorylation Mechanism Is Involved in the Antifungal Activity of Citral against Penicillium digitatum.

PubMed

OuYang, Qiuli; Tao, Nengguo; Zhang, Miaoling

2018-01-01

Citral exhibits strong antifungal activity against Penicillium digitatum . In this study, 41 over-expressed and 84 repressed proteins in P. digitatum after 1.0 μL/mL of citral exposure for 30 min were identified by the iTRAQ technique. The proteins were closely related with oxidative phosphorylation, the TCA cycle and RNA transport. The mitochondrial complex I, complex II, complex III, complex IV and complex V, which are involved in oxidative phosphorylation were drastically affected. Among of them, the activities of mitochondrial complex I and complex IV were apparently suppressed, whereas those of mitochondrial complex II, complex III and complex V were significantly induced. Meanwhile, citral apparently triggered a reduction in the intracellular ATP, the mitochondrial membrane potential (MMP) and glutathione content, in contrast to an increase in the glutathione S-transferase activity and the accumulation of reactive oxygen species (ROS). Addition of exogenous cysteine decreased the antifungal activity. In addition, cysteine maintained the basal ROS level, deferred the decrease of MMP and the membrane damage. These results indicate that citral inhibited the growth of P. digitatum by damaging oxidative phosphorylation and cell membranes through the massive accumulation of ROS.

Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mena, Natalia P.; Millennium Institute of Cell Dynamics and Biotechnology, Santiago; Bulteau, Anne Laure

2011-06-03

Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters aremore » involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex I and iron accumulation are hallmarks of idiopathic Parkinson's disease, the findings reported here may have relevance for understanding the pathophysiology of this disease.« less

Cannabinoid-Induced Changes in the Activity of Electron Transport Chain Complexes of Brain Mitochondria.

PubMed

Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

2015-08-01

The aim of this study was to investigate changes in the activity of individual mitochondrial respiratory chain complexes (I, II/III, IV) and citrate synthase induced by pharmacologically different cannabinoids. In vitro effects of selected cannabinoids on mitochondrial enzymes were measured in crude mitochondrial fraction isolated from pig brain. Both cannabinoid receptor agonists, Δ(9)-tetrahydrocannabinol, anandamide, and R-(+)-WIN55,212-2, and antagonist/inverse agonists of cannabinoid receptors, AM251, and cannabidiol were examined in pig brain mitochondria. Different effects of these cannabinoids on mitochondrial respiratory chain complexes and citrate synthase were found. Citrate synthase activity was decreased only by Δ(9)-tetrahydrocannabinol and AM251. Significant increase in the complex I activity was induced by anandamide. At micromolar concentration, all the tested cannabinoids inhibited the activity of electron transport chain complexes II/III and IV. Stimulatory effect of anandamide on activity of complex I may participate on distinct physiological effects of endocannabinoids compared to phytocannabinoids or synthetic cannabinoids. Common inhibitory effect of cannabinoids on activity of complex II/III and IV confirmed a non-receptor-mediated mechanism of cannabinoid action on individual components of system of oxidative phosphorylation.

Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei.

PubMed

Verner, Zdeněk; Cermáková, Petra; Skodová, Ingrid; Kováčová, Bianka; Lukeš, Julius; Horváth, Anton

2014-01-01

Trypanosomatids are unicellular parasites living in a wide range of host environments, which to large extent shaped their mitochondrial energy metabolism, resulting in quite large differences even among closely related flagellates. In a comparative manner, we analyzed the activities and composition of mitochondrial respiratory complexes in four species (Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and Trypanosoma brucei), which represent the main model trypanosomatids. Moreover, we measured the activity of mitochondrial glycerol-3-phosphate dehydrogenase, the overall oxygen consumption and the mitochondrial membrane potential in each species. The comparative analysis suggests an inverse relationship between the activities of respiratory complexes I and II, as well as the overall activity of the canonical complexes and glycerol-3-phosphate dehydrogenase. Our comparative analysis shows that mitochondrial functions are highly variable in these versatile parasites. Copyright © 2014 Elsevier B.V. All rights reserved.

The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

PubMed Central

Braun, H P; Emmermann, M; Kruft, V; Schmitz, U K

1992-01-01

The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase. Images PMID:1324169

Diminished superoxide generation is associated with respiratory chain dysfunction and changes in the mitochondrial proteome of sensory neurons from diabetic rats.

PubMed

Akude, Eli; Zherebitskaya, Elena; Chowdhury, Subir K Roy; Smith, Darrell R; Dobrowsky, Rick T; Fernyhough, Paul

2011-01-01

Impairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome. Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in mitochondria from dorsal root ganglia (DRG) of control, 22-week-old streptozotocin (STZ)-diabetic rats, and diabetic rats treated with insulin. Rates of oxygen consumption and complex activities in mitochondria from DRG were measured. Fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix-specific reactive oxygen species (ROS). Proteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. For example, cytochrome c oxidase subunit IV (COX IV; a complex IV protein) and NADH dehydrogenase Fe-S protein 3 (NDUFS3; a complex I protein) were reduced by 29 and 36% (P < 0.05), respectively, in diabetes and confirmed previous Western blot studies. Respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin-induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control. Abnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. Alternative pathways involving polyol pathway activity appear to contribute to raised ROS in axons of diabetic neurons under high glucose concentration.

Selective inhibition of deactivated mitochondrial complex I by biguanides.

PubMed

Matsuzaki, Satoshi; Humphries, Kenneth M

2015-03-24

Biguanides are widely used antihyperglycemic agents for diabetes mellitus and prediabetes treatment. Complex I is the rate-limiting step of the mitochondrial electron transport chain (ETC), a major source of mitochondrial free radical production, and a known target of biguanides. Complex I has two reversible conformational states, active and de-active. The deactivated state is promoted in the absence of substrates but is rapidly and fully reversed to the active state in the presence of NADH. The objective of this study was to determine the relative sensitivity of active/de-active complex I to biguanide-mediated inhibition and resulting superoxide radical (O₂(•⁻)) production. Using isolated rat heart mitochondria, we show that deactivation of complex I sensitizes it to metformin and phenformin (4- and 3-fold, respectively), but not to other known complex I inhibitors, such as rotenone. Mitochondrial O₂(•⁻) production by deactivated complex I was measured fluorescently by NADH-dependent 2-hydroxyethidium formation at alkaline pH to impede reactivation. Superoxide production was 260.4% higher than in active complex I at pH 9.4. However, phenformin treatment of de-active complex I decreased O₂(•⁻) production by 14.9%, while rotenone increased production by 42.9%. Mitochondria isolated from rat hearts subjected to cardiac ischemia, a condition known to induce complex I deactivation, were sensitized to phenformin-mediated complex I inhibition. This supports the idea that the effects of biguanides are likely to be influenced by the complex I state in vivo. These results demonstrate that the complex I active and de-active states are a determinant in biguanide-mediated inhibition.

Anti-cancer analogues ME-143 and ME-344 exert toxicity by directly inhibiting mitochondrial NADH: ubiquinone oxidoreductase (Complex I).

PubMed

Lim, Sze Chern; Carey, Kirstyn T; McKenzie, Matthew

2015-01-01

Isoflavonoids have been shown to inhibit tumor proliferation and metastasis by activating cell death pathways. As such, they have been widely studied as potential therapies for cancer prevention. The second generation synthetic isoflavan analogues ME-143 and ME-344 also exhibit anti-cancer effects, however their specific molecular targets have not been completely defined. To identify these targets, we examined the effects of ME-143 and ME-344 on cellular metabolism and found that they are potent inhibitors of mitochondrial oxidative phosphorylation (OXPHOS) complex I (NADH: ubiquinone oxidoreductase) activity. In isolated HEK293T mitochondria, ME-143 and ME-344 reduced complex I activity to 14.3% and 28.6% of control values respectively. In addition to the inhibition of complex I, ME-344 also significantly inhibited mitochondrial complex III (ubiquinol: ferricytochrome-c oxidoreductase) activity by 10.8%. This inhibition of complex I activity (and to a lesser extent complex III activity) was associated with a reduction in mitochondrial oxygen consumption. In permeabilized HEK293T cells, ME-143 and ME-344 significantly reduced the maximum ADP-stimulated respiration rate to 62.3% and 70.0% of control levels respectively in the presence of complex I-linked substrates. Conversely, complex II-linked respiration was unaffected by either drug. We also observed that the inhibition of complex I-linked respiration caused the dissipation of the mitochondrial membrane potential (ΔΨm). Blue native (BN-PAGE) analysis revealed that prolonged loss of ΔΨm results in the destabilization of the native OXPHOS complexes. In particular, treatment of 143B osteosarcoma, HeLa and HEK293T human embryonic kidney cells with ME-344 for 4 h resulted in reduced steady-state levels of mature complex I. Degradation of the complex I subunit NDUFA9, as well as the complex IV (ferrocytochrome c: oxygen oxidoreductase) subunit COXIV, was also evident. The identification of OXPHOS complex I as a target of ME-143 and ME-344 advances our understanding of how these drugs induce cell death by disrupting mitochondrial metabolism, and will direct future work to maximize the anti-cancer capacity of these and other isoflavone-based compounds.

High fat, high sucrose diet causes cardiac mitochondrial dysfunction due in part to oxidative post-translational modification of mitochondrial complex II

PubMed Central

Sverdlov, Aaron L.; Elezaby, Aly; Behring, Jessica B.; Bachschmid, Markus M.; Luptak, Ivan; Tu, Vivian H.; Siwik, Deborah A.; Miller, Edward J.; Liesa, Marc; Shirihai, Orian S; Pimentel, David R.; Cohen, Richard A.; Colucci, Wilson S.

2014-01-01

Background Diet-induced obesity leads to metabolic heart disease (MHD) characterized by increased oxidative stress that may cause oxidative post-translational modifications (OPTM) of cardiac mitochondrial proteins. The functional consequences of OPTM of cardiac mitochondrial proteins in MHD are unknown. Our objective was to determine whether cardiac mitochondrial dysfunction in MHD due to diet-induced obesity is associated with cysteine OPTM. Methods and results Male C57Bl/6J mice were fed either a high-fat, high-sucrose (HFHS) or control diet for 8 months. Cardiac mitochondria from HFHS-fed mice (vs. control diet) had an increased rate of H2O2 production, a decreased GSH/GSSG ratio, a decreased rate of complex II substrate-driven ATP synthesis and decreased complex II activity. Complex II substrate-driven ATP synthesis and complex II activity were partially restored ex-vivo by reducing conditions. A biotin switch assay showed that HFHS feeding increased cysteine OPTM in complex II subunits A (SDHA) and B (SDHB). Using iodo-TMT multiplex tags we found that HFHS feeding is associated with reversible oxidation of cysteines 89 and 231 in SDHA, and 100, 103 and 115 in SDHB. Conclusions MHD due to consumption of a HFHS “Western” diet causes increased H2O2 production and oxidative stress in cardiac mitochondria associated with decreased ATP synthesis and decreased complex II activity. Impaired complex II activity and ATP production are associated with reversible cysteine OPTM of complex II. Possible sites of reversible cysteine OPTM in SDHA and SDHB were identified by iodo-TMT tag labeling. Mitochondrial ROS may contribute to the pathophysiology of MHD by impairing the function of complex II. PMID:25109264

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yadav, N.; Kumar, S.; Marlowe, T.

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrialmore » biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency whereas a reverse trend was observed with apicidin. Together, these finding provide a new strategy for differential mitochondrial targeting in cancer therapy.« less

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents

DOE PAGES

Yadav, N.; Kumar, S.; Marlowe, T.; ...

2015-11-05

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrialmore » biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency whereas a reverse trend was observed with apicidin. Together, these finding provide a new strategy for differential mitochondrial targeting in cancer therapy.« less

High fat, high sucrose diet causes cardiac mitochondrial dysfunction due in part to oxidative post-translational modification of mitochondrial complex II.

PubMed

Sverdlov, Aaron L; Elezaby, Aly; Behring, Jessica B; Bachschmid, Markus M; Luptak, Ivan; Tu, Vivian H; Siwik, Deborah A; Miller, Edward J; Liesa, Marc; Shirihai, Orian S; Pimentel, David R; Cohen, Richard A; Colucci, Wilson S

2015-01-01

Diet-induced obesity leads to metabolic heart disease (MHD) characterized by increased oxidative stress that may cause oxidative post-translational modifications (OPTM) of cardiac mitochondrial proteins. The functional consequences of OPTM of cardiac mitochondrial proteins in MHD are unknown. Our objective was to determine whether cardiac mitochondrial dysfunction in MHD due to diet-induced obesity is associated with cysteine OPTM. Male C57BL/6J mice were fed either a high-fat, high-sucrose (HFHS) or control diet for 8months. Cardiac mitochondria from HFHS-fed mice (vs. control diet) had an increased rate of H2O2 production, a decreased GSH/GSSG ratio, a decreased rate of complex II substrate-driven ATP synthesis and decreased complex II activity. Complex II substrate-driven ATP synthesis and complex II activity were partially restored ex-vivo by reducing conditions. A biotin switch assay showed that HFHS feeding increased cysteine OPTM in complex II subunits A (SDHA) and B (SDHB). Using iodo-TMT multiplex tags we found that HFHS feeding is associated with reversible oxidation of cysteines 89 and 231 in SDHA, and 100, 103 and 115 in SDHB. MHD due to consumption of a HFHS "Western" diet causes increased H2O2 production and oxidative stress in cardiac mitochondria associated with decreased ATP synthesis and decreased complex II activity. Impaired complex II activity and ATP production are associated with reversible cysteine OPTM of complex II. Possible sites of reversible cysteine OPTM in SDHA and SDHB were identified by iodo-TMT tag labeling. Mitochondrial ROS may contribute to the pathophysiology of MHD by impairing the function of complex II. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease". Copyright © 2014 Elsevier Ltd. All rights reserved.

The Candida albicans Inhibitory Activity of the Extract from Papaya (Carica papaya L.) Seed Relates to Mitochondria Dysfunction.

PubMed

Zhang, Tao; Chen, Weijun

2017-08-25

The inhibitory activity of the papaya seed extract (PSE) on Candida albicans ( C. albicans ) was determined by turbidimetry method. The inhibitory mechanisms were also evaluated from the prospective of reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) decrease, and the activities of four complex enzymes in mitochondria respiratory chain. Results obtained from this study indicated that the PSE exhibited an effective inhibitory activity on C. albicans and induced significant accumulation of ROS and collapse of MMP. The Complex I and Complex III exhibited continues significant decrease in mitochondrial enzyme activity assays, but the Complex II and Complex IV activities were not positively correlated. Furthermore, the GC-MS analysis demonstrated that the PSE represents a rich and high-purity source of benzyl isothiocyanate (BITC), which indicated the BITC may be responsible for the mitochondrial dysfunction.

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at

2012-01-20

Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less

Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

PubMed

Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

2015-06-01

Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic derangement. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Selective Inhibition of Deactivated Mitochondrial Complex I by Biguanides †

PubMed Central

Matsuzaki, Satoshi; Humphries, Kenneth M.

2015-01-01

Biguanides are widely used antihyperglycemic agents for diabetes mellitus and prediabetes treatment. Complex I is the rate limiting step of the mitochondrial electron transport chain (ETC), a major source of mitochondrial free radical production, and a known target of biguanides. Complex I has two reversible conformational states, active and de-active. The deactivated state is promoted in the absence of substrates, but is rapidly and fully reversed to the active state in the presence of NADH. The objective of this study was to determine the relative sensitivity of active/de-active complex I to biguanide-mediated inhibition and resulting superoxide radical (O2•−) production. Using isolated rat heart mitochondria, we show that deactivation of complex I sensitizes it to metformin and phenformin (4- and 3-fold, respectively), but not to other known complex I inhibitors, such as rotenone. Mitochondrial O2•− production by deactivated complex I was measured fluorescently by the NADH-dependent 2-hydroxyethidium formation at alkaline pH to impede reactivation. Superoxide production was 260.4% higher than in active complex I at pH 9.4. However, phenformin treatment of de-active complex I decreased O2•− production by 14.9% while rotenone increased production by 42.9%. Mitochondria isolated from rat hearts subjected to cardiac ischemia, a condition known to induce complex I deactivation, were sensitized to phenformin:mediated complex I inhibition. This supports that the effects of biguanides are likely to be influenced by the complex I state in vivo. These results demonstrate that the complex I active/de-active states are a determinant in biguanide-mediated inhibition. PMID:25719498

Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae).

PubMed

VenkatRao, V; Chaitanya, R K; Naresh Kumar, D; Bramhaiah, M; Dutta-Gupta, A

2016-12-01

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F 1 F 0 ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein kinase C-ε activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules

PubMed Central

Bakajsova, Diana; Samarel, Allen M.

2011-01-01

PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307–F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F0F1-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F0F1-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC. PMID:21289057

Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify their functional properties.

PubMed

Drahota, Z; Palenickova, E; Endlicher, R; Milerova, M; Brejchova, J; Vosahlikova, M; Svoboda, P; Kazdova, L; Kalous, M; Cervinkova, Z; Cahova, M

2014-01-01

In this study, we focused on an analysis of biguanides effects on mitochondrial enzyme activities, mitochondrial membrane potential and membrane permeability transition pore function. We used phenformin, which is more efficient than metformin, and evaluated its effect on rat liver mitochondria and isolated hepatocytes. In contrast to previously published data, we found that phenformin, after a 5 min pre-incubation, dose-dependently inhibits not only mitochondrial complex I but also complex II and IV activity in isolated mitochondria. The enzymes complexes inhibition is paralleled by the decreased respiratory control index and mitochondrial membrane potential. Direct measurements of mitochondrial swelling revealed that phenformin increases the resistance of the permeability transition pore to Ca(2+) ions. Our data might be in agreement with the hypothesis of Schäfer (1976) that binding of biguanides to membrane phospholipids alters membrane properties in a non-specific manner and, subsequently, different enzyme activities are modified via lipid phase. However, our measurements of anisotropy of fluorescence of hydrophobic membrane probe diphenylhexatriene have not shown a measurable effect of membrane fluidity with the 1 mM concentration of phenformin that strongly inhibited complex I activity. Our data therefore suggest that biguanides could be considered as agents with high efficacy but low specifity.

m-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics.

PubMed

Consolato, Francesco; Maltecca, Francesca; Tulli, Susanna; Sambri, Irene; Casari, Giorgio

2018-04-09

The proteolytic processing of dynamin-like GTPase OPA1, mediated by the activity of both YME1L1 [intermembrane (i)-AAA protease complex] and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 [matrix (m) - AAA complex] as the major protease mediating this event, which acts by maturing the 60 kDa pre-pro-OMA1 to the 40 kDa pro-OMA1 form by severing the N-terminal portion without recognizing a specific consensus sequence. Therefore, m - AAA and i - AAA complexes coordinately regulate OMA1 processing and turnover, and consequently control which OPA1 isoforms are present, thus adding new information on the molecular mechanisms of mitochondrial dynamics and neurodegenerative diseases affected by these phenomena.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

Adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors

PubMed Central

Lund, Kaleb C.; Wallace, Kendall B.

2008-01-01

Nucleoside analog reverse transcriptase inhibitors (NRTI) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3′-azido-3′-deoxythymidine (AZT; 10 and 50 μM), AZT monophosphate (150 μM), and 2′,3′-dideoxycytidine (ddC; 1μM) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2′,3′-dideoxyinosine (ddI; 10 μM) and ddC (1 μM). In the presence of succinate + cAMP AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-γ activity; in the case of AZT these observations may provide a mechanism for the observed long-term toxicity with this drug. PMID:17904600

Altered mitochondrial function and oxidative stress in leukocytes of anorexia nervosa patients.

PubMed

Victor, Victor M; Rovira-Llopis, Susana; Saiz-Alarcon, Vanessa; Sangüesa, Maria C; Rojo-Bofill, Luis; Bañuls, Celia; Falcón, Rosa; Castelló, Raquel; Rojo, Luis; Rocha, Milagros; Hernández-Mijares, Antonio

2014-01-01

Anorexia nervosa is a common illness among adolescents and is characterised by oxidative stress. The effects of anorexia on mitochondrial function and redox state in leukocytes from anorexic subjects were evaluated. A multi-centre, cross-sectional case-control study was performed. Our study population consisted of 20 anorexic patients and 20 age-matched controls, all of which were Caucasian women. Anthropometric and metabolic parameters were evaluated in the study population. To assess whether anorexia nervosa affects mitochondrial function and redox state in leukocytes of anorexic patients, we measured mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels, mitochondrial mass, and complex I and III activity in polymorphonuclear cells. Mitochondrial function was impaired in the leukocytes of the anorexic patients. This was evident in a decrease in mitochondrial O2 consumption (P<0.05), mitochondrial membrane potential (P<0.01) and GSH levels (P<0.05), and an increase in ROS production (P<0.05) with respect to control subjects. Furthermore, a reduction of mitochondrial mass was detected in leukocytes of the anorexic patients (P<0.05), while the activity of mitochondrial complex I (P<0.001), but not that of complex III, was found to be inhibited in the same population. Oxidative stress is produced in the leukocytes of anorexic patients and is closely related to mitochondrial dysfunction. Our results lead us to propose that the oxidative stress that occurs in anorexia takes place at mitochondrial complex I. Future research concerning mitochondrial dysfunction and oxidative stress should aim to determine the physiological mechanism involved in this effect and the physiological impact of anorexia.

SIRT3 aggravates metformin-induced energy stress and apoptosis in ovarian cancer cells.

PubMed

Wu, Yao; Gao, Wei-Nan; Xue, Ya-Nan; Zhang, Li-Chao; Zhang, Juan-Juan; Lu, Sheng-Yao; Yan, Xiao-Yu; Yu, Hui-Mei; Su, Jing; Sun, Lian-Kun

2018-06-15

Increasing evidence suggests that mitochondrial respiratory chain complex I participates in carcinogenesis and cancer progression by providing energy and maintaining mitochondrial function. However, the role of complex I in ovarian cancer is largely unknown. In this study we showed that metformin, considered to be an inhibitor of complex I, simultaneously inhibited cell growth and induced mitochondrial-related apoptosis in human ovarian cancer cells. Metformin interrupted cellular energy metabolism mainly by causing damage to complex I that impacted mitochondrial function. Additionally, treatment with metformin increased the activation of sirtuin 3 (SIRT3), a mitochondrial deacetylase. We demonstrated that SIRT3 overexpression aggravated metformin-induced apoptosis, energy stress and mitochondrial dysfunction. Moreover, treatment with metformin or SIRT3 overexpression increased activation of AMP-activated protein kinase (AMPK), a major sensor of cellular energy status. AMPK compensated for energy loss by increasing glycolysis. The impact of this was assessed by reducing glucose levels in the media or by using inhibitors (2-deoxyglucose, Compound C) of glycolysis and AMPK. The combination of these factors with metformin intensified cytotoxicity through further downregulation of ATP. Our study outlines an important role for SIRT3 in the antitumor effect of mitochondrial complex I inhibitors in human ovarian cancer cells. This effect appears to be mediated by induction of energy stress and apoptosis. Strategies that target the mitochondria could be enhanced by modulating glycolysis to further aggravate energy stress that may increase the antitumor effect. Copyright © 2018 Elsevier Inc. All rights reserved.

Reduced Mitochondrial Activity is Early and Steady in the Entorhinal Cortex but it is Mainly Unmodified in the Frontal Cortex in Alzheimer's Disease.

PubMed

Armand-Ugon, Mercedes; Ansoleaga, Belen; Berjaoui, Sara; Ferrer, Isidro

2017-01-01

It is well established that mitochondrial damage plays a role in the pathophysiology of Alzheimer's disease (AD). However, studies carried out in humans barely contemplate regional differences with disease progression. To study the expression of selected nuclear genes encoding subunits of the mitochondrial complexes and the activity of mitochondrial complexes in AD, in two regions: the entorhinal cortex (EC) and frontal cortex area 8 (FC). Frozen samples from 148 cases processed for gene expression by qRT-PCR and determination of individual activities of mitochondrial complexes I, II, IV and V using commercial kits and home-made assays. Decreased expression of NDUFA2, NDUFB3, UQCR11, COX7C, ATPD, ATP5L and ATP50, covering subunits of complex I, II, IV and V, occurs in total homogenates of the EC in AD stages V-VI when compared with stages I-II. However reduced activity of complexes I, II and V of isolated mitochondria occurs as early as stages I-II when compared with middle-aged individuals in the EC. In contrast, no alterations in the expression of the same genes and no alterations in the activity of mitochondrial complexes are found in the FC in the same series. Different mechanisms of impaired energy metabolism may occur in AD, one of them, represented by the EC, is the result of primary and early alteration of mitochondria; the other one is probably the result, at least in part, of decreased functional input and is represented by hypometabolism in the FC in AD patients aged 86 or younger. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Anti-cancer analogues ME-143 and ME-344 exert toxicity by directly inhibiting mitochondrial NADH: ubiquinone oxidoreductase (Complex I)

PubMed Central

Lim, Sze Chern; Carey, Kirstyn T; McKenzie, Matthew

2015-01-01

Isoflavonoids have been shown to inhibit tumor proliferation and metastasis by activating cell death pathways. As such, they have been widely studied as potential therapies for cancer prevention. The second generation synthetic isoflavan analogues ME-143 and ME-344 also exhibit anti-cancer effects, however their specific molecular targets have not been completely defined. To identify these targets, we examined the effects of ME-143 and ME-344 on cellular metabolism and found that they are potent inhibitors of mitochondrial oxidative phosphorylation (OXPHOS) complex I (NADH: ubiquinone oxidoreductase) activity. In isolated HEK293T mitochondria, ME-143 and ME-344 reduced complex I activity to 14.3% and 28.6% of control values respectively. In addition to the inhibition of complex I, ME-344 also significantly inhibited mitochondrial complex III (ubiquinol: ferricytochrome-c oxidoreductase) activity by 10.8%. This inhibition of complex I activity (and to a lesser extent complex III activity) was associated with a reduction in mitochondrial oxygen consumption. In permeabilized HEK293T cells, ME-143 and ME-344 significantly reduced the maximum ADP-stimulated respiration rate to 62.3% and 70.0% of control levels respectively in the presence of complex I-linked substrates. Conversely, complex II-linked respiration was unaffected by either drug. We also observed that the inhibition of complex I-linked respiration caused the dissipation of the mitochondrial membrane potential (ΔΨm). Blue native (BN-PAGE) analysis revealed that prolonged loss of ΔΨm results in the destabilization of the native OXPHOS complexes. In particular, treatment of 143B osteosarcoma, HeLa and HEK293T human embryonic kidney cells with ME-344 for 4 h resulted in reduced steady-state levels of mature complex I. Degradation of the complex I subunit NDUFA9, as well as the complex IV (ferrocytochrome c: oxygen oxidoreductase) subunit COXIV, was also evident. The identification of OXPHOS complex I as a target of ME-143 and ME-344 advances our understanding of how these drugs induce cell death by disrupting mitochondrial metabolism, and will direct future work to maximize the anti-cancer capacity of these and other isoflavone-based compounds. PMID:25973307

In Vitro Effects of Cognitives and Nootropics on Mitochondrial Respiration and Monoamine Oxidase Activity.

PubMed

Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

2017-10-01

Impairment of mitochondrial metabolism, particularly the electron transport chain (ETC), as well as increased oxidative stress might play a significant role in pathogenesis of Alzheimer's disease (AD). Some effects of drugs used for symptomatic AD treatment may be related to their direct action on mitochondrial function. In vitro effects of pharmacologically different cognitives (galantamine, donepezil, rivastigmine, 7-MEOTA, memantine) and nootropic drugs (latrepirdine, piracetam) were investigated on selected mitochondrial parameters: activities of ETC complexes I, II + III, and IV, citrate synthase, monoamine oxidase (MAO), oxygen consumption rate, and hydrogen peroxide production of pig brain mitochondria. Complex I activity was decreased by galantamine, donepezil, and memantine; complex II + III activity was increased by galantamine. None of the tested drugs caused significant changes in the rate of mitochondrial oxygen consumption, even at high concentrations. Except galantamine, all tested drugs were selective MAO-A inhibitors. Latrepirdine, donepezil, and 7-MEOTA were found to be the most potent MAO-A inhibitors. Succinate-induced mitochondrial hydrogen peroxide production was not significantly affected by the drugs tested. The direct effect of cognitives and nootropics used in the treatment of AD on mitochondrial respiration is relatively small. The safest drugs in terms of disturbing mitochondrial function appear to be piracetam and rivastigmine. The MAO-A inhibition by cognitives and nootropics may also participate in mitochondrial neuroprotection. The results support the future research aimed at measuring the effects of currently used drugs or newly synthesized drugs on mitochondrial functioning in order to understand their mechanism of action.

Mitochondrial respiratory chain enzyme assay and DNA analysis in peripheral blood leukocytes for the etiological study of Chinese children with Leigh syndrome due to complex I deficiency.

PubMed

Ma, Yan Yan; Wu, Tong Fei; Liu, Yu Peng; Wang, Qiao; Li, Xi Yuan; Zhang, Yao; Song, Jin Qing; Wang, Yu Jie; Yang, Yan Ling

2013-02-01

Mitochondrial respiratory chain complex I enzyme deficiency is the most commonly seen mitochondrial respiratory chain disorder. Although screening and diagnostic methods are available overseas, clinically feasible diagnostic methods have not yet been established in China. In this study, four Chinese boys with Leigh syndrome due to complex I deficiency were diagnosed by mitochondrial respiratory chain enzyme assay and DNA analysis using peripheral blood leukocytes. Four patients were admitted at the age of 5-14 years because of unexplained progressive neuromuscular symptoms, including motor developmental delay or regression, weakness, and seizures. Their cranial magnetic resonance imaging revealed typical finding as Leigh syndrome. Peripheral leukocyte mitochondrial respiratory chain complex I activities were found decreased to 9.6-33.1 nmol/min/mg mitochondrial protein(control 44.0 ± 5.4 nmol/min/mg). The ratios of complex I to citrate synthase activity were also decreased (8.9-19.8% in patients vs. control 48 ± 11%). Three mtDNA mutations were identified from three out of four patients, supporting the diagnosis of complex I deficiency. Point mutations m.10191T>C in mitochondrial ND3 gene, m.13513G>A in ND5 gene and m.14,453G>A in ND6 gene were detected in three patients.

Microglial activation and the nitric oxide/cGMP/PKG pathway underlie enhanced neuronal vulnerability to mitochondrial dysfunction in experimental multiple sclerosis.

PubMed

Mancini, Andrea; Tantucci, Michela; Mazzocchetti, Petra; de Iure, Antonio; Durante, Valentina; Macchioni, Lara; Giampà, Carmela; Alvino, Alessandra; Gaetani, Lorenzo; Costa, Cinzia; Tozzi, Alessandro; Calabresi, Paolo; Di Filippo, Massimiliano

2018-05-01

During multiple sclerosis (MS), a close link has been demonstrated to occur between inflammation and neuro-axonal degeneration, leading to the hypothesis that immune mechanisms may promote neurodegeneration, leading to irreversible disease progression. Energy deficits and inflammation-driven mitochondrial dysfunction seem to be involved in this process. In this work we investigated, by the use of striatal electrophysiological field-potential recordings, if the inflammatory process associated with experimental autoimmune encephalomyelitis (EAE) is able to influence neuronal vulnerability to the blockade of mitochondrial complex IV, a crucial component for mitochondrial activity responsible of about 90% of total cellular oxygen consumption. We showed that during the acute relapsing phase of EAE, neuronal susceptibility to mitochondrial complex IV inhibition is markedly enhanced. This detrimental effect was counteracted by the pharmacological inhibition of microglia, of nitric oxide (NO) synthesis and its intracellular pathway (involving soluble guanylyl cyclase, sGC, and protein kinase G, PKG). The obtained results suggest that mitochondrial complex IV exerts an important role in maintaining neuronal energetic homeostasis during EAE. The pathological processes associated with experimental MS, and in particular the activation of microglia and of the NO pathway, lead to an increased neuronal vulnerability to mitochondrial complex IV inhibition, representing promising pharmacological targets. Copyright © 2018 Elsevier Inc. All rights reserved.

Mitochondrial Complex 1 Activity Measured by Spectrophotometry Is Reduced across All Brain Regions in Ageing and More Specifically in Neurodegeneration.

PubMed

Pollard, Amelia Kate; Craig, Emma Louise; Chakrabarti, Lisa

2016-01-01

Mitochondrial function, in particular complex 1 of the electron transport chain (ETC), has been shown to decrease during normal ageing and in neurodegenerative disease. However, there is some debate concerning which area of the brain has the greatest complex 1 activity. It is important to identify the pattern of activity in order to be able to gauge the effect of age or disease related changes. We determined complex 1 activity spectrophotometrically in the cortex, brainstem and cerebellum of middle aged mice (70-71 weeks), a cerebellar ataxic neurodegeneration model (pcd5J) and young wild type controls. We share our updated protocol on the measurements of complex1 activity and find that mitochondrial fractions isolated from frozen tissues can be measured for robust activity. We show that complex 1 activity is clearly highest in the cortex when compared with brainstem and cerebellum (p<0.003). Cerebellum and brainstem mitochondria exhibit similar levels of complex 1 activity in wild type brains. In the aged brain we see similar levels of complex 1 activity in all three-brain regions. The specific activity of complex 1 measured in the aged cortex is significantly decreased when compared with controls (p<0.0001). Both the cerebellum and brainstem mitochondria also show significantly reduced activity with ageing (p<0.05). The mouse model of ataxia predictably has a lower complex 1 activity in the cerebellum, and although reductions are measured in the cortex and brain stem, the remaining activity is higher than in the aged brains. We present clear evidence that complex 1 activity decreases across the brain with age and much more specifically in the cerebellum of the pcd5j mouse. Mitochondrial impairment can be a region specific phenomenon in disease, but in ageing appears to affect the entire brain, abolishing the pattern of higher activity in cortical regions.

Tempol, a Superoxide Dismutase Mimetic Agent, Ameliorates Cisplatin-Induced Nephrotoxicity through Alleviation of Mitochondrial Dysfunction in Mice

PubMed Central

Ahmed, Lamiaa A.; Shehata, Nagwa I.; Abdelkader, Noha F.; Khattab, Mahmoud M.

2014-01-01

Background Mitochondrial dysfunction is a crucial mechanism by which cisplatin, a potent chemotherapeutic agent, causes nephrotoxicity where mitochondrial electron transport complexes are shifted mostly toward imbalanced reactive oxygen species versus energy production. In the present study, the protective role of tempol, a membrane-permeable superoxide dismutase mimetic agent, was evaluated on mitochondrial dysfunction and the subsequent damage induced by cisplatin nephrotoxicity in mice. Methods and Findings Nephrotoxicity was assessed 72 h after a single i.p. injection of cisplatin (25 mg/kg) with or without oral administration of tempol (100 mg/kg/day). Serum creatinine and urea as well as glucosuria and proteinuria were evaluated. Both kidneys were isolated for estimation of oxidative stress markers, adenosine triphosphate (ATP) content and caspase-3 activity. Moreover, mitochondrial oxidative phosphorylation capacity, complexes I–IV activities and mitochondrial nitric oxide synthase (mNOS) protein expression were measured along with histological examinations of renal tubular damage and mitochondrial ultrastructural changes. Tempol was effective against cisplatin-induced elevation of serum creatinine and urea as well as glucosuria and proteinuria. Moreover, pretreatment with tempol notably inhibited cisplatin-induced oxidative stress and disruption of mitochondrial function by restoring mitochondrial oxidative phosphorylation, complexes I and III activities, mNOS protein expression and ATP content. Tempol also provided significant protection against apoptosis, tubular damage and mitochondrial ultrastructural changes. Interestingly, tempol did not interfere with the cytotoxic effect of cisplatin against the growth of solid Ehrlich carcinoma. Conclusion This study highlights the potential role of tempol in inhibiting cisplatin-induced nephrotoxicity without affecting its antitumor activity via amelioration of oxidative stress and mitochondrial dysfunction. PMID:25271439

Chronic exposure to nitric oxide alters the free iron pool in endothelial cells: Role of mitochondrial respiratory complexes and heat shock proteins

PubMed Central

Ramachandran, Anup; Ceaser, Erin; Darley-Usmar, Victor M.

2004-01-01

The mechanisms of nitric oxide (NO) signaling include binding to the iron centers in soluble guanylate cyclase and cytochrome c oxidase and posttranslational modification of proteins by S-nitrosation. Low levels of NO control mitochondrial number in cells, but little is known of the impact of chronic exposure to high levels of NO on mitochondrial function in endothelial cells. The focus of this study is the interaction of NO with mitochondrial respiratory complexes in cell culture and the effect this has on iron homeostasis. We demonstrate that chronic exposure of endothelial cells to NO decreased activity and protein levels of complexes I, II, and IV, whereas citrate synthase and ATP synthase were unaffected. Inhibition of these respiratory complexes was accompanied by an increase in cellular S-nitrosothiol levels, modification of cysteines residues, and an increase in the labile iron pool. The NO-dependent increase in the free iron pool and inhibition of complex II was prevented by inhibition of mitochondrial protein synthesis, consistent with a major contribution of the organelle to iron homeostasis. In addition, inhibition of mitochondrial protein synthesis was associated with an increase in heat shock protein 60 levels, which may be an additional mechanism leading to preservation of complex II activity. PMID:14691259

Lipoic acid metabolism and mitochondrial redox regulation.

PubMed

Solmonson, Ashley D; DeBerardinis, Ralph J

2017-11-30

Lipoic acid is an essential cofactor for mitochondrial metabolism and is synthesized de novo using intermediates from mitochondrial fatty acid synthesis type II, S-adenosylmethionine and iron-sulfur clusters. This cofactor is required for catalysis by multiple mitochondrial 2-ketoacid dehydrogenase complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase. Lipoic acid also plays a critical role in stabilizing and regulating these multi-enzyme complexes.  Many of these dehydrogenases are regulated by reactive oxygen species, mediated through the disulfide bond of the prosthetic lipoyl moiety.  Collectively, its functions explain why lipoic acid is required for cell growth, mitochondrial activity and coordination of fuel metabolism. Lipoic acid is an essential cofactor for mitochondrial metabolism and is synthesized de novo using intermediates from mitochondrial fatty acid synthesis type II, S-adenosylmethionine and iron-sulfur clusters. This cofactor is required for catalysis by multiple mitochondrial 2-ketoacid dehydrogenase complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase. Lipoic acid also plays a critical role in stabilizing and regulating these multi-enzyme complexes.  Many of these dehydrogenases are regulated by reactive oxygen species, mediated through the disulfide bond of the prosthetic lipoyl moiety.  Collectively, its functions explain why lipoic acid is required for cell growth, mitochondrial activity and coordination of fuel metabolism. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

Hydroxytyrosol prevents diet-induced metabolic syndrome and attenuates mitochondrial abnormalities in obese mice.

PubMed

Cao, Ke; Xu, Jie; Zou, Xuan; Li, Yuan; Chen, Cong; Zheng, Adi; Li, Hao; Li, Hua; Szeto, Ignatius Man-Yau; Shi, Yujie; Long, Jiangang; Liu, Jiankang; Feng, Zhihui

2014-02-01

A Mediterranean diet rich in olive oil has profound influence on health outcomes including metabolic syndrome. However, the active compound and detailed mechanisms still remain unclear. Hydroxytyrosol (HT), a major polyphenolic compound in virgin olive oil, has received increased attention for its antioxidative activity and regulation of mitochondrial function. Here, we investigated whether HT is the active compound in olive oil exerting a protective effect against metabolic syndrome. In this study, we show that HT could prevent high-fat-diet (HFD)-induced obesity, hyperglycemia, hyperlipidemia, and insulin resistance in C57BL/6J mice after 17 weeks supplementation. Within liver and skeletal muscle tissues, HT could decrease HFD-induced lipid deposits through inhibition of the SREBP-1c/FAS pathway, ameliorate HFD-induced oxidative stress by enhancing antioxidant enzyme activities, normalize expression of mitochondrial complex subunits and mitochondrial fission marker Drp1, and eventually inhibit apoptosis activation. Moreover, in muscle tissue, the levels of mitochondrial carbonyl protein were decreased and mitochondrial complex activities were significantly improved by HT supplementation. In db/db mice, HT significantly decreased fasting glucose, similar to metformin. Notably, HT decreased serum lipid, at which metformin failed. Also, HT was more effective at decreasing the oxidation levels of lipids and proteins in both liver and muscle tissue. Similar to the results in the HFD model, HT decreased muscle mitochondrial carbonyl protein levels and improved mitochondrial complex activities in db/db mice. Our study links the olive oil component HT to diabetes and metabolic disease through changes that are not limited to decreases in oxidative stress, suggesting a potential pharmaceutical or clinical use of HT in metabolic syndrome treatment. Copyright © 2013 Elsevier Inc. All rights reserved.

Mitochondrial Changes in Platelets Are Not Related to Those in Skeletal Muscle during Human Septic Shock

PubMed Central

Protti, Alessandro; Fortunato, Francesco; Caspani, Maria L.; Pluderi, Mauro; Lucchini, Valeria; Grimoldi, Nadia; Solimeno, Luigi P.; Fagiolari, Gigliola; Ciscato, Patrizia; Zella, Samis M. A.; Moggio, Maurizio; Comi, Giacomo P.; Gattinoni, Luciano

2014-01-01

Platelets can serve as general markers of mitochondrial (dys)function during several human diseases. Whether this holds true even during sepsis is unknown. Using spectrophotometry, we measured mitochondrial respiratory chain biochemistry in platelets and triceps brachii muscle of thirty patients with septic shock (within 24 hours from admission to Intensive Care) and ten surgical controls (during surgery). Results were expressed relative to citrate synthase (CS) activity, a marker of mitochondrial density. Patients with septic shock had lower nicotinamide adenine dinucleotide dehydrogenase (NADH)/CS (p = 0.015), complex I/CS (p = 0.018), complex I and III/CS (p<0.001) and complex IV/CS (p = 0.012) activities in platelets but higher complex I/CS activity (p = 0.021) in triceps brachii muscle than controls. Overall, NADH/CS (r2 = 0.00; p = 0.683) complex I/CS (r2 = 0.05; p = 0.173), complex I and III/CS (r2 = 0.01; p = 0.485), succinate dehydrogenase (SDH)/CS (r2 = 0.00; p = 0.884), complex II and III/CS (r2 = 0.00; p = 0.927) and complex IV/CS (r2 = 0.00; p = 0.906) activities in platelets were not associated with those in triceps brachii muscle. In conclusion, several respiratory chain enzymes were variably inhibited in platelets, but not in triceps brachii muscle, of patients with septic shock. Sepsis-induced mitochondrial changes in platelets do not reflect those in other organs. PMID:24787741

Tongluo Xingnao Effervescent Tablet preserves mitochondrial energy metabolism and attenuates cognition deficits in APPswe/PS1De9 mice.

PubMed

Dai, Yuan; Ma, Tao; Ren, Xiangyi; Wei, Jiangping; Fu, Wenjun; Ma, Yuntong; Xu, Shijun; Zhang, Zhanjun

2016-09-06

Tongluo Xingnao Effervescent Tablet (TXET), a traditional Chinese herbal formula composed of Ligusticum chuanxiong hor, Scutellaria baicalensis Georgi and Angelica sinensis, has been widely used to treat Alzheimer's disease (AD) and related dementias for decades in China. In the present study, we investigated the effects of TXET on mitochondrial function, energy metabolism and cognitive amelioration in the APPswe/PS1De9 transgenetic mouse model of AD. The energy charge and phosphocreatine, activity of the mitochondrial electron transport chain complexes, mitochondrial membrane potential, activity of Na(+)-K(+) ATPase and the expression levels of Bcl-2 and Bax in the brains were measured, respectively. TXET exhibits significant protection on mitochondrial function and energy supply in addition to ameliorating cognitive decline in APPswe/PS1De9 mice. TXET rescues mitochondrial function by increasing the mitochondrial membrane potential, energy charge levels, activity of respiratory chain complexes and Na(+)-K(+) ATPase activity. These findings suggest that TXET may attenuate cognition impairment through the restoration of mitochondrial function and energy metabolism in the brains in APPswe/PS1De9 mice. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Apolipoprotein E4 (1-272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells.

PubMed

Nakamura, Toshiyuki; Watanabe, Atsushi; Fujino, Takahiro; Hosono, Takashi; Michikawa, Makoto

2009-08-20

Apolipoprotein E allele epsilon4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1-272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1-272) more strongly than intact apoE4(1-299). Further analysis showed that in Neuro-2a cells expressing apoE4(1-272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1-299). ApoE4(1-272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1-272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration.

Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure.

PubMed

Rurek, Michal; Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

2015-01-01

The biogenesis of the cauliflower curd mitochondrial proteome was investigated under cold, heat and the recovery. For the first time, two dimensional fluorescence difference gel electrophoresis was used to study the plant mitochondrial complexome in heat and heat recovery. Particularly, changes in the complex I and complex III subunits and import proteins, and the partial disintegration of matrix complexes were observed. The presence of unassembled subunits of ATP synthase was accompanied by impairment in mitochondrial translation of its subunit. In cold and heat, the transcription profiles of mitochondrial genes were uncorrelated. The in-gel activities of respiratory complexes were particularly affected after stress recovery. Despite a general stability of respiratory chain complexes in heat, functional studies showed that their activity and the ATP synthesis yield were affected. Contrary to cold stress, heat stress resulted in a reduced efficiency of oxidative phosphorylation likely due to changes in alternative oxidase (AOX) activity. Stress and stress recovery differently modulated the protein level and activity of AOX. Heat stress induced an increase in AOX activity and protein level, and AOX1a and AOX1d transcript level, while heat recovery reversed the AOX protein and activity changes. Conversely, cold stress led to a decrease in AOX activity (and protein level), which was reversed after cold recovery. Thus, cauliflower AOX is only induced by heat stress. In heat, contrary to the AOX activity, the activity of rotenone-insensitive internal NADH dehydrogenase was diminished. The relevance of various steps of plant mitochondrial biogenesis to temperature stress response and recovery is discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

In female rat heart mitochondria, oophorectomy results in loss of oxidative phosphorylation.

PubMed

Pavón, Natalia; Cabrera-Orefice, Alfredo; Gallardo-Pérez, Juan Carlos; Uribe-Alvarez, Cristina; Rivero-Segura, Nadia A; Vazquez-Martínez, Edgar Ricardo; Cerbón, Marco; Martínez-Abundis, Eduardo; Torres-Narvaez, Juan Carlos; Martínez-Memije, Raúl; Roldán-Gómez, Francisco-Javier; Uribe-Carvajal, Salvador

2017-02-01

Oophorectomy in adult rats affected cardiac mitochondrial function. Progression of mitochondrial alterations was assessed at one, two and three months after surgery: at one month, very slight changes were observed, which increased at two and three months. Gradual effects included decrease in the rates of oxygen consumption and in respiratory uncoupling in the presence of complex I substrates, as well as compromised Ca 2+ buffering ability. Malondialdehyde concentration increased, whereas the ROS-detoxifying enzyme Mn 2+ superoxide dismutase (MnSOD) and aconitase lost activity. In the mitochondrial respiratory chain, the concentration and activity of complex I and complex IV decreased. Among other mitochondrial enzymes and transporters, adenine nucleotide carrier and glutaminase decreased. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase also decreased. Data strongly suggest that in the female rat heart, estrogen depletion leads to progressive, severe mitochondrial dysfunction. © 2017 Society for Endocrinology.

Mitochondrial function in diaphragm of emphysematous hamsters after treatment with nandrolone.

PubMed

Wijnhoven, Hanneke J H; Ennen, Leo; Rodenburg, Richard J T; Dekhuijzen, P N Richard

2006-01-01

Respiratory failure in patients with COPD may be caused by insufficient force production or insufficient endurance capacity of the respiratory muscles. Anabolic steroids may improve respiratory muscle function in COPD. The effect of anabolic steroids on mitochondrial function in the diaphragm in emphysema is unknown. In an emphysematous male hamster model, we investigated whether administration of the anabolic steroid nandrolone decanoate (ND) altered the activity of mitochondrial respiratory chain complexes in the diaphragm. The bodyweight of hamsters treated with ND was decreased after treatment compared with initial values, and serum testosterone levels were significantly lower in hamsters treated with ND than in control hamsters. No difference in the activity of mitochondrial respiratory chain complexes in the diaphragm between normal and emphysematous hamsters was observed. Treatment with ND did not change the activity of mitochondrial respiratory chain complexes in the diaphragm of both normal and emphysematous hamsters. In emphysematous hamsters, administration of ND decreased the activity of succinate:cytochrome c oxidoreductase compared with ND treatment in normal hamsters. We conclude that anabolic steroids have negative effects on the activity of succinate:cytochrome c oxidoreductase and anabolic status in this emphysematous hamster model.

Mitochondrial function in diaphragm of emphysematous hamsters after treatment with nandrolone

PubMed Central

Wijnhoven, Hanneke JH; Ennen, Leo; Rodenburg, Richard JT; Dekhuijzen, PN Richard

2006-01-01

Respiratory failure in patients with COPD may be caused by insufficient force production or insufficient endurance capacity of the respiratory muscles. Anabolic steroids may improve respiratory muscle function in COPD. The effect of anabolic steroids on mitochondrial function in the diaphragm in emphysema is unknown. In an emphysematous male hamster model, we investigated whether administration of the anabolic steroid nandrolone decanoate (ND) altered the activity of mitochondrial respiratory chain complexes in the diaphragm. The bodyweight of hamsters treated with ND was decreased after treatment compared with initial values, and serum testosterone levels were significantly lower in hamsters treated with ND than in control hamsters. No difference in the activity of mitochondrial respiratory chain complexes in the diaphragm between normal and emphysematous hamsters was observed. Treatment with ND did not change the activity of mitochondrial respiratory chain complexes in the diaphragm of both normal and emphysematous hamsters. In emphysematous hamsters, administration of ND decreased the activity of succinate:cytochrome c oxidoreductase compared with ND treatment in normal hamsters. We conclude that anabolic steroids have negative effects on the activity of succinate:cytochrome c oxidoreductase and anabolic status in this emphysematous hamster model. PMID:18046906

Mitochondrial gene polymorphisms that protect mice from colitis.

PubMed

Bär, Florian; Bochmann, Wiebke; Widok, Andrea; von Medem, Kilian; Pagel, Rene; Hirose, Misa; Yu, Xinhua; Kalies, Kathrin; König, Peter; Böhm, Ruwen; Herdegen, Thomas; Reinicke, Anna T; Büning, Jürgen; Lehnert, Hendrik; Fellermann, Klaus; Ibrahim, Saleh; Sina, Christian

2013-11-01

Dysregulated energy homeostasis in the intestinal mucosa frequently is observed in patients with ulcerative colitis (UC). Intestinal tissues from these patients have reduced activity of the mitochondrial oxidative phosphorylation (OXPHOS) complex, so mitochondrial dysfunction could contribute to the pathogenesis of UC. However, little is known about the mechanisms by which OXPHOS activity could be altered. We used conplastic mice, which have identical nuclear but different mitochondrial genomes, to investigate activities of the OXPHOS complex. Colitis was induced in C57BL/6J wild-type (B6.B6) and 3 strains of conplastic mice (B6.NZB, B6.NOD, and B6.AKR) by administration of dextran sodium sulfate or rectal application of trinitrobenzene sulfonate. Colon tissues were collected and analyzed by histopathology, immunohistochemical analysis, and immunoblot analysis; we also measured mucosal levels of adenosine triphosphate (ATP) and reactive oxygen species, OXPHOS complex activity, and epithelial cell proliferation and apoptosis. We identified mice with increased mucosal OXPHOS complex activities and levels of ATP. These mice developed less-severe colitis after administration of dextran sodium sulfate or trinitrobenzene sulfonate than mice with lower mucosal levels of ATP. Colon tissues from these mice also had increased enterocyte proliferation and transcription factor nuclear factor-κB activity, which have been shown to protect the mucosal barrier-defects in these processes have been associated with inflammatory bowel disease. Variants in mitochondrial DNA that increase mucosal levels of ATP protect mice from colitis. Increasing mitochondrial ATP synthesis in intestinal epithelial cells could be a therapeutic approach for UC. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

Discrete mitochondrial aberrations in the spinal cord of sporadic ALS patients.

PubMed

Delic, Vedad; Kurien, Crupa; Cruz, Josean; Zivkovic, Sandra; Barretta, Jennifer; Thomson, Avery; Hennessey, Daniel; Joseph, Jaheem; Ehrhart, Jared; Willing, Alison E; Bradshaw, Patrick; Garbuzova-Davis, Svitlana

2018-08-01

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by progressive motor neuron degeneration in the brain and spinal cord leading to muscle atrophy, paralysis, and death. Mitochondrial dysfunction is a major contributor to motor neuron degeneration associated with ALS progression. Mitochondrial abnormalities have been determined in spinal cords of animal disease models and ALS patients. However, molecular mechanisms leading to mitochondrial dysfunction in sporadic ALS (sALS) patients remain unclear. Also, segmental or regional variation in mitochondrial activity in the spinal cord has not been extensively examined in ALS. In our study, the activity of mitochondrial electron transport chain complex IV was examined in post-mortem gray and white matter of the cervical and lumbar spinal cords from male and female sALS patients and controls. Mitochondrial distribution and density in spinal cord motor neurons, lateral funiculus, and capillaries in gray and white matter were analyzed by immunohistochemistry. Results showed that complex IV activity was significantly decreased only in gray matter in both cervical and lumbar spinal cords from ALS patients. In ALS cervical and lumbar spinal cords, significantly increased mitochondrial density and altered distribution were observed in motor neurons, lateral funiculus, and cervical white matter capillaries. Discrete decreased complex IV activity in addition to changes in mitochondria distribution and density determined in the spinal cord in sALS patients are novel findings. These explicit mitochondrial defects in the spinal cord may contribute to ALS pathogenesis and should be considered in development of therapeutic approaches for this disease. © 2018 Wiley Periodicals, Inc.

Redox imbalance and mitochondrial abnormalities in the diabetic lung.

PubMed

Wu, Jinzi; Jin, Zhen; Yan, Liang-Jun

2017-04-01

Although the lung is one of the least studied organs in diabetes, increasing evidence indicates that it is an inevitable target of diabetic complications. Nevertheless, the underlying biochemical mechanisms of lung injury in diabetes remain largely unexplored. Given that redox imbalance, oxidative stress, and mitochondrial dysfunction have been implicated in diabetic tissue injury, we set out to investigate mechanisms of lung injury in diabetes. The objective of this study was to evaluate NADH/NAD + redox status, oxidative stress, and mitochondrial abnormalities in the diabetic lung. Using STZ induced diabetes in rat as a model, we measured redox-imbalance related parameters including aldose reductase activity, level of poly ADP ribose polymerase (PAPR-1), NAD + content, NADPH content, reduced form of glutathione (GSH), and glucose 6-phophate dehydrogenase (G6PD) activity. For assessment of mitochondrial abnormalities in the diabetic lung, we measured the activities of mitochondrial electron transport chain complexes I to IV and complex V as well as dihydrolipoamide dehydrogenase (DLDH) content and activity. We also measured the protein content of NAD + dependent enzymes such as sirtuin3 (sirt3) and NAD(P)H: quinone oxidoreductase 1 (NQO1). Our results demonstrate that NADH/NAD + redox imbalance occurs in the diabetic lung. This redox imbalance upregulates the activities of complexes I to IV, but not complex V; and this upregulation is likely the source of increased mitochondrial ROS production, oxidative stress, and cell death in the diabetic lung. These results, together with the findings that the protein contents of DLDH, sirt3, and NQO1 all are decreased in the diabetic lung, demonstrate that redox imbalance, mitochondrial abnormality, and oxidative stress contribute to lung injury in diabetes. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Skeletal muscle mitochondrial bioenergetics and associations with myostatin genotypes in the Thoroughbred horse

PubMed Central

Porter, Richard K.; Katz, Lisa M.; Hill, Emmeline W.

2017-01-01

Variation in the myostatin (MSTN) gene has been reported to be associated with race distance, body composition and skeletal muscle fibre composition in the horse. The aim of the present study was to test the hypothesis that MSTN variation influences mitochondrial phenotypes in equine skeletal muscle. Mitochondrial abundance and skeletal muscle fibre types were measured in whole muscle biopsies from the gluteus medius of n = 82 untrained (21 ± 3 months) Thoroughbred horses. Skeletal muscle fibre type proportions were significantly (p < 0.01) different among the three MSTN genotypes and mitochondrial content was significantly (p < 0.01) lower in the combined presence of the C-allele of SNP g.66493737C>T (C) and the SINE insertion 227 bp polymorphism (I). Evaluation of mitochondrial complex activities indicated higher combined mitochondrial complex I+III and II+III activities in the presence of the C-allele / I allele (p ≤ 0.05). The restoration of complex I+III and complex II+III activities following addition of exogenous coenzyme Q1 (ubiquinone1) (CoQ1) in vitro in the TT/NN (homozygous T allele/homozygous no insertion) cohort indicated decreased coenzyme Q in these animals. In addition, decreased gene expression in two coenzyme Q (CoQ) biosynthesis pathway genes (COQ4, p ≤ 0.05; ADCK3, p ≤ 0.01) in the TT/NN horses was observed. This study has identified several mitochondrial phenotypes associated with MSTN genotype in untrained Thoroughbred horses and in addition, our findings suggest that nutritional supplementation with CoQ may aid to restore coenzyme Q activity in TT/NN horses. PMID:29190290

Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria.

PubMed

Popov-Celeketić, Dusan; Mapa, Koyeli; Neupert, Walter; Mokranjac, Dejana

2008-05-21

The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments.

Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria

PubMed Central

Popov-Čeleketić, Dus̆an; Mapa, Koyeli; Neupert, Walter; Mokranjac, Dejana

2008-01-01

The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments. PMID:18418384

Deficiency of PHB complex impairs respiratory supercomplex formation and activates mitochondrial flashes.

PubMed

Jian, Chongshu; Xu, Fengli; Hou, Tingting; Sun, Tao; Li, Jinghang; Cheng, Heping; Wang, Xianhua

2017-08-01

Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins. PHBs form multimeric ring complexes acting as scaffolds in the inner mitochondrial membrane. Mitochondrial flashes (mitoflashes) are newly discovered mitochondrial signaling events that reflect electrical and chemical excitations of the organelle. Here, we investigate the possible roles of PHBs in the regulation of mitoflash signaling. Downregulation of PHBs increases mitoflash frequency by up to 5.4-fold due to elevated basal reactive oxygen species (ROS) production in the mitochondria. Mechanistically, PHB deficiency impairs the formation of mitochondrial respiratory supercomplexes (RSCs) without altering the abundance of individual respiratory complex subunits. These impairments induced by PHB deficiency are effectively rescued by co-expression of PHB1 and PHB2, indicating that the multimeric PHB complex acts as the functional unit. Furthermore, downregulating other RSC assembly factors, including SCAFI (also known as COX7A2L), RCF1a (HIGD1A), RCF1b (HIGD2A), UQCC3 and SLP2 (STOML2), all activate mitoflashes through elevating mitochondrial ROS production. Our findings identify the PHB complex as a new regulator of RSC formation and mitoflash signaling, and delineate a general relationship among RSC formation, basal ROS production and mitoflash biogenesis. © 2017. Published by The Company of Biologists Ltd.

Glutaredoxin-2 is required to control oxidative phosphorylation in cardiac muscle by mediating deglutathionylation reactions.

PubMed

Mailloux, Ryan J; Xuan, Jian Ying; McBride, Skye; Maharsy, Wael; Thorn, Stephanie; Holterman, Chet E; Kennedy, Christopher R J; Rippstein, Peter; deKemp, Robert; da Silva, Jean; Nemer, Mona; Lou, Marjorie; Harper, Mary-Ellen

2014-05-23

Glutaredoxin-2 (Grx2) modulates the activity of several mitochondrial proteins in cardiac tissue by catalyzing deglutathionylation reactions. However, it remains uncertain whether Grx2 is required to control mitochondrial ATP output in heart. Here, we report that Grx2 plays a vital role modulating mitochondrial energetics and heart physiology by mediating the deglutathionylation of mitochondrial proteins. Deletion of Grx2 (Grx2(-/-)) decreased ATP production by complex I-linked substrates to half that in wild type (WT) mitochondria. Decreased respiration was associated with increased complex I glutathionylation diminishing its activity. Tissue glucose uptake was concomitantly increased. Mitochondrial ATP output and complex I activity could be recovered by restoring the redox environment to that favoring the deglutathionylated states of proteins. Grx2(-/-) hearts also developed left ventricular hypertrophy and fibrosis, and mice became hypertensive. Mitochondrial energetics from Grx2 heterozygotes (Grx2(+/-)) were also dysfunctional, and hearts were hypertrophic. Intriguingly, Grx2(+/-) mice were far less hypertensive than Grx2(-/-) mice. Thus, Grx2 plays a vital role in modulating mitochondrial metabolism in cardiac muscle, and Grx2 deficiency leads to pathology. As mitochondrial ATP production was restored by the addition of reductants, these findings may be relevant to novel redox-related therapies in cardiac disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Aging impact on biochemical activities and gene expression of Drosophila melanogaster mitochondria.

PubMed

Dubessay, Pascal; Garreau-Balandier, Isabelle; Jarrousse, Anne-Sophie; Fleuriet, Annie; Sion, Benoit; Debise, Roger; Alziari, Serge

2007-08-01

The consequences of aging are characterized by a decline in the main cellular functions, including those of the mitochondria. Although these consequences have been much studied, efforts have often focused solely on a few parameters used to assess the "state" of mitochondrial function during aging. We performed comparative measurements of several parameters in young (a few days) and old (8 and 12 weeks) adult male Drosophila melanogaster: respiratory complex activities, mitochondrial respiration, ATP synthesis, lipid composition of the inner membrane, concentrations of respiratory complex subunits, expression of genes (nuclear and mitochondrial) coding for mitochondrial proteins. Our results show that, in the mitochondria of "old" flies, the activities of three respiratory complexes (I, III, IV) are greatly diminished, ATP synthesis is decreased, and the lipid composition of the inner membrane (fatty acids, cardiolipin) is modified. However, the respiration rate and subunit concentrations measured by Western blot are unaffected. Although cellular mitochondrial DNA (mtDNA) content remains constant, there is a decrease in concentrations of nuclear and mitochondrial transcripts apparently coordinated. The expression of nuclear genes encoding the transcription factors TFAM, TFB1, TFB2, and DmTTF, which are essential for the maintenance and expression of mtDNA are also decreased. The decrease in nuclear and mitochondrial transcript concentrations may be one of the principal effects of aging on mitochondria, and could explain observed decreases in mitochondrial efficiency.

Mitochondrial Reactive Oxygen Species Mediate Cardiac Structural, Functional, and Mitochondrial Consequences of Diet-Induced Metabolic Heart Disease.

PubMed

Sverdlov, Aaron L; Elezaby, Aly; Qin, Fuzhong; Behring, Jessica B; Luptak, Ivan; Calamaras, Timothy D; Siwik, Deborah A; Miller, Edward J; Liesa, Marc; Shirihai, Orian S; Pimentel, David R; Cohen, Richard A; Bachschmid, Markus M; Colucci, Wilson S

2016-01-11

Mitochondrial reactive oxygen species (ROS) are associated with metabolic heart disease (MHD). However, the mechanism by which ROS cause MHD is unknown. We tested the hypothesis that mitochondrial ROS are a key mediator of MHD. Mice fed a high-fat high-sucrose (HFHS) diet develop MHD with cardiac diastolic and mitochondrial dysfunction that is associated with oxidative posttranslational modifications of cardiac mitochondrial proteins. Transgenic mice that express catalase in mitochondria and wild-type mice were fed an HFHS or control diet for 4 months. Cardiac mitochondria from HFHS-fed wild-type mice had a 3-fold greater rate of H2O2 production (P=0.001 versus control diet fed), a 30% decrease in complex II substrate-driven oxygen consumption (P=0.006), 21% to 23% decreases in complex I and II substrate-driven ATP synthesis (P=0.01), and a 62% decrease in complex II activity (P=0.002). In transgenic mice that express catalase in mitochondria, all HFHS diet-induced mitochondrial abnormalities were ameliorated, as were left ventricular hypertrophy and diastolic dysfunction. In HFHS-fed wild-type mice complex II substrate-driven ATP synthesis and activity were restored ex vivo by dithiothreitol (5 mmol/L), suggesting a role for reversible cysteine oxidative posttranslational modifications. In vitro site-directed mutation of complex II subunit B Cys100 or Cys103 to redox-insensitive serines prevented complex II dysfunction induced by ROS or high glucose/high palmitate in the medium. Mitochondrial ROS are pathogenic in MHD and contribute to mitochondrial dysfunction, at least in part, by causing oxidative posttranslational modifications of complex I and II proteins including reversible oxidative posttranslational modifications of complex II subunit B Cys100 and Cys103. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

PubMed Central

Presley, Tennille; Vedam, Kaushik; Liu, Xiaoping; Zweier, Jay L.

2009-01-01

Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O2 binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO2 through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO2. ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO2, but it was unaffected at low pO2 in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42°C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O2 binding at CcO at complex IV. PMID:19412660

Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells.

PubMed

Sun, Wen; Wu, Xiaxia; Gao, Hongwei; Yu, Jie; Zhao, Wenwen; Lu, Jin-Jian; Wang, Jinhua; Du, Guanhua; Chen, Xiuping

2017-07-01

Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca 2+ ) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium accumulation is a critical mediator in MAM-induced necroptosis through sustained JNK activation and mitochondrial ROS production. Our study also provided new insights into the molecular regulation of necroptosis in human colon cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Mature DIABLO/Smac Is Produced by the IMP Protease Complex on the Mitochondrial Inner Membrane

PubMed Central

Burri, Lena; Strahm, Yvan; Hawkins, Christine J.; Gentle, Ian E.; Puryer, Michelle A.; Verhagen, Anne; Callus, Bernard; Vaux, David; Lithgow, Trevor

2005-01-01

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX5P in Imp1 and NX5S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space. PMID:15814844

Activation of a cryptic splice site in the mitochondrial elongation factor GFM1 causes combined OXPHOS deficiency☆

PubMed Central

Simon, Mariella T.; Ng, Bobby G.; Friederich, Marisa W.; Wang, Raymond Y.; Boyer, Monica; Kircher, Martin; Collard, Renata; Buckingham, Kati J.; Chang, Richard; Shendure, Jay; Nickerson, Deborah A.; Bamshad, Michael J.; Van Hove, Johan L.K.; Freeze, Hudson H.; Abdenur, Jose E.

2017-01-01

We report the clinical, biochemical, and molecular findings in two brothers with encephalopathy and multi-systemic disease. Abnormal transferrin glycoforms were suggestive of a type I congenital disorder of glycosylation (CDG). While exome sequencing was negative for CDG related candidate genes, the testing revealed compound heterozygous mutations in the mitochondrial elongation factor G gene (GFM1). One of the mutations had been reported previously while the second, novel variant was found deep in intron 6, activating a cryptic splice site. Functional studies demonstrated decreased GFM1 protein levels, suggested disrupted assembly of mitochondrial complexes III and V and decreased activities of mitochondrial complexes I and IV, all indicating combined OXPHOS deficiency. PMID:28216230

Contribution of dopamine to mitochondrial complex I inhibition and dopaminergic deficits caused by methylenedioxymethamphetamine in mice.

PubMed

Barros-Miñones, L; Goñi-Allo, B; Suquia, V; Beitia, G; Aguirre, N; Puerta, E

2015-06-01

Methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that MDMA-induced neurotoxicity is mediated by oxidative stress probably due to the inhibition of mitochondrial complex I activity. In this study we investigated the contribution of dopamine (DA) to such effects. For this, we modulated the dopaminergic system of mice at the synthesis, uptake or metabolism levels. Striatal mitochondrial complex I activity was decreased 1 h after MDMA; an effect not observed in the striatum of DA depleted mice or in the hippocampus, a dopamine spare region. The DA precursor, L-dopa, caused a significant reduction of mitochondrial complex I activity by itself and exacerbated the dopaminergic deficits when combined with systemic MDMA. By contrast, no damage was observed when L-dopa was combined with intrastriatal injections of MDMA. On the other hand, dopamine uptake blockade using GBR 12909, inhibited both, the acute inhibition of complex I activity and the long-term dopaminergic toxicity caused by MDMA. Moreover, the inhibition of DA metabolism with the monoamine oxidase (MAO) inhibitor, pargyline, afforded a significant protection against MDMA-induced complex I inhibition and neurotoxicity. Taken together, these findings point to the formation of hydrogen peroxide subsequent to DA metabolism by MAO, rather than a direct DA-mediated mitochondrial complex I inhibition, and the contribution of a peripheral metabolite of MDMA, as the key steps in the chain of biochemical events leading to DA neurotoxicity caused by MDMA in mice. Copyright © 2015 Elsevier Ltd. All rights reserved.

Stomatin-Like Protein 2 Binds Cardiolipin and Regulates Mitochondrial Biogenesis and Function▿

PubMed Central

Christie, Darah A.; Lemke, Caitlin D.; Elias, Isaac M.; Chau, Luan A.; Kirchhof, Mark G.; Li, Bo; Ball, Eric H.; Dunn, Stanley D.; Hatch, Grant M.; Madrenas, Joaquín

2011-01-01

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched microdomains in which electron transport complexes are optimally assembled. Likely through the prohibitin functional interactome, SLP-2 then regulates mitochondrial biogenesis and function. PMID:21746876

Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs After Exposure to Hyperoxia

PubMed Central

Audi, Said H.; Staniszewski, Kevin S.; Haworth, Steven T.; Jacobs, Elizabeth R.; Ranji, Mahsa; Zablocki, Clement J.

2013-01-01

Recently, we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes nicotinamide adenine dinucleotide (NADH) and oxidized form of flavin adenine dinucleotide \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$({\\rm FADH}_{2})$\\end{document} (FAD), as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this paper was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${>}{95\\%}~{\\rm O}_{2}$\\end{document} for 48 h) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, when compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change in mitochondrial redox state of hyperoxic lungs prior to histological changes characteristic of hyperoxia. PMID:25379360

Mitochondrial dysfunction in the gastrointestinal mucosa of children with autism: A blinded case-control study

PubMed Central

Rose, Shannon; Bennuri, Sirish C.; Murray, Katherine F.; Buie, Timothy; Winter, Harland

2017-01-01

Gastrointestinal (GI) symptoms are prevalent in autism spectrum disorder (ASD) but the pathophysiology is poorly understood. Imbalances in the enteric microbiome have been associated with ASD and can cause GI dysfunction potentially through disruption of mitochondrial function as microbiome metabolites modulate mitochondrial function and mitochondrial dysfunction is highly associated with GI symptoms. In this study, we compared mitochondrial function in rectal and cecum biopsies under the assumption that certain microbiome metabolites, such as butyrate and propionic acid, are more abundant in the cecum as compared to the rectum. Rectal and cecum mucosal biopsies were collected during elective diagnostic colonoscopy. Using a single-blind case-control design, complex I and IV and citrate synthase activities and complex I-V protein quantity from 10 children with ASD, 10 children with Crohn’s disease and 10 neurotypical children with nonspecific GI complaints were measured. The protein for all complexes, except complex II, in the cecum as compared to the rectum was significantly higher in ASD samples as compared to other groups. For both rectal and cecum biopsies, ASD samples demonstrated higher complex I activity, but not complex IV or citrate synthase activity, compared to other groups. Mitochondrial function in the gut mucosa from children with ASD was found to be significantly different than other groups who manifested similar GI symptomatology suggesting a unique pathophysiology for GI symptoms in children with ASD. Abnormalities localized to the cecum suggest a role for imbalances in the microbiome, potentially in the production of butyrate, in children with ASD. PMID:29028817

Exercise training improves vascular mitochondrial function

PubMed Central

Park, Song-Young; Rossman, Matthew J.; Gifford, Jayson R.; Bharath, Leena P.; Bauersachs, Johann; Richardson, Russell S.; Abel, E. Dale; Symons, J. David

2016-01-01

Exercise training is recognized to improve cardiac and skeletal muscle mitochondrial respiratory capacity; however, the impact of chronic exercise on vascular mitochondrial respiratory function is unknown. We hypothesized that exercise training concomitantly increases both vascular mitochondrial respiratory capacity and vascular function. Arteries from both sedentary (SED) and swim-trained (EX, 5 wk) mice were compared in terms of mitochondrial respiratory function, mitochondrial content, markers of mitochondrial biogenesis, redox balance, nitric oxide (NO) signaling, and vessel function. Mitochondrial complex I and complex I + II state 3 respiration and the respiratory control ratio (complex I + II state 3 respiration/complex I state 2 respiration) were greater in vessels from EX relative to SED mice, despite similar levels of arterial citrate synthase activity and mitochondrial DNA content. Furthermore, compared with the SED mice, arteries from EX mice displayed elevated transcript levels of peroxisome proliferative activated receptor-γ coactivator-1α and the downstream targets cytochrome c oxidase subunit IV isoform 1, isocitrate dehydrogenase (Idh) 2, and Idh3a, increased manganese superoxide dismutase protein expression, increased endothelial NO synthase phosphorylation (Ser1177), and suppressed reactive oxygen species generation (all P < 0.05). Although there were no differences in EX and SED mice concerning endothelium-dependent and endothelium-independent vasorelaxation, phenylephrine-induced vasocontraction was blunted in vessels from EX compared with SED mice, and this effect was normalized by NOS inhibition. These training-induced increases in vascular mitochondrial respiratory capacity and evidence of improved redox balance, which may, at least in part, be attributable to elevated NO bioavailability, have the potential to protect against age- and disease-related challenges to arterial function. PMID:26825520

Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity.

PubMed

Mena, Natalia P; Bulteau, Anne Laure; Salazar, Julio; Hirsch, Etienne C; Núñez, Marco T

2011-06-03

Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex I and iron accumulation are hallmarks of idiopathic Parkinson's disease, the findings reported here may have relevance for understanding the pathophysiology of this disease. Copyright © 2011 Elsevier Inc. All rights reserved.

Activation of mitochondrial calpain and increased cardiac injury: beyond AIF release

PubMed Central

Thompson, Jeremy; Hu, Ying; Lesnefsky, Edward J.

2015-01-01

Calpain 1 (CPN1) is a ubiquitous cysteine protease that exists in both cytosol and cardiac mitochondria. Mitochondrial CPN1 (mit-CPN1) is located in the intermembrane space and matrix. Activation of mit-CPN1 within the intermembrane space increases cardiac injury by releasing apoptosis-inducing factor from mitochondria during ischemia-reperfusion (IR). We asked if activation of mit-CPN1 is involved in mitochondrial injury during IR. MDL-28170 (MDL) was used to inhibit CPN1 in buffer-perfused hearts following 25-min ischemia and 30-min reperfusion. MDL treatment decreased the release of lactate dehydrogenase into coronary effluent compared with untreated hearts, indicating that inhibition of CPN1 decreases cardiac injury. MDL also prevented the cleavage of spectrin (a substrate of CPN1) in cytosol during IR, supporting that MDL treatment decreased cytosolic calpain activation. In addition, MDL markedly improved calcium retention capacity compared with untreated heart, suggesting that MDL treatment decreases mitochondrial permeability transition pore opening. In addition, we found that IR led to decreased complex I activity, whereas inhibition of mit-CPN1 using MDL protected complex I. Pyruvate dehydrogenase content was decreased following IR. However, pyruvate dehydrogenase content was preserved in MDL-treated mitochondria. Taken together, MDL treatment decreased cardiac injury during IR by inhibiting both cytosolic and mit-CPN1. Activation of mit-CPN1 increases cardiac injury during IR by sensitizing mitochondrial permeability transition pore opening and impairing mitochondrial metabolism through damage of complex I. PMID:26637561

Exercise in claudicants increase or decrease walking ability and the response relates to mitochondrial function.

PubMed

van Schaardenburgh, Michel; Wohlwend, Martin; Rognmo, Øivind; Mattsson, Erney J R

2017-06-07

Exercise of patients with intermittent claudication improves walking performance. Exercise does not usually increase blood flow, but seems to increase muscle mitochondrial enzyme activities. Although exercise is beneficial in most patients, it might be harmful in some. The mitochondrial response to exercise might therefore differ between patients. Our hypothesis was that changes in walking performance relate to changes in mitochondrial function after 8 weeks of exercise. At a subgroup level, negative responders decrease and positive responders increase mitochondrial capacity. Two types of exercise were studied, calf raising and walking (n = 28). We wanted to see whether there were negative and positive responders, independent of type of exercise. Measurements of walking performance, peripheral hemodynamics, mitochondrial respiration and content (citrate synthase activity) were obtained on each patient before and after the intervention period. Multiple linear regression was used to test whether changes in peak walking time relate to mitochondrial function. Subgroups of negative (n = 8) and positive responders (n = 8) were defined as those that either decreased or increased peak walking time following exercise. Paired t test and analysis of covariance was used to test changes within and between subgroups. Changes in peak walking time were related to changes in mitochondrial respiration supported by electron transferring flavoprotein (ETF + CI) P (p = 0.004), complex I (CI + ETF) P (p = 0.003), complex I + complex II (CI + CII + ETF) P (p = 0.037) and OXPHOS coupling efficiency (p = 0.046) in the whole group. Negative responders had more advanced peripheral arterial disease. Mitochondrial respiration supported by electron transferring flavoprotein (ETF + CI) P (p = 0.0013), complex I (CI + ETF) P (p = 0.0005), complex I + complex II (CI + CII + ETF) P (p = 0.011) and electron transfer system capacity (CI + CII + ETF) E (p = 0.021) and OXPHOS coupling efficiency decreased in negative responders (p = 0.0007) after exercise. Positive responders increased citrate synthase activity (p = 0.010). Changes in walking performance seem to relate to changes in mitochondrial function after exercise. Negative responders have more advanced peripheral arterial disease and decrease, while positive responders increase mitochondrial capacity. Trial registration ClinicalTrials.gov ID: NCT023110256.

Mitochondrial modulation-induced activation of vagal sensory neuronal subsets by antimycin A, but not CCCP or rotenone, correlates with mitochondrial superoxide production.

PubMed

Stanford, Katherine R; Taylor-Clark, Thomas E

2018-01-01

Inflammation causes nociceptive sensory neuron activation, evoking debilitating symptoms and reflexes. Inflammatory signaling pathways are capable of modulating mitochondrial function, resulting in reactive oxygen species (ROS) production, mitochondrial depolarization and calcium release. Previously we showed that mitochondrial modulation with antimycin A, a complex III inhibitor, selectively stimulated nociceptive bronchopulmonary C-fibers via the activation of transient receptor potential (TRP) ankyrin 1 (A1) and vanilloid 1 (V1) cation channels. TRPA1 is ROS-sensitive, but there is little evidence that TRPV1 is activated by ROS. Here, we used dual imaging of dissociated vagal neurons to investigate the correlation of mitochondrial superoxide production (mitoSOX) or mitochondrial depolarization (JC-1) with cytosolic calcium (Fura-2AM), following mitochondrial modulation by antimycin A, rotenone (complex I inhibitor) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP, mitochondrial uncoupling agent). Mitochondrial modulation by all agents selectively increased cytosolic calcium in a subset of TRPA1/TRPV1-expressing (A1/V1+) neurons. There was a significant correlation between antimycin A-induced calcium responses and mitochondrial superoxide in wild-type 'responding' A1/V1+ neurons, which was eliminated in TRPA1-/- neurons, but not TRPV1-/- neurons. Nevertheless, antimycin A-induced superoxide production did not always increase calcium in A1/V1+ neurons, suggesting a critical role of an unknown factor. CCCP caused both superoxide production and mitochondrial depolarization but neither correlated with calcium fluxes in A1/V1+ neurons. Rotenone-induced calcium responses in 'responding' A1/V1+ neurons correlated with mitochondrial depolarization but not superoxide production. Our data are consistent with the hypothesis that mitochondrial dysfunction causes calcium fluxes in a subset of A1/V1+ neurons via ROS-dependent and ROS-independent mechanisms.

Differential responses of targeted lung redox enzymes to rat exposure to 60 or 85% oxygen

PubMed Central

Gan, Zhuohui; Roerig, David L.; Clough, Anne V.

2011-01-01

Rat exposure to 60% O2 (hyper-60) or 85% O2 (hyper-85) for 7 days confers susceptibility or tolerance, respectively, of the otherwise lethal effects of exposure to 100% O2. The objective of this study was to determine whether activities of the antioxidant cytosolic enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex III are differentially altered in hyper-60 and hyper-85 lungs. Duroquinone (DQ), an NQO1 substrate, or its hydroquinone (DQH2), a complex III substrate, was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of DQH2 and DQ were measured. Based on inhibitor effects and kinetic modeling, capacities of NQO1-mediated DQ reduction (Vmax1) and complex III-mediated DQH2 oxidation (Vmax2) increased by ∼140 and ∼180% in hyper-85 lungs, respectively, compared with rates in lungs of rats exposed to room air (normoxic). In hyper-60 lungs, Vmax1 increased by ∼80%, with no effect on Vmax2. Additional studies revealed that mitochondrial complex I activity in hyper-60 and hyper-85 lung tissue homogenates was ∼50% lower than in normoxic lung homogenates, whereas mitochondrial complex IV activity was ∼90% higher in only hyper-85 lung tissue homogenates. Thus NQO1 activity increased in both hyper-60 and hyper-85 lungs, whereas complex III activity increased in hyper-85 lungs only. This increase, along with the increase in complex IV activity, may counter the effects the depression in complex I activity might have on tissue mitochondrial function and/or reactive oxygen species production and may be important to the tolerance of 100% O2 observed in hyper-85 rats. PMID:21551015

Differential responses of targeted lung redox enzymes to rat exposure to 60 or 85% oxygen.

PubMed

Gan, Zhuohui; Roerig, David L; Clough, Anne V; Audi, Said H

2011-07-01

Rat exposure to 60% O(2) (hyper-60) or 85% O(2) (hyper-85) for 7 days confers susceptibility or tolerance, respectively, of the otherwise lethal effects of exposure to 100% O(2). The objective of this study was to determine whether activities of the antioxidant cytosolic enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex III are differentially altered in hyper-60 and hyper-85 lungs. Duroquinone (DQ), an NQO1 substrate, or its hydroquinone (DQH(2)), a complex III substrate, was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of DQH(2) and DQ were measured. Based on inhibitor effects and kinetic modeling, capacities of NQO1-mediated DQ reduction (V(max1)) and complex III-mediated DQH(2) oxidation (V(max2)) increased by ∼140 and ∼180% in hyper-85 lungs, respectively, compared with rates in lungs of rats exposed to room air (normoxic). In hyper-60 lungs, V(max1) increased by ∼80%, with no effect on V(max2). Additional studies revealed that mitochondrial complex I activity in hyper-60 and hyper-85 lung tissue homogenates was ∼50% lower than in normoxic lung homogenates, whereas mitochondrial complex IV activity was ∼90% higher in only hyper-85 lung tissue homogenates. Thus NQO1 activity increased in both hyper-60 and hyper-85 lungs, whereas complex III activity increased in hyper-85 lungs only. This increase, along with the increase in complex IV activity, may counter the effects the depression in complex I activity might have on tissue mitochondrial function and/or reactive oxygen species production and may be important to the tolerance of 100% O(2) observed in hyper-85 rats.

Effects of OXPHOS complex deficiencies and ESA dysfunction in working intact skeletal muscle: implications for mitochondrial myopathies.

PubMed

Korzeniewski, Bernard

2015-10-01

The effects of inborn oxidative phosphorylation (OXPHOS) complex deficiencies or possible each-step activation (ESA) dysfunction on the bioenergetic system in working intact skeletal muscle are studied using a computer model of OXPHOS published previously. The curves representing the dependencies of V˙O2 and metabolite concentrations on single complex activity, entire OXPHOS activity or ESA intensity exhibit a characteristic threshold at some OXPHOS complex activity/ESA intensity. This threshold for V˙O2 of single complex activities is significantly lower in intact muscle during moderate and heavy work, than in isolated mitochondria in state 3. Metabolite concentrations and pH in working muscle start to change significantly at much higher OXPHOS complex activities/ESA intensities than V˙O2. The effect of entire OXPHOS deficiency or ESA dysfunction is potentially much stronger than the effect of a single complex deficiency. Implications of these findings for the genesis of mitochondrial myopathies are discussed. It is concluded that V˙O2 in state 3 and its dependence on complex activity in isolated mitochondria is not a universal quantitative determinant of the effect of mitochondrial dysfunctions in vivo. Moderate and severe mitochondria dysfunctions are defined: the former affect significantly only metabolite concentrations and pH, while the latter also decrease significantly V˙O2 in intact skeletal muscle during work. The dysfunction-caused decrease in V˙O2/oxidative ATP synthesis flux, disturbance of metabolite homeostasis, elevated ROS production and anaerobic glycolysis recruitment can account for such mitochondrial myopathy symptoms as muscle weakness, exercise intolerance (exertional fatigue) and lactic acidosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Assessment of mitochondrial functions in Daphnia pulex clones using high-resolution respirometry.

PubMed

Kake-Guena, Sandrine A; Touisse, Kamal; Vergilino, Roland; Dufresne, France; Blier, Pierre U; Lemieux, Hélène

2015-06-01

The objectives of our study were to adapt a method to measure mitochondrial function in intact mitochondria from the small crustacean Daphnia pulex and to validate if this method was sensitive enough to characterize mitochondrial metabolism in clones of the pulex complex differing in ploidy levels, mitochondrial DNA haplotypes, and geographic origins. Daphnia clones belonging to the Daphnia pulex complex represent a powerful model to delineate the link between mitochondrial DNA evolution and mitochondrial phenotypes, as single genotypes with divergent mtDNA can be grown under various experimental conditions. Our study included two diploid clones from temperate environments and two triploid clones from subarctic environments. The whole animal permeabilization and measurement of respiration with high-resolution respirometry enabled the measurement of the functional capacity of specific mitochondrial complexes in four clones. When expressing the activity as ratios, our method detected significant interclonal variations. In the triploid subarctic clone from Kuujjurapik, a higher proportion of the maximal physiological oxidative phosphorylation (OXPHOS) capacity of mitochondria was supported by complex II, and a lower proportion by complex I. The triploid subarctic clone from Churchill (Manitoba) showed the lowest proportion of the maximal OXPHOS supported by complex II. Additional studies are required to determine if these differences in mitochondrial functions are related to differences in mitochondrial haplotypes or ploidy level and if they might be associated with fitness divergences and therefore selective value. © 2015 Wiley Periodicals, Inc.

Activation of Akt is essential for the propagation of mitochondrial respiratory stress signaling and activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

PubMed

Guha, Manti; Fang, Ji-Kang; Monks, Robert; Birnbaum, Morris J; Avadhani, Narayan G

2010-10-15

Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFκB c-Rel/p50, C/EBPδ, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in up-regulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

Low-concentration exposure to glyphosate-based herbicide modulates the complexes of the mitochondrial respiratory chain and induces mitochondrial hyperpolarization in the Danio rerio brain.

PubMed

Pereira, Aline G; Jaramillo, Michael L; Remor, Aline P; Latini, Alexandra; Davico, Carla E; da Silva, Mariana L; Müller, Yara M R; Ammar, Dib; Nazari, Evelise M

2018-06-11

Glyphosate (N-phosphonomethyl-glycine) (GLY) is the active ingredient of the most used herbicides in the world. GLY is applied in formulated products known as glyphosate-based herbicides (GBH), which could induce effects that are not predicted by toxicity assays with pure GLY. This herbicide is classified as organophosphorus compound, which is known to induce neurotoxic effects. Although this compound is classified as non-neurotoxic by regulatory agencies, acute exposure to GBH causes neurological symptoms in humans. However, there is no consensus in relation to neurotoxic effects of GBH. Thus, the aim of this study was to investigate the neurotoxic effects of the GBH in the zebrafish Danio rerio, focusing on acute toxicity, the activity and transcript levels of mitochondrial respiratory chain complexes, mitochondrial membrane potential, reactive species (RS) formation, and behavioral repertoire. Adult zebrafish were exposed in vivo to three concentrations of GBH Scout ® , which contained GLY in formulation (fGLY) (0.065, 1.0 and 10.0 mg L -1 fGLY) for 7 d, and an in vitro assay was performed using also pure GLY. Our results show that GBH induced in zebrafish brain a decrease in cell viability, inhibited mitochondrial complex enzymatic activity, modulated gene expression related to mitochondrial complexes, induced an increase in RS production, promoted hyperpolarization of mitochondrial membrane, and induced behavioral impairments. Together, our data contributes to the knowledge of the neurotoxic effects of GBH. Mitochondrial dysfunction has been recognized as a relevant cellular response that should not be disregarded. Moreover, this study pointed to the mitochondria as an important target of GBH. Copyright © 2018 Elsevier Ltd. All rights reserved.

Combined Inhibition of the Renin-Angiotensin System and Neprilysin Positively Influences Complex Mitochondrial Adaptations in Progressive Experimental Heart Failure

PubMed Central

Reinders, Jörg; Schröder, Josef; Dietl, Alexander; Schmid, Peter M.; Jungbauer, Carsten; Resch, Markus; Maier, Lars S.; Luchner, Andreas; Birner, Christoph

2017-01-01

Background Inhibitors of the renin angiotensin system and neprilysin (RAS-/NEP-inhibitors) proved to be extraordinarily beneficial in systolic heart failure. Furthermore, compelling evidence exists that impaired mitochondrial pathways are causatively involved in progressive left ventricular (LV) dysfunction. Consequently, we aimed to assess whether RAS-/NEP-inhibition can attenuate mitochondrial adaptations in experimental heart failure (HF). Methods and Results By progressive right ventricular pacing, distinct HF stages were induced in 15 rabbits, and 6 animals served as controls (CTRL). Six animals with manifest HF (CHF) were treated with the RAS-/NEP-inhibitor omapatrilat. Echocardiographic studies and invasive blood pressure measurements were undertaken during HF progression. Mitochondria were isolated from LV tissue, respectively, and further worked up for proteomic analysis using the SWATH technique. Enzymatic activities of citrate synthase and the electron transfer chain (ETC) complexes I, II, and IV were assessed. Ultrastructural analyses were performed by transmission electron microscopy. During progression to overt HF, intricate expression changes were mainly detected for proteins belonging to the tricarboxylic acid cycle, glucose and fat metabolism, and the ETC complexes, even though ETC complex I, II, or IV enzymatic activities were not significantly influenced. Treatment with a RAS-/NEP-inhibitor then reversed some maladaptive metabolic adaptations, positively influenced the decline of citrate synthase activity, and altered the composition of each respiratory chain complex, even though this was again not accompanied by altered ETC complex enzymatic activities. Finally, ultrastructural evidence pointed to a reduction of autophagolytic and degenerative processes with omapatrilat-treatment. Conclusions This study describes complex adaptations of the mitochondrial proteome in experimental tachycardia-induced heart failure and shows that a combined RAS-/NEP-inhibition can beneficially influence mitochondrial key pathways. PMID:28076404

Iron overload promotes mitochondrial fragmentation in mesenchymal stromal cells from myelodysplastic syndrome patients through activation of the AMPK/MFF/Drp1 pathway.

PubMed

Zheng, Qingqing; Zhao, Youshan; Guo, Juan; Zhao, Sida; Fei, Chengming; Xiao, Chao; Wu, Dong; Wu, Lingyun; Li, Xiao; Chang, Chunkang

2018-05-03

Iron overload (IO) has been reported to contribute to mesenchymal stromal cell (MSC) damage, but the precise mechanism has yet to be clearly elucidated. In this study, we found that IO increased cell apoptosis and lowered cell viability in MSCs, accompanied by extensive mitochondrial fragmentation and autophagy enhancement. All these effects were reactive oxygen species (ROS) dependent. In MSCs with IO, the ATP concentrations were significantly reduced due to high ROS levels and low electron respiratory chain complex (ETC) II/III activity. Reduced ATP phosphorylated AMP-activated protein kinase (AMPK). Activation of AMPK kinase complexes triggered mitochondrial fission. Moreover, gene knockout of AMPK via CRISPR/Cas9 reduced cell apoptosis, enhanced cell viability and attenuated mitochondrial fragmentation and autophagy caused by IO in MSCs. Further, AMPK-induced mitochondrial fragmentation of MSCs with IO was mediated via phosphorylation of mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for the GTPase dynamin-related protein 1 (Drp1). Gene knockdown of MFF reversed AMPK-induced mitochondrial fragmentation in MSCs with IO. In addition, MSCs from IO patients with myelodysplastic syndrome (MDS) showed increased cell apoptosis, decreased cell viability, higher ROS levels, lower ATP concentrations and increased mitochondrial fragmentation compared with MSCs from non-IO patients. In addition, iron chelation or antioxidant weakened the activity of the AMPK/MFF/Drp1 pathway in MDS-MSCs with IO from several patients, accompanied by attenuation of mitochondrial fragmentation and autophagy. Taken together, the AMPK/MFF/Drp1 pathway has an important role in the damage to MDS-MSCs caused by IO.

Apolipoprotein E4 (1–272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells

PubMed Central

Nakamura, Toshiyuki; Watanabe, Atsushi; Fujino, Takahiro; Hosono, Takashi; Michikawa, Makoto

2009-01-01

Background Apolipoprotein E allele ε4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1–272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. Results To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1–272) more strongly than intact apoE4(1–299). Further analysis showed that in Neuro-2a cells expressing apoE4(1–272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1–299). Conclusion ApoE4(1–272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1–272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration. PMID:19695092

Mitochondrial generation of superoxide and hydrogen peroxide as the source of mitochondrial redox signaling.

PubMed

Brand, Martin D

2016-11-01

This review examines the generation of reactive oxygen species by mammalian mitochondria, and the status of different sites of production in redox signaling and pathology. Eleven distinct mitochondrial sites associated with substrate oxidation and oxidative phosphorylation leak electrons to oxygen to produce superoxide or hydrogen peroxide: oxoacid dehydrogenase complexes that feed electrons to NAD + ; respiratory complexes I and III, and dehydrogenases, including complex II, that use ubiquinone as acceptor. The topologies, capacities, and substrate dependences of each site have recently clarified. Complex III and mitochondrial glycerol 3-phosphate dehydrogenase generate superoxide to the external side of the mitochondrial inner membrane as well as the matrix, the other sites generate superoxide and/or hydrogen peroxide exclusively in the matrix. These different site-specific topologies are important for redox signaling. The net rate of superoxide or hydrogen peroxide generation depends on the substrates present and the antioxidant systems active in the matrix and cytosol. The rate at each site can now be measured in complex substrate mixtures. In skeletal muscle mitochondria in media mimicking muscle cytosol at rest, four sites dominate, two in complex I and one each in complexes II and III. Specific suppressors of two sites have been identified, the outer ubiquinone-binding site in complex III (site III Qo ) and the site in complex I active during reverse electron transport (site I Q ). These suppressors prevent superoxide/hydrogen peroxide production from a specific site without affecting oxidative phosphorylation, making them excellent tools to investigate the status of the sites in redox signaling, and to suppress the sites to prevent pathologies. They allow the cellular roles of mitochondrial superoxide/hydrogen peroxide production to be investigated without catastrophic confounding bioenergetic effects. They show that sites III Qo and I Q are active in cells and have important roles in redox signaling (e.g. hypoxic signaling and ER-stress) and in causing oxidative damage in a variety of biological contexts. Copyright © 2016 Elsevier Inc. All rights reserved.

MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging.

PubMed

Lustgarten, Michael S; Jang, Youngmok C; Liu, Yuhong; Qi, Wenbo; Qin, Yuejuan; Dahia, Patricia L; Shi, Yun; Bhattacharya, Arunabh; Muller, Florian L; Shimizu, Takahiko; Shirasawa, Takuji; Richardson, Arlan; Van Remmen, Holly

2011-06-01

In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2(fl/fl) mice). In this study, we used TnIFastCreSod2(fl/fl) mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2(fl/fl) mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2(fl/fl) mice, when compared with control mice. Complex II activity is reduced by 47% in young and by approximately 90% in old TnIFastCreSod2(fl/fl) mice, and was found to be associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2(fl/fl) mice. Complex II-linked mitochondrial Adenosine-Tri-Phosphate (ATP) production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2(fl/fl) mice. Furthermore, in old TnIFastCreSod2(fl/fl) mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains > 2-fold elevated; and oxidative damage (measured as F(2) - isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage, and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2(fl/fl) mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy. No claim to original US government works. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart.

PubMed

Gong, Guohua; Liu, Xiaoyun; Zhang, Huiliang; Sheu, Shey-Shing; Wang, Wang

2015-10-01

Mitochondrial respiration through electron transport chain (ETC) activity generates ATP and reactive oxygen species in eukaryotic cells. The modulation of mitochondrial respiration in vivo or under physiological conditions remains elusive largely due to the lack of appropriate approach to monitor ETC activity in a real-time manner. Here, we show that ETC-coupled mitochondrial flash is a novel biomarker for monitoring mitochondrial respiration under pathophysiological conditions in cultured adult cardiac myocyte and perfused beating heart. Through real-time confocal imaging, we follow the frequency of a transient bursting fluorescent signal, named mitochondrial flash, from individual mitochondria within intact cells expressing a mitochondrial matrix-targeted probe, mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein). This mt-cpYFP recorded mitochondrial flash has been shown to be composed of a major superoxide signal with a minor alkalization signal within the mitochondrial matrix. Through manipulating physiological substrates for mitochondrial respiration, we find a close coupling between flash frequency and the ETC electron flow, as measured by oxygen consumption rate in cardiac myocyte. Stimulating electron flow under physiological conditions increases flash frequency. On the other hand, partially block or slowdown electron flow by inhibiting the F0F1 ATPase, which represents a pathological condition, transiently increases then decreases flash frequency. Limiting electron entrance at complex I by knocking out Ndufs4, an assembling subunit of complex I, suppresses mitochondrial flash activity. These results suggest that mitochondrial electron flow can be monitored by real-time imaging of mitochondrial flash. The mitochondrial flash frequency could be used as a novel biomarker for mitochondrial respiration under physiological and pathological conditions. Copyright © 2015 the American Physiological Society.

Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency.

PubMed

Stein, Colleen S; Jadiya, Pooja; Zhang, Xiaoming; McLendon, Jared M; Abouassaly, Gabrielle M; Witmer, Nathan H; Anderson, Ethan J; Elrod, John W; Boudreau, Ryan L

2018-06-26

Mitochondria are composed of many small proteins that control protein synthesis, complex assembly, metabolism, and ion and reactive oxygen species (ROS) handling. We show that a skeletal muscle- and heart-enriched long non-coding RNA, LINC00116, encodes a highly conserved 56-amino-acid microprotein that we named mitoregulin (Mtln). Mtln localizes to the inner mitochondrial membrane, where it binds cardiolipin and influences protein complex assembly. In cultured cells, Mtln overexpression increases mitochondrial membrane potential, respiration rates, and Ca 2+ retention capacity while decreasing mitochondrial ROS and matrix-free Ca 2+ . Mtln-knockout mice display perturbations in mitochondrial respiratory (super)complex formation and activity, fatty acid oxidation, tricarboxylic acid (TCA) cycle enzymes, and Ca 2+ retention capacity. Blue-native gel electrophoresis revealed that Mtln co-migrates alongside several complexes, including the complex I assembly module, complex V, and supercomplexes. Under denaturing conditions, Mtln remains in high-molecular-weight complexes, supporting its role as a sticky molecular tether that enhances respiratory efficiency by bolstering protein complex assembly and/or stability. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Xanthohumol induces generation of reactive oxygen species and triggers apoptosis through inhibition of mitochondrial electron transfer chain complex I.

PubMed

Zhang, Bo; Chu, Wei; Wei, Peng; Liu, Ying; Wei, Taotao

2015-12-01

Xanthohumol is a prenylflavonoid extracted from hops (Humulus lupulus). It possesses anti-cancer and anti-inflammatory activities in vitro and in vivo, and offers therapeutic benefits for treatment of metabolic syndromes. However, the precise mechanisms underlying its pharmacological effects remain to be elucidated, together with its cellular target. Here, we provide evidence that xanthohumol directly interacts with the mitochondrial electron transfer chain complex I (NADH dehydrogenase), inhibits the oxidative phosphorylation, triggers the production of reactive oxygen species, and induces apoptosis. In addition, we show that as a result of the inhibition of the mitochondrial oxidative phosphorylation, xanthohumol exposure causes a rapid decrease of mitochondrial transmembrane potential. Furthermore, we showed that xanthohumol up-regulates the glycolytic capacity in cells, and thus compensates cellular ATP generation. Dissection of the multiple steps of aerobic respiration by extracellular flux assays revealed that xanthohumol specifically inhibits the activity of mitochondrial complex I, but had little effect on that of complex II, III and IV. Inhibition of complex I by xanthohumol caused the overproduction of reactive oxygen species, which are responsible for the induction of apoptosis in cancer cells. We also found that isoxanthohumol, the structural isomer of xanthohumol, is inactive to cells, suggesting that the reactive 2-hydroxyl group of xanthohumol is crucial for its targeting to the mitochondrial complex I. Together, the remodeling of cell metabolism revealed here has therapeutic potential for the use of xanthohumol. Copyright © 2015 Elsevier Inc. All rights reserved.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake.

PubMed

Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

2017-05-01

Mitochondrial calcium ([Ca 2+ ] m ) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca 2+ ] m uptake upon SK channel activation as detected by time lapse mitochondrial Ca 2+ measurements with the Ca 2+ -binding mitochondria-targeted aequorin and FRET-based [Ca 2+ ] m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca 2+ ] m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake

PubMed Central

Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

2017-01-01

Mitochondrial calcium ([Ca2+]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca2+]m uptake upon SK channel activation as detected by time lapse mitochondrial Ca2+ measurements with the Ca2+-binding mitochondria-targeted aequorin and FRET-based [Ca2+]m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca2+]m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death. PMID:28282037

SiO2 nanoparticle-induced impairment of mitochondrial energy metabolism in hepatocytes directly and through a Kupffer cell-mediated pathway in vitro

PubMed Central

Xue, Yang; Chen, Qingqing; Ding, Tingting; Sun, Jiao

2014-01-01

The liver has been shown to be a primary target organ for SiO2 nanoparticles in vivo, and may be highly susceptible to damage by these nanoparticles. However, until now, research focusing on the potential toxic effects of SiO2 nanoparticles on mitochondria-associated energy metabolism in hepatocytes has been lacking. In this work, SiO2 nanoparticles 20 nm in diameter were evaluated for their ability to induce dysfunction of mitochondrial energy metabolism. First, a buffalo rat liver (BRL) cell line was directly exposed to SiO2 nanoparticles, which induced cytotoxicity and mitochondrial damage accompanied by decreases in mitochondrial dehydrogenase activity, mitochondrial membrane potential, enzymatic expression in the Krebs cycle, and activity of the mitochondrial respiratory chain complexes I, III and IV. Second, the role of rat-derived Kupffer cells was evaluated. The supernatants from Kupffer cells treated with SiO2 nanoparticles were transferred to stimulate BRL cells. We observed that SiO2 nanoparticles had the ability to activate Kupffer cells, leading to release of tumor necrosis factor-α, nitric oxide, and reactive oxygen species from these cells and subsequently to inhibition of mitochondrial respiratory chain complex I activity in BRL cells. PMID:24959077

Plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity.

PubMed

Joseph, Anna-Maria; Hood, David A

2012-03-01

We investigated the assembly of the TOM complex within skeletal muscle under conditions of chronic contractile activity-induced mitochondrial biogenesis. Tom40 import into mitochondria was increased by chronic contractile activity, as was its time-dependent assembly into the TOM complex. These changes coincided with contractile activity-induced augmentations in the expression of key protein import machinery components Tim17, Tim23, and Tom22, as well as the cytosolic chaperone Hsp90. These data indicate the adaptability of the TOM protein import complex and suggest a regulatory role for the assembly of this complex in exercise-induced mitochondrial biogenesis. Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.

Complex IV Deficient Surf1−/− Mice Initiate Mitochondrial Stress Responses

PubMed Central

Pulliam, Daniel A.; Deepa, Sathyaseelan S.; Liu, Yuhong; Hill, Shauna; Lin, Ai-Ling; Bhattacharya, Arunabh; Shi, Yun; Sloane, Lauren; Viscomi, Carlo; Zeviani, Massimo; Van Remmen, Holly

2014-01-01

Summary Mutations in SURF1 cytochrome c oxidase (COX) assembly protein are associated with Leigh’s syndrome, a human mitochondrial disorder that manifests as severe mitochondrial phenotypes and early lethality. In contrast, mice lacking the Surf1 protein (Surf1−/−) are viable and were previously shown to have enhanced longevity and a greater than 50% reduction in COX activity. We measured mitochondrial function in heart and skeletal muscle, and despite the significant reduction in COX activity, we found little or no difference in reactive oxygen species (ROS) generation, membrane potential, ATP production or respiration in isolated mitochondria from Surf1−/− mice compared to wild-type. However, blood lactate levels are elevated and Surf1−/− mice have reduced running endurance, suggesting compromised mitochondrial energy metabolism in vivo. Decreased COX activity in Surf1−/− mice is associated with increased markers of mitochondrial biogenesis (PGC-1α and VDAC) in both heart and skeletal muscle. While mitochondrial biogenesis is a common response in the two tissues, skeletal muscle have an up-regulation of the mitochondrial unfolded protein response (UPRMT) and heart exhibits induction of the Nrf2 antioxidant response pathway. These data are the first to report induction of the UPRMT in a mammalian model of diminished COX activity. In addition our results suggest that impaired mitochondrial function can lead to induction of mitochondrial stress pathways to confer protective effects on cellular homeostasis. Loss of complex IV assembly factor Surf1 in mice results in compensatory responses including mitochondrial biogenesis, the nrf2 pathway and the mitochondrial unfolded protein response. This compensatory response may contribute to the lack of deleterious phenotypes under basal conditions. PMID:24911525

ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

PubMed Central

Babot, Marion; Labarbuta, Paola; Birch, Amanda; Kee, Sara; Fuszard, Matthew; Botting, Catherine H.; Wittig, Ilka; Heide, Heinrich; Galkin, Alexander

2014-01-01

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III2 + IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover. PMID:24560811

Palladium alpha-lipoic acid complex formulation enhances activities of Krebs cycle dehydrogenases and respiratory complexes I-IV in the heart of aged rats.

PubMed

Sudheesh, N P; Ajith, T A; Janardhanan, K K; Krishnan, C V

2009-08-01

Age-related decline in the capacity to withstand stress, such as ischemia and reperfusion, results in congestive heart failure. Though the mechanisms underlying cardiac decay are not clear, age dependent somatic damages to mitochondrial DNA (mtDNA), loss of mitochondrial function, and a resultant increase in oxidative stress in heart muscle cells may be responsible for the increased risk for cardiovascular diseases. The effect of a safe nutritional supplement, POLY-MVA, containing the active ingredient palladium alpha-lipoic acid complex, was evaluated on the activities of the Krebs cycle enzymes such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV in heart mitochondria of aged male albino rats of Wistar strain. Administration of 0.05 ml/kg of POLY-MVA (which is equivalent to 0.38 mg complexed alpha-lipoic acid/kg, p.o), once daily for 30 days, was significantly (p<0.05) effective to enhance the Krebs cycle dehydrogenases, and mitochondrial electron transport chain complexes. The unique electronic and redox properties of palladium alpha-lipoic acid complex appear to be a key to this physiological effectiveness. The results strongly suggest that this formulation might be effective to protect the aging associated risk of cardiovascular and neurodegenerative diseases.

Maternal High Fat Diet Alters Skeletal Muscle Mitochondrial Catalytic Activity in Adult Male Rat Offspring

PubMed Central

Pileggi, Chantal A.; Hedges, Christopher P.; Segovia, Stephanie A.; Markworth, James F.; Durainayagam, Brenan R.; Gray, Clint; Zhang, Xiaoyuan D.; Barnett, Matthew P. G.; Vickers, Mark H.; Hickey, Anthony J. R.; Reynolds, Clare M.; Cameron-Smith, David

2016-01-01

A maternal high-fat (HF) diet during pregnancy can lead to metabolic compromise, such as insulin resistance in adult offspring. Skeletal muscle mitochondrial dysfunction is one mechanism contributing to metabolic impairments in insulin resistant states. Therefore, the present study aimed to investigate whether mitochondrial dysfunction is evident in metabolically compromised offspring born to HF-fed dams. Sprague-Dawley dams were randomly assigned to receive a purified control diet (CD; 10% kcal from fat) or a high fat diet (HFD; 45% kcal from fat) for 10 days prior to mating, throughout pregnancy and during lactation. From weaning, all male offspring received a standard chow diet and soleus muscle was collected at day 150. Expression of the mitochondrial transcription factors nuclear respiratory factor-1 (NRF1) and mitochondrial transcription factor A (mtTFA) were downregulated in HF offspring. Furthermore, genes encoding the mitochondrial electron transport system (ETS) respiratory complex subunits were suppressed in HF offspring. Moreover, protein expression of the complex I subunit, NDUFB8, was downregulated in HF offspring (36%), which was paralleled by decreased maximal catalytic linked activity of complex I and III (40%). Together, these results indicate that exposure to a maternal HF diet during development may elicit lifelong mitochondrial alterations in offspring skeletal muscle. PMID:27917127

Mitochondrial Respiratory Chain Dysfunction in Dorsal Root Ganglia of Streptozotocin-Induced Diabetic Rats and Its Correction by Insulin Treatment

PubMed Central

Chowdhury, Subir K. Roy; Zherebitskaya, Elena; Smith, Darrell R.; Akude, Eli; Chattopadhyay, Sharmila; Jolivalt, Corinne G.; Calcutt, Nigel A.; Fernyhough, Paul

2010-01-01

OBJECTIVE Impairments in mitochondrial physiology may play a role in diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in sensory neurons is due to abnormal mitochondrial respiratory function. RESEARCH DESIGN AND METHODS Rates of oxygen consumption were measured in mitochondria from dorsal root ganglia (DRG) of 12- to- 22-week streptozotocin (STZ)-induced diabetic rats, diabetic rats treated with insulin, and age-matched controls. Activities and expression of components of mitochondrial complexes and reactive oxygen species (ROS) were analyzed. RESULTS Rates of coupled respiration with pyruvate + malate (P + M) and with ascorbate + TMPD (Asc + TMPD) in DRG were unchanged after 12 weeks of diabetes. By 22 weeks of diabetes, respiration with P + M was significantly decreased by 31–44% and with Asc + TMPD by 29–39% compared with control. Attenuated mitochondrial respiratory activity of STZ-diabetic rats was significantly improved by insulin that did not correct other indices of diabetes. Activities of mitochondrial complexes I and IV and the Krebs cycle enzyme, citrate synthase, were decreased in mitochondria from DRG of 22-week STZ-diabetic rats compared with control. ROS levels in perikarya of DRG neurons were not altered by diabetes, but ROS generation from mitochondria treated with antimycin A was diminished compared with control. Reduced mitochondrial respiratory function was associated with downregulation of expression of mitochondrial proteins. CONCLUSIONS Mitochondrial dysfunction in sensory neurons from type 1 diabetic rats is associated with impaired rates of respiratory activity and occurs without a significant rise in perikaryal ROS. PMID:20103706

Indian Ginseng (Withania somnifera) supplementation ameliorates oxidative stress and mitochondrial dysfunctions in experimental model of stroke.

PubMed

Sood, Abhilasha; Mehrotra, Arpit; Dhawan, Devinder K; Sandhir, Rajat

2018-04-18

Stroke is an increasingly prevalent clinical condition and second leading cause of death globally. The present study evaluated the therapeutic potential of Indian Ginseng, also known as Withania somnifera (WS), supplementation on middle cerebral artery occlusion (MCAO) induced mitochondrial dysfunctions in experimental model of ischemic stroke. Stroke was induced in animals by occluding the middle cerebral artery, followed by reperfusion injury. Ischemia reperfusion injury resulted in increased oxidative stress indicated by increased reactive oxygen species and protein carbonyl levels; compromised antioxidant system; in terms of reduced superoxide dismutase and catalase activity, along with reduction in GSH levels and the redox ratio, impaired mitochondrial functions and enhanced expression of apoptosis markers. Ischemia reperfusion injury induced mitochondrial dysfunctions in terms of (i) reduced activity of the mitochondrial respiratory chain enzymes, (ii) reduced histochemical staining of complex-II and IV, (iii) reduced in-gel activity of mitochondrial complex-I to V, (iv) mitochondrial structural changes in terms of increased mitochondrial swelling, reduced mitochondrial membrane potential and ultrastructural changes. Additionally, an increase in the activity of caspase-3 and caspase-9 was also observed, along with altered expression of apoptotic proteins Bcl-2 and Bax in MCAO animals. MCAO animals also showed significant impairment in cognitive functions assessed using Y maze test. WS pre-supplementation, on the other hand ameliorated MCAO induced oxidative stress, mitochondrial dysfunctions, apoptosis and cognitive impairments. The results show protective effect of WS pre-supplementation in ischemic stroke and are suggestive of its potential application in stroke management.

Inhibitory Effects of Amorphigenin on the Mitochondrial Complex I of Culex pipiens pallens Coquillett (Diptera: Culicidae)

PubMed Central

Ji, Mingshan; Liang, Yaping; Gu, Zumin; Li, Xiuwei

2015-01-01

Previous studies in our laboratory found that the extract from seeds of Amorpha fruticosa in the Leguminosae family had lethal effects against mosquito larvae, and an insecticidal compound amorphigenin was isolated. In this study, the inhibitory effects of amorphigenin against the mitochondrial complex I of Culex pipiens pallens (Diptera: Culicidae) were investigated and compared with that of rotenone. The results showed that amorphigenin and rotenone can decrease the mitochondrial complex I activity both in vivo and in vitro as the in vivo IC50 values (the inhibitor concentrations leading to 50% of the enzyme activity lost) were determined to be 2.4329 and 2.5232 μmol/L, respectively, while the in vitro IC50 values were 2.8592 and 3.1375 μmol/L, respectively. Both amorphigenin and rotenone were shown to be reversible and mixed-I type inhibitors of the mitochondrial complex I of Cx. pipiens pallens, indicating that amorphigenin and rotenone inhibited the enzyme activity not only by binding with the free enzyme but also with the enzyme-substrate complex, and the values of KI and KIS for amorphigenin were determined to be 20.58 and 87.55 μM, respectively, while the values for rotenone were 14.04 and 69.23 μM, respectively. PMID:26307964

Oxidative phosphorylation in Debaryomyces hansenii: physiological uncoupling at different growth phases.

PubMed

Cabrera-Orefice, Alfredo; Guerrero-Castillo, Sergio; Díaz-Ruíz, Rodrigo; Uribe-Carvajal, Salvador

2014-07-01

Physiological uncoupling of mitochondrial oxidative phosphorylation (OxPhos) was studied in Debaryomyces hansenii. In other species, such as Yarrowia lipolytica and Saccharomyces cerevisiae, OxPhos can be uncoupled through differential expression of branched respiratory chain enzymes or by opening of a mitochondrial unspecific channel (ScMUC), respectively. However D. hansenii mitochondria, which contain both a branched respiratory chain and a mitochondrial unspecific channel (DhMUC), selectively uncouple complex I-dependent rate of oxygen consumption in the stationary growth phase. The uncoupled complex I-dependent respiration was only 20% of the original activity. Inhibition was not due to inactivation of complex I, lack of protein expression or to differential expression of alternative oxidoreductases. Furthermore, all other respiratory chain activities were normal. Decrease of complex I-dependent respiration was due to NAD(+) loss from the matrix, probably through an open of DhMUC. When NAD(+) was added back, coupled complex I-activity was recovered. NAD(+) re-uptake was independent of DhMUC opening and seemed to be catalyzed by a NAD(+)-specific transporter, which was sensitive to bathophenanthroline, bromocresol purple or pyridoxal-5'-phosphate as described for S. cerevisiae mitochondrial NAD(+) transporters. Loss of NAD(+) from the matrix through an open MUC is proposed as an additional mechanism to uncouple OxPhos. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells.

PubMed

Garofalo, Tina; Ferri, Alberto; Sorice, Maurizio; Azmoon, Pardis; Grasso, Maria; Mattei, Vincenzo; Capozzi, Antonella; Manganelli, Valeria; Misasi, Roberta

2018-04-01

Since stressing conditions induce a relocalization of endogenous human neuroglobin (NGB) to mitochondria, this research is aimed to evaluate the protective role of NGB overexpression against neurotoxic stimuli, through mitochondrial lipid raft-associated complexes. To this purpose, we built a neuronal model of oxidative stress by the use of human dopaminergic neuroblastoma cells, SK-N-BE2, stably overexpressing NGB by transfection and treated with 1-methyl-4-phenylpyridinium ion (MPP+). We preliminary observed the redistribution of NGB to mitochondria following MPP+ treatment. The analysis of mitochondrial raft-like microdomains revealed that, following MPP+ treatment, NGB translocated to raft fractions (Triton X-100-insoluble), where it interacts with ganglioside GD3. Interestingly, the administration of agents capable of perturbating microdomain before MPP+ treatment, significantly affected viability in SK-N-BE2-NGB cells. The overexpression of NGB was able to abrogate the mitochondrial injuries on complex IV activity or mitochondrial morphology induced by MPP+ administration. The protective action of NGB on mitochondria only takes place if the mitochondrial lipid(s) rafts-like microdomains are intact, indeed NGB fails to protect complex IV activity when purified mitochondria were treated with the lipid rafts disruptor methyl-β-cyclodextrin. Thus, our unique in vitro model of stably transfected cells overexpressing endogenous NGB allowed us to suggest that the role in neuroprotection played by NGB is reliable only through interaction with mitochondrial lipid raft-associated complexes. Copyright © 2018 Elsevier Inc. All rights reserved.

Genetic reduction of mitochondrial complex I function does not lead to loss of dopamine neurons in vivo.

PubMed

Kim, Hyung-Wook; Choi, Won-Seok; Sorscher, Noah; Park, Hyung Joon; Tronche, François; Palmiter, Richard D; Xia, Zhengui

2015-09-01

Inhibition of mitochondrial complex I activity is hypothesized to be one of the major mechanisms responsible for dopaminergic neuron death in Parkinson's disease. However, loss of complex I activity by systemic deletion of the Ndufs4 gene, one of the subunits comprising complex I, does not cause dopaminergic neuron death in culture. Here, we generated mice with conditional Ndufs4 knockout in dopaminergic neurons (Ndufs4 conditional knockout mice [cKO]) to examine the effect of complex I inhibition on dopaminergic neuron function and survival during aging and on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in vivo. Ndufs4 cKO mice did not show enhanced dopaminergic neuron loss in the substantia nigra pars compacta or dopamine-dependent motor deficits over the 24-month life span. These mice were just as susceptible to MPTP as control mice. However, compared with control mice, Ndufs4 cKO mice exhibited an age-dependent reduction of dopamine in the striatum and increased α-synuclein phosphorylation in dopaminergic neurons of the substantia nigra pars compacta. We also used an inducible Ndufs4 knockout mouse strain (Ndufs4 inducible knockout) in which Ndufs4 is conditionally deleted in all cells in adult to examine the effect of adult onset, complex I inhibition on MPTP sensitivity of dopaminergic neurons. The Ndufs4 inducible knockout mice exhibited similar sensitivity to MPTP as control littermates. These data suggest that mitochondrial complex I inhibition in dopaminergic neurons does contribute to dopamine loss and the development of α-synuclein pathology. However, it is not sufficient to cause cell-autonomous dopaminergic neuron death during the normal life span of mice. Furthermore, mitochondrial complex I inhibition does not underlie MPTP toxicity in vivo in either cell autonomous or nonautonomous manner. These results provide strong evidence that inhibition of mitochondrial complex I activity is not sufficient to cause dopaminergic neuron death during aging nor does it contribute to dopamine neuron toxicity in the MPTP model of Parkinson's disease. These findings suggest the existence of alternative mechanisms of dopaminergic neuron death independent of mitochondrial complex I inhibition. Copyright © 2015 Elsevier Inc. All rights reserved.

Tumor cell death induced by the inhibition of mitochondrial electron transport: The effect of 3-hydroxybakuchiol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaña, Fabián; Faini, Francesca; Lapier, Michel

Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line andmore » induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells. - Highlights: • We studied the anticancer activity of a natural compound, 3-OHbk, on TA3/Ha cells. • 3-OHbk inhibited mitochondrial electron flow by interacting with Complex I. • Complex I inhibition did not induce ROS generation. • 3-OHbk induced apoptosis in tumor cells with no effect on mammary epithelial cells. • Mitochondrial bioenergetics is implicated in anticancer action of 3-OHbk.« less

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

PubMed

Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

2008-05-02

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

Loss of Pink1 modulates synaptic mitochondrial bioenergetics in the rat striatum prior to motor symptoms: concomitant complex I respiratory defects and increased complex II-mediated respiration.

PubMed

Stauch, Kelly L; Villeneuve, Lance M; Purnell, Phillip R; Ottemann, Brendan M; Emanuel, Katy; Fox, Howard S

2016-12-01

Mutations in PTEN-induced putative kinase 1 (Pink1), a mitochondrial serine/threonine kinase, cause a recessive inherited form of Parkinson's disease (PD). Pink1 deletion in rats results in a progressive PD-like phenotype, characterized by significant motor deficits starting at 4 months of age. Despite the evidence of mitochondrial dysfunction, the pathogenic mechanism underlying disease due to Pink1-deficiency remains obscure. Striatal synaptic mitochondria from 3-month-old Pink1-deficient rats were characterized using bioenergetic and mass spectroscopy (MS)-based proteomic analyses. Striatal synaptic mitochondria from Pink1-deficient rats exhibit decreased complex I-driven respiration and increased complex II-mediated respiration compared with wild-type rats. MS-based proteomics revealed 69 of the 811 quantified mitochondrial proteins were differentially expressed between Pink1-deficient rats and controls. Down-regulation of several electron carrier proteins, which shuttle electrons to reduce ubiquinone at complex III, in the Pink1-knockouts suggests disruption of the linkage between fatty acid, amino acid, and choline metabolism and the mitochondrial respiratory system. These results suggest that complex II activity is increased to compensate for loss of electron transfer mechanisms due to reduced complex I activity and loss of electron carriers within striatal nerve terminals early during disease progression. This may contribute to the pathogenesis of PD. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phellinus rimosus improves mitochondrial energy status and attenuates nephrotoxicity in diabetic rats.

PubMed

Rony, K A; Ajith, T A; Kuttikadan, Tony A; Blaze, R; Janardhanan, K K

2017-09-26

Mitochondrial dysfunction and increase in reactive oxygen species during diabetes can lead to pathological consequences in kidneys. The present study was aimed to investigate the effect of Phellinus rimosus in the streptozotocin (STZ)-induced diabetic rat renal mitochondria and the possible mechanism of protection. Phellinus rimosus (50 and 250 mg/kg, p.o) was treated after inducing diabetes by STZ (45 mg/kg, i.p) in rats. The serum samples were subjected to creatinine and urea estimation. Mitochondrial antioxidant status such as mitochondrial superoxide dismutase, glutathione peroxidase, and reduced glutathione; adenosine triphosphate level; and lipid peroxidation were measured. The activities of Krebs cycle enzymes such as isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, III, and IV in kidney mitochondria were also determined. Administration of P. rimosus (250 mg/kg b.wt) once daily for 30 days, significantly (p<0.05) enhanced the activities of Krebs cycle dehydrogenases, mitochondrial electron transport chain complexes, and ATP level. Further, P. rimosus had significantly protected the renal mitochondrial antioxidant status and lipid peroxidation. The results of the study concluded that by limiting the extent of renal mitochondrial damage in the hyperglycemic state, P. rimosus alleviated nephrotoxicity.

Oxidative Inactivation of Liver Mitochondria in High Fructose Diet-Induced Metabolic Syndrome in Rats: Effect of Glycyrrhizin Treatment.

PubMed

Sil, Rajarshi; Chakraborti, Abhay Sankar

2016-09-01

Metabolic syndrome is a serious health problem in the present world. Glycyrrhizin, a triterpenoid saponin of licorice (Glycyrrhiza glabra) root, has been reported to ameliorate the primary complications and hepatocellular damage in rats with the syndrome. In this study, we have explored metabolic syndrome-induced changes in liver mitochondrial function and effect of glycyrrhizin against the changes. Metabolic syndrome was induced in rats by high fructose (60%) diet for 6 weeks. The rats were then treated with glycyrrhizin (50 mg/kg body weight) by single intra-peritoneal injection. After 2 weeks of the treatment, the rats were sacrificed to collect liver tissue. Elevated mitochondrial ROS, lipid peroxidation and protein carbonyl, and decreased reduced glutathione content indicated oxidative stress in metabolic syndrome. Loss of mitochondrial inner membrane cardiolipin was observed. Mitochondrial complex I activity did not change but complex IV activity decreased significantly. Mitochondrial MTT reduction ability, membrane potential, phosphate utilisation and oxygen consumption decreased in metabolic syndrome. Reduced mitochondrial aconitase activity and increased aconitase carbonyl content suggested oxidative damage of the enzyme. Elevated Fe(2+) ion level in mitochondria might be associated with increased ROS generation in metabolic syndrome. Glycyrrhizin effectively attenuated mitochondrial oxidative stress and aconitase degradation, and improved electron transport chain activity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Mitochondrial phenotype during torpor: Modulation of mitochondrial electron transport system in the Chilean mouse-opossum Thylamys elegans.

PubMed

Cortes, Pablo A; Bozinovic, Francisco; Blier, Pierre U

2018-07-01

Mammalian torpor is a phenotype characterized by a controlled decline of metabolic rate, generally followed by a reduction in body temperature. During arousal from torpor, both metabolic rate and body temperature rapidly returns to resting levels. Metabolic rate reduction experienced by torpid animals is triggered by active suppression of mitochondrial respiration, which is rapidly reversed during rewarming process. In this study, we analyzed the changes in the maximal activity of key enzymes related to electron transport system (complexes I, III and IV) in six tissues of torpid, arousing and euthermic Chilean mouse-opossums (Thylamys elegans). We observed higher maximal activities of complexes I and IV during torpor in brain, heart and liver, the most metabolically active organs in mammals. On the contrary, higher enzymatic activities of complexes III were observed during torpor in kidneys and lungs. Moreover, skeletal muscle was the only tissue without significant differences among stages in all complexes evaluated, suggesting no modulation of oxidative capacities of electron transport system components in this thermogenic tissue. In overall, our data suggest that complexes I and IV activity plays a major role in initiation and maintenance of metabolic suppression during torpor in Chilean mouse-opossum, whereas improvement of oxidative capacities in complex III might be critical to sustain metabolic machinery in organs that remains metabolically active during torpor. Copyright © 2018 Elsevier Inc. All rights reserved.

Cell Cycle Regulators Guide Mitochondrial Activity in Radiation-Induced Adaptive Response

PubMed Central

Alexandrou, Aris T.

2014-01-01

Abstract Significance: There are accruing concerns on potential genotoxic agents present in the environment including low-dose ionizing radiation (LDIR) that naturally exists on earth's surface and atmosphere and is frequently used in medical diagnosis and nuclear industry. Although its long-term health risk is being evaluated and remains controversial, LDIR is shown to induce temporary but significant adaptive responses in mammalian cells and animals. The mechanisms guiding the mitochondrial function in LDIR-induced adaptive response represent a unique communication between DNA damage and cellular metabolism. Elucidation of the LDIR-regulated mitochondrial activity may reveal new mechanisms adjusting cellular function to cope with hazardous environmental stress. Recent Advances: Key cell cycle regulators, including Cyclin D1/CDK4 and Cyclin B1/cyclin-dependent kinase 1 (CDK1) complexes, are actively involved in the regulation of mitochondrial functions via phosphorylation of their mitochondrial targets. Accumulating new evidence supports a concept that the Cyclin B1/CDK1 complex acts as a mediator in the cross talk between radiation-induced DNA damage and mitochondrial functions to coordinate cellular responses to low-level genotoxic stresses. Critical Issues: The LDIR-mediated mitochondrial activity via Cyclin B1/CDK1 regulation is an irreplaceable network that is able to harmonize vital cellular functions with adjusted mitochondrial metabolism to enhance cellular homeostasis. Future Directions: Further investigation of the coordinative mechanism that regulates mitochondrial activities in sublethal stress conditions, including LDIR, will reveal new insights of how cells cope with genotoxic injury and will be vital for future targeted therapeutic interventions that reduce environmental injury and cancer risk. Antioxid. Redox Signal. 20, 1463–1480. PMID:24180340

Initiation of Electron Transport Chain Activity in the Embryonic Heart Coincides with the Activation of Mitochondrial Complex 1 and the Formation of Supercomplexes

PubMed Central

Beutner, Gisela; Eliseev, Roman A.; Porter, George A.

2014-01-01

Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes. PMID:25427064

Initiation of electron transport chain activity in the embryonic heart coincides with the activation of mitochondrial complex 1 and the formation of supercomplexes.

PubMed

Beutner, Gisela; Eliseev, Roman A; Porter, George A

2014-01-01

Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes.

Thyrotropin-releasing hormone controls mitochondrial biology in human epidermis.

PubMed

Knuever, Jana; Poeggeler, Burkhard; Gáspár, Erzsébet; Klinger, Matthias; Hellwig-Burgel, Thomas; Hardenbicker, Celine; Tóth, Balázs I; Bíró, Tamás; Paus, Ralf

2012-03-01

Mitochondrial capacity and metabolic potential are under the control of hormones, such as thyroid hormones. The most proximal regulator of the hypothalamic-pituitary-thyroid (HPT) axis, TRH, is the key hypothalamic integrator of energy metabolism via its impact on thyroid hormone secretion. Here, we asked whether TRH directly modulates mitochondrial functions in normal, TRH-receptor-positive human epidermis. Organ-cultured human skin was treated with TRH (5-100 ng/ml) for 12-48 h. TRH significantly increased epidermal immunoreactivity for the mitochondria-selective subunit I of respiratory chain complex IV (MTCO1). This resulted from an increased MTCO1 transcription and protein synthesis and a stimulation of mitochondrial biogenesis as demonstrated by transmission electron microscopy and TRH-enhanced mitochondrial DNA synthesis. TRH also significantly stimulated the transcription of several other mitochondrial key genes (TFAM, HSP60, and BMAL1), including the master regulator of mitochondrial biogenesis (PGC-1α). TRH significantly enhanced mitochondrial complex I and IV enzyme activity and enhanced the oxygen consumption of human skin samples, which shows that the stimulated mitochondria are fully vital because the main source for cellular oxygen consumption is mitochondrial endoxidation. These findings identify TRH as a potent, novel neuroendocrine stimulator of mitochondrial activity and biogenesis in human epidermal keratinocytes in situ. Thus, human epidermis offers an excellent model for dissecting neuroendocrine controls of human mitochondrial biology under physiologically relevant conditions and for exploring corresponding clinical applications.

Tributyltin (TBT) and mitochondrial respiration in mussel digestive gland.

PubMed

Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Pagliarani, Alessandra

2011-06-01

The toxicity of organotins and especially tri-n-butyltin (TBT) on mitochondria is well known. However as far as we are aware, effects on mitochondrial respiration are unexplored in mollusks. In this work mitochondria isolated from the digestive gland of Mytilus galloprovincialis and susceptive to the classical respiratory chain inhibitors, were assayed in the presence of micromolar TBT concentrations to investigate mitochondrial respiratory activities. Intact and freeze-thawed mitochondria were used. TBT significantly inhibited oxygen consumption in the presence of glutamate/malate or succinate as substrates. Conversely cytochrome c oxidase activity (complex IV), assayed both polarographically and spectrophotometrically, was unaffected. The addition of 1,4-dithioerythritol (DTE) decreased the TBT-driven inhibition of complexes I and III. The TBT capability of covalent binding to thiol groups of mitochondrial proteins in a dose-dependent manner was confirmed by the aid of Ellman's reagent. Data strongly suggests that TBT may prevent the electron transfer from complexes I and III to downhill respiratory chain complexes by binding to critical SH residues. Copyright © 2011 Elsevier Ltd. All rights reserved.

Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals.

PubMed

Hosseini, Seyed H; Kohler, James J; Haase, Chad P; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-03-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance

PubMed Central

Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity. PMID:17322372

SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila.

PubMed

Zanon, Alessandra; Kalvakuri, Sreehari; Rakovic, Aleksandar; Foco, Luisa; Guida, Marianna; Schwienbacher, Christine; Serafin, Alice; Rudolph, Franziska; Trilck, Michaela; Grünewald, Anne; Stanslowsky, Nancy; Wegner, Florian; Giorgio, Valentina; Lavdas, Alexandros A; Bodmer, Rolf; Pramstaller, Peter P; Klein, Christine; Hicks, Andrew A; Pichler, Irene; Seibler, Philip

2017-07-01

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Depressed mitochondrial function and electron transport Complex II-mediated H2O2 production in the cortex of type 1 diabetic rodents.

PubMed

Chowdhury, Subir Roy; Djordjevic, Jelena; Thomson, Ella; Smith, Darrell R; Albensi, Benedict C; Fernyhough, Paul

2018-05-23

Abnormalities in mitochondrial function under diabetic conditions can lead to deficits in function of cortical neurons and their support cells exhibiting a pivotal role in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. We aimed to assess simultaneously mitochondrial respiration rates and membrane potential or H 2 O 2 generation and proteins involved in mitochondrial dynamics, antioxidants and AMPK/SIRT/PGC-1α pathway activity in cortex under diabetic conditions. Cortical mitochondria from streptozotocin (STZ)-induced type 1 diabetic rats or mice, and aged-match controls were used for simultaneous measurements of mitochondrial respiration rates and mitochondrial membrane potential (mtMP) or H 2 O 2 using OROBOROS oxygraph and measurements of enzymatic activities by a spectrophotometer. Protein levels in cortical mitochondria and homogenates were determined by Western blotting. Mitochondrial coupled respiration rates and FCCP-induced uncoupled respiration rates were significantly decreased in mitochondria of STZ-diabetic cortical rats compared to controls. The mtMP in the presence of ADP was significantly depolarized and succinate-dependent respiration rates and H 2 O 2 were significantly diminished in mitochondria of diabetic animals compared to controls, accompanied with reduced expression of CuZn- and Mn-superoxide dismutase. The enzymatic activities of Complex I, II, and IV and protein levels of certain components of Complex I and II, mitofusin 2 (Mfn2), dynamin-related protein 1 (DRP1), P-AMPK, SIRT2 and PGC-1α were significantly diminished in diabetic cortex. Deficits in mitochondrial function, dynamics, and antioxidant capabilities putatively mediated through sub-optimal AMPK/SIRT/PGC-1α signaling, are involved in the development of early sub-clinical neurodegeneration in the cortex under diabetic conditions. Copyright © 2017. Published by Elsevier Inc.

Thiabendazole inhibits ubiquinone reduction activity of mitochondrial respiratory complex II via a water molecule mediated binding feature.

PubMed

Zhou, Qiangjun; Zhai, Yujia; Lou, Jizhong; Liu, Man; Pang, Xiaoyun; Sun, Fei

2011-07-01

The mitochondrial respiratory complex II or succinate: ubiquinone oxidoreductase (SQR) is a key membrane complex in both the tricarboxylic acid cycle and aerobic respiration. Five disinfectant compounds were investigated with their potent inhibition effects on the ubiquinone reduction activity of the porcine mitochondrial SQR by enzymatic assay and crystallography. Crystal structure of the SQR bound with thiabendazole (TBZ) reveals a different inhibitor-binding feature at the ubiquinone binding site where a water molecule plays an important role. The obvious inhibitory effect of TBZ based on the biochemical data (IC(50) ~100 μmol/L) and the significant structure-based binding affinity calculation (~94 μmol/L) draw the suspicion of using TBZ as a good disinfectant compound for nematode infections treatment and fruit storage.

In vitro reconstitution and characterization of the yeast mitochondrial degradosome complex unravels tight functional interdependence.

PubMed

Malecki, Michal; Jedrzejczak, Robert; Stepien, Piotr P; Golik, Pawel

2007-09-07

The mitochondrial degradosome (mtEXO), the main RNA-degrading complex of yeast mitochondria, is composed of two subunits: an exoribonuclease encoded by the DSS1 gene and an RNA helicase encoded by the SUV3 gene. We expressed both subunits of the yeast mitochondrial degradosome in Escherichia coli, reconstituted the complex in vitro and analyzed the RNase, ATPase and helicase activities of the two subunits separately and in complex. The results reveal a very strong functional interdependence. For every enzymatic activity, we observed significant changes when the relevant protein was present in the complex, compared to the activity measured for the protein alone. The ATPase activity of Suv3p is stimulated by RNA and its background activity in the absence of RNA is reduced greatly when the protein is in the complex with Dss1p. The Suv3 protein alone does not display RNA-unwinding activity and the 3' to 5' directional helicase activity requiring a free 3' single-stranded substrate becomes apparent only when Suv3p is in complex with Dss1p. The Dss1 protein alone does have some basal exoribonuclease activity, which is not ATP-dependent, but in the presence of Suv3p the activity of the entire complex is enhanced greatly and is entirely ATP-dependent, with no residual activity observed in the absence of ATP. Such absolute ATP-dependence is unique among known exoribonuclease complexes. On the basis of these results, we propose a model in which the Suv3p RNA helicase acts as a molecular motor feeding the substrate to the catalytic centre of the RNase subunit.

Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction

PubMed Central

Sanz, Alberto; Soikkeli, Mikko; Portero-Otín, Manuel; Wilson, Angela; Kemppainen, Esko; McIlroy, George; Ellilä, Simo; Kemppainen, Kia K.; Tuomela, Tea; Lakanmaa, Matti; Kiviranta, Essi; Stefanatos, Rhoda; Dufour, Eric; Hutz, Bettina; Naudí, Alba; Jové, Mariona; Zeb, Akbar; Vartiainen, Suvi; Matsuno-Yagi, Akemi; Yagi, Takao; Rustin, Pierre; Pamplona, Reinald; Jacobs, Howard T.

2010-01-01

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling. PMID:20435911

Shilajit attenuates behavioral symptoms of chronic fatigue syndrome by modulating the hypothalamic-pituitary-adrenal axis and mitochondrial bioenergetics in rats.

PubMed

Surapaneni, Dinesh Kumar; Adapa, Sree Rama Shiva Shanker; Preeti, Kumari; Teja, Gangineni Ravi; Veeraragavan, Muruganandam; Krishnamurthy, Sairam

2012-08-30

Shilajit has been used as a rejuvenator for ages in Indian ancient traditional medicine and has been validated for a number of pharmacological activities. The effect of processed shilajit which was standardized to dibenzo-α-pyrones (DBPs;0.43% w/w), DBP-chromoproteins (DCPs; 20.45% w/w) and fulvic acids (56.75% w/w) was evaluated in a rat model of chronic fatigue syndrome (CFS). The mitochondrial bioenergetics and the activity of hypothalamus-pituitary-adrenal (HPA) axis were evaluated for the plausible mechanism of action of shilajit. CFS was induced by forcing the rats to swim for 15mins for 21 consecutive days. The rats were treated with shilajit (25, 50 and 100mg/kg) for 21 days before exposure to stress procedure. The behavioral consequence of CFS was measured in terms of immobility and the climbing period. The post-CFS anxiety level was assessed by elevated plus maze (EPM) test. Plasma corticosterone and adrenal gland weight were estimated as indices of HPA axis activity. Analysis of mitochondrial complex chain enzymes (Complex I, II, IV and V) and mitochondrial membrane potential (MMP) in prefrontal cortex (PFC) were performed to evaluate the mitochondrial bioenergetics and integrity respectively. Shilajit reversed the CFS-induced increase in immobility period and decrease in climbing behavior as well as attenuated anxiety in the EPM test. Shilajit reversed CFS-induced decrease in plasma corticosterone level and loss of adrenal gland weight indicating modulation of HPA axis. Shilajit prevented CFS-induced mitochondrial dysfunction by stabilizing the complex enzyme activities and the loss of MMP. Shilajit reversed CFS-induced mitochondrial oxidative stress in terms of NO concentration and, LPO, SOD and catalase activities. The results indicate that shilajit mitigates the effects of CFS in this model possibly through the modulation of HPA axis and preservation of mitochondrial function and integrity. The reversal of CFS-induced behavioral symptoms and mitochondrial bioenergetics by shilajit indicates mitochondria as a potential target for treatment of CFS. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Role of mitochondrial DNA damage and dysfunction in veterans with Gulf War Illness.

PubMed

Chen, Yang; Meyer, Joel N; Hill, Helene Z; Lange, Gudrun; Condon, Michael R; Klein, Jacquelyn C; Ndirangu, Duncan; Falvo, Michael J

2017-01-01

Gulf War Illness (GWI) is a chronic multi-symptom illness not currently diagnosed by standard medical or laboratory test that affects 30% of veterans who served during the 1990-1991 Gulf War. The clinical presentation of GWI is comparable to that of patients with certain mitochondrial disorders-i.e., clinically heterogeneous multisystem symptoms. Therefore, we hypothesized that mitochondrial dysfunction may contribute to both the symptoms of GWI as well as its persistence over time. We recruited 21 cases of GWI (CDC and Kansas criteria) and 7 controls to participate in this study. Peripheral blood samples were obtained in all participants and a quantitative polymerase chain reaction (QPCR) based assay was performed to quantify mitochondrial and nuclear DNA lesion frequency and mitochondrial DNA (mtDNA) copy number (mtDNAcn) from peripheral blood mononuclear cells. Samples were also used to analyze nuclear DNA lesion frequency and enzyme activity for mitochondrial complexes I and IV. Both mtDNA lesion frequency (p = 0.015, d = 1.13) and mtDNAcn (p = 0.001; d = 1.69) were elevated in veterans with GWI relative to controls. Nuclear DNA lesion frequency was also elevated in veterans with GWI (p = 0.344; d = 1.41), but did not reach statistical significance. Complex I and IV activity (p > 0.05) were similar between groups and greater mtDNA lesion frequency was associated with reduced complex I (r2 = -0.35, p = 0.007) and IV (r2 = -0.28, p < 0.01) enzyme activity. In conclusion, veterans with GWI exhibit greater mtDNA damage which is consistent with mitochondrial dysfunction.

Chronic aerobic exercise training attenuates aortic stiffening and endothelial dysfunction through preserving aortic mitochondrial function in aged rats.

PubMed

Gu, Qi; Wang, Bing; Zhang, Xiao-Feng; Ma, Yan-Ping; Liu, Jian-Dong; Wang, Xiao-Ze

2014-08-01

Aging leads to large vessel arterial stiffening and endothelial dysfunction, which are important determinants of cardiovascular risk. The aim of present work was to assess the effects of chronic aerobic exercise training on aortic stiffening and endothelial dysfunction in aged rats and investigate the underlying mechanism about mitochondrial function. Chronic aerobic exercise training attenuated aortic stiffening with age marked by reduced collagen concentration, increased elastin concentration and reduced pulse wave velocity (PWV), and prevented aging-related endothelial dysfunction marked by improved endothelium-mediated vascular relaxation of aortas in response to acetylcholine. Chronic aerobic exercise training abated oxidative stress and nitrosative stress in aortas of aged rats. More importantly, we found that chronic aerobic exercise training in old rats preserved aortic mitochondrial function marked by reduced reactive oxygen species (ROS) formation and mitochondrial swelling, increased ATP formation and mitochondrial DNA content, and restored activities of complexes I and III and electron-coupling capacity between complexes I and III and between complexes II and III. In addition, it was found that chronic aerobic exercise training in old rats enhanced protein expression of uncoupling protein 2 (UCP-2), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), manganese superoxide dismutase (Mn-SOD), aldehyde dehydrogenase 2 (ALDH-2), prohibitin (PHB) and AMP-activated kinase (AMPK) phosphorylation in aortas. In conclusion, chronic aerobic exercise training preserved mitochondrial function in aortas, which, at least in part, explained the aorta-protecting effects of exercise training in aging. Copyright © 2014 Elsevier Inc. All rights reserved.

Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress.

PubMed

Hiller, Sylvia; DeKroon, Robert; Hamlett, Eric D; Xu, Longquan; Osorio, Cristina; Robinette, Jennifer; Winnik, Witold; Simington, Stephen; Maeda, Nobuyo; Alzate, Oscar; Yi, Xianwen

2016-01-01

S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

Protective Effects of Myricetin on Acute Hypoxia-Induced Exercise Intolerance and Mitochondrial Impairments in Rats

PubMed Central

Zou, Dan; Liu, Peng; Chen, Ka; Xie, Qi; Liang, Xinyu; Bai, Qian; Zhou, Qicheng; Liu, Kai; Zhang, Ting; Zhu, Jundong; Mi, Mantian

2015-01-01

Purpose Exercise tolerance is impaired in hypoxia. The aim of this study was to evaluate the effects of myricetin, a dietary flavonoid compound widely found in fruits and vegetables, on acute hypoxia-induced exercise intolerance in vivo and in vitro. Methods Male rats were administered myricetin or vehicle for 7 days and subsequently spent 24 hours at a barometric pressure equivalent to 5000 m. Exercise capacity was then assessed through the run-to-fatigue procedure, and mitochondrial morphology in skeletal muscle cells was observed by transmission electron microscopy (TEM). The enzymatic activities of electron transfer complexes were analyzed using an enzyme-linked immuno-sorbent assay (ELISA). mtDNA was quantified by real-time-PCR. Mitochondrial membrane potential was measured by JC-1 staining. Protein expression was detected through western blotting, immunohistochemistry, and immunofluorescence. Results Myricetin supplementation significantly prevented the decline of run-to-fatigue time of rats in hypoxia, and attenuated acute hypoxia-induced mitochondrial impairment in skeletal muscle cells in vivo and in vitro by maintaining mitochondrial structure, mtDNA content, mitochondrial membrane potential, and activities of the respiratory chain complexes. Further studies showed that myricetin maintained mitochondrial biogenesis in skeletal muscle cells under hypoxic conditions by up-regulating the expressions of mitochondrial biogenesis-related regluators, in addition, AMP-activated protein kinase(AMPK) plays a crucial role in this process. Conclusions Myricetin may have important applications for improving physical performance under hypoxic environment, which may be attributed to the protective effect against mitochondrial impairment by maintaining mitochondrial biogenesis. PMID:25919288

Succinyl-CoA synthetase (SUCLA2) deficiency in two siblings with impaired activity of other mitochondrial oxidative enzymes in skeletal muscle without mitochondrial DNA depletion.

PubMed

Huang, Xiaoping; Bedoyan, Jirair K; Demirbas, Didem; Harris, David J; Miron, Alexander; Edelheit, Simone; Grahame, George; DeBrosse, Suzanne D; Wong, Lee-Jun; Hoppel, Charles L; Kerr, Douglas S; Anselm, Irina; Berry, Gerard T

2017-03-01

Mutations in SUCLA2 result in succinyl-CoA ligase (ATP-forming) or succinyl-CoA synthetase (ADP-forming) (A-SCS) deficiency, a mitochondrial tricarboxylic acid cycle disorder. The phenotype associated with this gene defect is largely encephalomyopathy. We describe two siblings compound heterozygous for SUCLA2 mutations, c.985A>G (p.M329V) and c.920C>T (p.A307V), with parents confirmed as carriers of each mutation. We developed a new LC-MS/MS based enzyme assay to demonstrate the decreased SCS activity in the siblings with this unique genotype. Both siblings shared bilateral progressive hearing loss, encephalopathy, global developmental delay, generalized myopathy, and dystonia with choreoathetosis. Prior to diagnosis and because of lactic acidosis and low activity of muscle pyruvate dehydrogenase complex (PDC), sibling 1 (S1) was placed on dichloroacetate, while sibling 2 (S2) was on a ketogenic diet. S1 developed severe cyclic vomiting refractory to therapy, while S2 developed Leigh syndrome, severe GI dysmotility, intermittent anemia, hypogammaglobulinemia and eventually succumbed to his disorder. The mitochondrial DNA contents in skeletal muscle (SM) were normal in both siblings. Pyruvate dehydrogenase complex, ketoglutarate dehydrogenase complex, and several mitochondrial electron transport chain (ETC) activities were low or at the low end of the reference range in frozen SM from S1 and/or S2. In contrast, activities of PDC, other mitochondrial enzymes of pyruvate metabolism, ETC and, integrated oxidative phosphorylation, in skin fibroblasts were not significantly impaired. Although we show that propionyl-CoA inhibits PDC, it does not appear to account for decreased PDC activity in SM. A better understanding of the mechanisms of phenotypic variability and the etiology for tissue-specific secondary deficiencies of mitochondrial enzymes of oxidative metabolism, and independently mitochondrial DNA depletion (common in other cases of A-SCS deficiency), is needed given the implications for control of lactic acidosis and possible clinical management. Copyright © 2016 Elsevier Inc. All rights reserved.

Tetrahydrocannabinol induces brain mitochondrial respiratory chain dysfunction and increases oxidative stress: a potential mechanism involved in cannabis-related stroke.

PubMed

Wolff, Valérie; Schlagowski, Anna-Isabel; Rouyer, Olivier; Charles, Anne-Laure; Singh, François; Auger, Cyril; Schini-Kerth, Valérie; Marescaux, Christian; Raul, Jean-Sébastien; Zoll, Joffrey; Geny, Bernard

2015-01-01

Cannabis has potential therapeutic use but tetrahydrocannabinol (THC), its main psychoactive component, appears as a risk factor for ischemic stroke in young adults. We therefore evaluate the effects of THC on brain mitochondrial function and oxidative stress, key factors involved in stroke. Maximal oxidative capacities V max (complexes I, III, and IV activities), V succ (complexes II, III, and IV activities), V tmpd (complex IV activity), together with mitochondrial coupling (V max/V 0), were determined in control conditions and after exposure to THC in isolated mitochondria extracted from rat brain, using differential centrifugations. Oxidative stress was also assessed through hydrogen peroxide (H2O2) production, measured with Amplex Red. THC significantly decreased V max (-71%; P < 0.0001), V succ (-65%; P < 0.0001), and V tmpd (-3.5%; P < 0.001). Mitochondrial coupling (V max/V 0) was also significantly decreased after THC exposure (1.8±0.2 versus 6.3±0.7; P < 0.001). Furthermore, THC significantly enhanced H2O2 production by cerebral mitochondria (+171%; P < 0.05) and mitochondrial free radical leak was increased from 0.01±0.01 to 0.10±0.01% (P < 0.001). Thus, THC increases oxidative stress and induces cerebral mitochondrial dysfunction. This mechanism may be involved in young cannabis users who develop ischemic stroke since THC might increase patient's vulnerability to stroke.

4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Meili, E-mail: fumeilidrlinyi@tom.com; Wan, Fuqiang; Li, Zhengling

The aim of the present study is to investigate the potential anti-hepatocellular carcinoma (HCC) cell activity by 4SC-202, a novel class I HDAC inhibitor (HDACi). The associated signaling mechanisms were also analyzed. We showed that 4SC-202 treatment induced potent cytotoxic and proliferation–inhibitory activities against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, adding 4SC-202 in HCC cells activated mitochondrial apoptosis pathway, which was evidenced by mitochondrial permeability transition pore (mPTP) opening, cytochrome C cytosol release and caspase-3/-9 activation. Inhibition of this apoptosis pathway, by caspase-3/-9 inhibitors, mPTP blockers, or by shRNA-mediated knockdown of cyclophilin-D (Cyp-D,more » a key component of mPTP), significantly attenuated 4SC-202-induced HCC cell death and apoptosis. Reversely, over-expression of Cyp-D enhanced 4SC-202's sensitivity in HCC cells. Further studies showed that 4SC-202 induced apoptosis signal-regulating kinase 1 (ASK1) activation, causing it translocation to mitochondria and physical association with Cyp-D. This mitochondrial ASK1-Cyp-D complexation appeared required for mediating 4SC-202-induced apoptosis activation. ASK1 stable knockdown by targeted-shRNAs largely inhibited 4SC-202-induced mPTP opening, cytochrome C release, and following HCC cell apoptotic death. Together, we suggest that 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to potently inhibit human HCC cells. - Highlights: • 4SC-202 exerts potent anti-proliferative and cytotoxic activity against established/primary HCC cells. • SC-202-induced anti-HCC cell activity relies on caspase-dependent apoptosis activation. • 4SC-202 activates Cyp-D-dependent mitochondrial apoptosis pathway in HCC cells. • 4SC-202 activates ASK1 in HCC cells, causing it translocation to mitochondria. • Mitochondrial ASK1-Cyp-D complexation mediates 4SC-202's activity in HCC cells.« less

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis

PubMed Central

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O.; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-01-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim–Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. PMID:28982759

Low-intensity laser irradiation at 660 nm stimulates cytochrome c oxidase in stressed fibroblast cells.

PubMed

Houreld, Nicolette N; Masha, Roland T; Abrahamse, Heidi

2012-07-01

Low-intensity laser irradiation (LILI) has been used to modulate a variety of biological processes, including diabetic wound healing. The mechanism of action is thought to exist primarily with the mitochondria. This study aimed to determine the effect of irradiation on normal, diabetic, and ischemic mitochondrial electron transport chain (ETC) complexes. Normal, diabetic and ischemic human skin fibroblast mitochondria were irradiated in vitro at a wavelength of 660 nm and a fluence of either 5 or 15 J/cm(2). Non-irradiated mitochondria served as controls. Enzyme activities of mitochondrial complexes I, II, III, and IV were determined immediately post-irradiation. Normal, diabetic, and ischemic cells were irradiated and adenosine triphosphate (ATP) and active mitochondria were determined by luminescence and fluorescent microscopy, respectively. Irradiated diabetic mitochondria at a fluence of 15 J/cm(2) showed a significant decrease in complex III activity (P < 0.05). Normal (P < 0.01) and diabetic (P < 0.05) mitochondria irradiated at either 5 or 15 J/cm(2) showed a significant increase in complex IV activity. ATP results showed a significant increase in irradiated normal cells (5 J/cm(2); P < 0.05) and diabetic cells (15 J/cm(2); P < 0.01). There was a higher accumulation of active mitochondria in irradiated cells than non-irradiated cells. Irradiation at 660 nm has the ability to influence mitochondrial enzyme activity, in particular cytochrome c oxidase. This leads to increased mitochondrial activity and ATP synthesis. Copyright © 2012 Wiley Periodicals, Inc.

Effect of Simvastatin, Coenzyme Q10, Resveratrol, Acetylcysteine and Acetylcarnitine on Mitochondrial Respiration.

PubMed

Fišar, Z; Hroudová, J; Singh, N; Kopřivová, A; Macečková, D

2016-01-01

Some therapeutic and/or adverse effects of drugs may be related to their effects on mitochondrial function. The effects of simvastatin, resveratrol, coenzyme Q10, acetylcysteine, and acetylcarnitine on Complex I-, Complex II-, or Complex IV-linked respiratory rate were determined in isolated brain mitochondria. The protective effects of these biologically active compounds on the calcium-induced decrease of the respiratory rate were also studied. We observed a significant inhibitory effect of simvastatin on mitochondrial respiration (IC50 = 24.0 μM for Complex I-linked respiration, IC50 = 31.3 μM for Complex II-linked respiration, and IC50 = 42.9 μM for Complex IV-linked respiration); the inhibitory effect of resveratrol was found at very high concentrations (IC50 = 162 μM for Complex I-linked respiration, IC50 = 564 μM for Complex II-linked respiration, and IC50 = 1454 μM for Complex IV-linked respiration). Concentrations required for effective simvastatin- or resveratrol-induced inhibition of mitochondrial respiration were found much higher than concentrations achieved under standard dosing of these drugs. Acetylcysteine and acetylcarnitine did not affect the oxygen consumption rate of mitochondria. Coenzyme Q10 induced an increase of Complex I-linked respiration. The increase of free calcium ions induced partial inhibition of the Complex I+II-linked mitochondrial respiration, and all tested drugs counteracted this inhibition. None of the tested drugs showed mitochondrial toxicity (characterized by respiratory rate inhibition) at drug concentrations achieved at therapeutic drug intake. Resveratrol, simvastatin, and acetylcarnitine had the greatest neuroprotective potential (characterized by protective effects against calcium-induced reduction of the respiratory rate).

Investigating mitochondrial dysfunction in human lung cells exposed to redox-active PM components.

PubMed

Lavrich, Katelyn S; Corteselli, Elizabeth M; Wages, Phillip A; Bromberg, Philip A; Simmons, Steven O; Gibbs-Flournoy, Eugene A; Samet, James M

2018-03-01

Exposure to ambient particulate matter (PM) causes cardiopulmonary morbidity and mortality through mechanisms that involve oxidative stress. 1,2-naphthoquinone (1,2-NQ) is a ubiquitous component of PM and a potent redox-active electrophile. We previously reported that 1,2-NQ increases mitochondrial H 2 O 2 production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H 2 O 2 in human airway epithelial cells. We measured the effects of acute exposure to 1,2-NQ on oxygen consumption rate (OCR) in the human bronchial epithelial cell line BEAS-2B and mitochondrial preparations using extracellular flux analysis. Complex-specific assays and NADPH depletion by glucose deprivation distinguished between mitochondrial and non-mitochondrial oxygen utilization. 1,2-NQ exposure of BEAS cells caused a rapid, marked dose-dependent increase in OCR that was independent of mitochondrial respiration, exceeded the OCR observed after mitochondrial uncoupling, and remained sensitive to NADPH depletion, implicating extra-mitochondrial redox cycling processes. Similar effects were observed with the environmentally relevant redox-cycling quinones 1,4-naphthoquinone and 9,10-phenanthrenequinone, but not with quinones that do not redox cycle, such as 1,4-benzoquinone. In mitochondrial preparations, 1,2-NQ caused a decrease in Complex I-linked substrate oxidation, suggesting impairment of pyruvate utilization or transport, a novel mechanism of mitochondrial inhibition by an environmental exposure. This study also highlights the methodological utility and challenges in the use of extracellular flux analysis to elucidate the mechanisms of action of redox-active electrophiles present in ambient air. Published by Elsevier Inc.

Troxerutin attenuates diet-induced oxidative stress, impairment of mitochondrial biogenesis and respiratory chain complexes in mice heart.

PubMed

Rajagopalan, Geetha; Chandrasekaran, Sathiya Priya; Carani Venkatraman, Anuradha

2017-01-01

Mitochondrial abnormality is thought to play a key role in cardiac disease originating from the metabolic syndrome (MS). We evaluated the effect of troxerutin (TX), a semi-synthetic derivative of the natural bioflavanoid rutin, on the respiratory chain complex activity, oxidative stress, mitochondrial biogenesis and dynamics in heart of high fat, high fructose diet (HFFD) -induced mouse model of MS. Adult male Mus musculus mice of body weight 25-30 g were fed either control diet or HFFD for 60 days. Mice from each dietary regimen were divided into two groups on the 16th day and were treated or untreated with TX (150 mg/kg body weight [bw], per oral) for the next 45 days. At the end of experimental period, respiratory chain complex activity, uncoupling proteins (UCP)-2 and -3, mtDNA content, mitochondrial biogenesis and dynamics, oxidative stress markers and reactive oxygen species (ROS) generation were analyzed. Reduced mtDNA abundance with alterations in the expression of genes related to mitochondrial biogenesis and fission and fusion processes were observed in HFFD-fed mice. Disorganized and smaller mitochondria, reduction in complexes I, III and IV activities (by about 55%) and protein levels of UCP-2 (52%) and UCP-3 (46%) were noted in these mice. TX administration suppressed oxidative stress, improved the oxidative capacity and biogenesis and restored fission/fusion imbalance in the cardiac mitochondria of HFFD-fed mice. TX protects the myocardium by modulating the putative molecules of mitochondrial biogenesis and dynamics and by its anti-oxidant function in a mouse model of MS. © 2016 John Wiley & Sons Australia, Ltd.

Mitochondrial oxidative stress caused by Sod2 deficiency promotes cellular senescence and aging phenotypes in the skin

PubMed Central

Velarde, Michael C.; Flynn, James M.; Day, Nicholas U.; Melov, Simon; Campisi, Judith

2012-01-01

Cellular senescence arrests the proliferation of mammalian cells at risk for neoplastic transformation, and is also associated with aging. However, the factors that cause cellular senescence during aging are unclear. Excessive reactive oxygen species (ROS) have been shown to cause cellular senescence in culture, and accumulated molecular damage due to mitochondrial ROS has long been thought to drive aging phenotypes in vivo. Here, we test the hypothesis that mitochondrial oxidative stress can promote cellular senescence in vivo and contribute to aging phenotypes in vivo, specifically in the skin. We show that the number of senescent cells, as well as impaired mitochondrial (complex II) activity increase in naturally aged mouse skin. Using a mouse model of genetic Sod2 deficiency, we show that failure to express this important mitochondrial anti-oxidant enzyme also impairs mitochondrial complex II activity, causes nuclear DNA damage, and induces cellular senescence but not apoptosis in the epidermis. Sod2 deficiency also reduced the number of cells and thickness of the epidermis, while increasing terminal differentiation. Our results support the idea that mitochondrial oxidative stress and cellular senescence contribute to aging skin phenotypes in vivo. PMID:22278880

In Vitro Evaluation of Mitochondrial Function and Estrogen Signaling in Cell Lines Exposed to the Antiseptic Cetylpyridinium Chloride

PubMed Central

Datta, Sandipan; He, Guochun; Tomilov, Alexey; Sahdeo, Sunil; Denison, Michael S.

2017-01-01

Background: Quaternary ammonium salts (QUATS), such as cetylpyridinium chloride (CPC) and benzalkonium chloride (BAK), are frequently used in antiseptic formulations, including toothpastes, mouthwashes, lozenges, throat and nasal sprays, and as biocides. Although in a recent ruling, the U.S. Food and Drug Administration (FDA) banned CPC from certain products and requested more data on BAK’s efficacy and safety profile, QUATS, in general, and CPC and BAK, in particular, continue to be used in personal health care, food, and pharmaceutical and cleaning industries. Objectives: We aimed to assess CPC's effects on mitochondrial toxicity and endocrine disruption in vitro. Method: Mitochondrial O2 consumption and adenosine triphosphate (ATP) synthesis rates of osteosarcoma cybrid cells were measured before and after CPC and BAK treatment. Antiestrogenic effects of the compounds were measured by a luciferase-based assay using recombinant human breast carcinoma cells (VM7Luc4E2, ERalpha-positive). Results: CPC inhibited both mitochondrial O2 consumption [half maximal inhibitory concentration (IC50): 3.8μM] and ATP synthesis (IC50: 0.9μM), and additional findings supported inhibition of mitochondrial complex 1 as the underlying mechanism for these effects. In addition, CPC showed concentration-dependent antiestrogenic activity half maximal effective concentration [(EC50): 4.5μM)]. BAK, another antimicrobial QUATS that is structurally similar to CPC, and the pesticide rotenone, a known complex 1 inhibitor, also showed mitochondrial inhibitory and antiestrogenic effects. In all three cases, there was overlap of the antiestrogenic activity with the mitochondrial inhibitory activity. Conclusions: Mitochondrial inhibition in vitro occurred at a CPC concentration that may be relevant to human exposures. The antiestrogenic activity of CPC, BAK, rotenone, and triclosan may be related to their mitochondrial inhibitory activity. Our findings support the need for additional research on the mitochondrial inhibitory and antiestrogenic effects of QUATS, including CPC and BAK. https://doi.org/10.1289/EHP1404 PMID:28885978

Ganoderma lucidum ameliorate mitochondrial damage in isoproterenol-induced myocardial infarction in rats by enhancing the activities of TCA cycle enzymes and respiratory chain complexes.

PubMed

Sudheesh, N P; Ajith, T A; Janardhanan, K K

2013-04-30

Decreased mitochondrial function has been suggested to be one of the important pathological events in isoproterenol (ISO)-induced cardiotoxicity. In this communication, we have evaluated the protective effect of Ganoderma lucidum against ISO induced cardiac toxicity and mitochondrial dysfunction. Cardiac toxicity was assessed by determining the activities of creatine kinase (CK) and lactate dehydrogenases (LDH) after subcutaneous injection of ISO (85 mg/kg) at an interval of 24h for 2 days. The animals were sacrificed 24h after last ISO administration. G. lucidum (100 and 250 mg/kg, p.o.) was given to the rats once daily for 15 days prior to the ISO challenge. Similarly, α-Tocopherol (100mg/kg, p.o) was kept as the standard. To assess the extent of cardiac mitochondrial damage, the activities of Krebs cycle dehydrogenases and mitochondrial complexes I, II, III, and IV as well as the level of ROS and mitochondrial membrane potential (ΔΨmt) were evaluated. Administration of G. lucidum and α-tocopherol significantly protected the elevated activities of CK and LDH. Further, the activities of mitochondrial enzymes and the level of ΔΨmt were significantly enhanced and the level of ROS was significantly declined in the G. lucidum and α-tocopherol treatments. The present study concluded that the cardiac mitochondrial enzymes are markedly declined by the ISO challenge and the administration G. lucidum and α-Tocopherol significantly protected mitochondria by preventing the decline of antioxidant status and ΔΨmt or by directly scavenging the free radicals. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Differential Gene Expression Reveals Mitochondrial Dysfunction in an Imprinting Center Deletion Mouse Model of Prader–Willi Syndrome

PubMed Central

Yazdi, Puya G.; Su, Hailing; Ghimbovschi, Svetlana; Fan, Weiwei; Coskun, Pinar E.; Nalbandian, Angèle; Knoblach, Susan; Resnick, James L.; Hoffman, Eric; Wallace, Douglas C.

2013-01-01

Abstract Prader–Willi syndrome (PWS) is a genetic disorder caused by deficiency of imprinted gene expression from the paternal chromosome 15q11–15q13 and clinically characterized by neonatal hypotonia, short stature, cognitive impairment, hypogonadism, hyperphagia, morbid obesity, and diabetes. Previous clinical studies suggest that a defect in energy metabolism may be involved in the pathogenesis of PWS. We focused our attention on the genes associated with energy metabolism and found that there were 95 and 66 mitochondrial genes differentially expressed in PWS muscle and brain, respectively. Assessment of enzyme activities of mitochondrial oxidative phosphorylation complexes in the brain, heart, liver, and muscle were assessed. We found the enzyme activities of the cardiac mitochondrial complexes II‫III were up‐regulated in the PWS imprinting center deletion mice compared to the wild‐type littermates. These studies suggest that differential gene expression, especially of the mitochondrial genes may contribute to the pathophysiology of PWS. PMID:24127921

Differential gene expression reveals mitochondrial dysfunction in an imprinting center deletion mouse model of Prader-Willi syndrome.

PubMed

Yazdi, Puya G; Su, Hailing; Ghimbovschi, Svetlana; Fan, Weiwei; Coskun, Pinar E; Nalbandian, Angèle; Knoblach, Susan; Resnick, James L; Hoffman, Eric; Wallace, Douglas C; Kimonis, Virginia E

2013-10-01

Prader-Willi syndrome (PWS) is a genetic disorder caused by deficiency of imprinted gene expression from the paternal chromosome 15q11-15q13 and clinically characterized by neonatal hypotonia, short stature, cognitive impairment, hypogonadism, hyperphagia, morbid obesity, and diabetes. Previous clinical studies suggest that a defect in energy metabolism may be involved in the pathogenesis of PWS. We focused our attention on the genes associated with energy metabolism and found that there were 95 and 66 mitochondrial genes differentially expressed in PWS muscle and brain, respectively. Assessment of enzyme activities of mitochondrial oxidative phosphorylation complexes in the brain, heart, liver, and muscle were assessed. We found the enzyme activities of the cardiac mitochondrial complexes II+‫III were up-regulated in the PWS imprinting center deletion mice compared to the wild-type littermates. These studies suggest that differential gene expression, especially of the mitochondrial genes may contribute to the pathophysiology of PWS. © 2013 Wiley Periodicals, Inc.

Changes in Whole-Body Oxygen Consumption and Skeletal Muscle Mitochondria During Linezolid-Induced Lactic Acidosis.

PubMed

Protti, Alessandro; Ronchi, Dario; Bassi, Gabriele; Fortunato, Francesco; Bordoni, Andreina; Rizzuti, Tommaso; Fumagalli, Roberto

2016-07-01

To better clarify the pathogenesis of linezolid-induced lactic acidosis. Case report. ICU. A 64-year-old man who died with linezolid-induced lactic acidosis. Skeletal muscle was sampled at autopsy to study mitochondrial function. Lactic acidosis developed during continuous infusion of linezolid while oxygen consumption and oxygen extraction were diminishing from 172 to 52 mL/min/m and from 0.27 to 0.10, respectively. Activities of skeletal muscle respiratory chain complexes I, III, and IV, encoded by nuclear and mitochondrial DNA, were abnormally low, whereas activity of complex II, entirely encoded by nuclear DNA, was not. Protein studies confirmed stoichiometric imbalance between mitochondrial (cytochrome c oxidase subunits 1 and 2) and nuclear (succinate dehydrogenase A) DNA-encoded respiratory chain subunits. These findings were not explained by defects in mitochondrial DNA or transcription. There were no compensatory mitochondrial biogenesis (no induction of nuclear respiratory factor 1 and mitochondrial transcript factor A) or adaptive unfolded protein response (reduced concentration of heat shock proteins 60 and 70). Linezolid-induced lactic acidosis is associated with diminished global oxygen consumption and extraction. These changes reflect selective inhibition of mitochondrial protein synthesis (probably translation) with secondary mitonuclear imbalance. One novel aspect of linezolid toxicity that needs to be confirmed is blunting of reactive mitochondrial biogenesis and unfolded protein response.

Naked mole-rats maintain healthy skeletal muscle and Complex IV mitochondrial enzyme function into old age

PubMed Central

Stoll, Elizabeth A; Karapavlovic, Nevena; Rosa, Hannah; Woodmass, Michael; Rygiel, Karolina; White, Kathryn; Turnbull, Douglass M; Faulkes, Chris G

2016-01-01

The naked mole-rat (NMR) Heterocephalus glaber is an exceptionally long-lived rodent, living up to 32 years in captivity. This extended lifespan is accompanied by a phenotype of negligible senescence, a phenomenon of very slow changes in the expected physiological characteristics with age. One of the many consequences of normal aging in mammals is the devastating and progressive loss of skeletal muscle, termed sarcopenia, caused in part by respiratory enzyme dysfunction within the mitochondria of skeletal muscle fibers. Here we report that NMRs avoid sarcopenia for decades. Muscle fiber integrity and mitochondrial ultrastructure are largely maintained in aged animals. While mitochondrial Complex IV expression and activity remains stable, Complex I expression is significantly decreased. We show that aged naked mole-rat skeletal muscle tissue contains some mitochondrial DNA rearrangements, although the common mitochondrial DNA deletions associated with aging in human and other rodent skeletal muscles are not present. Interestingly, NMR skeletal muscle fibers demonstrate a significant increase in mitochondrial DNA copy number. These results have intriguing implications for the role of mitochondria in aging, suggesting Complex IV, but not Complex I, function is maintained in the long-lived naked mole rat, where sarcopenia is avoided and healthy muscle function is maintained for decades. PMID:27997359

Glucose Modulates Respiratory Complex I Activity in Response to Acute Mitochondrial Dysfunction

PubMed Central

Cannino, Giuseppe; El-Khoury, Riyad; Pirinen, Marja; Hutz, Bettina; Rustin, Pierre; Jacobs, Howard T.; Dufour, Eric

2012-01-01

Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an “emergency shutdown” system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases. PMID:23007390

Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jeong Eun; Hanyang Biomedical Research Institute, Seoul; Park, Jae Hyeon

2012-09-01

Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity asmore » well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.« less

Reactive oxygen species produced by NADPH oxidase and mitochondrial dysfunction in lung after an acute exposure to Residual Oil Fly Ashes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Magnani, Natalia D.; Marchini, Timoteo; Vanasco, Virginia

2013-07-01

Reactive O{sub 2} species production triggered by particulate matter (PM) exposure is able to initiate oxidative damage mechanisms, which are postulated as responsible for increased morbidity along with the aggravation of respiratory diseases. The aim of this work was to quantitatively analyse the major sources of reactive O{sub 2} species involved in lung O{sub 2} metabolism after an acute exposure to Residual Oil Fly Ashes (ROFAs). Mice were intranasally instilled with a ROFA suspension (1.0 mg/kg body weight), and lung samples were analysed 1 h after instillation. Tissue O{sub 2} consumption and NADPH oxidase (Nox) activity were evaluated in tissuemore » homogenates. Mitochondrial respiration, respiratory chain complexes activity, H{sub 2}O{sub 2} and ATP production rates, mitochondrial membrane potential and oxidative damage markers were assessed in isolated mitochondria. ROFA exposure was found to be associated with 61% increased tissue O{sub 2} consumption, a 30% increase in Nox activity, a 33% increased state 3 mitochondrial O{sub 2} consumption and a mitochondrial complex II activity increased by 25%. During mitochondrial active respiration, mitochondrial depolarization and a 53% decreased ATP production rate were observed. Neither changes in H{sub 2}O{sub 2} production rate, nor oxidative damage in isolated mitochondria were observed after the instillation. After an acute ROFA exposure, increased tissue O{sub 2} consumption may account for an augmented Nox activity, causing an increased O{sub 2}{sup ·−} production. The mitochondrial function modifications found may prevent oxidative damage within the organelle. These findings provide new insights to the understanding of the mechanisms involving reactive O{sub 2} species production in the lung triggered by ROFA exposure. - Highlights: • Exposure to ROFA alters the oxidative metabolism in mice lung. • The augmented Nox activity contributes to the high tissue O{sub 2} consumption. • Exposure to ROFA produces alterations in mitochondrial function. • ΔΨ{sub m} decrease in state 3 may be responsible for the decreased ATP production. • Mild uncoupling prevents mitochondrial oxidative damage.« less

Methylenedioxymethamphetamine inhibits mitochondrial complex I activity in mice: a possible mechanism underlying neurotoxicity

PubMed Central

Puerta, Elena; Hervias, Isabel; Goñi-Allo, Beatriz; Zhang, Steven F; Jordán, Joaquín; Starkov, Anatoly A; Aguirre, Norberto

2010-01-01

Background and purpose: 3,4-methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that such neurotoxicity is due to oxidative stress but the source of free radicals remains unknown. Inhibition of mitochondrial electron transport chain complexes by MDMA was assessed as a possible source. Experimental approach: Activities of mitochondrial complexes after MDMA were evaluated spectrophotometrically. In situ visualization of superoxide production in the striatum was assessed by ethidium fluorescence and striatal dopamine levels were determined by HPLC as an index of dopaminergic toxicity. Key results: 3,4-methylenedioxymethamphetamine decreased mitochondrial complex I activity in the striatum of mice, an effect accompanied by an increased production of superoxide radicals and the inhibition of endogenous aconitase. α-Lipoic acid prevented superoxide generation and long-term toxicity independent of any effect on complex I inhibition. These effects of α-lipoic acid were also associated with a significant increase of striatal glutathione levels. The relevance of glutathione was supported by reducing striatal glutathione content with L-buthionine-(S,R)-sulfoximine, which exacerbated MDMA-induced dopamine deficits, effects suppressed by α-lipoic acid. The nitric oxide synthase inhibitor, NG-nitro-L-arginine, partially prevented MDMA-induced dopamine depletions, an effect reversed by L-arginine but not D-arginine. Finally, a direct relationship between mitochondrial complex I inhibition and long-term dopamine depletions was found in animals treated with MDMA in combination with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Conclusions and implications: Inhibition of mitochondrial complex I following MDMA could be the source of free radicals responsible for oxidative stress and the consequent neurotoxicity of this drug in mice. This article is commented on by Moncada, pp. 217–219 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00706.x and to view related papers in this issue by Pravdic et al. and Kurz et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00698.x and http://dx.doi.org/10.1111/j.1476-5381.2010.00656.x PMID:20423338

Effects of tramadol, clonazepam, and their combination on brain mitochondrial complexes.

PubMed

Mohamed, Tarek Mostafa; Ghaffar, Hamdy M Abdel; El Husseiny, Rabee M R

2015-12-01

The present study is an unsubstantiated qualitative assessment of the abused drugs-tramadol and clonazepam. The aim of this study is to evaluate whether the effects of tramadol, clonazepam, and their combination on mitochondrial electron transport chain (ETC) complexes were influential at therapeutic or at progressively increasing doses. The study comprised of a total of 70 healthy male rats, aged 3 months. According to the drug intake regimen, animals were divided into seven groups: control, tramadol therapeutic, clonazepam therapeutic, combination therapeutic, tramadol abuse, clonazepam abuse, and combination abuse group. At the end of the experiment, brain mitochondrial ETC complexes (I, II, III, and IV) were evaluated. Histopathological examinations were also performed on brain tissues. The results showed that groups that received tramadol (therapeutic and abuse) suffered from weight loss. Tramadol abuse group and combination abuse group showed significant decrease in the activities of I, III, and IV complexes but not in the activity of complex II. In conclusion, tramadol but not clonazepam has been found to partially inhibit the activities of respiratory chain complexes I, III, and IV but not the activity of complex II and such inhibition occurred only at doses that exceeded the maximum recommended adult human daily therapeutic doses. This result explains the clinical and histopathological effects of tramadol, such as seizures and red neurons (marker for apoptosis), respectively. © The Author(s) 2012.

Altered zinc transport disrupts mitochondrial protein processing/import in fragile X-associated tremor/ataxia syndrome

PubMed Central

Napoli, Eleonora; Ross-Inta, Catherine; Wong, Sarah; Omanska-Klusek, Alicja; Barrow, Cedrick; Iwahashi, Christine; Garcia-Arocena, Dolores; Sakaguchi, Danielle; Berry-Kravis, Elizabeth; Hagerman, Randi; Hagerman, Paul J.; Giulivi, Cecilia

2011-01-01

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that affects individuals who are carriers of small CGG premutation expansions in the fragile X mental retardation 1 (FMR1) gene. Mitochondrial dysfunction was observed as an incipient pathological process occurring in individuals who do not display overt features of FXTAS ( 1). Fibroblasts from premutation carriers had lower oxidative phosphorylation capacity (35% of controls) and Complex IV activity (45%), and higher precursor-to-mature ratios (P:M) of nDNA-encoded mitochondrial proteins (3.1-fold). However, fibroblasts from carriers with FXTAS symptoms presented higher FMR1 mRNA expression (3-fold) and lower Complex V (38%) and aconitase activities (43%). Higher P:M of ATPase β-subunit (ATPB) and frataxin were also observed in cortex from patients that died with FXTAS symptoms. Biochemical findings observed in FXTAS cells (lower mature frataxin, lower Complex IV and aconitase activities) along with common phenotypic traits shared by Friedreich's ataxia and FXTAS carriers (e.g. gait ataxia, loss of coordination) are consistent with a defective iron homeostasis in both diseases. Higher P:M, and lower ZnT6 and mature frataxin protein expression suggested defective zinc and iron metabolism arising from altered ZnT protein expression, which in turn impairs the activity of mitochondrial Zn-dependent proteases, critical for the import and processing of cytosolic precursors, such as frataxin. In support of this hypothesis, Zn-treated fibroblasts showed a significant recovery of ATPB P:M, ATPase activity and doubling time, whereas Zn and desferrioxamine extended these recoveries and rescued Complex IV activity. PMID:21558427

TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury

PubMed Central

Miller, Barbara A.; Hoffman, Nicholas E.; Merali, Salim; Zhang, Xue-Qian; Wang, JuFang; Rajan, Sudarsan; Shanmughapriya, Santhanam; Gao, Erhe; Barrero, Carlos A.; Mallilankaraman, Karthik; Song, Jianliang; Gu, Tongda; Hirschler-Laszkiewicz, Iwona; Koch, Walter J.; Feldman, Arthur M.; Madesh, Muniswamy; Cheung, Joseph Y.

2014-01-01

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels. PMID:24492610

Fetal programming alters reactive oxygen species production in sheep cardiac mitochondria.

PubMed

von Bergen, Nicholas H; Koppenhafer, Stacia L; Spitz, Douglas R; Volk, Kenneth A; Patel, Sonali S; Roghair, Robert D; Lamb, Fred S; Segar, Jeffrey L; Scholz, Thomas D

2009-04-01

Exposure to an adverse intrauterine environment is recognized as an important risk factor for the development of cardiovascular disease later in life. Although oxidative stress has been proposed as a mechanism for the fetal programming phenotype, the role of mitochondrial O(2)(*-) (superoxide radical) production has not been explored. To determine whether mitochondrial ROS (reactive oxygen species) production is altered by in utero programming, pregnant ewes were given a 48-h dexamethasone (dexamethasone-exposed, 0.28 mg.kg(-1) of body weight.day(-1)) or saline (control) infusion at 27-28 days gestation (term=145 days). Intact left ventricular mitochondria and freeze-thaw mitochondrial membranes were studied from offspring at 4-months of age. AmplexRed was used to measure H(2)O(2) production. Activities of the antioxidant enzymes Mn-SOD (manganese superoxide dismutase), GPx (glutathione peroxidase) and catalase were measured. Compared with controls, a significant increase in Complex I H(2)O(2) production was found in intact mitochondria from dexamethasone-exposed animals. The treatment differences in Complex I-driven H(2)O(2) production were not seen in mitochondrial membranes. Consistent changes in H(2)O(2) production from Complex III in programmed animals were not found. Despite the increase in H(2)O(2) production in intact mitochondria from programmed animals, dexamethasone exposure significantly increased mitochondrial catalase activity, whereas Mn-SOD and GPx activities were unchanged. The results of the present study point to an increase in the rate of release of H(2)O(2) from programmed mitochondria despite an increase in catalase activity. Greater mitochondrial H(2)O(2) release into the cell may play a role in the development of adult disease following exposure to an adverse intrauterine environment.

Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice.

PubMed

Paul, Rajib; Borah, Anupom

2017-12-20

There exists an intricate relationship between hypercholesterolemia (elevated plasma cholesterol) and brain functions. The present study aims to understand the impact of hypercholesterolemia on pathological consequences in mouse brain. A chronic mouse model of hypercholesterolemia was induced by giving high-cholesterol diet for 12 weeks. The hypercholesterolemic mice developed cognitive impairment as evident from object recognition memory test. Cholesterol accumulation was observed in four discrete brain regions, such as cortex, striatum, hippocampus and substantia nigra along with significantly damaged blood-brain barrier by hypercholesterolemia. The crucial finding is the loss of acetylcholinesterase activity with mitochondrial dysfunction globally in the brain of hypercholesterolemic mice, which is related to the levels of cholesterol. Moreover, the levels of hydroxyl radical were elevated in the regions of brain where the activity of mitochondrial complexes was found to be reduced. Intriguingly, elevations of inflammatory stress markers in the cholesterol-rich brain regions were observed. As cognitive impairment, diminished brain acetylcholinesterase activity, mitochondrial dysfunctions, and inflammation are the prima facie pathologies of neurodegenerative diseases, the findings impose hypercholesterolemia as potential risk factor towards brain dysfunction.

Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase)

PubMed Central

Miyadera, Hiroko; Shiomi, Kazuro; Ui, Hideaki; Yamaguchi, Yuichi; Masuma, Rokuro; Tomoda, Hiroshi; Miyoshi, Hideto; Osanai, Arihiro; Kita, Kiyoshi; Ōmura, Satoshi

2003-01-01

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues. PMID:12515859

Brain alpha-ketoglutarate dehydrogenase complex: kinetic properties, regional distribution, and effects of inhibitors.

PubMed

Lai, J C; Cooper, A J

1986-11-01

The substrate and cofactor requirements and some kinetic properties of the alpha-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for alpha-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex greater than cerebellum greater than or equal to midbrain greater than striatum = hippocampus greater than hypothalamus greater than pons and medulla greater than olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2-2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both beta-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 microM, calcium slightly stimulated (by 10-15%) the brain mitochondrial KGDHC. At concentrations above 100 microM, calcium (IC50 = 1 mM) inhibited both brain mitochondrial and purified heart KGDHC. The present results suggest that some of the kinetic properties of the rat brain mitochondrial KGDHC differ from those of the purified bovine heart and rat heart mitochondrial enzyme complexes. They also suggest that the inhibition of KGDHC by ammonia and the consequent effect on the citric acid cycle fluxes may be of pathophysiological and/or pathogenetic importance in hyperammonemia and in diseases (e.g., hepatic encephalopathy, inborn errors of urea metabolism, Reye's syndrome) where hyperammonemia is a consistent feature. Brain accumulation of calcium occurs in a number of pathological conditions. Therefore, it is possible that such a calcium accumulation may have a deleterious effect on KGDHC activity.

Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver.

PubMed

Gusdon, Aaron M; Fernandez-Bueno, Gabriel A; Wohlgemuth, Stephanie; Fernandez, Jenelle; Chen, Jing; Mathews, Clayton E

2015-09-10

Aberrant mitochondrial function, including excessive reactive oxygen species (ROS) production, has been implicated in the pathogenesis of human diseases. The use of mitochondrial inhibitors to ascertain the sites in the electron transport chain (ETC) resulting in altered ROS production can be an important tool. However, the response of mouse mitochondria to ETC inhibitors has not been thoroughly assessed. Here we set out to characterize the differences in phenotypic response to ETC inhibitors between the more energetically demanding brain mitochondria and less energetically demanding liver mitochondria in commonly utilized C57BL/6J mice. We show that in contrast to brain mitochondria, inhibiting distally within complex I or within complex III does not increase liver mitochondrial ROS production supported by complex I substrates, and liver mitochondrial ROS production supported by complex II substrates occurred primarily independent of membrane potential. Complex I, II, and III enzymatic activities and membrane potential were equivalent between liver and brain and responded to ETC. inhibitors similarly. Brain mitochondria exhibited an approximately two-fold increase in complex I and II supported respiration compared with liver mitochondria while exhibiting similar responses to inhibitors. Elevated NADH transport and heightened complex II-III coupled activity accounted for increased complex I and II supported respiration, respectively in brain mitochondria. We conclude that important mechanistic differences exist between mouse liver and brain mitochondria and that mouse mitochondria exhibit phenotypic differences compared with mitochondria from other species.

Impaired Muscle Mitochondrial Biogenesis and Myogenesis in Spinal Muscular Atrophy

PubMed Central

Ripolone, Michela; Ronchi, Dario; Violano, Raffaella; Vallejo, Dionis; Fagiolari, Gigliola; Barca, Emanuele; Lucchini, Valeria; Colombo, Irene; Villa, Luisa; Berardinelli, Angela; Balottin, Umberto; Morandi, Lucia; Mora, Marina; Bordoni, Andreina; Fortunato, Francesco; Corti, Stefania; Parisi, Daniela; Toscano, Antonio; Sciacco, Monica; DiMauro, Salvatore; Comi, Giacomo P.; Moggio, Maurizio

2016-01-01

IMPORTANCE The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively). OBJECTIVE To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA. DESIGN, SETTING, AND PARTICIPANTS We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca’ Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015. EXPOSURES We used histochemical, biochemical, and molecular techniques to examine the muscle samples. MAIN OUTCOMES AND MEASURES Respiratory chain activity and mitochondrial content. RESULTS Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P < .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P < .05). We linked these alterations to downregulation of peroxisome proliferator–activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors—muscle-specificmyogenic factor 5, myoblast determination 1, and myogenin—were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05). CONCLUSIONS AND RELEVANCE Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes. PMID:25844556

m-AAA Complexes Are Not Crucial for the Survival of Arabidopsis Under Optimal Growth Conditions Despite Their Importance for Mitochondrial Translation.

PubMed

Kolodziejczak, Marta; Skibior-Blaszczyk, Renata; Janska, Hanna

2018-05-01

For optimal mitochondrial activity, the mitochondrial proteome must be properly maintained or altered in response to developmental and environmental stimuli. Based on studies of yeast and humans, one of the key players in this control are m-AAA proteases, mitochondrial inner membrane-bound ATP-dependent metalloenzymes. This study focuses on the importance of m-AAA proteases in plant mitochondria, providing their first experimentally proven physiological substrate. We found that the Arabidopsis m- AAA complexes composed of AtFTSH3 and/or AtFTSH10 are involved in the proteolytic maturation of ribosomal subunit L32. Consequently, in the double Arabidopsis ftsh3/10 mutant, mitoribosome biogenesis, mitochondrial translation and functionality of OXPHOS (oxidative phosphorylation) complexes are impaired. However, in contrast to their mammalian or yeast counterparts, plant m-AAA complexes are not critical for the survival of Arabidopsis under optimal conditions; ftsh3/10 plants are only slightly smaller in size at the early developmental stage compared with plants containing m-AAA complexes. Our data suggest that a lack of significant visible morphological alterations under optimal growth conditions involves mechanisms which rely on existing functional redundancy and induced functional compensation in Arabidopsis mitochondria.

Loss of protohaem IX farnesyltransferase in mature dentate granule cells impairs short-term facilitation at mossy fibre to CA3 pyramidal cell synapses.

PubMed

Booker, Sam A; Campbell, Graham R; Mysiak, Karolina S; Brophy, Peter J; Kind, Peter C; Mahad, Don J; Wyllie, David J A

2017-03-15

Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity. Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low-frequency dentate to CA3 glutamatergic synaptic transmission. High-frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase-deficient mice. Intact presynaptic mitochondrial function is critical for the short-term dynamics of mossy fibre to CA3 synaptic function. Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole-cell patch-clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV-deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy-fibre synapse because the amplitude, input-output relationship and 50 ms paired-pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short-term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired-pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses with CA3 pyramidal cells via large mossy-fibre boutons, but rather to all synapses formed by dentate granule cells. Therefore, presynaptic mitochondrial function is critical for the short-term dynamics of synapse function, which may contribute to the cognitive deficits observed in pathological mitochondrial dysfunction. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Altered mitochondrial bioenergetics and ultrastructure in the skeletal muscle of young adults with type 1 diabetes.

PubMed

Monaco, Cynthia M F; Hughes, Meghan C; Ramos, Sofhia V; Varah, Nina E; Lamberz, Christian; Rahman, Fasih A; McGlory, Chris; Tarnopolsky, Mark A; Krause, Matthew P; Laham, Robert; Hawke, Thomas J; Perry, Christopher G R

2018-06-01

A comprehensive assessment of skeletal muscle ultrastructure and mitochondrial bioenergetics has not been undertaken in individuals with type 1 diabetes. This study aimed to systematically assess skeletal muscle mitochondrial phenotype in young adults with type 1 diabetes. Physically active, young adults (men and women) with type 1 diabetes (HbA 1c 63.0 ± 16.0 mmol/mol [7.9% ± 1.5%]) and without type 1 diabetes (control), matched for sex, age, BMI and level of physical activity, were recruited (n = 12/group) to undergo vastus lateralis muscle microbiopsies. Mitochondrial respiration (high-resolution respirometry), site-specific mitochondrial H 2 O 2 emission and Ca 2+ retention capacity (CRC) (spectrofluorometry) were assessed using permeabilised myofibre bundles. Electron microscopy and tomography were used to quantify mitochondrial content and investigate muscle ultrastructure. Skeletal muscle microvasculature was assessed by immunofluorescence. Mitochondrial oxidative capacity was significantly lower in participants with type 1 diabetes vs the control group, specifically at Complex II of the electron transport chain, without differences in mitochondrial content between groups. Muscles of those with type 1 diabetes also exhibited increased mitochondrial H 2 O 2 emission at Complex III and decreased CRC relative to control individuals. Electron tomography revealed an increase in the size and number of autophagic remnants in the muscles of participants with type 1 diabetes. Despite this, levels of the autophagic regulatory protein, phosphorylated AMP-activated protein kinase (p-AMPKα Thr172 ), and its downstream targets, phosphorylated Unc-51 like autophagy activating kinase 1 (p-ULK1 Ser555 ) and p62, was similar between groups. In addition, no differences in muscle capillary density or platelet aggregation were observed between the groups. Alterations in mitochondrial ultrastructure and bioenergetics are evident within the skeletal muscle of active young adults with type 1 diabetes. It is yet to be elucidated whether more rigorous exercise may help to prevent skeletal muscle metabolic deficiencies in both active and inactive individuals with type 1 diabetes.

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

PubMed

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-09-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-X L , recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-X L inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

Single channel recording of a mitochondrial calcium uniporter.

PubMed

Wu, Guangyan; Li, Shunjin; Zong, Guangning; Liu, Xiaofen; Fei, Shuang; Shen, Linda; Guan, Xiangchen; Yang, Xue; Shen, Yuequan

2018-01-29

Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the entire uniporter complex and plays an important role in mitochondrial calcium uptake. However, the single channel recording of MCU remains controversial. Here, we expressed and purified different MCU proteins and then reconstituted them into planar lipid bilayers for single channel recording. We showed that MCU alone from Pyronema omphalodes (pMCU) is active with prominent single channel Ca 2+ currents. In sharp contrast, MCU alone from Homo sapiens (hMCU) is inactive. The essential MCU regulator (EMRE) activates hMCU, and therefore, the complex (hMCU-hEMRE) shows prominent single channel Ca 2+ currents. These single channel currents are sensitive to the specific MCU inhibitor Ruthenium Red. Our results clearly demonstrate that active MCU can conduct large amounts of calcium into the mitochondria. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of the alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular calcium regulation.

PubMed

Huang, Hsueh-Meei; Zhang, Hui; Xu, Hui; Gibson, Gary E

2003-01-20

Mitochondrial dysfunction occurs in many neurodegenerative diseases. The alpha-ketoglutarate dehydrogenase complex (KGDHC) catalyzes a key and arguably rate-limiting step of the tricarboxylic acid cycle (TCA). A reduction in the activity of the KGDHC occurs in brains and cells of patients with many of these disorders and may underlie the abnormal mitochondrial function. Abnormalities in calcium homeostasis also occur in fibroblasts from Alzheimer's disease (AD) patients and in cells bearing mutations that lead to AD. Thus, the present studies test whether the reduction of KGDHC activity can lead to the alterations in mitochondrial function and calcium homeostasis. alpha-Keto-beta-methyl-n-valeric acid (KMV) inhibits KGDHC activity in living N2a cells in a dose- and time-dependent manner. Surprisingly, concentration of KMV that inhibit in situ KGDHC by 80% does not alter the mitochondrial membrane potential (MMP). However, similar concentrations of KMV induce the release of cytochrome c from mitochondria into the cytosol, reduce basal [Ca(2+)](i) by 23% (P<0.005), and diminish the bradykinin (BK)-induced calcium release from the endoplasmic reticulum (ER) by 46% (P<0.005). This result suggests that diminished KGDHC activities do not lead to the Ca(2+) abnormalities in fibroblasts from AD patients or cells bearing PS-1 mutations. The increased release of cytochrome c with diminished KGDHC activities will be expected to activate other pathways including cell death cascades. Reductions in this key mitochondrial enzyme will likely make the cells more vulnerable to metabolic insults that promote cell death.

Mild Hypothermia Attenuates Mitochondrial Oxidative Stress by Protecting Respiratory Enzymes and Upregulating MnSOD in a Pig Model of Cardiac Arrest

PubMed Central

Gong, Ping; Li, Chun-Sheng; Hua, Rong; Zhao, Hong; Tang, Zi-Ren; Mei, Xue; Zhang, Ming-Yue; Cui, Juan

2012-01-01

Mild hypothermia is the only effective treatment confirmed clinically to improve neurological outcomes for comatose patients with cardiac arrest. However, the underlying mechanism is not fully elucidated. In this study, our aim was to determine the effect of mild hypothermia on mitochondrial oxidative stress in the cerebral cortex. We intravascularly induced mild hypothermia (33°C), maintained this temperature for 12 h, and actively rewarmed in the inbred Chinese Wuzhishan minipigs successfully resuscitated after 8 min of untreated ventricular fibrillation. Cerebral samples were collected at 24 and 72 h following return of spontaneous circulation (ROSC). We found that mitochondrial malondialdehyde and protein carbonyl levels were significantly increased in the cerebral cortex in normothermic pigs even at 24 h after ROSC, whereas mild hypothermia attenuated this increase. Moreover, mild hypothermia attenuated the decrease in Complex I and Complex III (i.e., major sites of reactive oxygen species production) activities of the mitochondrial respiratory chain and increased antioxidant enzyme manganese superoxide dismutase (MnSOD) activity. This increase in MnSOD activity was consistent with the upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and protein expressions, and with the increase of Nrf2 nuclear translocation in normothermic pigs at 24 and 72 h following ROSC, whereas mild hypothermia enhanced these tendencies. Thus, our findings indicate that mild hypothermia attenuates mitochondrial oxidative stress in the cerebral cortex, which may be associated with reduced impairment of mitochondrial respiratory chain enzymes, and enhancement of MnSOD activity and expression via Nrf2 activation. PMID:22532848

OCIAD1 Controls Electron Transport Chain Complex I Activity to Regulate Energy Metabolism in Human Pluripotent Stem Cells.

PubMed

Shetty, Deeti K; Kalamkar, Kaustubh P; Inamdar, Maneesha S

2018-06-14

Pluripotent stem cells (PSCs) derive energy predominantly from glycolysis and not the energy-efficient oxidative phosphorylation (OXPHOS). Differentiation is initiated with energy metabolic shift from glycolysis to OXPHOS. We investigated the role of mitochondrial energy metabolism in human PSCs using molecular, biochemical, genetic, and pharmacological approaches. We show that the carcinoma protein OCIAD1 interacts with and regulates mitochondrial complex I activity. Energy metabolic assays on live pluripotent cells showed that OCIAD1-depleted cells have increased OXPHOS and may be poised for differentiation. OCIAD1 maintains human embryonic stem cells, and its depletion by CRISPR/Cas9-mediated knockout leads to rapid and increased differentiation upon induction, whereas OCIAD1 overexpression has the opposite effect. Pharmacological alteration of complex I activity was able to rescue the defects of OCIAD1 modulation. Thus, hPSCs can exist in energy metabolic substates. OCIAD1 provides a target to screen for additional modulators of mitochondrial activity to promote transient multipotent precursor expansion or enhance differentiation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Reprogramming of leukemic cell metabolism through the naphthoquinonic compound Quambalarine B

PubMed Central

Vališ, Karel; Grobárová, Valéria; Hernychová, Lucie; Bugáňová, Martina; Kavan, Daniel; Kalous, Martin; Černý, Jiří; Stodůlková, Eva; Kuzma, Marek; Flieger, Miroslav; Černý, Jan; Novák, Petr

2017-01-01

Abnormalities in cancer metabolism represent potential targets for cancer therapy. We have recently identified a natural compound Quambalarine B (QB), which inhibits proliferation of several leukemic cell lines followed by cell death. We have predicted ubiquinone binding sites of mitochondrial respiratory complexes as potential molecular targets of QB in leukemia cells. Hence, we tracked the effect of QB on leukemia metabolism by applying several omics and biochemical techniques. We have confirmed the inhibition of respiratory complexes by QB and found an increase in the intracellular AMP levels together with respiratory substrates. Inhibition of mitochondrial respiration by QB triggered reprogramming of leukemic cell metabolism involving disproportions in glycolytic flux, inhibition of proteins O-glycosylation, stimulation of glycine synthesis pathway, and pyruvate kinase activity, followed by an increase in pyruvate and a decrease in lactate levels. Inhibition of mitochondrial complex I by QB suppressed folate metabolism as determined by a decrease in formate production. We have also observed an increase in cellular levels of several amino acids except for aspartate, indicating the dependence of Jurkat (T-ALL) cells on aspartate synthesis. These results indicate blockade of mitochondrial complex I and II activity by QB and reduction in aspartate and folate metabolism as therapeutic targets in T-ALL cells. Anti-cancer activity of QB was also confirmed during in vivo studies, suggesting the therapeutic potential of this natural compound. PMID:29262552

Metabolites from invasive pests inhibit mitochondrial complex II: A potential strategy for the treatment of human ovarian carcinoma?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ferramosca, Alessandra, E-mail: alessandra.ferramosca@unisalento.it; Conte, Annalea; Guerra, Flora

The red pigment caulerpin, a secondary metabolite from the marine invasive green algae Caulerpa cylindracea can be accumulated and transferred along the trophic chain, with detrimental consequences on biodiversity and ecosystem functioning. Despite increasing research efforts to understand how caulerpin modifies fish physiology, little is known on the effects of algal metabolites on mammalian cells. Here we report for the first time the mitochondrial targeting activity of both caulerpin, and its closely related derivative caulerpinic acid, by using as experimental model rat liver mitochondria, a system in which bioenergetics mechanisms are not altered. Mitochondrial function was tested by polarographic andmore » spectrophotometric methods. Both compounds were found to selectively inhibit respiratory complex II activity, while complexes I, III, and IV remained functional. These results led us to hypothesize that both algal metabolites could be used as antitumor agents in cell lines with defects in mitochondrial complex I. Ovarian cancer cisplatin-resistant cells are a good example of cell lines with a defective complex I function on which these molecules seem to have a toxic effect on proliferation. This provided novel insight toward the potential use of metabolites from invasive Caulerpa species for the treatment of human ovarian carcinoma cisplatin-resistant cells. -- Highlights: •Novel insight toward the potential use of the algal metabolites for the treatment of human diseases. •Caulerpin and caulerpinic acid inhibit respiratory complex II activity. •Both algal metabolites could be used as antitumor agents in ovarian cancer cisplatin-resistant cells.« less

A cannabinoid link between mitochondria and memory.

PubMed

Hebert-Chatelain, Etienne; Desprez, Tifany; Serrat, Román; Bellocchio, Luigi; Soria-Gomez, Edgar; Busquets-Garcia, Arnau; Pagano Zottola, Antonio Christian; Delamarre, Anna; Cannich, Astrid; Vincent, Peggy; Varilh, Marjorie; Robin, Laurie M; Terral, Geoffrey; García-Fernández, M Dolores; Colavita, Michelangelo; Mazier, Wilfrid; Drago, Filippo; Puente, Nagore; Reguero, Leire; Elezgarai, Izaskun; Dupuy, Jean-William; Cota, Daniela; Lopez-Rodriguez, Maria-Luz; Barreda-Gómez, Gabriel; Massa, Federico; Grandes, Pedro; Bénard, Giovanni; Marsicano, Giovanni

2016-11-24

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB 1 ) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB 1 receptors. Genetic exclusion of CB 1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB 1 receptors signal through intra-mitochondrial Gα i protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB 1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

Selective downregulation of mitochondrial electron transport chain activity and increased oxidative stress in human atrial fibrillation.

PubMed

Emelyanova, Larisa; Ashary, Zain; Cosic, Milanka; Negmadjanov, Ulugbek; Ross, Gracious; Rizvi, Farhan; Olet, Susan; Kress, David; Sra, Jasbir; Tajik, A Jamil; Holmuhamedov, Ekhson L; Shi, Yang; Jahangir, Arshad

2016-07-01

Mitochondria are critical for maintaining normal cardiac function, and a deficit in mitochondrial energetics can lead to the development of the substrate that promotes atrial fibrillation (AF) and its progression. However, the link between mitochondrial dysfunction and AF in humans is still not fully defined. The aim of this study was to elucidate differences in the functional activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes and oxidative stress in right atrial tissue from patients without (non-AF) and with AF (AF) who were undergoing open-heart surgery and were not significantly different for age, sex, major comorbidities, and medications. The overall functional activity of the electron transport chain (ETC), NADH:O2 oxidoreductase activity, was reduced by 30% in atrial tissue from AF compared with non-AF patients. This was predominantly due to a selective reduction in complex I (0.06 ± 0.007 vs. 0.09 ± 0.006 nmol·min(-1)·citrate synthase activity(-1), P = 0.02) and II (0.11 ± 0.012 vs. 0.16 ± 0.012 nmol·min(-1)·citrate synthase activity(-1), P = 0.003) functional activity in AF patients. Conversely, complex V activity was significantly increased in AF patients (0.21 ± 0.027 vs. 0.12 ± 0.01 nmol·min(-1)·citrate synthase activity(-1), P = 0.005). In addition, AF patients exhibited a higher oxidative stress with increased production of mitochondrial superoxide (73 ± 17 vs. 11 ± 2 arbitrary units, P = 0.03) and 4-hydroxynonenal level (77.64 ± 30.2 vs. 9.83 ± 2.83 ng·mg(-1) protein, P = 0.048). Our findings suggest that AF is associated with selective downregulation of ETC activity and increased oxidative stress that can contribute to the progression of the substrate for AF. Copyright © 2016 the American Physiological Society.

Quantifying small molecule phenotypic effects using mitochondrial morpho-functional fingerprinting and machine learning.

PubMed

Blanchet, Lionel; Smeitink, Jan A M; van Emst-de Vries, Sjenet E; Vogels, Caroline; Pellegrini, Mina; Jonckheere, An I; Rodenburg, Richard J T; Buydens, Lutgarde M C; Beyrath, Julien; Willems, Peter H G M; Koopman, Werner J H

2015-01-26

In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that in vitro potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.

Quantifying small molecule phenotypic effects using mitochondrial morpho-functional fingerprinting and machine learning

NASA Astrophysics Data System (ADS)

Blanchet, Lionel; Smeitink, Jan A. M.; van Emst-de Vries, Sjenet E.; Vogels, Caroline; Pellegrini, Mina; Jonckheere, An I.; Rodenburg, Richard J. T.; Buydens, Lutgarde M. C.; Beyrath, Julien; Willems, Peter H. G. M.; Koopman, Werner J. H.

2015-01-01

In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that in vitro potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.

Mitochondrial fragmentation in excitotoxicity requires ROCK activation.

PubMed

Martorell-Riera, Alejandro; Segarra-Mondejar, Marc; Reina, Manuel; Martínez-Estrada, Ofelia M; Soriano, Francesc X

2015-01-01

Mitochondria morphology constantly changes through fission and fusion processes that regulate mitochondrial function, and it therefore plays a prominent role in cellular homeostasis. Cell death progression is associated with mitochondrial fission. Fission is mediated by the mainly cytoplasmic Drp1, which is activated by different post-translational modifications and recruited to mitochondria to perform its function. Our research and other studies have shown that in the early moments of excitotoxic insult Drp1 must be nitrosylated to mediate mitochondrial fragmentation in neurons. Nonetheless, mitochondrial fission is a multistep process in which filamentous actin assembly/disassembly and myosin-mediated mitochondrial constriction play prominent roles. Here we establish that in addition to nitric oxide production, excitotoxicity-induced mitochondrial fragmentation also requires activation of the actomyosin regulator ROCK. Although ROCK1 has been shown to phosphorylate and activate Drp1, experiments using phosphor-mutant forms of Drp1 in primary cortical neurons indicate that in excitotoxic conditions, ROCK does not act directly on Drp1 to mediate fission, but may act on the actomyosin complex. Thus, these data indicate that a wider range of signaling pathways than those that target Drp1 are amenable to be inhibited to prevent mitochondrial fragmentation as therapeutic option.

Mitochondrial Dysfunction and Oxidative Stress Promote Apoptotic Cell Death in the Striatum via Cytochrome c/Caspase-3 Signaling Cascade Following Chronic Rotenone Intoxication in Rats

PubMed Central

Lin, Tsu-Kung; Cheng, Ching-Hsiao; Chen, Shang-Der; Liou, Chia-Wei; Huang, Chi-Ren; Chuang, Yao-Chung

2012-01-01

Parkinson’s disease (PD) is a progressive neurological disorder marked by nigrostriatal dopaminergic degeneration. Evidence suggests that mitochondrial dysfunction may be linked to PD through a variety of different pathways, including free-radical generation and dysfunction of the mitochondrial Complex I activity. In Lewis rats, chronic systemic administration of a specific mitochondrial Complex I inhibitor, rotenone (3 mg/kg/day) produced parkinsonism-like symptoms. Increased oxidized proteins and peroxynitrite, and mitochondrial or cytosol translocation of Bim, Bax or cytochrome c in the striatum was observed after 2–4 weeks of rotenone infusion. After 28 days of systemic rotenone exposure, imunohistochemical staining for tyrosine hydroxylase indicated nigrostriatal dopaminergic neuronal cell degeneration. Characteristic histochemical (TUNEL or activated caspase-3 staining) or ultrastructural (electron microscopy) features of apoptotic cell death were present in the striatal neuronal cell after chronic rotenone intoxication. We conclude that chronic rotenone intoxication may enhance oxidative and nitrosative stress that induces mitochondrial dysfunction and ultrastructural damage, resulting in translocation of Bim and Bax from cytosol to mitochondria that contributes to apoptotic cell death in the striatum via cytochrome c/caspase-3 signaling cascade. PMID:22942730

Mutation screening of 75 candidate genes in 152 complex I deficiency cases identifies pathogenic variants in 16 genes including NDUFB9.

PubMed

Haack, Tobias B; Madignier, Florence; Herzer, Martina; Lamantea, Eleonora; Danhauser, Katharina; Invernizzi, Federica; Koch, Johannes; Freitag, Martin; Drost, Rene; Hillier, Ingo; Haberberger, Birgit; Mayr, Johannes A; Ahting, Uwe; Tiranti, Valeria; Rötig, Agnes; Iuso, Arcangela; Horvath, Rita; Tesarova, Marketa; Baric, Ivo; Uziel, Graziella; Rolinski, Boris; Sperl, Wolfgang; Meitinger, Thomas; Zeviani, Massimo; Freisinger, Peter; Prokisch, Holger

2012-02-01

Mitochondrial complex I deficiency is the most common cause of mitochondrial disease in childhood. Identification of the molecular basis is difficult given the clinical and genetic heterogeneity. Most patients lack a molecular definition in routine diagnostics. A large-scale mutation screen of 75 candidate genes in 152 patients with complex I deficiency was performed by high-resolution melting curve analysis and Sanger sequencing. The causal role of a new disease allele was confirmed by functional complementation assays. The clinical phenotype of patients carrying mutations was documented using a standardised questionnaire. Causative mutations were detected in 16 genes, 15 of which had previously been associated with complex I deficiency: three mitochondrial DNA genes encoding complex I subunits, two mitochondrial tRNA genes and nuclear DNA genes encoding six complex I subunits and four assembly factors. For the first time, a causal mutation is described in NDUFB9, coding for a complex I subunit, resulting in reduction in NDUFB9 protein and both amount and activity of complex I. These features were rescued by expression of wild-type NDUFB9 in patient-derived fibroblasts. Mutant NDUFB9 is a new cause of complex I deficiency. A molecular diagnosis related to complex I deficiency was established in 18% of patients. However, most patients are likely to carry mutations in genes so far not associated with complex I function. The authors conclude that the high degree of genetic heterogeneity in complex I disorders warrants the implementation of unbiased genome-wide strategies for the complete molecular dissection of mitochondrial complex I deficiency.

Insulin and IGF-1 improve mitochondrial function in a PI-3K/Akt-dependent manner and reduce mitochondrial generation of reactive oxygen species in Huntington's disease knock-in striatal cells.

PubMed

Ribeiro, Márcio; Rosenstock, Tatiana R; Oliveira, Ana M; Oliveira, Catarina R; Rego, A Cristina

2014-09-01

Oxidative stress and mitochondrial dysfunction have been described in Huntington's disease, a disorder caused by expression of mutant huntingtin (mHtt). IGF-1 was previously shown to protect HD cells, whereas insulin prevented neuronal oxidative stress. In this work we analyzed the role of insulin and IGF-1 in striatal cells derived from HD knock-in mice on mitochondrial production of reactive oxygen species (ROS) and related antioxidant and signaling pathways influencing mitochondrial function. Insulin and IGF-1 decreased mitochondrial ROS induced by mHtt and normalized mitochondrial SOD activity, without affecting intracellular glutathione levels. IGF-1 and insulin promoted Akt phosphorylation without changing the nuclear levels of phosphorylated Nrf2 or Nrf2/ARE activity. Insulin and IGF-1 treatment also decreased mitochondrial Drp1 phosphorylation, suggesting reduced mitochondrial fragmentation, and ameliorated mitochondrial function in HD cells in a PI-3K/Akt-dependent manner. This was accompanied by increased total and phosphorylated Akt, Tfam, and mitochondrial-encoded cytochrome c oxidase II, as well as Tom20 and Tom40 in mitochondria of insulin- and IGF-1-treated mutant striatal cells. Concomitantly, insulin/IGF-1-treated mutant cells showed reduced apoptotic features. Hence, insulin and IGF-1 improve mitochondrial function and reduce mitochondrial ROS caused by mHtt by activating the PI-3K/Akt signaling pathway, in a process independent of Nrf2 transcriptional activity, but involving enhanced mitochondrial levels of Akt and mitochondrial-encoded complex IV subunit. Copyright © 2014 Elsevier Inc. All rights reserved.

Regulation of NADH/CoQ oxidoreductase: do phosphorylation events affect activity?

PubMed

Maj, Mary C; Raha, Sandeep; Myint, Tomoko; Robinson, Brian H

2004-01-01

We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.

Rat liver mitochondrial damage under acute or chronic carbon tetrachloride-induced intoxication: Protection by melatonin and cranberry flavonoids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheshchevik, V.T.; Department of Biochemistry, Yanka Kupala Grodno State University, Len. Kom. Blvd. - 50, 230017 Grodno; Lapshina, E.A.

In current societies, the risk of toxic liver damage has markedly increased. The aim of the present work was to carry out further research into the mechanism(s) of liver mitochondrial damage induced by acute (0.8 g/kg body weight, single injection) or chronic (1.6 g/ kg body weight, 30 days, biweekly injections) carbon tetrachloride – induced intoxication and to evaluate the hepatoprotective potential of the antioxidant, melatonin, as well as succinate and cranberry flavonoids in rats. Acute intoxication resulted in considerable impairment of mitochondrial respiratory parameters in the liver. The activity of mitochondrial succinate dehydrogenase (complex II) decreased (by 25%, pmore » < 0.05). Short-term melatonin treatment (10 mg/kg, three times) of rats did not reduce the degree of toxic mitochondrial dysfunction but decreased the enhanced NO production. After 30-day chronic intoxication, no significant change in the respiratory activity of liver mitochondria was observed, despite marked changes in the redox-balance of mitochondria. The activities of the mitochondrial enzymes, succinate dehydrogenase and glutathione peroxidase, as well as that of cytoplasmic catalase in liver cells were inhibited significantly. Mitochondria isolated from the livers of the rats chronically treated with CCl{sub 4} displayed obvious irreversible impairments. Long-term melatonin administration (10 mg/kg, 30 days, daily) to chronically intoxicated rats diminished the toxic effects of CCl{sub 4}, reducing elevated plasma activities of alanine aminotransferase and aspartate aminotransferase and bilirubin concentration, prevented accumulation of membrane lipid peroxidation products in rat liver and resulted in apparent preservation of the mitochondrial ultrastructure. The treatment of the animals by the complex of melatonin (10 mg/kg) plus succinate (50 mg/kg) plus cranberry flavonoids (7 mg/kg) was even more effective in prevention of toxic liver injury and liver mitochondria damage. Highlights: ► After 30-day chronic CCl{sub 4} intoxication mitochondria displayed considerable changes. ► The functional parameters of mitochondria were similar to the control values. ► Melatonin + succinate + flavonoids prevented mitochondrial ultrastructure damage. ► The above complex enhanced regenerative processes in the liver.« less

Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

PubMed

Haendeler, Judith; Dröse, Stefan; Büchner, Nicole; Jakob, Sascha; Altschmied, Joachim; Goy, Christine; Spyridopoulos, Ioakim; Zeiher, Andreas M; Brandt, Ulrich; Dimmeler, Stefanie

2009-06-01

The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

MitoQ regulates autophagy by inducing a pseudo-mitochondrial membrane potential

PubMed Central

Sun, Chao; Liu, Xiongxiong; Di, Cuixia; Wang, Zhenhua; Mi, Xiangquan; Liu, Yang; Zhao, Qiuyue; Mao, Aihong; Chen, Weiqiang; Gan, Lu; Zhang, Hong

2017-01-01

ABSTRACT During the process of oxidative phosphorylation, protons are pumped into the mitochondrial intermembrane space to establish a mitochondrial membrane potential (MMP). The electrochemical gradient generated allows protons to return to the matrix through the ATP synthase complex and generates ATP in the process. MitoQ is a lipophilic cationic drug that is adsorbed to the inner mitochondrial membrane; however, the cationic moiety of MitoQ remains in the intermembrane space. We found that the positive charges in MitoQ inhibited the activity of respiratory chain complexes I, III, and IV, reduced proton production, and decreased oxygen consumption. Therefore, a pseudo-MMP (PMMP) was formed via maintenance of exogenous positive charges. Proton backflow was severely impaired, leading to a decrease in ATP production and an increase in AMP production. Excess AMP activates AMP kinase, which inhibits the MTOR (mechanistic target of rapamycin) pathway and induces macroautophagy/autophagy. Therefore, we conclude that MitoQ increases PMMP via proton displacement with exogenous positive charges. In addition, PMMP triggered autophagy in hepatocellular carcinoma HepG2 cells via modification of mitochondrial bioenergetics pathways. PMID:28121478

MitoQ regulates autophagy by inducing a pseudo-mitochondrial membrane potential.

PubMed

Sun, Chao; Liu, Xiongxiong; Di, Cuixia; Wang, Zhenhua; Mi, Xiangquan; Liu, Yang; Zhao, Qiuyue; Mao, Aihong; Chen, Weiqiang; Gan, Lu; Zhang, Hong

2017-04-03

During the process of oxidative phosphorylation, protons are pumped into the mitochondrial intermembrane space to establish a mitochondrial membrane potential (MMP). The electrochemical gradient generated allows protons to return to the matrix through the ATP synthase complex and generates ATP in the process. MitoQ is a lipophilic cationic drug that is adsorbed to the inner mitochondrial membrane; however, the cationic moiety of MitoQ remains in the intermembrane space. We found that the positive charges in MitoQ inhibited the activity of respiratory chain complexes I, III, and IV, reduced proton production, and decreased oxygen consumption. Therefore, a pseudo-MMP (PMMP) was formed via maintenance of exogenous positive charges. Proton backflow was severely impaired, leading to a decrease in ATP production and an increase in AMP production. Excess AMP activates AMP kinase, which inhibits the MTOR (mechanistic target of rapamycin) pathway and induces macroautophagy/autophagy. Therefore, we conclude that MitoQ increases PMMP via proton displacement with exogenous positive charges. In addition, PMMP triggered autophagy in hepatocellular carcinoma HepG2 cells via modification of mitochondrial bioenergetics pathways.

Ameliorative efficacy of quercetin against cisplatin‑induced mitochondrial dysfunction: Study on isolated rat liver mitochondria.

PubMed

Waseem, Mohammad; Tabassum, Heena; Bhardwaj, Monica; Parvez, Suhel

2017-09-01

The present study aimed to investigate the hepatoprotective effects of the bioflavonoid quercetin (QR) on cisplatin (CP)‑induced mitochondrial oxidative stress in the livers of rats, to elucidate the role of mitochondria in CP‑induced hepatotoxicity, and its underlying mechanism. Isolated liver mitochondria were incubated with 100 µg/ml CP and/or 50 µM QR in vitro. CP treatment triggered a significant increase in membrane lipid peroxidation (LPO) levels, protein carbonyl (PC) contents, and a decrease in reduced glutathione (GSH) and non‑protein thiol (NP‑SH) levels. In addition, CP caused a marked decline in the activities of enzymatic antioxidants and mitochondrial complexes (I, II, III and V) in liver mitochondria. QR pre‑treatment significantly modulated the activities of enzymatic antioxidants and mitochondrial complex enzymes. Furthermore, QR reversed the alterations in LPO and PC levels, and GSH and NP‑SH contents in liver mitochondria. The results of the present study suggested that QR supplementation may suppress CP‑induced mitochondrial toxicity during chemotherapy, and provides a potential prophylactic and defensive candidate for anticancer agent‑induced oxidative stress.

Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function

PubMed Central

Mitsopoulos, Panagiotis; Chang, Yu-Han; Wai, Timothy; König, Tim; Dunn, Stanley D.; Langer, Thomas

2015-01-01

Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III2 and I-III2-IV1-3 RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function. PMID:25776552

Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart.

PubMed

Stepanova, Anna; Shurubor, Yevgeniya; Valsecchi, Federica; Manfredi, Giovanni; Galkin, Alexander

2016-09-01

Mitochondrial Complex II is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd~10(-8)M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30.2±6.0% and 56.4±5.6% in the heart and brain, respectively) is in the free, active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form. Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues, but it did not change the amount of free complex II. Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Beating oxygen: chronic anoxia exposure reduces mitochondrial F1FO-ATPase activity in turtle (Trachemys scripta) heart

PubMed Central

Galli, Gina L. J.; Lau, Gigi Y.; Richards, Jeffrey G.

2013-01-01

SUMMARY The freshwater turtle Trachemys scripta can survive in the complete absence of O2 (anoxia) for periods lasting several months. In mammals, anoxia leads to mitochondrial dysfunction, which culminates in cellular necrosis and apoptosis. Despite the obvious clinical benefits of understanding anoxia tolerance, little is known about the effects of chronic oxygen deprivation on the function of turtle mitochondria. In this study, we compared mitochondrial function in hearts of T. scripta exposed to either normoxia or 2 weeks of complete anoxia at 5°C and during simulated acute anoxia/reoxygenation. Mitochondrial respiration, electron transport chain activities, enzyme activities, proton conductance and membrane potential were measured in permeabilised cardiac fibres and isolated mitochondria. Two weeks of anoxia exposure at 5°C resulted in an increase in lactate, and decreases in ATP, glycogen, pH and phosphocreatine in the heart. Mitochondrial proton conductance and membrane potential were similar between experimental groups, while aerobic capacity was dramatically reduced. The reduced aerobic capacity was the result of a severe downregulation of the F1FO-ATPase (Complex V), which we assessed as a decrease in enzyme activity. Furthermore, in stark contrast to mammalian paradigms, isolated turtle heart mitochondria endured 20 min of anoxia followed by reoxygenation without any impact on subsequent ADP-stimulated O2 consumption (State III respiration) or State IV respiration. Results from this study demonstrate that turtle mitochondria remodel in response to chronic anoxia exposure and a reduction in Complex V activity is a fundamental component of mitochondrial and cellular anoxia survival. PMID:23926310

Mitochondrial Complex I Activity Suppresses Inflammation and Enhances Bone Resorption by Tipping the Balance of Macrophage-Osteoclast Polarization

PubMed Central

Jin, Zixue; Wei, Wei; Yang, Marie; Du, Yang; Wan, Yihong

2014-01-01

SUMMARY Mitochondrial complex I (CI) deficiency is associated with multiple neurological and metabolic disorders. However, its effect on innate immunity and bone remodeling is unclear. Using deletion of the essential CI subunit Ndufs4 as a model for mitochondrial dysfunction, we report that mitochondria suppress macrophage activation and inflammation while promoting osteoclast differentiation and bone resorption via both cell-autonomous and systemic regulation. Global Ndufs4 deletion causes systemic inflammation and osteopetrosis. Hematopoietic Ndufs4 deletion causes an intrinsic lineage shift from osteoclast to macrophage. Liver Ndufs4 deletion causes a metabolic shift from fatty acid oxidation to glycolysis, accumulating fatty acids and lactate (FA/LAC) in circulation. FA/LAC further activates Ndufs4−/− macrophages via ROS induction, and diminishes osteoclast lineage commitment in Ndufs4−/− progenitors; both inflammation and osteopetrosis in Ndufs4−/− mice are attenuated by TLR4/2 deletion. Together, these findings reveal mitochondrial CI as a critical rheostat of innate immunity and skeletal homeostasis. PMID:25130399

Mitochondrial protection by low doses of insulin-like growth factor- I in experimental cirrhosis.

PubMed

Pérez, Raquel; García-Fernández, María; Díaz-Sánchez, Matías; Puche, Juan E; Delgado, Gloria; Conchillo, Marian; Muntané, Jordi; Castilla-Cortázar, Inma

2008-05-07

To characterize the mitochondrial dysfunction in experimental cirrhosis and to study whether insulin-like growth factor-I (IGF- I) therapy (4 wk) is able to induce beneficial effects on damaged mitochondria leading to cellular protection. Wistar rats were divided into three groups: Control group, untreated cirrhotic rats and cirrhotic rats treated with IGF- I treatment (2 microg/100 g bw/d). Mitochondrial function was analyzed by flow cytometry in isolated hepatic mitochondria, caspase 3 activation was assessed by Western blot and apoptosis by TUNEL in the three experimental groups. Untreated cirrhotic rats showed a mitochondrial dysfunction characterized by a significant reduction of mitochondrial membrane potential (in status 4 and 3); an increase of intramitochondrial reactive oxigen species (ROS) generation and a significant reduction of ATPase activity. IGF- I therapy normalized mitochondrial function by increasing the membrane potential and ATPase activity and reducing the intramitochondrial free radical production. Activity of the electron transport complexes I and III was increased in both cirrhotic groups. In addition, untreated cirrhotic rats showed an increase of caspase 3 activation and apoptosis. IGF- I therapy reduced the expression of the active peptide of caspase 3 and resulted in reduced apoptosis. These results show that IGF- I exerts a mitochondrial protection in experimental cirrhosis leading to reduced apoptosis and increased ATP production.

Mitochondrial protection by low doses of insulin-like growth factor-Iin experimental cirrhosis

PubMed Central

Pérez, Raquel; García-Fernández, María; Díaz-Sánchez, Matías; Puche, Juan E; Delgado, Gloria; Conchillo, Marian; Muntané, Jordi; Castilla-Cortázar, Inma

2008-01-01

AIM: To characterize the mitochondrial dysfunction in experimental cirrhosis and to study whether insulin-like growth factor-I(IGF-I) therapy (4 wk) is able to induce beneficial effects on damaged mitochondria leading to cellular protection. METHODS: Wistar rats were divided into three groups: Control group, untreated cirrhotic rats and cirrhotic rats treated with IGF-Itreatment (2 μg/100 g bw/d). Mitochondrial function was analyzed by flow cytometry in isolated hepatic mitochondria, caspase 3 activation was assessed by Western blot and apoptosis by TUNEL in the three experimental groups. RESULTS: Untreated cirrhotic rats showed a mitochondrial dysfunction characterized by a significant reduction of mitochondrial membrane potential (in status 4 and 3); an increase of intramitochondrial reactive oxigen species (ROS) generation and a significant reduction of ATPase activity. IGF-Itherapy normalized mitochondrial function by increasing the membrane potential and ATPase activity and reducing the intramitochondrial free radical production. Activity of the electron transport complexes Iand III was increased in both cirrhotic groups. In addition, untreated cirrhotic rats showed an increase of caspase 3 activation and apoptosis. IGF-Itherapy reduced the expression of the active peptide of caspase 3 and resulted in reduced apoptosis. CONCLUSION: These results show that IGF-Iexerts a mitochondrial protection in experimental cirrhosis leading to reduced apoptosis and increased ATP production. PMID:18461658

Regulation of succinate-ubiquinone reductase and fumarate reductase activities in human complex II by phosphorylation of its flavoprotein subunit.

PubMed

Tomitsuka, Eriko; Kita, Kiyoshi; Esumi, Hiroyasu

2009-01-01

Complex II (succinate-ubiquinone reductase; SQR) is a mitochondrial respiratory chain enzyme that is directly involved in the TCA cycle. Complex II exerts a reverse reaction, fumarate reductase (FRD) activity, in various species such as bacteria, parasitic helminths and shellfish, but the existence of FRD activity in humans has not been previously reported. Here, we describe the detection of FRD activity in human cancer cells. The activity level was low, but distinct, and it increased significantly when the cells were cultured under hypoxic and glucose-deprived conditions. Treatment with phosphatase caused the dephosphorylation of flavoprotein subunit (Fp) with a concomitant increase in SQR activity, whereas FRD activity decreased. On the other hand, treatment with protein kinase caused an increase in FRD activity and a decrease in SQR activity. These data suggest that modification of the Fp subunit regulates both the SQR and FRD activities of complex II and that the phosphorylation of Fp might be important for maintaining mitochondrial energy metabolism within the tumor microenvironment.

Streptozotocin-Induced Adaptive Modification of Mitochondrial Supercomplexes in Liver of Wistar Rats and the Protective Effect of Moringa oleifera Lam.

PubMed

Alejandra Sánchez-Muñoz, María; Valdez-Solana, Mónica Andrea; Campos-Almazán, Mara Ibeth; Flores-Herrera, Óscar; Esparza-Perusquía, Mercedes; Olvera-Sánchez, Sofia; García-Arenas, Guadalupe; Avitia-Domínguez, Claudia; Téllez-Valencia, Alfredo; Sierra-Campos, Erick

2018-01-01

The increasing prevalence of diabetes continues to be a major health issue worldwide. Alteration of mitochondrial electron transport chain is a recognized hallmark of the diabetic-associated decline in liver bioenergetics; however, the molecular events involved are only poorly understood. Moringa oleifera is used for the treatment of diabetes. However, its role on mitochondrial functionality is not yet established. This study was aimed to evaluate the effect of M. oleifera extract on supercomplex formation, ATPase activity, ROS production, GSH levels, lipid peroxidation, and protein carbonylation. The levels of lipid peroxidation and protein carbonylation were increased in diabetic group. However, the levels were decreased in Moringa -treated diabetic rats. Analysis of in-gel activity showed an increase in all complex activities in the diabetic group, but spectrophotometric determinations of complex II and IV activities were unaffected in this treatment. However, we found an oxygen consumption abolition through complex I-III-IV pathway in the diabetic group treated with Moringa . While respiration with succinate feeding into complex II-III-IV was increased in the diabetic group. These findings suggest that hyperglycemia modifies oxygen consumption, supercomplexes formation, and increases ROS levels in mitochondria from the liver of STZ-diabetic rats, whereas M. oleifera may have a protective role against some alterations.

Streptozotocin-Induced Adaptive Modification of Mitochondrial Supercomplexes in Liver of Wistar Rats and the Protective Effect of Moringa oleifera Lam

PubMed Central

Alejandra Sánchez-Muñoz, María; Flores-Herrera, Óscar; Esparza-Perusquía, Mercedes; Olvera-Sánchez, Sofia; García-Arenas, Guadalupe; Téllez-Valencia, Alfredo

2018-01-01

The increasing prevalence of diabetes continues to be a major health issue worldwide. Alteration of mitochondrial electron transport chain is a recognized hallmark of the diabetic-associated decline in liver bioenergetics; however, the molecular events involved are only poorly understood. Moringa oleifera is used for the treatment of diabetes. However, its role on mitochondrial functionality is not yet established. This study was aimed to evaluate the effect of M. oleifera extract on supercomplex formation, ATPase activity, ROS production, GSH levels, lipid peroxidation, and protein carbonylation. The levels of lipid peroxidation and protein carbonylation were increased in diabetic group. However, the levels were decreased in Moringa-treated diabetic rats. Analysis of in-gel activity showed an increase in all complex activities in the diabetic group, but spectrophotometric determinations of complex II and IV activities were unaffected in this treatment. However, we found an oxygen consumption abolition through complex I-III-IV pathway in the diabetic group treated with Moringa. While respiration with succinate feeding into complex II-III-IV was increased in the diabetic group. These findings suggest that hyperglycemia modifies oxygen consumption, supercomplexes formation, and increases ROS levels in mitochondria from the liver of STZ-diabetic rats, whereas M. oleifera may have a protective role against some alterations. PMID:29686903

Mitochondrial respiratory chain complexes as sources and targets of thiol-based redox-regulation.

PubMed

Dröse, Stefan; Brandt, Ulrich; Wittig, Ilka

2014-08-01

The respiratory chain of the inner mitochondrial membrane is a unique assembly of protein complexes that transfers the electrons of reducing equivalents extracted from foodstuff to molecular oxygen to generate a proton-motive force as the primary energy source for cellular ATP-synthesis. Recent evidence indicates that redox reactions are also involved in regulating mitochondrial function via redox-modification of specific cysteine-thiol groups in subunits of respiratory chain complexes. Vice versa the generation of reactive oxygen species (ROS) by respiratory chain complexes may have an impact on the mitochondrial redox balance through reversible and irreversible thiol-modification of specific target proteins involved in redox signaling, but also pathophysiological processes. Recent evidence indicates that thiol-based redox regulation of the respiratory chain activity and especially S-nitrosylation of complex I could be a strategy to prevent elevated ROS production, oxidative damage and tissue necrosis during ischemia-reperfusion injury. This review focuses on the thiol-based redox processes involving the respiratory chain as a source as well as a target, including a general overview on mitochondria as highly compartmentalized redox organelles and on methods to investigate the redox state of mitochondrial proteins. This article is part of a Special Issue entitled: Thiol-Based Redox Processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Phosphatidylserine transport by Ups2-Mdm35 in respiration-active mitochondria.

PubMed

Miyata, Non; Watanabe, Yasunori; Tamura, Yasushi; Endo, Toshiya; Kuge, Osamu

2016-07-04

Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2-Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria. UPS2- and MDM35-null mutations greatly attenuated conversion of PS to PE in yeast cells growing logarithmically under nonfermentable conditions, but not fermentable conditions. A recombinant Ups2-Mdm35 fusion protein exhibited phospholipid-transfer activity between liposomes in vitro. Furthermore, UPS2 expression was elevated under nonfermentable conditions and at the diauxic shift, the metabolic transition from glycolysis to oxidative phosphorylation. These results demonstrate that Ups2-Mdm35 functions as a PS transfer protein and enhances mitochondrial PE synthesis in response to the cellular metabolic state. © 2016 Miyata et al.

A novel deficiency of mitochondrial ATPase of nuclear origin.

PubMed

Houstek, J; Klement, P; Floryk, D; Antonická, H; Hermanská, J; Kalous, M; Hansíková, H; Hout'ková, H; Chowdhury, S K; Rosipal, T; Kmoch, S; Stratilová, L; Zeman, J

1999-10-01

We report a new type of fatal mitochondrial disorder caused by selective deficiency of mitochondrial ATP synthase (ATPase). A hypotrophic newborn from a consanguineous marriage presented severe lactic acidosis, cardiomegaly and hepatomegaly and died from heart failure after 2 days. The activity of oligomycin-sensitive ATPase was only 31-34% of the control, both in muscle and heart, but the activities of cytochrome c oxidase, citrate synthase and pyruvate dehydrogenase were normal. Electrophoretic and western blot analysis revealed selective reduction of ATPase complex but normal levels of the respiratory chain complexes I, III and IV. The same selective deficiency of ATPase was found in cultured skin fibroblasts which showed similar decreases in ATPase content, ATPase hydrolytic activity and level of substrate-dependent ATP synthesis (20-25, 18 and 29-33% of the control, respectively). Pulse-chase labelling of patient fibroblasts revealed low incorporation of [(35)S]methionine into assembled ATPase complexes, but increased incorporation into immunoprecipitated ATPase subunit beta, which had a very short half-life. In contrast, no difference was found in the size and subunit composition of the assembled and newly produced ATPase complex. Transmitochondrial cybrids prepared from enucleated fibroblasts of the patient and rho degrees cells derived from 143B. TK(-)human osteosarcoma cells fully restored the ATPase activity, ATP synthesis and ATPase content, when compared with control cybrids. Likewise, the pattern of [(35)S]methionine labelling of ATPase was found to be normal in patient cybrids. We conclude that the generalized deficiency of mitochondrial ATPase described is of nuclear origin and is caused by altered biosynthesis of the enzyme.

Metabolic flexibility of mitochondrial respiratory chain disorders predicted by computer modelling.

PubMed

Zieliński, Łukasz P; Smith, Anthony C; Smith, Alexander G; Robinson, Alan J

2016-11-01

Mitochondrial respiratory chain dysfunction causes a variety of life-threatening diseases affecting about 1 in 4300 adults. These diseases are genetically heterogeneous, but have the same outcome; reduced activity of mitochondrial respiratory chain complexes causing decreased ATP production and potentially toxic accumulation of metabolites. Severity and tissue specificity of these effects varies between patients by unknown mechanisms and treatment options are limited. So far most research has focused on the complexes themselves, and the impact on overall cellular metabolism is largely unclear. To illustrate how computer modelling can be used to better understand the potential impact of these disorders and inspire new research directions and treatments, we simulated them using a computer model of human cardiomyocyte mitochondrial metabolism containing over 300 characterised reactions and transport steps with experimental parameters taken from the literature. Overall, simulations were consistent with patient symptoms, supporting their biological and medical significance. These simulations predicted: complex I deficiencies could be compensated using multiple pathways; complex II deficiencies had less metabolic flexibility due to impacting both the TCA cycle and the respiratory chain; and complex III and IV deficiencies caused greatest decreases in ATP production with metabolic consequences that parallel hypoxia. Our study demonstrates how results from computer models can be compared to a clinical phenotype and used as a tool for hypothesis generation for subsequent experimental testing. These simulations can enhance understanding of dysfunctional mitochondrial metabolism and suggest new avenues for research into treatment of mitochondrial disease and other areas of mitochondrial dysfunction. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

DNA recombination activity in soybean mitochondria.

PubMed

Manchekar, Medha; Scissum-Gunn, Karyn; Song, Daqing; Khazi, Fayaz; McLean, Stephanie L; Nielsen, Brent L

2006-02-17

Mitochondrial genomes in higher plants are much larger and more complex as compared to animal mitochondrial genomes. There is growing evidence that plant mitochondrial genomes exist predominantly as a collection of linear and highly branched DNA molecules and replicate by a recombination-dependent mechanism. However, biochemical evidence of mitochondrial DNA (mtDNA) recombination activity in plants has previously been lacking. We provide the first report of strand-invasion activity in plant mitochondria. Similar to bacterial RecA, this activity from soybean is dependent on the presence of ATP and Mg(2+). Western blot analysis using an antibody against the Arabidopsis mitochondrial RecA protein shows cross-reaction with a soybean protein of about 44 kDa, indicating conservation of this protein in at least these two plant species. mtDNA structure was analyzed by electron microscopy of total soybean mtDNA and molecules recovered after field-inversion gel electrophoresis (FIGE). While most molecules were found to be linear, some molecules contained highly branched DNA structures and a small but reproducible proportion consisted of circular molecules (many with tails) similar to recombination intermediates. The presence of recombination intermediates in plant mitochondria preparations is further supported by analysis of mtDNA molecules by 2-D agarose gel electrophoresis, which indicated the presence of complex recombination structures along with a considerable amount of single-stranded DNA. These data collectively provide convincing evidence for the occurrence of homologous DNA recombination in plant mitochondria.

Coenzyme Q10 Prevents Mitochondrial Dysfunction and Facilitates Pharmacological Activity of Atorvastatin in 6-OHDA Induced Dopaminergic Toxicity in Rats.

PubMed

Prajapati, Santosh Kumar; Garabadu, Debapriya; Krishnamurthy, Sairam

2017-05-01

Atorvastatin (ATV) generally used to treat dyslipidemia is also reported to have effect against 6-hydroxydopamine (6-OHDA) induced neurotoxicity. Additionally, atorvastatin can interfere with mitochondrial function by reducing the level of Q10. Therefore, the therapeutic effect of atorvastatin (20 mg/kg) could be compromised. In this context, the present study evaluated the effect of ATV supplemented with Q10. 6-OHDA was unilaterally injected into the right striatum of male rats. On day 8 of 6-OHDA infusion, ATV (20 mg/kg), Q10 (200 mg/kg), and their combination were administered per oral for 14 days. On day 21, there was significant loss of striatal dopamine indicating neurotoxicity. The combination of ATV+Q10 showed significant amelioration of dopamine (DA) toxicity compared to individual treatments. Similarly, ATV+Q10 compared to individual treatment significantly decreased the motor deficits induced by 6-OHDA. Further, 6-OHDA induced mitochondrial dysfunction in the substantia nigra pars compacta (SNpc). There was significant decrease in mitochondrial complex enzyme activities and mitochondrial membrane potential (MMP). Treatment with ATV and ATV+Q10 ameliorated mitochondrial dysfunction by increasing complex enzyme activities; however, only ATV+Q10 were able to stabilize MMP and maintained mitochondrial integrity. Moreover, there was significant induction of oxidative stress as observed from increase in lipid peroxidases (LPO) and nitrite (NO), and decrease in super oxide dismutase (SOD). Treatment with ATV+Q10 significantly altered the above effects indicating antioxidant activity. Furthermore, only combination of ATV and Q10 decreased the 6-OHDA induced expression of cytochrome-C, caspase-9 and caspase-3. Therefore, current results provide evidence that supplementation of Q10 with ATV shows synergistic effect in reducing dopamine toxicity.

Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle*

PubMed Central

De La Fuente, Sergio; Fernandez-Sanz, Celia; Vail, Caitlin; Agra, Elorm J.; Holmstrom, Kira; Sun, Junhui; Mishra, Jyotsna; Williams, Dewight; Finkel, Toren; Murphy, Elizabeth; Joseph, Suresh K.; Sheu, Shey-Shing; Csordás, György

2016-01-01

Control of myocardial energetics by Ca2+ signal propagation to the mitochondrial matrix includes local Ca2+ delivery from sarcoplasmic reticulum (SR) ryanodine receptors (RyR2) to the inner mitochondrial membrane (IMM) Ca2+ uniporter (mtCU). mtCU activity in cardiac mitochondria is relatively low, whereas the IMM surface is large, due to extensive cristae folding. Hence, stochastically distributed mtCU may not suffice to support local Ca2+ transfer. We hypothesized that mtCU concentrated at mitochondria-SR associations would promote the effective Ca2+ transfer. mtCU distribution was determined by tracking MCU and EMRE, the proteins essential for channel formation. Both proteins were enriched in the IMM-outer mitochondrial membrane (OMM) contact point submitochondrial fraction and, as super-resolution microscopy revealed, located more to the mitochondrial periphery (inner boundary membrane) than inside the cristae, indicating high accessibility to cytosol-derived Ca2+ inputs. Furthermore, MCU immunofluorescence distribution was biased toward the mitochondria-SR interface (RyR2), and this bias was promoted by Ca2+ signaling activity in intact cardiomyocytes. The SR fraction of heart homogenate contains mitochondria with extensive SR associations, and these mitochondria are highly enriched in EMRE. Size exclusion chromatography suggested for EMRE- and MCU-containing complexes a wide size range and also revealed MCU-containing complexes devoid of EMRE (thus disabled) in the mitochondrial but not the SR fraction. Functional measurements suggested more effective mtCU-mediated Ca2+ uptake activity by the mitochondria of the SR than of the mitochondrial fraction. Thus, mtCU “hot spots” can be formed at the cardiac muscle mitochondria-SR associations via localization and assembly bias, serving local Ca2+ signaling and the excitation-energetics coupling. PMID:27637331

The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria.

PubMed

König, Tim; Tröder, Simon E; Bakka, Kavya; Korwitz, Anne; Richter-Dennerlein, Ricarda; Lampe, Philipp A; Patron, Maria; Mühlmeister, Mareike; Guerrero-Castillo, Sergio; Brandt, Ulrich; Decker, Thorsten; Lauria, Ines; Paggio, Angela; Rizzuto, Rosario; Rugarli, Elena I; De Stefani, Diego; Langer, Thomas

2016-10-06

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca 2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca 2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca 2+ homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitochondrial Metabolism in Aging Heart

PubMed Central

Lesnefsky, Edward J.; Chen, Qun; Hoppel, Charles L.

2016-01-01

Altered mitochondrial metabolism is the underlying basis for the increased sensitivity in the aged heart to stress. The aged heart exhibits impaired metabolic flexibility, with a decreased capacity to oxidize fatty acids and enhanced dependence on glucose metabolism. Aging impairs mitochondrial oxidative phosphorylation, with a greater role played by the mitochondria located between the myofibrils, the interfibrillar mitochondria. With aging, there is a decrease in activity of complexes III and IV, which account for the decrease in respiration. Furthermore, aging decreases mitochondrial content among the myofibrils. The end result is that in the interfibrillar area there is an approximate 50% decrease in mitochondrial function, affecting all substrates. The defective mitochondria persist in the aged heart, leading to enhanced oxidant production and oxidative injury and the activation of oxidant signaling for cell death. Aging defects in mitochondria represent new therapeutic targets, whether by manipulation of the mitochondrial proteome, modulation of electron transport, activation of biogenesis or mitophagy, or the regulation of mitochondrial fission and fusion. These mechanisms provide new ways to attenuate cardiac disease in elders by preemptive treatment of age-related defects, in contrast to the treatment of disease-induced dysfunction. PMID:27174952

Correction of Mitochondrial Enzyme Activities in the Skeletal Muscles of Old Rats in Response to Addition of Olive Oil to the Ration.

PubMed

Bronnikov, G E; Kulagina, T P; Aripovskii, A V; Kramarova, L I

2015-06-01

Activities of mitochondrial electron transport chain enzymes NADH-CoQ oxidoreductase (complex I), cytochrome C-oxidase (complex IV), and citrate synthase were measured by spectrophotometry in m. quadriceps femoris homogenate from old rats receiving olive oil with the ration. Reduced activities of complexes I and IV in old animals were restored to the level of young animals after 6-week consumption of olive oil. Activity of citrate synthase did not change with age. Positive effect of olive oil on fatty-acid composition of the muscle tissue in old animals was demonstrated. The content of summary monounsaturated fatty acids, reduced with aging, and of summary polyunsaturated ones, increasing with age, were restored in old rats to the levels virtually not differing from the levels of young animals.

Mechanism of triclosan toxicity: Mitochondrial dysfunction including complex II inhibition, superoxide release and uncoupling of oxidative phosphorylation.

PubMed

Teplova, Vera V; Belosludtsev, Konstantin N; Kruglov, Alexey G

2017-06-05

Triclosan (5-chloro-2'-(2,4-dichlorophenoxy)phenol), a widely used antibacterial agent, exerts adverse effects on the organism of mammals. Recent research reviled that triclosan at low micromolar concentrations causes mitochondrial dysfunction in many cell types, but the mechanisms of its effect are not fully understood. Here we show that exposure to triclosan disrupted membrane potential, prevented the calcium uptake-driven high-amplitude mitochondrial swelling, stimulated the respiration in the presence of complex I substrates, and suppressed the ADP-stimulated respiration in the presence of complex II substrate, succinate. Triclosan directly inhibited complex II activity. Similar to the complex II inhibitor thenoyltrifluoroacetone, triclosan induced the oxidation of the cytochromes b566 and b562 and caused the release of mitochondrial superoxide. Opposite to thenoyltrifluoroacetone, triclosan increased superoxide release synergistically with myxothiazol but not with antimycin A, indicating different topology of superoxide-producing sites. We concluded that triclosan is unique by its capability of acting as both a protonophore and an unusual complex II inhibitor, which interferes with the mitochondrial respiration by blocking the electron transfer between ubiquinone at the Q d -binding site and heme b. Our data can provide an insight into the mechanisms of the carcinogenic effect of triclosan in the liver and other tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP-kinase.

PubMed

Bridges, Hannah R; Sirviö, Ville A; Agip, Ahmed-Noor A; Hirst, Judy

2016-08-09

The biguanides are a family of drugs with diverse clinical applications. Metformin, a widely used anti-hyperglycemic biguanide, suppresses mitochondrial respiration by inhibiting respiratory complex I. Phenformin, a related anti-hyperglycemic biguanide, also inhibits respiration, but proguanil, which is widely used for the prevention of malaria, does not. The molecular structures of phenformin and proguanil are closely related and both inhibit isolated complex I. Proguanil does not inhibit respiration in cells and mitochondria because it is unable to access complex I. The molecular features that determine which biguanides accumulate in mitochondria, enabling them to inhibit complex I in vivo, are not known. Here, a family of seven biguanides are used to reveal the molecular features that determine why phenformin enters mitochondria and inhibits respiration whereas proguanil does not. All seven biguanides inhibit isolated complex I, but only four of them inhibit respiration in cells and mitochondria. Direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. This high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only those biguanides that enter mitochondria and inhibit complex I activate AMP kinase, strengthening links between complex I and the downstream effects of biguanide treatments. Biguanides inhibit mitochondrial complex I, but specific molecular features control the uptake of substituted biguanides into mitochondria, so only some biguanides inhibit mitochondrial respiration in vivo. Biguanides with restricted intracellular access may be used to determine physiologically relevant targets of biguanide action, and for the rational design of substituted biguanides for diverse clinical applications.

Sexual Dimorphism in the Alterations of Cardiac Muscle Mitochondrial Bioenergetics Associated to the Ageing Process.

PubMed

Colom, Bartomeu; Oliver, Jordi; Garcia-Palmer, Francisco J

2015-11-01

The incidence of cardiac disease is age and sex dependent, but the mechanisms governing these associations remain poorly understood. Mitochondria are the organelles in charge of producing energy for the cells, and their malfunction has been linked to cardiovascular disease and heart failure. Interestingly, heart mitochondrial content and functionality are also age and sex dependent. Here we investigated the combinatory effects of age and sex in mitochondrial bioenergetics that could help to understand their role on cardiac disease. Cardiac mitochondria from 6- and 24-month-old male and female Wistar rats were isolated, and the enzymatic activities of the oxidative-phosphorylative complexes I, III, and IV and ATPase, as well as the protein levels of complex IV, β-ATPase, and mitochondrial transcription factor A (TFAM), were measured. Furthermore, heart DNA content, citrate synthase activity, mitochondrial protein content, oxygen consumption, and H2O2 generation were also determined. Results showed a reduction in heart mitochondrial mass and functionality with age that correlated with increased H2O2 generation. Moreover, sex-dependent differences were found in several of these parameters. In particular, old females exhibited a significant loss of mitochondrial function and increased relative H2O2 production compared with their male counterparts. The results demonstrate a sex dimorphism in the age-associated defects on cardiac mitochondrial function. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Protective effect of curcumin (Curcuma longa) against D-galactose-induced senescence in mice.

PubMed

Kumar, Anil; Prakash, Atish; Dogra, Samrita

2011-01-01

Brain senescence plays an important role in cognitive dysfunction and neurodegenerative disorders. Curcumin was reported to have beneficial effect against several neurodegenerative disorders including Alzheimer's disease. Therefore, the present study was conducted in order to explore the possible role of curcumin against D-galactose-induced cognitive dysfunction, oxidative damage, and mitochondrial dysfunction in mice. Chronic administration of D-galactose for 6 weeks significantly impaired cognitive function (both in Morris water maze and elevated plus maze), locomotor activity, oxidative defense (raised lipid peroxidation, nitrite concentration, depletion of reduced glutathione and catalase activity), and mitochondrial enzyme complex activities (I, II, and III) as compared to vehicle treated group. Curcumin (15 and 30 mg/kg) and galantamine (5 mg/kg) treatment for 6 weeks significantly improved cognitive tasks, locomotor activity, oxidative defense, and restored mitochondrial enzyme complex activity as compared to control (D-galactose). Chronic D-galactose treatment also significantly increased acetylcholine esterase activity that was attenuated by curcumin (15 and 30 mg/kg) and galantamine (5 mg/kg) treatment. In conclusion, the present study highlights the therapeutic potential of curcumin against d-galactose induced senescence in mice.

AMPK Is Essential to Balance Glycolysis and Mitochondrial Metabolism to Control T-ALL Cell Stress and Survival.

PubMed

Kishton, Rigel J; Barnes, Carson E; Nichols, Amanda G; Cohen, Sivan; Gerriets, Valerie A; Siska, Peter J; Macintyre, Andrew N; Goraksha-Hicks, Pankuri; de Cubas, Aguirre A; Liu, Tingyu; Warmoes, Marc O; Abel, E Dale; Yeoh, Allen Eng Juh; Gershon, Timothy R; Rathmell, W Kimryn; Richards, Kristy L; Locasale, Jason W; Rathmell, Jeffrey C

2016-04-12

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with Notch pathway mutations. While both normal activated and leukemic T cells can utilize aerobic glycolysis to support proliferation, it is unclear to what extent these cell populations are metabolically similar and if differences reveal T-ALL vulnerabilities. Here we show that aerobic glycolysis is surprisingly less active in T-ALL cells than proliferating normal T cells and that T-ALL cells are metabolically distinct. Oncogenic Notch promoted glycolysis but also induced metabolic stress that activated 5' AMP-activated kinase (AMPK). Unlike stimulated T cells, AMPK actively restrained aerobic glycolysis in T-ALL cells through inhibition of mTORC1 while promoting oxidative metabolism and mitochondrial Complex I activity. Importantly, AMPK deficiency or inhibition of Complex I led to T-ALL cell death and reduced disease burden. Thus, AMPK simultaneously inhibits anabolic growth signaling and is essential to promote mitochondrial pathways that mitigate metabolic stress and apoptosis in T-ALL. Copyright © 2016 Elsevier Inc. All rights reserved.

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria

PubMed Central

Karicheva, Olga Z.; Kolesnikova, Olga A.; Schirtz, Tom; Vysokikh, Mikhail Y.; Mager-Heckel, Anne-Marie; Lombès, Anne; Boucheham, Abdeldjalil; Krasheninnikov, Igor A.; Martin, Robert P.; Entelis, Nina; Tarassov, Ivan

2011-01-01

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNALeu(UUR). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNALeu(UUR) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders. PMID:21724600

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria.

PubMed

Karicheva, Olga Z; Kolesnikova, Olga A; Schirtz, Tom; Vysokikh, Mikhail Y; Mager-Heckel, Anne-Marie; Lombès, Anne; Boucheham, Abdeldjalil; Krasheninnikov, Igor A; Martin, Robert P; Entelis, Nina; Tarassov, Ivan

2011-10-01

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.

Azoxystrobin, a mitochondrial complex III Qo site inhibitor, exerts beneficial metabolic effects in vivo and in vitro.

PubMed

Gao, An-Hui; Fu, Yan-Yun; Zhang, Kun-Zhi; Zhang, Mei; Jiang, Hao-Wen; Fan, Li-Xia; Nan, Fa-Jun; Yuan, Chong-Gang; Li, Jia; Zhou, Yu-Bo; Li, Jing-Ya

2014-07-01

Several anti-diabetes drugs exert beneficial effects against metabolic syndrome by inhibiting mitochondrial function. Although much progress has been made toward understanding the role of mitochondrial function inhibitors in treating metabolic diseases, the potential effects of these inhibitors on mitochondrial respiratory chain complex III remain unclear. We investigated the metabolic effects of azoxystrobin (AZOX), a Qo inhibitor of complex III, in a high-fat diet-fed mouse model with insulin resistance in order to elucidate the mechanism by which AZOX improves glucose and lipid metabolism at the metabolic cellular level. Acute administration of AZOX in mice increased the respiratory exchange ratio. Chronic treatment with AZOX reduced body weight and significantly improved glucose tolerance and insulin sensitivity in high-fat diet-fed mice. AZOX treatment resulted in decreased triacylglycerol accumulation and down-regulated the expression of genes involved in liver lipogenesis. AZOX increased glucose uptake in L6 myotubes and 3T3-L1 adipocytes and inhibited de novo lipogenesis in HepG2 cells. The findings indicate that AZOX-mediated alterations to lipid and glucose metabolism may depend on AMP-activated protein kinase (AMPK) signaling. AZOX, a Qo inhibitor of mitochondrial respiratory complex III, exerts whole-body beneficial effects on the regulation of glucose and lipid homeostasis in high-fat diet-fed mice. These findings provide evidence that a Qo inhibitor of mitochondrial respiratory complex III could represent a novel approach for the treatment of obesity. Copyright © 2014 Elsevier B.V. All rights reserved.

A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria

PubMed Central

Hoppins, Suzanne; Collins, Sean R.; Cassidy-Stone, Ann; Hummel, Eric; DeVay, Rachel M.; Lackner, Laura L.; Westermann, Benedikt; Schuldiner, Maya

2011-01-01

To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane–associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria. PMID:21987634

Age-and Brain Region-Specific Differences in Mitochondrial ...

EPA Pesticide Factsheets

Mitochondria are central regulators of energy homeostasis and play a pivotal role in mechanisms of cellular senescence. The objective of the present study was to evaluate mitochondrial bio­-energetic parameters in five brain regions [brainstem (BS), frontal cortex (FC), cerebellum (CER), striatum (STR), hippocampus (HIP)] of four diverse age groups [1 Month (young), 4 Month (adult), 12 Month (middle-aged), 24 Month (old age)] to understand age-related differences in selected brain regions and their contribution to age-related chemical sensitivity. Mitochondrial bioenergetics parameters and enzyme activity were measured under identical conditions across multiple age groups and brain regions in Brown Norway rats (n = 5). The results indicate age- and brain region-specific patterns in mitochondrial functional endpoints. For example, an age-specific decline in ATP synthesis (State 111 respiration) was observed in BS and HIP. Similarly, the maximal respiratory capacities (State V1 and V2) showed age-specific declines in all brain regions examined (young > adult > middle-aged > old age). Amongst all regions, HIP had the greatest change in mitochondrial bioenergetics, showing declines in the 4, 12 and 24 Month age groups. Activities of mitochondrial pyruvate dehydrogenase complex (PDHC) and electron transport chain (ETC) complexes I, II, and IV enzymes were also age- and brain-region specific. In general changes associated with age were more pronounced, with

Mitochondrial events responsible for morphine's cardioprotection against ischemia/reperfusion injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Haiyan; Department of Pharmacology, Tianjin Medical University, Tianjin 300070; Huh, Jin

Morphine may induce cardioprotection by targeting mitochondria, but little is known about the exact mitochondrial events that mediate morphine's protection. We aimed to address the role of the mitochondrial Src tyrosine kinase in morphine's protection. Isolated rat hearts were subjected to 30 min ischemia and 2 h of reperfusion. Morphine was given before the onset of ischemia. Infarct size and troponin I release were measured to evaluate cardiac injury. Oxidative stress was evaluated by measuring mitochondrial protein carbonylation and mitochondrial ROS generation. HL-1 cells were subjected to simulated ischemia/reperfusion and LDH release and mitochondrial membrane potential (ΔΨm) were measured. Morphinemore » reduced infarct size as well as cardiac troponin I release which were aborted by the selective Src tyrosine kinase inhibitors PP2 and Src-I1. Morphine also attenuated LDH release and prevented a loss of ΔΨm at reperfusion in a Src tyrosine kinase dependent manner in HL-1 cells. However, morphine failed to reduce LDH release in HL-1 cells transfected with Src siRNA. Morphine increased mitochondrial Src phosphorylation at reperfusion and this was abrogated by PP2. Morphine attenuated mitochondrial protein carbonylation and mitochondrial superoxide generation at reperfusion through Src tyrosine kinase. The inhibitory effect of morphine on the mitochondrial complex I activity was reversed by PP2. These data suggest that morphine induces cardioprotection by preventing mitochondrial oxidative stress through mitochondrial Src tyrosine kinase. Inhibition of mitochondrial complex I at reperfusion by Src tyrosine kinase may account for the prevention of mitochondrial oxidative stress by morphine. - Highlights: • Morphine induced mito-Src phosphorylation and reduced infarct size in rat hearts. • Morphine failed to reduce I/R-induced LDH release in Src-silencing HL-1 cells. • Morphine prevented mitochondria damage caused by I/R through Src. • Morphine reduced mitochondrial ROS generation by inhibiting complex I via Src.« less

Mitochondrial Division Inhibitor 1 (mdivi-1) Protects Neurons against Excitotoxicity through the Modulation of Mitochondrial Function and Intracellular Ca2+ Signaling.

PubMed

Ruiz, Asier; Alberdi, Elena; Matute, Carlos

2018-01-01

Excessive dynamin related protein 1 (Drp1)-triggered mitochondrial fission contributes to apoptosis under pathological conditions and therefore it has emerged as a promising therapeutic target. Mitochondrial division inhibitor 1 (mdivi-1) inhibits Drp1-dependent mitochondrial fission and is neuroprotective in several models of brain ischemia and neurodegeneration. However, mdivi-1 also modulates mitochondrial function and oxidative stress independently of Drp1, and consequently the mechanisms through which it protects against neuronal injury are more complex than previously foreseen. In this study, we have analyzed the effects of mdivi-1 on mitochondrial dynamics, Ca 2+ signaling, mitochondrial bioenergetics and cell viability during neuronal excitotoxicity in vitro . Time-lapse fluorescence microscopy revealed that mdivi-1 blocked NMDA-induced mitochondrial fission but not that triggered by sustained AMPA receptor activation, showing that mdivi-1 inhibits excitotoxic mitochondrial fragmentation in a source specific manner. Similarly, mdivi-1 strongly reduced NMDA-triggered necrotic-like neuronal death and, to a lesser extent, AMPA-induced toxicity. Interestingly, neuroprotection provided by mdivi-1 against NMDA, but not AMPA, correlated with a reduction in cytosolic Ca 2+ ([Ca 2+ ] cyt ) overload and calpain activation indicating additional cytoprotective mechanisms. Indeed, mdivi-1 depolarized mitochondrial membrane and depleted ER Ca 2+ content, leading to attenuation of mitochondrial [Ca 2+ ] increase and enhancement of the integrated stress response (ISR) during NMDA receptor activation. Finally, lentiviral knockdown of Drp1 did not rescue NMDA-induced mitochondrial fission and toxicity, indicating that neuroprotective activity of mdivi-1 is Drp1-independent. Together, these results suggest that mdivi-1 induces a Drp1-independent protective phenotype that prevents predominantly NMDA receptor-mediated excitotoxicity through the modulation of mitochondrial function and intracellular Ca 2+ signaling.

Effect of simvastatin on vascular tone in porcine coronary artery: Potential role of the mitochondria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Almukhtar, H.; Garle, M.J.; Smith, P.A.

2016-08-15

Statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. As statins have been shown to alter mitochondrial function, in this study we investigated the role of mitochondria in the relaxation to simvastatin. Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Mitochondrial activity was determined by measuring changes in rhodamine 123 fluorescence. Changes in intracellular calcium levels were determined in freshly isolated smooth muscle cells with Fluo-4 using standard epifluorescent imaging techniques. Simvastatin, but not pravastatin, produced amore » slow relaxation of the coronary artery, which was independent of the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone (10 μM) and the complex III inhibitor myxothiazol (10 μM), or a combination of the two. The complex III inhibitor antimycin A (10 μM) produced a similar time-dependent relaxation of the porcine coronary artery, which was attenuated by rotenone. Changes in rhodamine 123 fluorescence showed that simvastatin (10 μM) depolarized the membrane potential of mitochondria in both isolated mitochondria and intact blood vessels. Simvastatin and antimycin A both inhibited calcium-induced contractions in isolated blood vessels and calcium influx in smooth muscle cells and this inhibition was prevented by rotenone. In conclusion, simvastatin produces an endothelium-independent relaxation of the porcine coronary artery which is dependent, in part, upon effects on the mitochondria. The effects on the mitochondria may lead to a reduction in calcium influx and hence relaxation of the blood vessel. - Highlights: • Simvastatin produces a relaxation of the porcine coronary artery. • This relaxation is inhibited by mitochondrial complex inhibitors. • Simvastatin alters mitochondrial membrane potential in intact blood vessels. • Simvastatin inhibits calcium influx in smooth muscle cells, prevented by a mitochondrial inhibitor. • Simvastatin-induced relaxations are dependent upon mitochondrial activity.« less

Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion

PubMed Central

Nacarelli, Timothy; Azar, Ashley; Sell, Christian

2014-01-01

The regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis. PMID:25104948

Peroxisome proliferator-activated receptors γ/mitochondrial uncoupling protein 2 signaling protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus

PubMed Central

2012-01-01

Background Status epilepticus induces subcellular changes that may lead to neuronal cell death in the hippocampus. However, the mechanism of seizure-induced neuronal cell death remains unclear. The mitochondrial uncoupling protein 2 (UCP2) is expressed in selected regions of the brain and is emerged as an endogenous neuroprotective molecule in many neurological disorders. We evaluated the neuroprotective role of UCP2 against seizure-induced hippocampal neuronal cell death under experimental status epilepticus. Methods In Sprague–Dawley rats, kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 subfield to induce prolonged bilateral seizure activity. Oxidized protein level, translocation of Bcl-2, Bax and cytochrome c between cytosol and mitochondria, and expression of peroxisome proliferator-activated receptors γ (PPARγ) and UCP2 were examined in the hippocampal CA3 subfield following KA-induced status epilepticus. The effects of microinjection bilaterally into CA3 area of a PPARγ agonist, rosiglitazone or a PPARγ antagonist, GW9662 on UCP2 expression, induced superoxide anion (O2· -) production, oxidized protein level, mitochondrial respiratory chain enzyme activities, translocation of Bcl-2, Bax and cytochrome c, and DNA fragmentation in bilateral CA3 subfields were examined. Results Increased oxidized proteins and mitochondrial or cytosol translocation of Bax or cytochrome c in the hippocampal CA3 subfield was observed 3–48 h after experimental status epilepticus. Expression of PPARγ and UCP2 increased 12–48 h after KA-induced status epilepticus. Pretreatment with rosiglitazone increased UCP2 expression, reduced protein oxidation, O2· - overproduction and dysfunction of mitochondrial Complex I, hindered the translocation of Bax and cytochrome c, and reduced DNA fragmentation in the CA3 subfield. Pretreatment with GW9662 produced opposite effects. Conclusions Activation of PPARγ upregulated mitochondrial UCP2 expression, which decreased overproduction of reactive oxygen species, improved mitochondrial Complex I dysfunction, inhibited mitochondrial translocation of Bax and prevented cytosolic release of cytochrome c by stabilizing the mitochondrial transmembrane potential, leading to amelioration of apoptotic neuronal cell death in the hippocampus following status epilepticus. PMID:22849356

Synergism of antifungal activity between mitochondrial respiration inhibitors and kojic acid

USDA-ARS?s Scientific Manuscript database

Co-application of certain types of compounds with conventional antimicrobial drugs results in the enhancement of efficacy of drugs through a mechanism termed chemosensitization. We show that kojic acid (KA), a natural product, is a potent chemosensitizer to complex III inhibitors of mitochondrial re...

Control of mitochondrial biogenesis and function by the ubiquitin-proteasome system.

PubMed

Bragoszewski, Piotr; Turek, Michal; Chacinska, Agnieszka

2017-04-01

Mitochondria are pivotal organelles in eukaryotic cells. The complex proteome of mitochondria comprises proteins that are encoded by nuclear and mitochondrial genomes. The biogenesis of mitochondrial proteins requires their transport in an unfolded state with a high risk of misfolding. The mislocalization of mitochondrial proteins is deleterious to the cell. The electron transport chain in mitochondria is a source of reactive oxygen species that damage proteins. Mitochondrial dysfunction is linked to many pathological conditions and, together with the loss of cellular protein homeostasis (proteostasis), are hallmarks of ageing and ageing-related degeneration diseases. The pathogenesis of neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, has been associated with mitochondrial and proteostasis failure. Thus, mitochondrial proteins require sophisticated surveillance mechanisms. Although mitochondria form a proteasome-exclusive compartment, multiple lines of evidence indicate a crucial role for the cytosolic ubiquitin-proteasome system (UPS) in the quality control of mitochondrial proteins. The proteasome affects mitochondrial proteins at stages of their biogenesis and maturity. The effects of the UPS go beyond the removal of damaged proteins and include the adjustment of mitochondrial proteome composition, the regulation of organelle dynamics and the protection of cellular homeostasis against mitochondrial failure. In turn, mitochondrial activity and mitochondrial dysfunction adjust the activity of the UPS, with implications at the cellular level. © 2017 The Authors.

Control of mitochondrial biogenesis and function by the ubiquitin–proteasome system

PubMed Central

Bragoszewski, Piotr; Turek, Michal

2017-01-01

Mitochondria are pivotal organelles in eukaryotic cells. The complex proteome of mitochondria comprises proteins that are encoded by nuclear and mitochondrial genomes. The biogenesis of mitochondrial proteins requires their transport in an unfolded state with a high risk of misfolding. The mislocalization of mitochondrial proteins is deleterious to the cell. The electron transport chain in mitochondria is a source of reactive oxygen species that damage proteins. Mitochondrial dysfunction is linked to many pathological conditions and, together with the loss of cellular protein homeostasis (proteostasis), are hallmarks of ageing and ageing-related degeneration diseases. The pathogenesis of neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, has been associated with mitochondrial and proteostasis failure. Thus, mitochondrial proteins require sophisticated surveillance mechanisms. Although mitochondria form a proteasome-exclusive compartment, multiple lines of evidence indicate a crucial role for the cytosolic ubiquitin–proteasome system (UPS) in the quality control of mitochondrial proteins. The proteasome affects mitochondrial proteins at stages of their biogenesis and maturity. The effects of the UPS go beyond the removal of damaged proteins and include the adjustment of mitochondrial proteome composition, the regulation of organelle dynamics and the protection of cellular homeostasis against mitochondrial failure. In turn, mitochondrial activity and mitochondrial dysfunction adjust the activity of the UPS, with implications at the cellular level. PMID:28446709

Mangifera indica L. extract (Vimang) inhibits Fe2+-citrate-induced lipoperoxidation in isolated rat liver mitochondria.

PubMed

Pardo Andreu, Gilberto; Delgado, René; Velho, Jesus; Inada, Natalia M; Curti, Carlos; Vercesi, Anibal E

2005-05-01

The extract of Mangifera indica L. (Vimang) is able to prevent iron mediated mitochondrial damage by means of oxidation of reduced transition metals required for the production of superoxide and hydroxyl radicals and direct free radical scavenging activity. In this study we report for the first time the iron-complexing ability of Vimang as a primary mechanism for protection of rat liver mitochondria against Fe2+ -citrate-induced lipoperoxidation. Thiobarbituric acid reactive substances (TBARS) and antimycin A-insensitive oxygen consumption were used as quantitative measures of lipoperoxidation. Vimang at 10 microM mangiferin concentration equivalent induced near-full protection against 50 microM Fe2+ -citrate-induced mitochondrial swelling and loss of mitochondrial transmembrane potential (DeltaPsi). The IC50 value for Vimang protection against Fe2+ -citrate-induced mitochondrial TBARS formation (7.89+/-1.19 microM) was around 10 times lower than that for tert-butylhydroperoxide mitochondrial induction of TBARS formation. The extract also inhibited the iron citrate induction of mitochondrial antimycin A-insensitive oxygen consumption, stimulated oxygen consumption due to Fe2+ autoxidation and prevented Fe3+ ascorbate reduction. The extracted polyphenolic compound, mainly mangiferin, could form a complex with Fe2+, accelerating Fe2+ oxidation and the formation of more stable Fe3+ -polyphenol complexes, unable to participate in Fenton-type reactions and lipoperoxidation propagation phase. The strong DPPH radical scavenging activity with an apparent IC50 of 2.45+/-0.08 microM suggests that besides its iron-complexing capacity, Vimang could also protect mitochondria from Fe2+ -citrate lipoperoxidation through direct free radical scavenging ability, mainly lipoperoxyl and alcoxyl radicals, acting as both a chain-breaking and iron-complexing antioxidant. These results are of pharmacological relevance since Vimang could be a potential candidate for antioxidant therapy in diseases related to abnormal intracellular iron distribution or iron overload.

Age-dependent reductions in mitochondrial respiration are exacerbated by calcium in the female rat heart.

PubMed

Hunter, J Craig; Machikas, Alexandra M; Korzick, Donna H

2012-06-01

Cardiovascular disease mortality increases rapidly after menopause by poorly defined mechanisms. Because mitochondrial function and Ca(2+) sensitivity are important regulators of cell death after myocardial ischemia, we sought to determine whether aging and/or estrogen deficiency (ovariectomy) increased mitochondrial Ca(2+) sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6 months; n = 26) and aged (24 months; n = 25), intact or ovariectomized female rats using the substrates α-ketoglutarate/malate (complex I); succinate/rotenone (complex II); ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/antimycin (complex IV). State 2 and 3 respiration was initiated by sequential addition of mitochondria and adenosine diphosphate. Ca(2+) sensitivity was assessed by Ca(2+)-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered intraperitoneally 45 minutes before euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for complexes I and II by 12% and 8%, respectively, independent of ovary status (P < 0.05). Of interest, Ca(2+) induced a greater decrease (18%-30%; P < 0.05) in complex I state 3 respiration in aged and ovariectomized animals, and mitochondrial swelling occurred twice as quickly in aged (vs adult) female rats (P < 0.05). Pretreatment with PPT increased RCI by 8% and 7% at complexes I and II, respectively (P < 0.05) but surprisingly increased Ca(2+) sensitivity. Age-dependent decreases in RCI and sensitization to Ca(2+) may explain in part the age-associated reductions in female ischemic tolerance; however, protection afforded by ER agonism involves more complex mechanisms. Copyright © 2012 Elsevier HS Journals, Inc. All rights reserved.

Age-Dependent Reductions in Mitochondrial Respiration are Exacerbated by Calcium in the Female Rat Heart

PubMed Central

Hunter, J. Craig; Machikas, Alexandra M.; Korzick, Donna H.

2012-01-01

Cardiovascular disease mortality increases rapidly following menopause by poorly defined mechanisms. Since mitochondrial function and Ca2+ sensitivity are important regulators of cell death following myocardial ischemia, we sought to determine if aging and/or estrogen deficiency (ovx) increased mitochondrial Ca2+ sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6mo; n=26) and aged (24mo; n=25), intact or ovariectomized female rats using the substrates: α-ketoglutarate/malate (Complex I); succinate/rotenone (Complex II); ascorbate/TMPD/Antimycin (Complex IV). State 2 and State 3 respiration was initiated by sequential addition of mitochondria and ADP. Ca2+ sensitivity was assessed by Ca2+-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered i.p. 45 min prior to euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for Complexes I and II by 12% and 8%, respectively, independent of ovary status (p<0.05). Of interest, Ca2+ induced a greater decrease (18–30%; p<0.05) in Complex I state 3 respiration in aged and ovx animals, and mitochondrial swelling occurred twice as quickly in aged (vs. adult) female rats (p<0.05). Pretreatment with PPT increased RCI by 8% and 7% at Complexes I and II, respectively (p<0.05) but surprisingly increased Ca2+ sensitivity. Age-dependent decreases in RCI and sensitization to Ca2+ may explain in part the age-associated reductions in female ischemic tolerance; however protection afforded by ER agonism involves more complex mechanisms. PMID:22555015

Mitochondrial Targeting of Vitamin E Succinate Enhances Its Pro-apoptotic and Anti-cancer Activity via Mitochondrial Complex II*

PubMed Central

Dong, Lan-Feng; Jameson, Victoria J. A.; Tilly, David; Cerny, Jiri; Mahdavian, Elahe; Marín-Hernández, Alvaro; Hernández-Esquivel, Luz; Rodríguez-Enríquez, Sara; Stursa, Jan; Witting, Paul K.; Stantic, Bela; Rohlena, Jakub; Truksa, Jaroslav; Kluckova, Katarina; Dyason, Jeffrey C.; Ledvina, Miroslav; Salvatore, Brian A.; Moreno-Sánchez, Rafael; Coster, Mark J.; Ralph, Stephen J.; Smith, Robin A. J.; Neuzil, Jiri

2011-01-01

Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC50 of 80 μm, whereas the electron transfer from CII to CIII was inhibited with IC50 of 1.5 μm. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser68 within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug. PMID:21059645

TIMMDC1/C3orf1 functions as a membrane-embedded mitochondrial complex I assembly factor through association with the MCIA complex.

PubMed

Guarani, Virginia; Paulo, Joao; Zhai, Bo; Huttlin, Edward L; Gygi, Steven P; Harper, J Wade

2014-03-01

Complex I (CI) of the electron transport chain, a large membrane-embedded NADH dehydrogenase, couples electron transfer to the release of protons into the mitochondrial inner membrane space to promote ATP production through ATP synthase. In addition to being a central conduit for ATP production, CI activity has been linked to neurodegenerative disorders, including Parkinson's disease. CI is built in a stepwise fashion through the actions of several assembly factors. We employed interaction proteomics to interrogate the molecular associations of 15 core subunits and assembly factors previously linked to human CI deficiency, resulting in a network of 101 proteins and 335 interactions (edges). TIMMDC1, a predicted 4-pass membrane protein, reciprocally associated with multiple members of the MCIA CI assembly factor complex and core CI subunits and was localized in the mitochondrial inner membrane, and its depletion resulted in reduced CI activity and cellular respiration. Quantitative proteomics demonstrated a role for TIMMDC1 in assembly of membrane-embedded and soluble arms of the complex. This study defines a new membrane-embedded CI assembly factor and provides a resource for further analysis of CI biology.

Suppression of BRCA2 by Mutant Mitochondrial DNA in Prostate Cancer

DTIC Science & Technology

2011-05-01

Briefly, the electron transfer activities of complex I/III (NADH dehydrogenase/cytochrome bc1 complex: catalyzes the electron transfer from NADH to...ferricytochrome c) and complex II/III (succinate dehydrogenase/cytochrome bc1 complex: catalyzes the electron transfer from succinate to ferricytochrome...The electron transfer activity of complex IV (cytochrome c oxidase: catalyzes the final step of the respiratory chain by transferring electrons from

Changes in mitochondrial function and mitochondria associated protein expression in response to 2-weeks of high intensity interval training

PubMed Central

Vincent, Grace; Lamon, Séverine; Gant, Nicholas; Vincent, Peter J.; MacDonald, Julia R.; Markworth, James F.; Edge, Johann A.; Hickey, Anthony J. R.

2015-01-01

Purpose: High-intensity short-duration interval training (HIT) stimulates functional and metabolic adaptation in skeletal muscle, but the influence of HIT on mitochondrial function remains poorly studied in humans. Mitochondrial metabolism as well as mitochondrial-associated protein expression were tested in untrained participants performing HIT over a 2-week period. Methods: Eight males performed a single-leg cycling protocol (12 × 1 min intervals at 120% peak power output, 90 s recovery, 4 days/week). Muscle biopsies (vastus lateralis) were taken pre- and post-HIT. Mitochondrial respiration in permeabilized fibers, citrate synthase (CS) activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and respiratory complex components were measured. Results: HIT training improved peak power and time to fatigue. Increases in absolute oxidative phosphorylation (OXPHOS) capacities and CS activity were observed, but not in the ratio of CCO to the electron transport system (CCO/ETS), the respiratory control ratios (RCR-1 and RCR-2) or mitochondrial-associated protein expression. Specific increases in OXPHOS flux were not apparent after normalization to CS, indicating that gross changes mainly resulted from increased mitochondrial mass. Conclusion: Over only 2 weeks HIT significantly increased mitochondrial function in skeletal muscle independently of detectable changes in mitochondrial-associated and mitogenic protein expression. PMID:25759671

Bnip3 mediates doxorubicin-induced cardiac myocyte necrosis and mortality through changes in mitochondrial signaling

PubMed Central

Dhingra, Rimpy; Margulets, Victoria; Chowdhury, Subir Roy; Thliveris, James; Jassal, Davinder; Fernyhough, Paul; Dorn, Gerald W.; Kirshenbaum, Lorrie A.

2014-01-01

Doxorubicin (DOX) is widely used for treating human cancers, but can induce heart failure through an undefined mechanism. Herein we describe a previously unidentified signaling pathway that couples DOX-induced mitochondrial respiratory chain defects and necrotic cell death to the BH3-only protein Bcl-2-like 19kDa-interacting protein 3 (Bnip3). Cellular defects, including vacuolization and disrupted mitochondria, were observed in DOX-treated mice hearts. This coincided with mitochondrial localization of Bnip3, increased reactive oxygen species production, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, and necrosis. Interestingly, a 3.1-fold decrease in maximal mitochondrial respiration was observed in cardiac mitochondria of mice treated with DOX. In vehicle-treated control cells undergoing normal respiration, the respiratory chain complex IV subunit 1 (COX1) was tightly bound to uncoupling protein 3 (UCP3), but this complex was disrupted in cells treated with DOX. Mitochondrial dysfunction induced by DOX was accompanied by contractile failure and necrotic cell death. Conversely, shRNA directed against Bnip3 or a mutant of Bnip3 defective for mitochondrial targeting abrogated DOX-induced loss of COX1-UCP3 complexes and respiratory chain defects. Finally, Bnip3−/− mice treated with DOX displayed relatively normal mitochondrial morphology, respiration, and mortality rates comparable to those of saline-treated WT mice, supporting the idea that Bnip3 underlies the cardiotoxic effects of DOX. These findings reveal a new signaling pathway in which DOX-induced mitochondrial respiratory chain defects and necrotic cell death are mutually dependent on and obligatorily linked to Bnip3 gene activation. Interventions that antagonize Bnip3 may prove beneficial in preventing mitochondrial injury and heart failure in cancer patients undergoing chemotherapy. PMID:25489073

Myopathy caused by mammalian target of rapamycin complex 1 (mTORC1) inactivation is not reversed by restoring mitochondrial function

PubMed Central

Romanino, Klaas; Mazelin, Laetitia; Albert, Verena; Conjard-Duplany, Agnès; Lin, Shuo; Bentzinger, C. Florian; Handschin, Christoph; Puigserver, Pere; Zorzato, Francesco; Schaeffer, Laurent; Gangloff, Yann-Gaël; Rüegg, Markus A.

2011-01-01

Mammalian target of rapamycin complex 1 (mTORC1) is central to the control of cell, organ, and body size. Skeletal muscle-specific inactivation of mTORC1 in mice results in smaller muscle fibers, fewer mitochondria, increased glycogen stores, and a progressive myopathy that causes premature death. In mTORC1-deficient muscles, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), which regulates mitochondrial biogenesis and glucose homeostasis, is strongly down-regulated. Here we tested whether induction of mitochondrial biogenesis pharmacologically or by the overexpression of PGC-1α is sufficient to reverse the phenotype of mice deficient for mTORC1. We show that both approaches normalize mitochondrial function, such as oxidative capacity and expression of mitochondrial genes. However, they do not prevent or delay the progressive myopathy. In addition, we find that mTORC1 has a much stronger effect than PGC-1α on the glycogen content in muscle. This effect is based on the strong activation of PKB/Akt in mTORC1-deficient mice. We also show that activation of PKB/Akt not only affects glycogen synthesis but also diminishes glycogen degradation. Thus, our work provides strong functional evidence that mitochondrial dysfunction in mice with inactivated mTORC1 signaling is caused by the down-regulation of PGC-1α. However, our data also show that the impairment of mitochondria does not lead directly to the lethal myopathy. PMID:22143799

Respiratory chain complex III deficiency in patients with tRNA-leu mutation.

PubMed

Jiang, J; Wang, X L; Ma, Y Y

2015-12-29

The aim of this study was to investigate the clinical and genetic profiles of mitochondrial disease resulting from deficiencies in the respiratory chain complex III. Three patients, aged between 8 months and 12 years, were recruited for this study. The activities of mitochondrial respiratory chain complexes in the peripheral leucocytes were spectrophotometrically measured. The entire mitochondrial DNA (mtDNA) sequence was analyzed. Samples obtained from the three patients and their families were subjected to restriction fragment length polymorphism and gene sequencing analyses. mtDNA copy numbers of all patients and their mothers were analyzed. The patients displayed nervous system impairment, including motor and mental developmental delay, hypotonia, and motor regression. Two patients also suffered from Leigh syndrome. Assay of the mitochondrial respiratory chain enzymes revealed an isolated complex III deficiency in the three patients. The m.3243 A>G mutation was detected in all patients and their mothers. The mutation loads were 48.3, 57.2, and 45.5% in the patients, and 20.5, 16.4, and 23.6% in their respective mothers. The leukocyte mtDNA copy numbers of the patients and their mothers were within the control range. The clinical manifestation and genetics were observed to be very heterogeneous. Patient carrying an m.3243 A>G mutation may biochemically display a deficiency in the mitochondrial respiratory chain complex III.

Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex.

PubMed

Boniecki, Michal T; Freibert, Sven A; Mühlenhoff, Ulrich; Lill, Roland; Cygler, Miroslaw

2017-11-03

Iron-sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status.

Triterpenic acids-enriched fraction from Cyclocarya paliurus attenuates non-alcoholic fatty liver disease via improving oxidative stress and mitochondrial dysfunction.

PubMed

Zhao, Meng-Ge; Sheng, Xue-Ping; Huang, Ya-Ping; Wang, Yi-Ting; Jiang, Cui-Hua; Zhang, Jian; Yin, Zhi-Qi

2018-08-01

The effects of triterpenic acids-enriched fraction from Cyclocarya paliurus (CPT) on nonalcoholic fatty liver disease (NAFLD) were investigated using in vivo and in vitro models. In high fat diet-induced Wister rats, CPT significantly increased superoxide dismutase (SOD) activity and glutathione/oxidized glutathione (GSH/GSSG) ratio, reduced malondialdehyde (MDA) and protein carbonyl (PCO) levels. Moreover, CPT restored mitochondrial membrane potential dysfunction, decreased cytochrome P450 enzyme 2E1 (CYP2E1) activity, improved nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-mediated antioxidant enzyme heme oxygenase1 (HO-1) expression. In free fatty acids-induced HepG2 cells, CPT dramatically decreased ROS content, increased mitochondrial NADH dehydrogenase (Complex I) and mitochondrial cytochrome C oxidase (Complex IV) levels. Furthermore, CPT could upregulate HO-1, quinine oxidoreductase 1 (NQO1) expression, and increase Nrf2 translocation from cytoplasm-to-nucleus. The results indicated CPT could protect mitochondria function and improve oxidative stress by activating Nrf2. Therefore, it can be inferred that CPT may be a potential agent against NAFLD. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Endothelial AMPK Activation Induces Mitochondrial Biogenesis and Stress Adaptation via eNOS-Dependent mTORC1 Signaling

PubMed Central

Li, Chunying; Reif, Michaella M; Craige, Siobhan; Kant, Shashi; Keaney, John F.

2016-01-01

Metabolic stress sensors like AMP-activated protein kinase (AMPK) are known to confer stress adaptation and promote longevity in lower organisms. This study demonstrates that activating the metabolic stress sensor AMP-activated protein kinase (AMPK) in endothelial cells helps maintain normal cellular function by promoting mitochondrial biogenesis and stress adaptation. To better define the mechanisms whereby AMPK promotes endothelial stress resistance, we used 5-aminoimidazole-4-carboxamide riboside (AICAR) to chronically activate AMPK and observed stimulation of mitochondrial biogenesis in wild type mouse endothelium, but not in endothelium from endothelial nitric oxide synthase knockout (eNOS-null) mice. Interestingly, AICAR-enhanced mitochondrial biogenesis was blocked by pretreatment with the mammalian target of rapamycin complex 1 (mTORC1) inhibitor, rapamycin. Further, AICAR stimulated mTORC1 as determined by phosphorylation of its known downstream effectors in wild type, but not eNOS-null, endothelial cells. Together these data indicate that eNOS is needed to couple AMPK activation to mTORC1 and thus promote mitochondrial biogenesis and stress adaptation in the endothelium. These data suggest a novel mechanism for mTORC1 activation that is significant for investigations in vascular dysfunction. PMID:26989010

'Mitochondrial energy imbalance and lipid peroxidation cause cell death in Friedreich's ataxia'.

PubMed

Abeti, R; Parkinson, M H; Hargreaves, I P; Angelova, P R; Sandi, C; Pook, M A; Giunti, P; Abramov, A Y

2016-05-26

Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease. The mutation consists of a GAA repeat expansion within the FXN gene, which downregulates frataxin, leading to abnormal mitochondrial iron accumulation, which may in turn cause changes in mitochondrial function. Although, many studies of FRDA patients and mouse models have been conducted in the past two decades, the role of frataxin in mitochondrial pathophysiology remains elusive. Are the mitochondrial abnormalities only a side effect of the increased accumulation of reactive iron, generating oxidative stress? Or does the progressive lack of iron-sulphur clusters (ISCs), induced by reduced frataxin, cause an inhibition of the electron transport chain complexes (CI, II and III) leading to reactive oxygen species escaping from oxidative phosphorylation reactions? To answer these crucial questions, we have characterised the mitochondrial pathophysiology of a group of disease-relevant and readily accessible neurons, cerebellar granule cells, from a validated FRDA mouse model. By using live cell imaging and biochemical techniques we were able to demonstrate that mitochondria are deregulated in neurons from the YG8R FRDA mouse model, causing a decrease in mitochondrial membrane potential (▵Ψm) due to an inhibition of Complex I, which is partially compensated by an overactivation of Complex II. This complex activity imbalance leads to ROS generation in both mitochondrial matrix and cytosol, which results in glutathione depletion and increased lipid peroxidation. Preventing this increase in lipid peroxidation, in neurons, protects against in cell death. This work describes the pathophysiological properties of the mitochondria in neurons from a FRDA mouse model and shows that lipid peroxidation could be an important target for novel therapeutic strategies in FRDA, which still lacks a cure.

Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin.

PubMed

Lazarou, Michael; Jin, Seok Min; Kane, Lesley A; Youle, Richard J

2012-02-14

Mutations in the mitochondrial kinase PINK1 and the cytosolic E3 ligase Parkin can cause Parkinson's disease. Damaged mitochondria accumulate PINK1 on the outer membrane where, dependent on kinase activity, it recruits and activates Parkin to induce mitophagy, potentially maintaining organelle fidelity. How PINK1 recruits Parkin is unknown. We show that endogenous PINK1 forms a 700 kDa complex with the translocase of the outer membrane (TOM) selectively on depolarized mitochondria whereas PINK1 ectopically targeted to the outer membrane retains association with TOM on polarized mitochondria. Inducibly targeting PINK1 to peroxisomes or lysosomes, which lack a TOM complex, recruits Parkin and activates ubiquitin ligase activity on the respective organelles. Once there, Parkin induces organelle selective autophagy of peroxisomes but not lysosomes. We propose that the association of PINK1 with the TOM complex allows rapid reimport of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation. Copyright © 2012 Elsevier Inc. All rights reserved.

A mouse model of mitochondrial complex III dysfunction induced by myxothiazol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davoudi, Mina; Kallijärvi, Jukka; Marjavaara, Sanna

2014-04-18

Highlights: • Reversible chemical inhibition of complex III in wild type mouse. • Myxothiazol causes decreased complex III activity in mouse liver. • The model is useful for therapeutic trials to improve mitochondrial function. - Abstract: Myxothiazol is a respiratory chain complex III (CIII) inhibitor that binds to the ubiquinol oxidation site Qo of CIII. It blocks electron transfer from ubiquinol to cytochrome b and thus inhibits CIII activity. It has been utilized as a tool in studies of respiratory chain function in in vitro and cell culture models. We developed a mouse model of biochemically induced and reversible CIIImore » inhibition using myxothiazol. We administered myxothiazol intraperitoneally at a dose of 0.56 mg/kg to C57Bl/J6 mice every 24 h and assessed CIII activity, histology, lipid content, supercomplex formation, and gene expression in the livers of the mice. A reversible CIII activity decrease to 50% of control value occurred at 2 h post-injection. At 74 h only minor histological changes in the liver were found, supercomplex formation was preserved and no significant changes in the expression of genes indicating hepatotoxicity or inflammation were found. Thus, myxothiazol-induced CIII inhibition can be induced in mice for four days in a row without overt hepatotoxicity or lethality. This model could be utilized in further studies of respiratory chain function and pharmacological approaches to mitochondrial hepatopathies.« less

Clinical, pathological and functional characterization of riboflavin-responsive neuropathy

PubMed Central

Manole, Andreea; Jaunmuktane, Zane; Hargreaves, Iain; Ludtmann, Marthe H R; Salpietro, Vincenzo; Bello, Oscar D; Pope, Simon; Pandraud, Amelie; Horga, Alejandro; Scalco, Renata S; Li, Abi; Ashokkumar, Balasubramaniem; Lourenço, Charles M; Heales, Simon; Horvath, Rita; Chinnery, Patrick F; Toro, Camilo; Singleton, Andrew B; Jacques, Thomas S; Abramov, Andrey Y; Muntoni, Francesco; Hanna, Michael G; Reilly, Mary M; Revesz, Tamas; Kullmann, Dimitri M

2017-01-01

Abstract Brown-Vialetto-Van Laere syndrome represents a phenotypic spectrum of motor, sensory, and cranial nerve neuropathy, often with ataxia, optic atrophy and respiratory problems leading to ventilator-dependence. Loss-of-function mutations in two riboflavin transporter genes, SLC52A2 and SLC52A3, have recently been linked to Brown-Vialetto-Van Laere syndrome. However, the genetic frequency, neuropathology and downstream consequences of riboflavin transporter mutations are unclear. By screening a large cohort of 132 patients with early-onset severe sensory, motor and cranial nerve neuropathy we confirmed the strong genetic link between riboflavin transporter mutations and Brown-Vialetto-Van Laere syndrome, identifying 22 pathogenic mutations in SLC52A2 and SLC52A3, 14 of which were novel. Brain and spinal cord neuropathological examination of two cases with SLC52A3 mutations showed classical symmetrical brainstem lesions resembling pathology seen in mitochondrial disease, including severe neuronal loss in the lower cranial nerve nuclei, anterior horns and corresponding nerves, atrophy of the spinothalamic and spinocerebellar tracts and posterior column–medial lemniscus pathways. Mitochondrial dysfunction has previously been implicated in an array of neurodegenerative disorders. Since riboflavin metabolites are critical components of the mitochondrial electron transport chain, we hypothesized that reduced riboflavin transport would result in impaired mitochondrial activity, and confirmed this using in vitro and in vivo models. Electron transport chain complex I and complex II activity were decreased in SLC52A2 patient fibroblasts, while global knockdown of the single Drosophila melanogaster riboflavin transporter homologue revealed reduced levels of riboflavin, downstream metabolites, and electron transport chain complex I activity. This in turn led to abnormal mitochondrial membrane potential, respiratory chain activity and morphology. Riboflavin transporter knockdown in Drosophila also resulted in severely impaired locomotor activity and reduced lifespan, mirroring patient pathology, and these phenotypes could be partially rescued using a novel esterified derivative of riboflavin. Our findings expand the genetic, clinical and neuropathological features of Brown-Vialetto-Van Laere syndrome, implicate mitochondrial dysfunction as a downstream consequence of riboflavin transporter gene defects, and validate riboflavin esters as a potential therapeutic strategy. PMID:29053833

Clinical, pathological and functional characterization of riboflavin-responsive neuropathy.

PubMed

Manole, Andreea; Jaunmuktane, Zane; Hargreaves, Iain; Ludtmann, Marthe H R; Salpietro, Vincenzo; Bello, Oscar D; Pope, Simon; Pandraud, Amelie; Horga, Alejandro; Scalco, Renata S; Li, Abi; Ashokkumar, Balasubramaniem; Lourenço, Charles M; Heales, Simon; Horvath, Rita; Chinnery, Patrick F; Toro, Camilo; Singleton, Andrew B; Jacques, Thomas S; Abramov, Andrey Y; Muntoni, Francesco; Hanna, Michael G; Reilly, Mary M; Revesz, Tamas; Kullmann, Dimitri M; Jepson, James E C; Houlden, Henry

2017-11-01

Brown-Vialetto-Van Laere syndrome represents a phenotypic spectrum of motor, sensory, and cranial nerve neuropathy, often with ataxia, optic atrophy and respiratory problems leading to ventilator-dependence. Loss-of-function mutations in two riboflavin transporter genes, SLC52A2 and SLC52A3, have recently been linked to Brown-Vialetto-Van Laere syndrome. However, the genetic frequency, neuropathology and downstream consequences of riboflavin transporter mutations are unclear. By screening a large cohort of 132 patients with early-onset severe sensory, motor and cranial nerve neuropathy we confirmed the strong genetic link between riboflavin transporter mutations and Brown-Vialetto-Van Laere syndrome, identifying 22 pathogenic mutations in SLC52A2 and SLC52A3, 14 of which were novel. Brain and spinal cord neuropathological examination of two cases with SLC52A3 mutations showed classical symmetrical brainstem lesions resembling pathology seen in mitochondrial disease, including severe neuronal loss in the lower cranial nerve nuclei, anterior horns and corresponding nerves, atrophy of the spinothalamic and spinocerebellar tracts and posterior column-medial lemniscus pathways. Mitochondrial dysfunction has previously been implicated in an array of neurodegenerative disorders. Since riboflavin metabolites are critical components of the mitochondrial electron transport chain, we hypothesized that reduced riboflavin transport would result in impaired mitochondrial activity, and confirmed this using in vitro and in vivo models. Electron transport chain complex I and complex II activity were decreased in SLC52A2 patient fibroblasts, while global knockdown of the single Drosophila melanogaster riboflavin transporter homologue revealed reduced levels of riboflavin, downstream metabolites, and electron transport chain complex I activity. This in turn led to abnormal mitochondrial membrane potential, respiratory chain activity and morphology. Riboflavin transporter knockdown in Drosophila also resulted in severely impaired locomotor activity and reduced lifespan, mirroring patient pathology, and these phenotypes could be partially rescued using a novel esterified derivative of riboflavin. Our findings expand the genetic, clinical and neuropathological features of Brown-Vialetto-Van Laere syndrome, implicate mitochondrial dysfunction as a downstream consequence of riboflavin transporter gene defects, and validate riboflavin esters as a potential therapeutic strategy. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.

Leigh syndrome in Drosophila melanogaster: morphological and biochemical characterization of Surf1 post-transcriptional silencing.

PubMed

Da-Rè, Caterina; von Stockum, Sophia; Biscontin, Alberto; Millino, Caterina; Cisotto, Paola; Zordan, Mauro A; Zeviani, Massimo; Bernardi, Paolo; De Pittà, Cristiano; Costa, Rodolfo

2014-10-17

Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LS(Surf1) patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. We established a Drosophila melanogaster model of LS based on the post-transcriptional silencing of CG9943, the Drosophila homolog of SURF1. Knockdown of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knockdown individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R(+) cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. We conclude that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

C1orf163/RESA1 is a novel mitochondrial intermembrane space protein connected to respiratory chain assembly.

PubMed

Kozjak-Pavlovic, Vera; Prell, Florian; Thiede, Bernd; Götz, Monika; Wosiek, Dominik; Ott, Christine; Rudel, Thomas

2014-02-20

Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60kDa and 150kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1). Copyright © 2013 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dwivedi, Nidhi; Mehta, Ashish; Yadav, Abhishek

Arsenicosis, due to contaminated drinking water, is a serious health hazard in terms of morbidity and mortality. Arsenic induced free radicals generated are known to cause cellular apoptosis through mitochondrial driven pathway. In the present study, we investigated the effect of arsenic interactions with various complexes of the electron transport chain and attempted to evaluate if there was any complex preference of arsenic that could trigger apoptosis. We also evaluated if chelation with monoisoamyl dimercaptosuccinic acid (MiADMSA) could reverse these detrimental effects. Our results indicate that arsenic exposure induced free radical generation in rat neuronal cells, which diminished mitochondrial potentialmore » and enzyme activities of all the complexes of the electron transport chain. Moreover, these complexes showed differential responses towards arsenic. These early events along with diminished ATP levels could be co-related with the later events of cytosolic migration of cytochrome c, altered bax/bcl{sub 2} ratio, and increased caspase 3 activity. Although MiADMSA could reverse most of these arsenic-induced altered variables to various extents, DNA damage remained unaffected. Our study for the first time demonstrates the differential effect of arsenic on the complexes leading to deficits in bioenergetics leading to apoptosis in rat brain. However, more in depth studies are warranted for better understanding of arsenic interactions with the mitochondria. -- Research highlights: Black-Right-Pointing-Pointer Arsenic impairs mitochondrial energy metabolism leading to neuronal apoptosis. Black-Right-Pointing-Pointer Arsenic differentially affects mitochondrial complexes, I - III and IV being more sensitive than complex II. Black-Right-Pointing-Pointer Arsenic-induced apoptosis initiates through ROS generation or impaired [Ca{sup 2+}]i homeostasis. Black-Right-Pointing-Pointer MiADMSA reverses arsenic toxicity via intracellular arsenic- chelation, antioxidant potential or both.« less

Genetic and biochemical findings in Chinese children with Leigh syndrome.

PubMed

Ma, Yan-Yan; Wu, Tong-Fei; Liu, Yu-Peng; Wang, Qiao; Song, Jin-Qing; Li, Xi-Yuan; Shi, Xiu-Yu; Zhang, Wei-Na; Zhao, Meng; Hu, Lin-Yan; Yang, Yan-Ling; Zou, Li-Ping

2013-11-01

This study investigated the genetic and enzymological features of Leigh syndrome due to respiratory chain complex deficiency in Chinese patients. The clinical features of 75 patients were recorded. Mitochondrial respiratory chain enzyme activities were determined via spectrophotometry. Mitochondrial gene sequence analysis was performed in 23 patients. Five core pedigrees were investigated via restriction fragment length polymorphism and gene sequencing. Psychomotor retardation (55%), motor regression (20%), weakness (29%), and epilepsy (25%) were the most frequent manifestations. Sixty-four patients (85.3%) had isolated respiratory complex deficiencies: complex I was seen in 28 patients (37.3%); complex II, seven (9.3%); complex III, six (8%); complex IV, ten (13.3%); and complex V, 13 patients (17.3%). Eleven patients (14.7%) had combined complex deficiencies. Mitochondrial DNA mutations were detected in 10 patients. Eight point mutations were found in mitochondrial structural genes: m.4833A>G in ND2, m.10191T>C in ND3, m.12338T>C and m.13513G>A in ND5, m.14502T>C and m.14487T>C in ND6, m.8108A>G in COXII, and m.8993T>G in ATPase6. Three mutations were found in tRNA genes: m.4395A>G in tRNA-Gln, m.10454T>C in tRNA-Arg, and m.5587T>C in tRNA-Ala. One patient and their mother both had the m.12338T>C and m.8993T>C mutations. In conclusion, mitochondrial respiratory chain complex I deficiency and structural gene mutations frequently occur in Chinese Leigh syndrome patients. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mitochondrial functionality in female reproduction.

PubMed

Gąsior, Łukasz; Daszkiewicz, Regina; Ogórek, Mateusz; Polański, Zbigniew

2017-01-04

In most animal species female germ cells are the source of mitochondrial genome for the whole body of individuals. As a source of mitochondrial DNA for future generations the mitochondria in the female germ line undergo dynamic quantitative and qualitative changes. In addition to maintaining the intact template of mitochondrial genome from one generation to another, mitochondrial role in oocytes is much more complex and pleiotropic. The quality of mitochondria determines the ability of meiotic divisions, fertilization ability, and activation after fertilization or sustaining development of a new embryo. The presence of normal number of functional mitochondria is also crucial for proper implantation and pregnancy maintaining. This article addresses issues of mitochondrial role and function in mammalian oocyte and presents new approaches in studies of mitochondrial function in female germ cells.

Mitochondria and ageing: role in heart, skeletal muscle and adipose tissue

PubMed Central

Boengler, Kerstin; Kosiol, Maik; Mayr, Manuel; Schulz, Rainer

2017-01-01

Abstract Age is the most important risk factor for most diseases. Mitochondria play a central role in bioenergetics and metabolism. In addition, several lines of evidence indicate the impact of mitochondria in lifespan determination and ageing. The best‐known hypothesis to explain ageing is the free radical theory, which proposes that cells, organs, and organisms age because they accumulate reactive oxygen species (ROS) damage over time. Mitochondria play a central role as the principle source of intracellular ROS, which are mainly formed at the level of complex I and III of the respiratory chain. Dysfunctional mitochondria generating less ATP have been observed in various aged organs. Mitochondrial dysfunction comprises different features including reduced mitochondrial content, altered mitochondrial morphology, reduced activity of the complexes of the electron transport chain, opening of the mitochondrial permeability transition pore, and increased ROS formation. Furthermore, abnormalities in mitochondrial quality control or defects in mitochondrial dynamics have also been linked to senescence. Among the tissues affected by mitochondrial dysfunction are those with a high‐energy demand and thus high mitochondrial content. Therefore, the present review focuses on the impact of mitochondria in the ageing process of heart and skeletal muscle. In this article, we review different aspects of mitochondrial dysfunction and discuss potential therapeutic strategies to improve mitochondrial function. Finally, novel aspects of adipose tissue biology and their involvement in the ageing process are discussed. PMID:28432755

Early mitochondrial dysfunction in glycolytic muscle, but not oxidative muscle, of the fructose-fed insulin-resistant rat

PubMed Central

Warren, Blair E.; Lou, Phing-How; Lucchinetti, Eliana; Zhang, Liyan; Clanachan, Alexander S.; Affolter, Andreas; Hersberger, Martin; Zaugg, Michael

2014-01-01

Although evidence that type 2 diabetes mellitus (T2DM) is accompanied by mitochondrial dysfunction in skeletal muscle has been accumulating, a causal link between mitochondrial dysfunction and the pathogenesis of the disease remains unclear. Our study focuses on an early stage of the disease to determine whether mitochondrial dysfunction contributes to the development of T2DM. The fructose-fed (FF) rat was used as an animal model of early T2DM. Mitochondrial respiration and acylcarnitine species were measured in oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] muscle. Although FF rats displayed characteristic signs of T2DM, including hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, mitochondrial content was preserved in both muscles from FF rats. The EDL muscle had reduced complex I and complex I and II respiration in the presence of pyruvate but not glutamate. The decrease in pyruvate-supported respiration was due to a decrease in pyruvate dehydrogenase activity. Accumulation of C14:1 and C14:2 acylcarnitine species and a decrease in respiration supported by long-chain acylcarnitines but not acetylcarnitine indicated dysfunctional β-oxidation in the EDL muscle. In contrast, the soleus muscle showed preserved mitochondrial respiration, pyruvate dehydrogenase activity, and increased fatty acid oxidation, as evidenced by overall reduced acylcarnitine levels. Aconitase activity, a sensitive index of reactive oxygen species production in mitochondria, was reduced exclusively in EDL muscle, which showed lower levels of the antioxidant enzymes thioredoxin reductase and glutathione peroxidase. Here, we show that the glycolytic EDL muscle is more prone to an imbalance between energy supply and oxidation caused by insulin resistance than the oxidative soleus muscle. PMID:24425766

Mitochondrial dysfunction and organophosphorus compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karami-Mohajeri, Somayyeh; Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Kerman University of Medical Sciences, Kerman; Abdollahi, Mohammad, E-mail: Mohammad.Abdollahi@UToronto.Ca

2013-07-01

Organophosphorous (OPs) pesticides are the most widely used pesticides in the agriculture and home. However, many acute or chronic poisoning reports about OPs have been published in the recent years. Mitochondria as a site of cellular oxygen consumption and energy production can be a target for OPs poisoning as a non-cholinergic mechanism of toxicity of OPs. In the present review, we have reviewed and criticized all the evidences about the mitochondrial dysfunctions as a mechanism of toxicity of OPs. For this purpose, all biochemical, molecular, and morphological data were retrieved from various studies. Some toxicities of OPs are arisen frommore » dysfunction of mitochondrial oxidative phosphorylation through alteration of complexes I, II, III, IV and V activities and disruption of mitochondrial membrane. Reductions of adenosine triphosphate (ATP) synthesis or induction of its hydrolysis can impair the cellular energy. The OPs disrupt cellular and mitochondrial antioxidant defense, reactive oxygen species generation, and calcium uptake and promote oxidative and genotoxic damage triggering cell death via cytochrome C released from mitochondria and consequent activation of caspases. The mitochondrial dysfunction induced by OPs can be restored by use of antioxidants such as vitamin E and C, alpha-tocopherol, electron donors, and through increasing the cytosolic ATP level. However, to elucidate many aspect of mitochondrial toxicity of Ops, further studies should be performed. - Highlights: • As a non-cholinergic mechanism of toxicity, mitochondria is a target for OPs. • OPs affect action of complexes I, II, III, IV and V in the mitochondria. • OPs reduce mitochondrial ATP. • OPs promote oxidative and genotoxic damage via release of cytochrome C from mitochondria. • OP-induced mitochondrial dysfunction can be restored by increasing the cytosolic ATP.« less

Respiratory chain inhibition: one more feature to propose MPTP intoxication as a Leigh syndrome model.

PubMed

Da Costa, Barbara; Dumon, Elodie; Le Moigno, Laurence; Bodard, Sylvie; Castelnau, Pierre; Letellier, Thierry; Rocher, Christophe

2016-10-01

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice have been widely used to model the loss of dopaminergic neurons. As this treatment leads to basal ganglia degeneration, it was proposed that MPTP mice could be used as a model of Leigh syndrome. However, this mitochondrial pathology is biochemically characterized by a respiratory chain dysfunction. To determine if MPTP can affect in vivo mitochondria function, we measured the activities of mitochondrial respiratory chain complexes in several tissues. Our results show that MPTP affects mainly mitochondrial respiratory chain complex IV, as found in Leigh Syndrome, confirming that acute MPTP intoxicated mice are a good model of Leigh Syndrome.

Rapeseed oil-rich diet alters in vitro menadione and nimesulide hepatic mitochondrial toxicity.

PubMed

Monteiro, João P; Silva, Ana M; Jurado, Amália S; Oliveira, Paulo J

2013-10-01

Diet-induced changes in the lipid composition of mitochondrial membranes have been shown to influence physiological processes. However, the modulation effect of diet on mitochondrially-active drugs has not yet received the deserved attention. Our hypothesis is that modulation of membrane dynamics by diet impacts drug-effects on liver mitochondrial functioning. In a previous work, we have shown that a diet rich in rapeseed oil altered mitochondrial membrane composition and bioenergetics in Wistar rats. In the present work, we investigated the influence of the modified diet on hepatic mitochondrial activity of two drugs, menadione and nimesulide, and FCCP, a classic protonophore, was used for comparison. The results showed that the effects of menadione and nimesulide were less severe on liver mitochondria for rats fed the modified diet than on rats fed the control diet. A specific effect on complex I seemed to be involved in drug-induced mitochondria dysfunction. Liver mitochondria from the modified diet group were more susceptible to nimesulide effects on MPT induction. The present work demonstrates that diet manipulation aimed at modifying mitochondrial membrane properties alters the toxicity of mitochondria active agents. This work highlights that diet may potentiate mitochondrial pharmacologic effects or increase drug-induced liabilities. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat

PubMed Central

Choi, Won-Seok; Kruse, Shane E.; Palmiter, Richard D.; Xia, Zhengui

2008-01-01

Inhibition of mitochondrial complex I is one of the leading hypotheses for dopaminergic neuron death associated with Parkinson's disease (PD). To test this hypothesis genetically, we used a mouse strain lacking functional Ndufs4, a gene encoding a subunit required for complete assembly and function of complex I. Deletion of the Ndufs4 gene abolished complex I activity in midbrain mesencephalic neurons cultured from embryonic day (E) 14 mice, but did not affect the survival of dopaminergic neurons in culture. Although dopaminergic neurons were more sensitive than other neurons in these cultures to cell death induced by rotenone, MPP+, or paraquat treatments, the absence of complex I activity did not protect the dopaminergic neurons, as would be expected if these compounds act by inhibiting complex 1. In fact, the dopaminergic neurons were more sensitive to rotenone. These data suggest that dopaminergic neuron death induced by treatment with rotenone, MPP+, or paraquat is independent of complex I inhibition. PMID:18812510

Light might directly affect retinal ganglion cell mitochondria to potentially influence function.

PubMed

del Olmo-Aguado, Susana; Manso, Alberto G; Osborne, Neville N

2012-01-01

Visible light (360-760 nm) entering the eye impinges on the many ganglion cell mitochondria in the non-myelinated part of their axons. The same light also disrupts isolated mitochondrial function in vitro and kills cells in culture with the blue light component being particularly lethal whereas red light has little effect. Significantly, a defined light insult only affects the survival of fibroblasts in vitro that contain functional mitochondria supporting the view that mitochondrial photosensitizers are influenced by light. Moreover, a blue light insult to cells in culture causes a change in mitochondrial structure and membrane potential and results in a release of cytochrome c. Blue light also causes an alteration in mitochondria located components of the OXPHOS (oxidative phosphorylation system). Complexes III and IV as well as complex V are significantly upregulated whereas complexes I and II are slightly but significantly up- and downregulated, respectively. Also, blue light causes Dexras1 and reactive oxygen species to be upregulated and for mitochondrial located apoptosis-inducing factor to be activated. A blue light detrimental insult to cells in culture does not involve the activation of caspases but is known to be attenuated by necrostatin-1, typical of a death mechanism named necroptosis. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

The function of mitochondrial F(O)F(1) ATP-synthase from the whiteleg shrimp Litopenaeus vannamei muscle during hypoxia.

PubMed

Martinez-Cruz, O; Calderon de la Barca, A M; Uribe-Carvajal, S; Muhlia-Almazan, A

2012-08-01

The effect of hypoxia and re-oxygenation on the mitochondrial complex F(O)F(1)-ATP synthase was investigated in the whiteleg shrimp Litopenaeus vannamei. A 660 kDa protein complex isolated from mitochondria of the shrimp muscle was identified as the ATP synthase complex. After 10h at hypoxia (1.5-2.0 mg oxygen/L), the concentration of L-lactate in plasma increased significantly, but the ATP amount and the concentration of ATPβ protein remained unaffected. Nevertheless, an increase of 70% in the ATPase activity was detected, suggesting that the enzyme may be regulated at a post-translational level. Thus, during hypoxia shrimp are able to maintain ATP amounts probably by using some other energy sources as phosphoarginine when an acute lack of energy occurs. During re-oxygenation, the ATPase activity decreased significantly and the ATP production continued via the electron transport chain and oxidative phosphorylation. The results obtained showed that shrimp faces hypoxia partially by hydrolyzing the ATP through the reaction catalyzed by the mitochondrial ATPase which increases its activity. Copyright © 2012 Elsevier Inc. All rights reserved.

Cocaine- and amphetamine-regulated transcript peptide increases mitochondrial respiratory chain complex II activity and protects against oxygen-glucose deprivation in neurons.

PubMed

Sha, Dujuan; Wang, Luna; Zhang, Jun; Qian, Lai; Li, Qiming; Li, Jin; Qian, Jian; Gu, Shuangshuang; Han, Ling; Xu, Peng; Xu, Yun

2014-09-25

The mechanisms of ischemic stroke, a main cause of disability and death, are complicated. Ischemic stroke results from the interaction of various factors including oxidative stress, a key pathological mechanism that plays an important role during the acute stage of ischemic brain injury. This study demonstrated that cocaine- and amphetamine-regulated transcript (CART) peptide, specifically CART55-102, increased the survival rate, but decreased the mortality of neurons exposed to oxygen-glucose deprivation (OGD), in a dose-dependent manner. The above-mentioned effects of CART55-102 were most significant at 0.4nM. These results indicated that CART55-102 suppressed neurotoxicity and enhanced neuronal survival after oxygen-glucose deprivation. CART55-102 (0.4nM) significantly diminished reactive oxygen species levels and markedly increased the activity of mitochondrial respiratory chain complex II in oxygen-glucose deprived neurons. In summary, CART55-102 suppressed oxidative stress in oxygen-glucose deprived neurons, possibly through elevating the activity of mitochondrial respiratory chain complex II. This result provides evidence for the development of CART55-102 as an antioxidant drug. Copyright © 2014 Elsevier B.V. All rights reserved.

Uncoupling Protein 2 and Metabolic Diseases

PubMed Central

Sreedhar, Annapoorna; Zhao, Yunfeng

2017-01-01

Mitochondria are fascinating organelles involved in various cellular-metabolic activities that are integral for mammalian development. Although they perform diverse, yet interconnected functions, mitochondria are remarkably regulated by complex signaling networks. Therefore, it is not surprising that mitochondrial dysfunction is involved in plethora of diseases, including neurodegenerative and metabolic disorders. One of the many factors that lead to mitochondrial-associated metabolic diseases is the uncoupling protein-2, a family of mitochondrial anion proteins present in the inner mitochondrial membrane. Since their discovery, uncoupling proteins have attracted considerable attention due to their involvement in mitochondrial-mediated oxidative stress and energy metabolism. This review attempts to provide a summary of recent developments in the field of uncoupling protein 2 relating to mitochondrial associated metabolic diseases. PMID:28351676

Glutaredoxin 2 prevents H(2)O(2)-induced cell apoptosis by protecting complex I activity in the mitochondria.

PubMed

Wu, Hongli; Xing, Kuiyi; Lou, Marjorie F

2010-10-01

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200muM hydrogen peroxide (H(2)O(2)) for 24h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H(2)O(2)-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H(2)O(2) treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H(2)O(2)-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H(2)O(2)-induced apoptosis is likely associated with its ability to preserve complex I. Published by Elsevier B.V.

Loss of Intralipid®- but Not Sevoflurane-Mediated Cardioprotection in Early Type-2 Diabetic Hearts of Fructose-Fed Rats: Importance of ROS Signaling

PubMed Central

Zhang, Liyan; Affolter, Andreas; Gandhi, Manoj; Hersberger, Martin; Warren, Blair E.; Lemieux, Hélène; Sobhi, Hany F.; Clanachan, Alexander S.; Zaugg, Michael

2014-01-01

Background Insulin resistance and early type-2 diabetes are highly prevalent. However, it is unknown whether Intralipid® and sevoflurane protect the early diabetic heart against ischemia-reperfusion injury. Methods Early type-2 diabetic hearts from Sprague-Dawley rats fed for 6 weeks with fructose were exposed to 15 min of ischemia and 30 min of reperfusion. Intralipid® (1%) was administered at the onset of reperfusion. Peri-ischemic sevoflurane (2 vol.-%) served as alternative protection strategy. Recovery of left ventricular function was recorded and the activation of Akt and ERK 1/2 was monitored. Mitochondrial function was assessed by high-resolution respirometry and mitochondrial ROS production was measured by Amplex Red and aconitase activity assays. Acylcarnitine tissue content was measured and concentration-response curves of complex IV inhibition by palmitoylcarnitine were obtained. Results Intralipid® did not exert protection in early diabetic hearts, while sevoflurane improved functional recovery. Sevoflurane protection was abolished by concomitant administration of the ROS scavenger N-2-mercaptopropionyl glycine. Sevoflurane, but not Intralipid® produced protective ROS during reperfusion, which activated Akt. Intralipid® failed to inhibit respiratory complex IV, while sevoflurane inhibited complex I. Early diabetic hearts exhibited reduced carnitine-palmitoyl-transferase-1 activity, but palmitoylcarnitine could not rescue protection and enhance postischemic functional recovery. Cardiac mitochondria from early diabetic rats exhibited an increased content of subunit IV-2 of respiratory complex IV and of uncoupling protein-3. Conclusions Early type-2 diabetic hearts lose complex IV-mediated protection by Intralipid® potentially due to a switch in complex IV subunit expression and increased mitochondrial uncoupling, but are amenable to complex I-mediated sevoflurane protection. PMID:25127027

Roles of Neuroglobin Binding to Mitochondrial Complex III Subunit Cytochrome c1 in Oxygen-Glucose Deprivation-Induced Neurotoxicity in Primary Neurons.

PubMed

Yu, Zhanyang; Zhang, Yu; Liu, Ning; Yuan, Jing; Lin, Li; Zhuge, Qichuan; Xiao, Jian; Wang, Xiaoying

2016-07-01

Neuroglobin (Ngb) is a tissue globin specifically expressed in brain neurons. Recent studies by our laboratory and others have demonstrated that Ngb is protective against stroke and related neurological disorders, but the mechanisms remain poorly understood. We previously identified cytochrome c1 (Cyc1) as an Ngb-interacting molecule by yeast two-hybrid screening. Cyc1 is a subunit of mitochondria complex III, which is a component of mitochondrial respiratory chain and a major source of reactive oxygen species (ROS) production under both physiological and pathological conditions. In this study, we for the first time defined Ngb-Cyc1 binding, and investigated its roles in oxygen-glucose deprivation (OGD)/reoxygenation-induced neurotoxicity and ROS production in primary neurons. Immunocytochemistry and co-immunoprecipitation validated Ngb-Cyc1 binding, which was significantly increased by OGD and Ngb overexpression. We found 4 h OGD with/without 4 h reoxygenation significantly increased complex III activity, but this activity elevation was significantly attenuated in three groups of neurons: Ngb overexpression, specific complex III inhibitor stigmatellin, or stigmatellin plus Ngb overexpression, whereas there was no significant differences between these three groups, suggesting Ngb-Cyc1 binding may function in suppressing OGD-mediated complex III activity elevation. Importantly, these three groups of neurons also showed significant decreases in OGD-induced superoxide anion generation and neurotoxicity. These results suggest that Ngb can bind to mitochondrial complex III subunit Cyc1, leading to suppression of OGD-mediated complex III activity and subsequent ROS production elevation, and eventually reduction of OGD-induced neurotoxicity. This molecular signaling cascade may be at least part of the mechanisms of Ngb neuroprotection against OGD-induced neurotoxicity.

Why to compare absolute numbers of mitochondria.

PubMed

Schmitt, Sabine; Schulz, Sabine; Schropp, Eva-Maria; Eberhagen, Carola; Simmons, Alisha; Beisker, Wolfgang; Aichler, Michaela; Zischka, Hans

2014-11-01

Prompted by pronounced structural differences between rat liver and rat hepatocellular carcinoma mitochondria, we suspected these mitochondrial populations to differ massively in their molecular composition. Aiming to reveal these mitochondrial differences, we came across the issue on how to normalize such comparisons and decided to focus on the absolute number of mitochondria. To this end, fluorescently stained mitochondria were quantified by flow cytometry. For rat liver mitochondria, this approach resulted in mitochondrial protein contents comparable to earlier reports using alternative methods. We determined similar protein contents for rat liver, heart and kidney mitochondria. In contrast, however, lower protein contents were determined for rat brain mitochondria and for mitochondria from the rat hepatocellular carcinoma cell line McA 7777. This result challenges mitochondrial comparisons that rely on equal protein amounts as a typical normalization method. Exemplarily, we therefore compared the activity and susceptibility toward inhibition of complex II of rat liver and hepatocellular carcinoma mitochondria and obtained significant discrepancies by either normalizing to protein amount or to absolute mitochondrial number. Importantly, the latter normalization, in contrast to the former, demonstrated a lower complex II activity and higher susceptibility toward inhibition in hepatocellular carcinoma mitochondria compared to liver mitochondria. These findings demonstrate that solely normalizing to protein amount may obscure essential molecular differences between mitochondrial populations. Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Ubiquinol-10 supplementation activates mitochondria functions to decelerate senescence in senescence-accelerated mice.

PubMed

Tian, Geng; Sawashita, Jinko; Kubo, Hiroshi; Nishio, Shin-ya; Hashimoto, Shigenari; Suzuki, Nobuyoshi; Yoshimura, Hidekane; Tsuruoka, Mineko; Wang, Yaoyong; Liu, Yingye; Luo, Hongming; Xu, Zhe; Mori, Masayuki; Kitano, Mitsuaki; Hosoe, Kazunori; Takeda, Toshio; Usami, Shin-ichi; Higuchi, Keiichi

2014-06-01

The present study was conducted to define the relationship between the anti-aging effect of ubiquinol-10 supplementation and mitochondrial activation in senescence-accelerated mouse prone 1 (SAMP1) mice. Here, we report that dietary supplementation with ubiquinol-10 prevents age-related decreases in the expression of sirtuin gene family members, which results in the activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a major factor that controls mitochondrial biogenesis and respiration, as well as superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2), which are major mitochondrial antioxidant enzymes. Ubiquinol-10 supplementation can also increase mitochondrial complex I activity and decrease levels of oxidative stress markers, including protein carbonyls, apurinic/apyrimidinic sites, malondialdehydes, and increase the reduced glutathione/oxidized glutathione ratio. Furthermore, ubiquinol-10 may activate Sirt1 and PGC-1α by increasing cyclic adenosine monophosphate (cAMP) levels that, in turn, activate cAMP response element-binding protein (CREB) and AMP-activated protein kinase (AMPK). These results show that ubiquinol-10 may enhance mitochondrial activity by increasing levels of SIRT1, PGC-1α, and SIRT3 that slow the rate of age-related hearing loss and protect against the progression of aging and symptoms of age-related diseases.

Mitochondrial mode of action of a thymidine-based cisplatin analogue breaks resistance in cancer cells.

PubMed

Onambele, Liliane A; Koth, Daniel; Czaplewska, Justyna A; Schubert, Ulrich S; Görls, Helmar; Yano, Shigenobu; Obata, Makoto; Gottschaldt, Michael; Prokop, Aram

2010-12-27

Cisplatin analogue complexes with platinum(II) and palladium(II) starting from 3',5'-diamino-3',5'-dideoxy-thymidines were synthesized, both with the D-erythro- and D-threo configurations. Complexes of the general formula [MCl(2)L] were obtained and characterized. NMR spectroscopic measurements and single crystal X-ray structure analysis showed that the metal centers are coordinated to the ligands by the amino groups in 3'- and 5'-positions and not through the thymine moiety. All ligands and complexes showed no significant in vitro activities except thymiplatin (cis-dichloro(3',5'-diamino-3',5'-dideoxy-D-threo-thymidine)platinum(II)). Detailed in vitro studies on the apoptosis pathway in lymphoma (BJAB), leukemia (NALM-6), and melanoma cells (Mel-HO) as well as on transfected or resistant cell lines were carried out. Thymiplatin significantly induced an apoptotic response, which was found to be associated with the loss of mitochondrial membrane potential and with caspase activation. The activity was shown to be independent of Fas-associated protein with death domain (FADD), but dependent on Bcl-2 expression. As a consequence, for thymiplatin a mitochondrial mode of action could be assigned. Moreover, the compound showed activity in cells resistant to common drugs, such as daunorubicin and vincristin, and showed synergistic effects with doxorubicin, vincristin, cytarabin, and daunorubicin.

Carbon monoxide shifts energetic metabolism from glycolysis to oxidative phosphorylation in endothelial cells.

PubMed

Kaczara, Patrycja; Motterlini, Roberto; Kus, Kamil; Zakrzewska, Agnieszka; Abramov, Andrey Y; Chlopicki, Stefan

2016-10-01

Carbon monoxide (CO) modulates mitochondrial respiration, but the mechanisms involved are not completely understood. The aim of the present study was to investigate the acute effects of CO on bioenergetics and metabolism in intact EA.hy926 endothelial cells using live cell imaging techniques. Our findings indicate that CORM-401, a compound that liberates CO, reduces ATP production from glycolysis, and induces a mild mitochondrial depolarization. In addition, CO from CORM-401 increases mitochondrial calcium and activates complexes I and II. The subsequent increase in mitochondrial respiration leads to ATP production through oxidative phosphorylation. Thus, our results show that nonactivated endothelial cells rely primarily on glycolysis, but in the presence of CO, mitochondrial Ca 2+ increases and activates respiration that shifts the metabolism of endothelial cells from glycolysis- to oxidative phosphorylation-dependent ATP production. © 2016 Federation of European Biochemical Societies.

No evidence of a role for mitochondrial complex I in Helicobacter pylori pathogenesis.

PubMed

Ng, Garrett Z; Ke, Bi-Xia; Laskowski, Adrienne; Thorburn, David R; Sutton, Philip

2017-06-01

Complex I is the first enzyme complex in the mitochondrial respiratory chain, responsible for generating a large fraction of energy during oxidative phosphorylation. Recently, it has been identified that complex I deficiency can result in increased inflammation due to the generation of reactive oxygen species by innate immune cells. As a reduction in complex I activity has been demonstrated in human stomachs with atrophic gastritis, we investigated whether complex I deficiency could influence Helicobacter pylori pathogenesis. Ndufs6 gt/gt mice have a partial complex I deficiency. Complex I activity was quantified in the stomachs and immune cells of Ndufs6 gt/gt mice by spectrophotometric assays. Ndufs6 gt/gt mice were infected with H. pylori and bacterial colonization assessed by colony-forming assay, gastritis assessed histologically, and H. pylori -specific humoral response quantified by ELISA. The immune cells and stomachs of Ndufs6 gt/gt mice were found to have significantly decreased complex I activity, validating the model for assessing the effects of complex I deficiency in H. pylori infection. However, there was no observable effect of complex I deficiency on either H. pylori colonization, the resulting gastritis, or the humoral response. Although complex I activity is described to suppress innate immune responses and is decreased during atrophic gastritis in humans, our data suggest it does not affect H. pylori pathogenesis. © 2017 John Wiley & Sons Ltd.

Alterations of motor performance and brain cortex mitochondrial function during ethanol hangover.

PubMed

Bustamante, Juanita; Karadayian, Analia G; Lores-Arnaiz, Silvia; Cutrera, Rodolfo A

2012-08-01

Ethanol has been known to affect various behavioral parameters in experimental animals, even several hours after ethanol (EtOH) is absent from blood circulation, in the period known as hangover. The aim of this study was to assess the effects of acute ethanol hangover on motor performance in association with the brain cortex energetic metabolism. Evaluation of motor performance and brain cortex mitochondrial function during alcohol hangover was performed in mice 6 hours after a high ethanol dose (hangover onset). Animals were injected i.p. either with saline (control group) or with ethanol (3.8 g/kg BW) (hangover group). Ethanol hangover group showed a bad motor performance compared with control animals (p < .05). Oxygen uptake in brain cortex mitochondria from hangover animals showed a 34% decrease in the respiratory control rate as compared with the control group. Mitochondrial complex activities were decreased being the complex I-III the less affected by the hangover condition; complex II-III was markedly decreased by ethanol hangover showing 50% less activity than controls. Complex IV was 42% decreased as compared with control animals. Hydrogen peroxide production was 51% increased in brain cortex mitochondria from the hangover group, as compared with the control animals. Quantification of the mitochondrial transmembrane potential indicated that ethanol injected animals presented 17% less ability to maintain the polarized condition as compared with controls. These results indicate that a clear decrease in proton motive force occurs in brain cortex mitochondria during hangover conditions. We can conclude that a decreased motor performance observed in the hangover group of animals could be associated with brain cortex mitochondrial dysfunction and the resulting impairment of its energetic metabolism. Copyright © 2012 Elsevier Inc. All rights reserved.

MLF1 is a proapoptotic antagonist of HOP complex-mediated survival.

PubMed

Sun, Yi; Chao, Jyh-Rong; Xu, Wu; Pourpak, Alan; Boyd, Kelli; Moshiach, Simon; Qi, Guo-Yan; Fu, Amina; Shao, Hua-Rong; Pounds, Stanley; Morris, Stephan W

2017-04-01

In the HAX1/HtrA2-OMI/PARL (HOP) mitochondrial protein complex, anti-apoptotic signals are generated by cleavage and activation of the serine protease HtrA2/OMI by the rhomboid protease PARL upon recruitment of both proteases to inner mitochondrial membrane protein HAX1 (HS1-associated protein X-1). Here we report the negative regulation of the HOP complex by human leukemia-associated myeloid leukemia factor 1 (MLF1). We demonstrate that MLF1 physically and functionally associates with HAX1 and HtrA2. Increased interaction of MLF1 with HAX1 and HtrA2 displaces HtrA2 from the HOP complex and inhibits HtrA2 cleavage and activation, resulting in the apoptotic cell death. Conversely, over-expressed HAX1 neutralizes MLF1's effect and inhibits MLF1-induced apoptosis. Importantly, Mlf1 deletion reverses B- and T-cell lymphopenia and significantly ameliorates the progressive striatal and cerebellar neurodegeneration observed in Hax1 -/- mice, with a doubling of the lifespan of Mlf1 -/- /Hax1 -/- animals compared to Hax1 -/- animals. Collectively, these data indicate that MLF1 serves as a proapoptotic antagonist that interacts with the HOP mitochondrial complex to modulate cell survival. Copyright © 2017 Elsevier B.V. All rights reserved.

Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production.

PubMed

Nogueira, Natália P; Saraiva, Francis M S; Oliveira, Matheus P; Mendonça, Ana Paula M; Inacio, Job D F; Almeida-Amaral, Elmo E; Menna-Barreto, Rubem F; Laranja, Gustavo A T; Torres, Eduardo J Lopes; Oliveira, Marcus F; Paes, Marcia C

2017-07-01

Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector-parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria-targeted antioxidant ((2-(2,2,6,6 Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme-induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously-indicating the hyperpolarization and increase of potential of the mitochondrial membrane (ΔΨm). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme-treated epimastigotes decreased their oxygen consumption, and increased the complex II-III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme-induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation. Copyright © 2017. Published by Elsevier Inc.

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

PubMed Central

Moreno-Beltrán, Blas; Guerra-Castellano, Alejandra; Del Conte, Rebecca; García-Mauriño, Sofía M.; Díaz-Moreno, Sofía; González-Arzola, Katiuska; Santos-Ocaña, Carlos; Velázquez-Campoy, Adrián; De la Rosa, Miguel A.; Turano, Paola; Díaz-Moreno, Irene

2017-01-01

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects. PMID:28348229

ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells.

PubMed

Arduino, Daniela M; Esteves, A Raquel; Domingues, A Filipa; Pereira, Claudia M F; Cardoso, Sandra M; Oliveira, Catarina R

2009-11-30

Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

Targeting mitochondrial respiration as a therapeutic strategy for cervical cancer.

PubMed

Tian, Shenglan; Chen, Heng; Tan, Wei

2018-05-23

Targeting mitochondrial respiration has been documented as an effective therapeutic strategy in cancer. However, the impact of mitochondrial respiration inhibition on cervical cancer cells are not well elucidated. Using a panel of cervical cancer cell lines, we show that an existing drug atovaquone is active against the cervical cancer cells with high profiling of mitochondrial biogenesis. Atovaquone inhibited proliferation and induced apoptosis with varying efficacy among cervical cancer cell lines regardless of HPV infection, cellular origin and their sensitivity to paclitaxel. We further demonstrated that atovaquone acts on cervical cancer cells via inhibiting mitochondrial respiration. In particular, atovaquone specifically inhibited mitochondrial complex III but not I, II or IV activity, leading to respiration inhibition and energy crisis. Importantly, we found that the different sensitivity of cervical cancer cell lines to atovaquone were due to their differential level of mitochondrial biogenesis and dependency to mitochondrial respiration. In addition, we demonstrated that the in vitro observations were translatable to in vivo cervical cancer xenograft mouse model. Our findings suggest that the mitochondrial biogenesis varies among patients with cervical cancer. Our work also suggests that atovaquone is a useful addition to cervical cancer treatment, particularly to those with high dependency on mitochondrial respiration. Copyright © 2018 Elsevier Inc. All rights reserved.

Melatonin and human mitochondrial diseases

PubMed Central

Sharafati-Chaleshtori, Reza; Shirzad, Hedayatollah; Rafieian-Kopaei, Mahmoud; Soltani, Amin

2017-01-01

Mitochondrial dysfunction is one of the main causative factors in a wide variety of complications such as neurodegenerative disorders, ischemia/reperfusion, aging process, and septic shock. Decrease in respiratory complex activity, increase in free radical production, increase in mitochondrial synthase activity, increase in nitric oxide production, and impair in electron transport system and/or mitochondrial permeability are considered as the main factors responsible for mitochondrial dysfunction. Melatonin, the pineal gland hormone, is selectively taken up by mitochondria and acts as a powerful antioxidant, regulating the mitochondrial bioenergetic function. Melatonin increases the permeability of membranes and is the stimulator of antioxidant enzymes including superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase. It also acts as an inhibitor of lipoxygenase. Melatonin can cause resistance to oxidation damage by fixing the microsomal membranes. Melatonin has been shown to retard aging and inhibit neurodegenerative disorders, ischemia/reperfusion, septic shock, diabetes, cancer, and other complications related to oxidative stress. The purpose of the current study, other than introducing melatonin, was to present the recent findings on clinical effects in diseases related to mitochondrial dysfunction including diabetes, cancer, gastrointestinal diseases, and diseases related to brain function. PMID:28400824

Functional and composition differences between mitochondrial complex II in Arabidopsis and rice are correlated with the complex genetic history of the enzyme.

PubMed

Huang, Shaobai; Taylor, Nicolas L; Narsai, Reena; Eubel, Holger; Whelan, James; Millar, A Harvey

2010-02-01

Complex II plays a central role in mitochondrial metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. However, the composition and function of the plant enzyme has been elusive and differs from the well-characterised enzymes in mammals and bacteria. Herewith, we demonstrate that mitochondrial Complex II from Arabidopsis and rice differ significantly in several aspects: (1) Stability-Rice complex II in contrast to Arabidopsis is not stable when resolved by native electrophoresis and activity staining. (2) Composition-Arabidopsis complex II contains 8 subunits, only 7 of which have homologs in the rice genome. SDH 1 and 2 subunits display high levels of amino acid identity between two species, while the remainder of the subunits are not well conserved at a sequence level, indicating significant divergence. (3) Gene expression-the pairs of orthologous SDH1 and SDH2 subunits were universally expressed in both Arabidopsis and rice. The very divergent genes for SDH3 and SDH4 were co-expressed in both species, consistent with their functional co-ordination to form the membrane anchor. The plant-specific SDH5, 6 and 7 subunits with unknown functions appeared to be differentially expressed in both species. (4) Biochemical regulation -succinate-dependent O(2) consumption and SDH activity of isolated Arabidopsis mitochondria were substantially stimulated by ATP, but a much more minor effect of ATP was observed for the rice enzyme. The ATP activation of succinate-dependent reduction of DCPIP in frozen-thawed and digitonin-solubilised mitochondrial samples, and with or without the uncoupler CCCP, indicate that the differential ATP effect on SDH is not via the protonmotive force but likely due to an allosteric effect on the plant SDH enzyme itself, in contrast to the enzyme in other organisms.

Analysis of regional brain mitochondrial bioenergetics and susceptibility to mitochondrial inhibition utilizing a microplate based system

PubMed Central

Sauerbeck, Andrew; Pandya, Jignesh; Singh, Indrapal; Bittman, Kevin; Readnower, Ryan; Bing, Guoying; Sullivan, Patrick

2012-01-01

The analysis of mitochondrial bioenergetic function typically has required 50–100 μg of protein per sample and at least 15 min per run when utilizing a Clark-type oxygen electrode. In the present work we describe a method utilizing the Seahorse Biosciences XF24 Flux Analyzer for measuring mitochondrial oxygen consumption simultaneously from multiple samples and utilizing only 5 μg of protein per sample. Utilizing this method we have investigated whether regionally based differences exist in mitochondria isolated from the cortex, striatum, hippocampus, and cerebellum. Analysis of basal mitochondrial bioenergetics revealed that minimal differences exist between the cortex, striatum, and hippocampus. However, the cerebellum exhibited significantly slower basal rates of Complex I and Complex II dependent oxygen consumption (p < 0.05). Mitochondrial inhibitors affected enzyme activity proportionally across all samples tested and only small differences existed in the effect of inhibitors on oxygen consumption. Investigation of the effect of rotenone administration on Complex I dependent oxygen consumption revealed that exposure to 10 pM rotenone led to a clear time dependent decrease in oxygen consumption beginning 12 min after administration (p < 0.05). These studies show that the utilization of this microplate based method for analysis of mitochondrial bioenergetics is effective at quantifying oxygen consumption simultaneously from multiple samples. Additionally, these studies indicate that minimal regional differences exist in mitochondria isolated from the cortex, striatum, or hippocampus. Furthermore, utilization of the mitochondrial inhibitors suggests that previous work indicating regionally specific deficits following systemic mitochondrial toxin exposure may not be the result of differences in the individual mitochondria from the affected regions. PMID:21402103

Measuring Interference of Drug-Like Molecules with the Respiratory Chain: Toward the Early Identification of Mitochondrial Uncouplers in Lead Finding

PubMed Central

Matter, Hans; Diekert, Kerstin; Dörner, Wolfgang; Dröse, Stefan; Licher, Thomas

2013-01-01

Abstract The electron transport chain (ETC) couples electron transfer between donors and acceptors with proton transport across the inner mitochondrial membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate (ATP). Proton transfer is based on the activity of complex I–V proteins in the ETC. The overall electrical activity of these proteins can be measured by proton transfer using Solid Supported Membrane technology. We tested the activity of complexes I, III, and V in a combined assay, called oxidative phosphorylation assay (oxphos assay), by activating each complex with the corresponding substrate. The oxphos assay was used to test in-house substances from different projects and several drugs currently available on the market that have reported effects on mitochondrial functions. The resulting data were compared to the influence of the respective compounds on mitochondria as determined by oxygen consumption and to data generated with an ATP depletion assay. The comparison shows that the oxidative phosphorylation assay provides both a rapid approach for detecting interaction of compounds with respiratory chain proteins and information on their mode of interaction. Therefore, the oxphos assay is a useful tool to support structure activity relationship studies by allowing early identification of mitotoxicity and for analyzing the outcome of phenotypic screens that are susceptible to the generation of mitotoxicity-related artifacts. PMID:23992120

Polypeptide Translocation Through the Mitochondrial TOM Channel: Temperature-Dependent Rates at the Single-Molecule Level.

PubMed

Mahendran, Kozhinjampara R; Lamichhane, Usha; Romero-Ruiz, Mercedes; Nussberger, Stephan; Winterhalter, Mathias

2013-01-03

The TOM protein complex facilitates the transfer of nearly all mitochondrial preproteins across outer mitochondrial membranes. Here we characterized the effect of temperature on facilitated translocation of a mitochondrial presequence peptide pF1β. Ion current fluctuations analysis through single TOM channels revealed thermodynamic and kinetic parameters of substrate binding and allowed determining the energy profile of peptide translocation. The activation energy for the on-rate and off-rate of the presequence peptide into the TOM complex was symmetric with respect to the electric field and estimated to be about 15 and 22 kT per peptide. These values are above that expected for free diffusion of ions in water (6 kT) and reflect the stronger interaction in the channel. Both values are in the range for typical enzyme kinetics and suggest one process without involving large conformational changes within the channel protein.

Mitochondrial mechanisms of neural cell death and neuroprotective interventions in Parkinson's disease.

PubMed

Fiskum, Gary; Starkov, Anatoly; Polster, Brian M; Chinopoulos, Christos

2003-06-01

Mitochondrial dysfunction, due to either environmental or genetic factors, can result in excessive production of reactive oxygen species, triggering the apoptotic death of dopaminergic cells in Parkinson's disease. Mitochondrial free radical production is promoted by the inhibition of electron transport at any point distal to the sites of superoxide production. Neurotoxins that induce parkinsonian neuropathology, such as MPP(+) and rotenone, stimulate superoxide production at complex I of the electron transport chain and also stimulate free radical production at proximal redox sites including mitochondrial matrix dehydrogenases. The oxidative stress caused by elevated mitochondrial production of reactive oxygen species promotes the expression and (or) intracellular distribution of the proapoptotic protein Bax to the mitochondrial outer membrane. Interactions between Bax and BH3 death domain proteins such as tBid result in Bax membrane integration, oligomerization, and permeabilization of the outer membrane to intermembrane proteins such as cytochrome c. Once released into the cytosol, cytochrome c together with other proteins activates the caspase cascade of protease activities that mediate the biochemical and morphological alterations characteristic of apoptosis. In addition, loss of mitochondrial cytochrome c stimulates mitochondrial free radical production, further promoting cell death pathways. Excessive mitochondrial Ca(2+) accumulation can also release cytochrome c and promote superoxide production through a mechanism distinctly different from that of Bax. Ca(2+) activates a mitochondrial inner membrane permeability transition causing osmotic swelling, rupture of the outer membrane, and complete loss of mitochondrial structural and functional integrity. While amphiphilic cations, such as dibucaine and propranolol, inhibit Bax-mediated cytochrome c release, transient receptor potential channel inhibitors inhibit mitochondrial swelling and cytochrome c release induced by the inner membrane permeability transition. These advances in the knowledge of mitochondrial cell death mechanisms and their inhibitors may lead to neuroprotective interventions applicable to Parkinsons's disease.

Impaired Mitochondrial Transcription Termination Disrupts the Stromal Redox Poise in Chlamydomonas1[OPEN

PubMed Central

Uhmeyer, Andreas

2017-01-01

In photosynthetic eukaryotes, the metabolite exchange between chloroplast and mitochondria ensures efficient photosynthesis under saturating light conditions. The Chlamydomonas reinhardtii mutant stm6 is devoid of the mitochondrial transcription termination factor MOC1 and aberrantly expresses the mitochondrial genome, resulting in enhanced photosynthetic hydrogen production and diminished light tolerance. We analyzed the modulation of mitochondrial and chlororespiration during the acclimation of stm6 and the MOC1-complemented strain to excess light. Although light stress stimulated mitochondrial respiration via the energy-conserving cytochrome c pathway in both strains, the mutant was unable to fine-tune the expression and activity of oxidative phosphorylation complex I in excess light, which was accompanied by an increased mitochondrial respiration via the alternative oxidase pathway. Furthermore, stm6 failed to fully activate chlororespiration and cyclic electron flow due to a more oxidized state of the chloroplast stroma, which is caused by an increased mitochondrial electron sink capacity. Increased susceptibility to photoinhibition of PSII in stm6 demonstrates that the MOC1-dependent modulation of mitochondrial respiration helps control the stromal redox poise as a crucial part of high-light acclimation in C. reinhardtii. PMID:28500267

Metformin selectively targets redox control of complex I energy transduction.

PubMed

Cameron, Amy R; Logie, Lisa; Patel, Kashyap; Erhardt, Stefan; Bacon, Sandra; Middleton, Paul; Harthill, Jean; Forteath, Calum; Coats, Josh T; Kerr, Calum; Curry, Heather; Stewart, Derek; Sakamoto, Kei; Repiščák, Peter; Paterson, Martin J; Hassinen, Ilmo; McDougall, Gordon; Rena, Graham

2018-04-01

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD + couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD + couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

NDUFA4L2 protects against ischaemia/reperfusion-induced cardiomyocyte apoptosis and mitochondrial dysfunction by inhibiting complex I.

PubMed

Li, Jianhua; Bai, Caiyan; Guo, Junxia; Liang, Wanqian; Long, Jingning

2017-07-01

Myocardial ischaemia/reperfusion (I/R) injury may cause the apoptosis of cardiomyocytes as well as mitochondrial dysfunction. The aims of the present study were to investigate whether NADH dehydrogenase 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2) on myocardial ischaemia-reperfusion (I/R) injury and the underlying molecular mechanism. The hypoxia-reperfusion (H/R) model was established in vitro using H9c2 cells to simulate I/R injury. NDUFA4L2 and complex I expression levels were detected using RT-PCR and western blot. The apoptosis of H9c2 cells was evaluated by flow cytometry and the expression of Bax and Bcl-2 was detected by western blot. The mitochondrial function was assessed by ATP concentration, mPTP opening and cytochrome c (cyto C) expression. Our data indicated that NDUFA4L2 expression was significantly down-regulated in myocardial H/R injury. Overexpression of NDUFA4L2 led to a dramatic prevention of H/R-induced apoptosis accompanied by a decrease in the expression of Bax and an increase in the expression of Bcl-2. Meanwhile, augmentation of NDUFA4L2 dramatically prevented mitochondrial dysfunction caused by H/R as reflecting in the increased ATP concentration, delayed mPTP opening, as well as down-regulated cyto C expression. Moreover, complex I activation was heightened and negatively regulated by NDUFA4L2. Silencing complex I conspicuously attenuated cell apoptosis and mitochondrial dysfunction. Taken together, our findings demonstrated that NDUFA4L2 protects against H/R injury by preventing myocardium apoptosis and mitochondrial dysfunction via the complex I, and may be a potential therapeutic approach for attenuating myocardial I/R injury. © 2017 John Wiley & Sons Australia, Ltd.

Induction of apoptosis in cells expressing exogenous Hippi, a molecular partner of huntingtin-interacting protein Hip1.

PubMed

Majumder, Pritha; Chattopadhyay, Biswanath; Mazumder, Arindam; Das, Pradeep; Bhattacharyya, Nitai P

2006-05-01

To decipher the pathway of apoptosis induction downstream to caspase-8 activation by exogenous expression of Hippi, an interactor of huntingtin-interacting protein Hip1, we studied apoptosis in HeLa and Neuro2A cells expressing GFP-tagged Hippi. Nuclear fragmentation, caspase-1, caspase-8, caspase-9/caspase-6 and caspase-3 activation were increased significantly in Hippi expressing cells. Cleavage of Bid, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria were also increased in GFP-Hippi expressing cells. It was observed that caspase-1 and caspase-8 activation was earlier than caspase-3 activation and nuclear fragmentation. Expression of caspase-1, caspase-3 and caspase-7 was increased while anti-apoptotic gene Bcl-2 and mitochondrial genes ND1 and ND4 were reduced in Hippi expressing cells. Besides, the expression SDHA and SDHB, nuclear genes, subunits of mitochondrial complex II were decreased in GFP-Hippi expressing cells. Taken together, we concluded that Hippi expression induced apoptosis by releasing AIF and cytochrome c from mitochondria, activation of caspase-1 and caspase-3, and altering the expression of apoptotic genes and genes involved in mitochondrial complex I and II.

Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

PubMed

Park, Song-Young; Gifford, Jayson R; Andtbacka, Robert H I; Trinity, Joel D; Hyngstrom, John R; Garten, Ryan S; Diakos, Nikolaos A; Ives, Stephen J; Dela, Flemming; Larsen, Steen; Drakos, Stavros; Richardson, Russell S

2014-08-01

Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s(-1)·mg(-1), P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g(-1)·min(-1), P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s(-1)·mg(-1), P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production.

Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart

PubMed Central

Ashmore, Tom; Fernandez, Bernadette O; Branco-Price, Cristina; West, James A; Cowburn, Andrew S; Heather, Lisa C; Griffin, Julian L; Johnson, Randall S; Feelisch, Martin; Murray, Andrew J

2014-01-01

Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l−1 NaCl (as control) or 0.7 mmol l−1 NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics. PMID:25172947

ATAD3 proteins: brokers of a mitochondria-endoplasmic reticulum connection in mammalian cells.

PubMed

Baudier, Jacques

2018-05-01

In yeast, a sequence of physical and genetic interactions termed the endoplasmic reticulum (ER)-mitochondria organizing network (ERMIONE) controls mitochondria-ER interactions and mitochondrial biogenesis. Several functions that characterize ERMIONE complexes are conserved in mammalian cells, suggesting that a similar tethering complex must exist in metazoans. Recent studies have identified a new family of nuclear-encoded ATPases associated with diverse cellular activities (AAA+-ATPase) mitochondrial membrane proteins specific to multicellular eukaryotes, called the ATPase family AAA domain-containing protein 3 (ATAD3) proteins (ATAD3A and ATAD3B). These proteins are crucial for normal mitochondrial-ER interactions and lie at the heart of processes underlying mitochondrial biogenesis. ATAD3A orthologues have been studied in flies, worms, and mammals, highlighting the widespread importance of this gene during embryonic development and in adulthood. ATAD3A is a downstream effector of target of rapamycin (TOR) signalling in Drosophila and exhibits typical features of proteins from the ERMIONE-like complex in metazoans. In humans, mutations in the ATAD3A gene represent a new link between altered mitochondrial-ER interaction and recognizable neurological syndromes. The primate-specific ATAD3B protein is a biomarker of pluripotent embryonic stem cells. Through negative regulation of ATAD3A function, ATAD3B supports mitochondrial stemness properties. © 2017 Cambridge Philosophical Society.

Cellular determinants involving mitochondrial dysfunction, oxidative stress and apoptosis correlate with the synergic cytotoxicity of epigallocatechin-3-gallate and menadione in human leukemia Jurkat T cells.

PubMed

Tofolean, Ioana Teodora; Ganea, Constanta; Ionescu, Diana; Filippi, Alexandru; Garaiman, Alexandru; Goicea, Alexandru; Gaman, Mihnea-Alexandru; Dimancea, Alexandru; Baran, Irina

2016-01-01

We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bioenergetic adaptation in response to autophagy regulators during rotenone exposure

PubMed Central

Giordano, Samantha; Dodson, Matthew; Ravi, Saranya; Redmann, Matthew; Ouyang, Xiaosen; Usmar, Victor M Darley; Zhang, Jianhua

2015-01-01

Parkinson’s disease (PD) is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, exposure to the environmental neurotoxin rotenone increases the probability of developing PD. We previously reported that in differentiated SH-SY5Y cells, rotenone-induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10–100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 hr, caused cell death at 24 hr with an LD50 of 10 nM. Overall autophagic flux was decreased by 10 nM rotenone at both 2 and 24 hr, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 hr, suggesting that a mitochondrial-specific lysosomal degradation pathway may be activated. Upregulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3-methyladenine (3-MA) exacerbated cell death. Interestingly, while 3-MA exacerbated the rotenone-dependent effects on bioenergetics, rapamycin did not prevent rotenone-induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor. PMID:25081478

Chemicals or mutations that target mitochondrial translation can rescue the respiratory deficiency of yeast bcs1 mutants.

PubMed

Panozzo, C; Laleve, A; Tribouillard-Tanvier, D; Ostojić, J; Sellem, C H; Friocourt, G; Bourand-Plantefol, A; Burg, A; Delahodde, A; Blondel, M; Dujardin, G

2017-12-01

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III 2 /IV in yeast or I/III 2 /IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes. Copyright © 2017. Published by Elsevier B.V.

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

PubMed Central

Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul

2010-01-01

Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane. PMID:20056103

Sirtuin signaling controls mitochondrial function in glycogen storage disease type Ia.

PubMed

Cho, Jun-Ho; Kim, Goo-Young; Mansfield, Brian C; Chou, Janice Y

2018-05-08

Glycogen storage disease type Ia (GSD-Ia) deficient in glucose-6-phosphatase-α (G6Pase-α) is a metabolic disorder characterized by impaired glucose homeostasis and a long-term complication of hepatocellular adenoma/carcinoma (HCA/HCC). Mitochondrial dysfunction has been implicated in GSD-Ia but the underlying mechanism and its contribution to HCA/HCC development remain unclear. We have shown that hepatic G6Pase-α deficiency leads to downregulation of sirtuin 1 (SIRT1) signaling that underlies defective hepatic autophagy in GSD-Ia. SIRT1 is a NAD + -dependent deacetylase that can deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), a master regulator of mitochondrial integrity, biogenesis, and function. We hypothesized that downregulation of hepatic SIRT1 signaling in G6Pase-α-deficient livers impairs PGC-1α activity, leading to mitochondrial dysfunction. Here we show that the G6Pase-α-deficient livers display defective PGC-1α signaling, reduced numbers of functional mitochondria, and impaired oxidative phosphorylation. Overexpression of hepatic SIRT1 restores PGC-1α activity, normalizes the expression of electron transport chain components, and increases mitochondrial complex IV activity. We have previously shown that restoration of hepatic G6Pase-α expression normalized SIRT1 signaling. We now show that restoration of hepatic G6Pase-α expression also restores PGC-1α activity and mitochondrial function. Finally, we show that HCA/HCC lesions found in G6Pase-α-deficient livers contain marked mitochondrial and oxidative DNA damage. Taken together, our study shows that downregulation of hepatic SIRT1/PGC-1α signaling underlies mitochondrial dysfunction and that oxidative DNA damage incurred by damaged mitochondria may contribute to HCA/HCC development in GSD-Ia.

Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

PubMed Central

Covian, Raul; Chess, David; Balaban, Robert S.

2012-01-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. PMID:22975200

Continuous monitoring of enzymatic activity within native electrophoresis gels: application to mitochondrial oxidative phosphorylation complexes.

PubMed

Covian, Raul; Chess, David; Balaban, Robert S

2012-12-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze the enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction medium recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high-resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase in which catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. Published by Elsevier Inc.

Ubiquinol-10 Supplementation Activates Mitochondria Functions to Decelerate Senescence in Senescence-Accelerated Mice

PubMed Central

Tian, Geng; Sawashita, Jinko; Kubo, Hiroshi; Nishio, Shin-ya; Hashimoto, Shigenari; Suzuki, Nobuyoshi; Yoshimura, Hidekane; Tsuruoka, Mineko; Wang, Yaoyong; Liu, Yingye; Luo, Hongming; Xu, Zhe; Mori, Masayuki; Kitano, Mitsuaki; Hosoe, Kazunori; Takeda, Toshio; Usami, Shin-ichi

2014-01-01

Abstract Aim: The present study was conducted to define the relationship between the anti-aging effect of ubiquinol-10 supplementation and mitochondrial activation in senescence-accelerated mouse prone 1 (SAMP1) mice. Results: Here, we report that dietary supplementation with ubiquinol-10 prevents age-related decreases in the expression of sirtuin gene family members, which results in the activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a major factor that controls mitochondrial biogenesis and respiration, as well as superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2), which are major mitochondrial antioxidant enzymes. Ubiquinol-10 supplementation can also increase mitochondrial complex I activity and decrease levels of oxidative stress markers, including protein carbonyls, apurinic/apyrimidinic sites, malondialdehydes, and increase the reduced glutathione/oxidized glutathione ratio. Furthermore, ubiquinol-10 may activate Sirt1 and PGC-1α by increasing cyclic adenosine monophosphate (cAMP) levels that, in turn, activate cAMP response element-binding protein (CREB) and AMP-activated protein kinase (AMPK). Innovation and Conclusion: These results show that ubiquinol-10 may enhance mitochondrial activity by increasing levels of SIRT1, PGC-1α, and SIRT3 that slow the rate of age-related hearing loss and protect against the progression of aging and symptoms of age-related diseases. Antioxid. Redox Signal. 20, 2606–2620 PMID:24124769

Neuroprotective activities of curcumin and quercetin with potential relevance to mitochondrial dysfunction induced by oxaliplatin.

PubMed

Waseem, Mohammad; Parvez, Suhel

2016-03-01

Peripheral neurotoxicity is one of the serious dose-limiting side effects of oxaliplatin (Oxa) when used in the treatment of malignant conditions. It is documented that it elicits major side effects specifically neurotoxicity due to oxidative stress forcing the patients to limit its clinical use in long-term treatment. Oxidative stress has been proven to be involved in Oxa-induced toxicity including neurotoxicity. The mitochondria have recently emerged as targets for anticancer drugs in various kinds of toxicity including neurotoxicity that can lead to neoplastic disease. However, there is paucity of literature involving the role of the mitochondria in mediating Oxa-induced neurotoxicity and its underlying mechanism is still debatable. The purpose of this study was to investigate the dose-dependent damage caused by Oxa on isolated brain mitochondria under in vitro conditions. The study was also designed to investigate the neuroprotective effects of nutraceuticals, curcumin (CMN), and quercetin (QR) on Oxa-induced mitochondrial oxidative stress and respiratory chain complexes in the brain of rats. Oxidative stress biomarkers, levels of nonenzymatic antioxidants, activities of enzymatic antioxidants, and mitochondrial complexes were evaluated against the neurotoxicity induced by Oxa. Pretreatment with CMN and QR significantly replenished the mitochondrial lipid peroxidation levels and protein carbonyl content induced by Oxa. CMN and QR ameliorated altered nonenzymatic and enzymatic antioxidants and complex enzymes of mitochondria. We conclude that CMN and QR, by attenuating oxidative stress as evident by mitochondrial dysfunction, hold promise as agents that can potentially reduce Oxa-induced adverse effects in the brain.

Mitochondrial genome-knockout cells demonstrate a dual mechanism of action for the electron transport complex I inhibitor mycothiazole.

PubMed

Meyer, Kirsten J; Singh, A Jonathan; Cameron, Alanna; Tan, An S; Leahy, Dora C; O'Sullivan, David; Joshi, Praneta; La Flamme, Anne C; Northcote, Peter T; Berridge, Michael V; Miller, John H

2012-04-01

Mycothiazole, a polyketide metabolite isolated from the marine sponge Cacospongia mycofijiensis, is a potent inhibitor of metabolic activity and mitochondrial electron transport chain complex I in sensitive cells, but other cells are relatively insensitive to the drug. Sensitive cell lines (IC(50) 0.36-13.8 nM) include HeLa, P815, RAW 264.7, MDCK, HeLa S3, 143B, 4T1, B16, and CD4/CD8 T cells. Insensitive cell lines (IC(50) 12.2-26.5 μM) include HL-60, LN18, and Jurkat. Thus, there is a 34,000-fold difference in sensitivity between HeLa and HL-60 cells. Some sensitive cell lines show a biphasic response, suggesting more than one mechanism of action. Mitochondrial genome-knockout ρ(0) cell lines are insensitive to mycothiazole, supporting a conditional mitochondrial site of action. Mycothiazole is cytostatic rather than cytotoxic in sensitive cells, has a long lag period of about 12 h, and unlike the complex I inhibitor, rotenone, does not cause G(2)/M cell cycle arrest. Mycothiazole decreases, rather than increases the levels of reactive oxygen species after 24 h. It is concluded that the cytostatic inhibitory effects of mycothiazole on mitochondrial electron transport function in sensitive cell lines may depend on a pre-activation step that is absent in insensitive cell lines with intact mitochondria, and that a second lower-affinity cytotoxic target may also be involved in the metabolic and growth inhibition of cells.

3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway.

PubMed

Peng, Xiaoli; Gan, Jing; Wang, Qian; Shi, Zhenqiang; Xia, Xiaodong

2016-11-30

3-Monochloro-1,2-propanediol (3-MCPD) is the most toxic chloropropanols compounds in foodstuff which mainly generated during thermal processing. Kidney is one of the primary target organs for 3-MCPD. Using human embryonic kidney cell (HEK293FT) as an in vitro model, we found that 3-MCPD caused concentration-dependent increase in cytoxicity as assessed by dye uptake, lactatedehydrogenase (LDH) leakage and MTT assays. HEK293FT cell treated with 3-MCPD suffered the decrease of mitochondrial membrane potential and the impairment of mitochondrial oxidative phosphorylation system, especially the reduced amount of mRNA expression and protein synthesis of electron transport chain complex II, complex IV, and complex III. More importantly, energy release (ATP synthesis) was significantly inhibited by 3-MCPD resulting from the down regulation expressions of ATP synthase (ATP6 and ATP8), as well as the loss of transmembrane potential required for synthesis of ATP. The decreased ratio of mitochondrial apoptogenic factors Bax/Bcl-2 and the cytochrome-c release from mitochondria to cytosol followed by the activation of apoptotic initiators caspase 9 and apoptotic executioners (caspase 3, caspase 6 and caspase 7) leading to apoptosis. The activation of caspase 8 and caspase 2 implied that there were probably other factors to induce the caspase-dependent apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Optical spectroscopy in turbid media utilizing an integrating sphere: mitochondrial chromophore analysis during metabolic transitions

PubMed Central

Chess, David J.; Billings, Eric; Covian, Raúl; Glancy, Brian; French, Stephanie; Taylor, Joni; de Bari, Heather; Murphy, Elizabeth; Balaban, Robert S.

2013-01-01

Recent evidence suggests that the activity of mitochondrial oxidative phosphorylation Complexes (MOPC) is modulated at multiple sites. Herein, a method of optically monitoring electron distribution within and between MOPC is described using a center-mounted sample in an integrating sphere (to minimize scattering effects) with a rapid-scanning spectrometer. The redox-sensitive MOPC absorbances (~465 to 630 nm) were modeled using linear least squares analysis with individual chromophore spectra. Classical mitochondrial activity transitions (e.g., ADP-induced increase in oxygen consumption) were used to characterize this approach. Most notable in these studies was the observation that intermediates of the catalytic cycle of cytochrome oxidase are dynamically modulated with metabolic state. The MOPC redox state, along with measurements of oxygen consumption and mitochondrial membrane potential, was used to evaluate the conductances of different sections of the electron transport chain. This analysis then was applied to mitochondria isolated from rabbit hearts subjected to ischemia-reperfusion (I/R). Surprisingly, I/R resulted in an inhibition of all measured MOPC conductances, suggesting a coordinated down-regulation of mitochondrial activity with this well-established cardiac perturbation. PMID:23665273

Increased mitochondrial matrix directed superoxide production by fatty acid hydroperoxides in skeletal muscle mitochondria

PubMed Central

Bhattacharya, Arunabh; Lustgarten, Michael; Shi, Yun; Liu, Yuhong; Jang, Youngmok C; Pulliam, Daniel; Jernigan, Amanda L; Van Remmen, Holly

2013-01-01

Previous studies have shown that muscle atrophy is associated with mitochondrial dysfunction and an increased rate of mitochondrial reactive oxygen species production. We recently demonstrated that fatty acid hydroperoxides (FA-OOH) are significantly elevated in mitochondria isolated from atrophied muscles. The purpose of the current study is to determine whether FA-OOH can alter skeletal muscle mitochondrial function. We found that FA-OOH (at low micromolar concentrations) induces mitochondrial dysfunction assessed by decrease in the rate of ATP production, oxygen consumption and activity of respiratory chain complexes I and III. Using methods to distinguish superoxide release towards the matrix and inter-membrane space, we demonstrate that FA-OOH significantly elevates oxidative stress in the mitochondrial matrix (and not the inter-membrane space) with complex I as the major site of superoxide production (most likely from a site upstream of the ubiquinone binding site but downstream from the flavin binding site-the iron sulfur clusters). Our results are the first to indicate that FA-OOH’s are important modulators of mitochondrial function and oxidative stress in skeletal muscle mitochondria and may play an important role in muscle atrophies that are associated with increased generation of FA-OOH’s, e.g., denervation-induced muscle atrophy. PMID:21172427

Effect of CoQ homologues on reactive oxygen generation by mitochondria.

PubMed

Imada, Isuke; Sato, Eisuke F; Kira, Yukimi; Inoue, Masayasu

2008-01-01

Effect of CoQ compounds (Qs) on reactive oxygen (ROS) generation by mitochondrial complex I was studied using rat liver mitochondria and chemiluminescence probe L012. Kinetic analysis revealed that short chain Qs, such as Q2 and idebenone enhanced ROS generation by mitochondrial NADH oxidase system by a succinate-inhibitable mechanism. Lipid peroxidation in mitochondrial membranes induced by NADH and iron was inhibited by short chain Qs. The inhibitory activity was enhanced by co-oxidation of succinate as determined by chemiluminescence method and by electron spin resonance spectroscopy. These results suggested that the reduced form of short chain Qs inhibited mitochondrial ROS generation and lipid peroxidation.

[Two patients with mitochondrial respiratory chain disease].

PubMed

Bangma, H R; Smit, G P A; Kuks, J B M; Grevink, R G; Wolffenbuttel, B H R

2008-10-18

A 23-year-old woman and a 13-year-old boy were diagnosed with mitochondrial respiratory chain disease. The woman had muscle pain, fatigue and bilateral ophthalmoplegia--symptoms consistent with Kearns-Sayre syndrome. The boy had aspecific symptoms; eventually, reduced activity of complex 1 was found to be the cause of the mitochondrial respiratory chain disease in the boy and his mother, who had suffered from unexplained fatigue and muscle pain for 15 years. Mitochondrial diseases often involve several organ systems. Diagnosis can be difficult, because laboratory tests such as serum and urinary lactate and creatine kinase have low sensitivity and specificity. Biochemical assessment of muscle biopsy can reveal reduced oxidation ATP synthesis and sometimes specific abnormalities in individual protein complexes. DNA analysis may be helpful in demonstrating mitochondrial or nuclear mutations or deletions. The goal of treatment is to increase mitochondrial ATP production, improve clinical symptoms and enhance stamina. Replacement of the following substances (also referred to as cofactors) may be attempted: co-enzyme Q10, antioxidants (lipoic acid, vitamins C and E), riboflavin, thiamine, creatine and carnitine. Evidence regarding the optimal treatment approach is lacking; one usually has to rely on observing effects in the individual patient.

Mitochondrial translocation of signal transducer and activator of transcription 5 (STAT5) in leukemic T cells and cytokine-stimulated cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chueh, Fu-Yu; Leong, King-Fu; Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu

2010-11-26

Research highlights: {yields} STAT5 interacts with a mitochondrial protein PDC-E2 in a leukemic T cell line LSTRA. {yields} Tyrosine-phosphorylated STAT5, but not STAT3, is present in LSTRA mitochondria. {yields} Cytokines induce mitochondrial translocation of STAT5, but not STAT1 or STAT3. {yields} Cytokine-induced mitochondrial translocation of tyrosine-phosphorylated STAT5 is transient. {yields} Mitochondrial STAT5 binds to a putative STAT5 site in the mitochondrial DNA in vitro. -- Abstract: Signal transducers and activators of transcription (STATs) were first identified as key signaling molecules in response to cytokines. Constitutive STAT activation also has been widely implicated in oncogenesis. We analyzed STAT5-associated proteins in amore » leukemic T cell line LSTRA, which exhibits constitutive tyrosine phosphorylation and activation of STAT5. A cellular protein was found to specifically interact with STAT5 in LSTRA cells by co-immunoprecipitation. Sequencing analysis and subsequent immunoblotting confirmed the identity of this STAT5-associated protein as the E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2). Consistent with this interaction, both subcellular fractionation and immunofluorescence microscopy revealed mitochondrial localization of STAT5 in LSTRA cells. Mitochondrial localization of tyrosine-phosphorylated STAT5 also occurred in cytokine-stimulated cells. A time course experiment further demonstrated the transient kinetics of STAT5 mitochondrial translocation after cytokine stimulation. In contrast, cytokine-induced STAT1 and STAT3 activation did not result in their translocation into mitochondria. Furthermore, we showed that mitochondrial STAT5 bound to the D-loop regulatory region of mitochondrial DNA in vitro. It suggests a potential role of STAT5 in regulating the mitochondrial genome. Proliferative metabolism toward aerobic glycolysis is well known in cancer cells as the Warburg effect and is also observed in cytokine-stimulated cells. Our novel findings of cytokine-induced STAT5 translocation into mitochondria and its link to oncogenesis provide important insights into the underlying mechanisms of this characteristic metabolic shift.« less

Absence of Complex I Is Associated with Diminished Respiratory Chain Function in European Mistletoe.

PubMed

Maclean, Andrew E; Hertle, Alexander P; Ligas, Joanna; Bock, Ralph; Balk, Janneke; Meyer, Etienne H

2018-05-21

Parasitism is a life history strategy found across all domains of life whereby nutrition is obtained from a host. It is often associated with reductive evolution of the genome, including loss of genes from the organellar genomes [1, 2]. In some unicellular parasites, the mitochondrial genome (mitogenome) has been lost entirely, with far-reaching consequences for the physiology of the organism [3, 4]. Recently, mitogenome sequences of several species of the hemiparasitic plant mistletoe (Viscum sp.) have been reported [5, 6], revealing a striking loss of genes not seen in any other multicellular eukaryotes. In particular, the nad genes encoding subunits of respiratory complex I are all absent and other protein-coding genes are also lost or highly diverged in sequence, raising the question what remains of the respiratory complexes and mitochondrial functions. Here we show that oxidative phosphorylation (OXPHOS) in European mistletoe, Viscum album, is highly diminished. Complex I activity and protein subunits of complex I could not be detected. The levels of complex IV and ATP synthase were at least 5-fold lower than in the non-parasitic model plant Arabidopsis thaliana, whereas alternative dehydrogenases and oxidases were higher in abundance. Carbon flux analysis indicates that cytosolic reactions including glycolysis are greater contributors to ATP synthesis than the mitochondrial tricarboxylic acid (TCA) cycle. Our results describe the extreme adjustments in mitochondrial functions of the first reported multicellular eukaryote without complex I. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells.

PubMed

Sakai, Chika; Tomitsuka, Eriko; Esumi, Hiroyasu; Harada, Shigeharu; Kita, Kiyoshi

2012-05-01

Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010. Copyright © 2011 Elsevier B.V. All rights reserved.

Genetics Home Reference: mitochondrial complex III deficiency

MedlinePlus

... DNA packaged in chromosomes within the cell nucleus (nuclear DNA). It is not clear why the severity ... deficiency Genetic Testing Registry: Mitochondrial complex III deficiency, nuclear type 2 Genetic Testing Registry: Mitochondrial complex III ...

Kalpaamruthaa ameliorates mitochondrial and metabolic alterations in diabetes mellitus induced cardiovascular damage.

PubMed

Latha, Raja; Shanthi, Palanivelu; Sachdanandam, Panchanadham

2014-12-01

Efficacy of Kalpaamruthaa on the activities of lipid and carbohydrate metabolic enzymes, electron transport chain complexes and mitochondrial ATPases were studied in heart and liver of experimental rats. Cardiovascular damage (CVD) was developed in 8 weeks after type 2 diabetes mellitus induction with high fat diet (2 weeks) and low dose of streptozotocin (2 × 35 mg/kg b.w. i.p. in 24 hr interval). In CVD-induced rats, the activities of total lipase, cholesterol ester hydrolase and cholesterol ester synthetase were increased, while lipoprotein lipase and lecithin-cholesterol acyltransferase activities were decreased. The activities of lipid-metabolizing enzymes were altered by Kalpaamruthaa in CVD-induced rats towards normal. Kalpaamruthaa modulated the activities of glycolytic enzymes (hexokinase, phosphogluco-isomerase, aldolase and glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1, 6-bisphosphatase) and glycogenolytic enzyme (glycogen phosphorylase) along with increased glycogen content in the liver of CVD-induced rats. The activities of isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, α-ketoglutarate dehydrogenase, Complexes and ATPases (Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase) were decreased in CVD-induced rats, which were ameliorated by the treatment with Kalpaamruthaa. This study ascertained the efficacy of Kalpaamruthaa for the treatment of CVD in diabetes through the modulation of metabolizing enzymes and mitochondrial dysfunction.

An Essential Role for ECSIT in Mitochondrial Complex I Assembly and Mitophagy in Macrophages.

PubMed

Carneiro, Flávia R G; Lepelley, Alice; Seeley, John J; Hayden, Matthew S; Ghosh, Sankar

2018-03-06

ECSIT is a mitochondrial complex I (CI)-associated protein that has been shown to regulate the production of mitochondrial reactive oxygen species (mROS) following engagement of Toll-like receptors (TLRs). We have generated an Ecsit conditional knockout (CKO) mouse strain to study the in vivo role of ECSIT. ECSIT deletion results in profound alteration of macrophage metabolism, leading to a striking shift to reliance on glycolysis, complete disruption of CI activity, and loss of the CI holoenzyme and multiple subassemblies. An increase in constitutive mROS production in ECSIT-deleted macrophages prevents further TLR-induced mROS production. Surprisingly, ECSIT-deleted cells accumulate damaged mitochondria because of defective mitophagy. ECSIT associates with the mitophagy regulator PINK1 and exhibits Parkin-dependent ubiquitination. However, upon ECSIT deletion, we observed increased mitochondrial Parkin without the expected increase in mitophagy. Taken together, these results demonstrate a key role of ECSIT in CI function, mROS production, and mitophagy-dependent mitochondrial quality control. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Anti-tumor activity and mechanism of apoptosis of A549 induced by ruthenium complex.

PubMed

Sun, Dongdong; Mou, Zhipeng; Li, Nuan; Zhang, Weiwei; Wang, Yazhe; Yang, Endong; Wang, Weiyun

2016-12-01

Two new ruthenium (II) polypyridyl complexes [Ru(MeIm) 4 (pip)] 2+ (1) and [Ru(MeIm) 4 (4-npip)] 2+ (2) were synthesized under the guidance of computational studies (DFT). Their binding property to human telomeric G-quadruplex studied by UV-Vis absorption spectroscopy, the fluorescent resonance energy transfer (FRET) melting assay and circular dichroism (CD) spectroscopy for validating the theoretical prediction. Both of them were evaluated for their potential anti-proliferative activity against four human tumor cell lines. Complex 2 shows growth inhibition against all the cell lines tested, especially the human lung tumor cell (A549). The RTCA analysis not only validated the inhibition activity but also showed the ability of reducing A549 cells' migration. DNA-flow cytometric analysis, mitochondrial membrane potential (ΔΨm) and the scavenger measurements of reactive oxygen species (ROS) analysis carried out to investigate the mechanism of cell growth inhibition and apoptosis-inducing effect of complex 2. The results demonstrated that complex 2 induces tumor cells apoptosis by acting on both mitochondrial homeostasis destruction and death receptor signaling pathways. And those suggested that complex 2 could be a candidate for further evaluation as a chemotherapeutic agent against human tumor.

Tributyltin (TBT) inhibition of oligomycin-sensitive Mg-ATPase activity in mussel mitochondria.

PubMed

Pagliarani, Alessandra; Bandiera, Patrizia; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Nesci, Salvatore; Borgatti, Anna Rosa

2008-06-01

Tributyltin (TBT), one of the most toxic lipophilic aquatic pollutants, can be efficiently incorporated from sea water and sediments by filter-feeding molluscs. As far as we are aware TBT effects on the mitochondrial oligomycin-sensitive Mg-ATPase, the enzymatic core of energy production and a known target of TBT toxicity in mammals, have not been yet investigated in molluscs; thus the hydrolytic capability of the mitochondrial complex in the presence of micromolar concentrations of TBT was assayed in the mussel Mytilus galloprovincialis. Gill and mantle ATPase activities were progressively depressed by increasing TBT doses up to a maximal inhibition (82% in the gills and 74% in the mantle) at 0.62 microM TBT. Non-cooperative inhibition kinetics (n(H) approximately -1) and a non-competitive mechanism with respect to ATP substrate were pointed out. The mitochondrial Mg-ATPase susceptivity to TBT in the marine mussel was consistent with the formation of a TBT-Mg-ATPase complex, apparently more stable in the gills than in the mantle. The complex shape of the dose-response curve and the partial release of Mg-ATPase inhibition within the 0.6-34.4 microM TBT range suggest multiple interactions of TBT with the enzyme complex putatively related to its molecular mechanism of toxicity.

Synergistic effects of melatonin and deprenyl against MPTP-induced mitochondrial damage and DA depletion.

PubMed

Khaldy, Hoda; Escames, Germaine; León, Josefa; Bikjdaouene, Leila; Acuña-Castroviejo, Darío

2003-01-01

Previous studies showed a synergistic effect of melatonin and deprenyl against dopamine (DA) autoxidation in vitro. Since oxidative stress is implicated in Parkinson's disease (PD), we explored the effects of melatonin plus deprenyl administration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD in C57/Bl6 mice. Melatonin, but not deprenyl prevents the inhibition of mitochondrial complex I and the oxidative damage in nigrostriatal neurons induced by MPTP. With the dose used deprenyl recovers 50% DA levels and tyrosine hydroxylase activity depressed by the neurotoxin, normalizing locomotor activity of mice. Melatonin, which was unable to counteract MPTP-induced DA depletion and inhibition of tyrosine hydroxylase activity, potentiates the effect of deprenyl on catecholamine turnover and mice ambulatory activity. These results suggest a dissociation of complex I inhibition from DA depletion in this model of Parkinson's disease. The data also support that a combination of melatonin, which improves mitochondrial electron transport chain and reduces oxidative damage, and deprenyl, which promotes the specific function of the rescued neurons, i.e. DA turnover, may be a promising strategy for the treatment of PD.

Control mechanisms in mitochondrial oxidative phosphorylation☆

PubMed Central

Hroudová, Jana; Fišar, Zdeněk

2013-01-01

Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5’- triphosphate production is regulated by many control mechanism–firstly by oxygen, substrate level, adenosine-5’-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by “second control mechanisms,” such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5’-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production. PMID:25206677

Control mechanisms in mitochondrial oxidative phosphorylation.

PubMed

Hroudová, Jana; Fišar, Zdeněk

2013-02-05

Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5'- triphosphate production is regulated by many control mechanism-firstly by oxygen, substrate level, adenosine-5'-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by "second control mechanisms," such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5'-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.

Functional deficiencies of subsarcolemmal mitochondria in the type 2 diabetic human heart

PubMed Central

Croston, Tara L.; Thapa, Dharendra; Holden, Anthony A.; Tveter, Kevin J.; Lewis, Sara E.; Shepherd, Danielle L.; Nichols, Cody E.; Long, Dustin M.; Olfert, I. Mark; Jagannathan, Rajaganapathi

2014-01-01

The mitochondrion has been implicated in the development of diabetic cardiomyopathy. Examination of cardiac mitochondria is complicated by the existence of spatially distinct subpopulations including subsarcolemmal (SSM) and interfibrillar (IFM). Dysfunction to cardiac SSM has been reported in murine models of type 2 diabetes mellitus; however, subpopulation-based mitochondrial analyses have not been explored in type 2 diabetic human heart. The goal of this study was to determine the impact of type 2 diabetes mellitus on cardiac mitochondrial function in the human patient. Mitochondrial subpopulations from atrial appendages of patients with and without type 2 diabetes were examined. Complex I- and fatty acid-mediated mitochondrial respiration rates were decreased in diabetic SSM compared with nondiabetic (P ≤ 0.05 for both), with no change in IFM. Electron transport chain (ETC) complexes I and IV activities were decreased in diabetic SSM compared with nondiabetic (P ≤ 0.05 for both), with a concomitant decline in their levels (P ≤ 0.05 for both). Regression analyses comparing comorbidities determined that diabetes mellitus was the primary factor accounting for mitochondrial dysfunction. Linear spline models examining correlative risk for mitochondrial dysfunction indicated that patients with diabetes display the same degree of state 3 and electron transport chain complex I dysfunction in SSM regardless of the extent of glycated hemoglobin (HbA1c) and hyperglycemia. Overall, the results suggest that independent of other pathologies, mitochondrial dysfunction is present in cardiac SSM of patients with type 2 diabetes and the degree of dysfunction is consistent regardless of the extent of elevated HbA1c or blood glucose levels. PMID:24778174

Structure-based design and mechanisms of allosteric inhibitors for mitochondrial branched-chain α-ketoacid dehydrogenase kinase

PubMed Central

Qi, Xiangbing; Gui, Wen-Jun; Morlock, Lorraine K.; Wallace, Amy L.; Ahmed, Kamran; Laxman, Sunil; Campeau, Philippe M.; Lee, Brendan H.; Hutson, Susan M.; Tu, Benjamin P.; Williams, Noelle S.; Tambar, Uttam K.; Wynn, R. Max; Chuang, David T.

2013-01-01

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-α-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure. PMID:23716694

Mitochondrial complex II is a source of the reserve respiratory capacity that is regulated by metabolic sensors and promotes cell survival.

PubMed

Pfleger, J; He, M; Abdellatif, M

2015-07-30

The survival of a cell depends on its ability to meet its energy requirements. We hypothesized that the mitochondrial reserve respiratory capacity (RRC) of a cell is a critical component of its bioenergetics that can be utilized during an increase in energy demand, thereby, enhancing viability. Our goal was to identify the elements that regulate and contribute to the development of RRC and its involvement in cell survival. The results show that activation of metabolic sensors, including pyruvate dehydrogenase and AMP-dependent kinase, increases cardiac myocyte RRC via a Sirt3-dependent mechanism. Notably, we identified mitochondrial complex II (cII) as a target of these metabolic sensors and the main source of RRC. Moreover, we show that RRC, via cII, correlates with enhanced cell survival after hypoxia. Thus, for the first time, we show that metabolic sensors via Sirt3 maximize the cellular RRC through activating cII, which enhances cell survival after hypoxia.

Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

PubMed

Floyd, Brendan J; Wilkerson, Emily M; Veling, Mike T; Minogue, Catie E; Xia, Chuanwu; Beebe, Emily T; Wrobel, Russell L; Cho, Holly; Kremer, Laura S; Alston, Charlotte L; Gromek, Katarzyna A; Dolan, Brendan K; Ulbrich, Arne; Stefely, Jonathan A; Bohl, Sarah L; Werner, Kelly M; Jochem, Adam; Westphall, Michael S; Rensvold, Jarred W; Taylor, Robert W; Prokisch, Holger; Kim, Jung-Ja P; Coon, Joshua J; Pagliarini, David J

2016-08-18

Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function. Copyright © 2016 Elsevier Inc. All rights reserved.

Dialogue between E. coli free radical pathways and the mitochondria of C. elegans.

PubMed

Govindan, J Amaranath; Jayamani, Elamparithi; Zhang, Xinrui; Mylonakis, Eleftherios; Ruvkun, Gary

2015-10-06

The microbial world presents a complex palette of opportunities and dangers to animals, which have developed surveillance and response strategies to hints of microbial intent. We show here that the mitochondrial homeostatic response pathway of the nematode Caenorhabditis elegans responds to Escherichia coli mutations that activate free radical detoxification pathways. Activation of C. elegans mitochondrial responses could be suppressed by additional mutations in E. coli, suggesting that C. elegans responds to products of E. coli to anticipate challenges to its mitochondrion. Out of 50 C. elegans gene inactivations known to mediate mitochondrial defense, we found that 7 genes were required for C. elegans response to a free radical producing E. coli mutant, including the bZip transcription factor atfs-1 (activating transcription factor associated with stress). An atfs-1 loss-of-function mutant was partially resistant to the effects of free radical-producing E. coli mutant, but a constitutively active atfs-1 mutant growing on wild-type E. coli inappropriately activated the pattern of mitochondrial responses normally induced by an E. coli free radical pathway mutant. Carbonylated proteins from free radical-producing E. coli mutant may directly activate the ATFS-1/bZIP transcription factor to induce mitochondrial stress response: feeding C. elegans with H2O2-treated E. coli induces the mitochondrial unfolded protein response, and inhibition of a gut peptide transporter partially suppressed C. elegans response to free radical damaged E. coli.

Mitochondrial 3 beta-hydroxysteroid dehydrogenase (HSD) is essential for the synthesis of progesterone by corpora lutea: An hypothesis

PubMed Central

Chapman, John C; Polanco, Jose R; Min, Soohong; Michael, Sandra D

2005-01-01

In mouse ovaries, the enzyme 3 beta-hydroxysteroid dehydrogenase (HSD) is distributed between microsomes and mitochondria. Throughout the follicular phase of the estrous cycle, the HSD activity in microsomes is predominant; whereas, after LH stimulation, HSD activity during the luteal phase is highest in the mitochondria. The current study examined whether or not LH stimulation always results in an increase in mitochondrial HSD activity. This was accomplished by measuring the HSD activity in microsomal and mitochondrial fractions from ovaries of pregnant mice. These animals have two peaks of LH during gestation, and one peak of LH after parturition. It was found that mitochondrial HSD activity was highest after each peak of LH. It is proposed that mitochondrial HSD is essential for the synthesis of high levels of progesterone. The increase in HSD activity in mitochondria after LH stimulation occurs because: 1) LH initiates the simultaneous synthesis of HSD and the cholesterol side-chain cleavage enzyme (P450scc); and, 2) HSD and P450scc bind together to form a complex, which becomes inserted into the inner membrane of the mitochondria. High levels of progesterone are synthesized by mitochondrial HSD because: 1) the requisite NAD+ cofactor for progesterone synthesis is provided directly by the mitochondria, rather than indirectly via the rate limiting malate-aspartate shuttle; and, 2) the end-product inhibition of P450scc by pregnenolone is eliminated because pregnenolone is converted to progesterone. PMID:15804366

Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint.

PubMed

Yamamori, Tohru; Yasui, Hironobu; Yamazumi, Masayuki; Wada, Yusuke; Nakamura, Yoshinari; Nakamura, Hideo; Inanami, Osamu

2012-07-15

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase. Copyright © 2012 Elsevier Inc. All rights reserved.

Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder.

PubMed

Coughlin, Curtis R; Scharer, Gunter H; Friederich, Marisa W; Yu, Hung-Chun; Geiger, Elizabeth A; Creadon-Swindell, Geralyn; Collins, Abigail E; Vanlander, Arnaud V; Coster, Rudy Van; Powell, Christopher A; Swanson, Michael A; Minczuk, Michal; Van Hove, Johan L K; Shaikh, Tamim H

2015-08-01

Mitochondrial disease is often suspected in cases of severe epileptic encephalopathy especially when a complex movement disorder, liver involvement and progressive developmental regression are present. Although mutations in either mitochondrial DNA or POLG are often present, other nuclear defects in mitochondrial DNA replication and protein translation have been associated with a severe epileptic encephalopathy. We identified a proband with an epileptic encephalopathy, complex movement disorder and a combined mitochondrial respiratory chain enzyme deficiency. The child presented with neurological regression, complex movement disorder and intractable seizures. A combined deficiency of mitochondrial complexes I, III and IV was noted in liver tissue, along with increased mitochondrial DNA content in skeletal muscle. Incomplete assembly of complex V, using blue native polyacrylamide gel electrophoretic analysis and complex I, using western blotting, suggested a disorder of mitochondrial transcription or translation. Exome sequencing identified compound heterozygous mutations in CARS2, a mitochondrial aminoacyl-tRNA synthetase. Both mutations affect highly conserved amino acids located within the functional ligase domain of the cysteinyl-tRNA synthase. A specific decrease in the amount of charged mt-tRNA(Cys) was detected in patient fibroblasts compared with controls. Retroviral transfection of the wild-type CARS2 into patient skin fibroblasts led to the correction of the incomplete assembly of complex V, providing functional evidence for the role of CARS2 mutations in disease aetiology. Our findings indicate that mutations in CARS2 result in a mitochondrial translational defect as seen in individuals with mitochondrial epileptic encephalopathy. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

The effects of 2 weeks of statin treatment on mitochondrial respiratory capacity in middle-aged males: the LIFESTAT study.

PubMed

Asping, Magnus; Stride, Nis; Søgaard, Ditte; Dohlmann, Tine Lovsø; Helge, Jørn W; Dela, Flemming; Larsen, Steen

2017-06-01

Statins are used to lower cholesterol in plasma and are one of the most used drugs in the world. Many statin users experience muscle pain, but the mechanisms are unknown at the moment. Many studies have hypothesized that mitochondrial function could be involved in these side effects. The aim of the study was to investigate mitochondrial function after 2 weeks of treatment with simvastatin (S; n = 10) or pravastatin (P; n = 10) in healthy middle-aged participants. Mitochondrial respiratory capacity and substrate sensitivity were measured in permeabilized muscle fibers by high-resolution respirometry. Mitochondrial content (citrate synthase (CS) activity), antioxidant content, as well as coenzyme Q 10 concentration (Q 10 ) were determined. Fasting plasma glucose and insulin concentrations were measured, and whole body maximal oxygen uptake (VO 2max ) was determined. No differences were seen in mitochondrial respiratory capacity although a tendency was observed for a reduction when complex IV respiration was analyzed in both S (229 (169; 289 (95% confidence interval)) vs. 179 (146; 211) pmol/s/mg, respectively; P = 0.062) and P (214 (143; 285) vs. 162 (104; 220) pmol/s/mg, respectively; P = 0.053) after treatment. A tendency (1.64 (1.28; 2.00) vs. 1.28 (0.99; 1.58) mM, respectively; P = 0.092) for an increased mitochondrial substrate sensitivity (complex I-linked substrate; glutamate) was seen only in S after treatment. No differences were seen in Q 10 , CS activity, or antioxidant content after treatment. Fasting glucose and insulin as well as VO 2max were not changed after treatment. Two weeks of statin (S or P) treatment have no major effect on mitochondrial function. The tendency for an increased mitochondrial substrate sensitivity after simvastatin treatment could be an early indication of the negative effects linked to statin treatment.

Effect of endogenous nitric oxide on mitochondrial respiration of rat hepatocytes in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stadler, J.; Curran, R.D.; Ochoa, J.B.

1991-02-01

Nitric oxide, a highly reactive radical, was recently identified as an intermediate of L-arginine metabolism in mammalian cells. We have shown that nitric oxide synthesis is induced in vitro in cultured hepatocytes by supernatants from activated Kupffer cells or in vivo by injecting rats with nonviable Corynebacterium parvum. In both cases, nitric oxide biosynthesis in hepatocytes was associated with suppression of total protein synthesis. This study attempts to determine the effect of nitric oxide biosynthesis on the activity of specific hepatocytic mitochondrial enzymes and to determine whether inhibition of protein synthesis is caused by suppression of energy metabolism. Exposure ofmore » hepatocytes to supernatants from activated Kupffer cells led to a 30% decrease of aconitase (Krebs cycle) and complex I (mitochondrial electron transport chain) activity. Using NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis, we demonstrated that the inhibition of mitochondrial aconitase activity was due, in part, to the action of nitric oxide. In contrast, in vivo nitric oxide synthesis of hepatocytes from Corynebacterium parvum-treated animals had no effect on mitochondrial respiration. This suggests that inhibition of protein synthesis by nitric oxide is not likely to be mediated by inhibition of energy metabolism.« less

Chronic plus binge ethanol feeding induces myocardial oxidative stress, mitochondrial and cardiovascular dysfunction, and steatosis

PubMed Central

Matyas, Csaba; Varga, Zoltan V.; Mukhopadhyay, Partha; Paloczi, Janos; Lajtos, Tamas; Erdelyi, Katalin; Nemeth, Balazs T.; Nan, Mintong; Hasko, Gyorgy; Gao, Bin

2016-01-01

Alcoholic cardiomyopathy in humans develops in response to chronic excessive alcohol consumption; however, good models of alcohol-induced cardiomyopathy in mice are lacking. Herein we describe mouse models of alcoholic cardiomyopathies induced by chronic and binge ethanol (EtOH) feeding and characterize detailed hemodynamic alterations, mitochondrial function, and redox signaling in these models. Mice were fed a liquid diet containing 5% EtOH for 10, 20, and 40 days (d) combined with single or multiple EtOH binges (5 g/kg body wt). Isocalorically pair-fed mice served as controls. Left ventricular (LV) function and morphology were assessed by invasive pressure-volume conductance approach and by echocardiography. Mitochondrial complex (I, II, IV) activities, 3-nitrotyrosine (3-NT) levels, gene expression of markers of oxidative stress (gp91phox, p47phox), mitochondrial biogenesis (PGC1α, peroxisome proliferator-activated receptor α), and fibrosis were examined. Cardiac steatosis and fibrosis were investigated by histological/immunohistochemical methods. Chronic and binge EtOH feeding (already in 10 days EtOH plus single binge group) was characterized by contractile dysfunction (decreased slope of end-systolic pressure-volume relationship and preload recruitable stroke work), impaired relaxation (decreased time constant of LV pressure decay and maximal slope of systolic pressure decrement), and vascular dysfunction (impaired arterial elastance and lower total peripheral resistance). This was accompanied by enhanced myocardial oxidative/nitrative stress (3-NT; gp91phox; p47phox; angiotensin II receptor, type 1a) and deterioration of mitochondrial complex I, II, IV activities and mitochondrial biogenesis, excessive cardiac steatosis, and higher mortality. Collectively, chronic plus binge EtOH feeding in mice leads to alcohol-induced cardiomyopathies (National Institute on Alcohol Abuse and Alcoholism models) characterized by increased myocardial oxidative/nitrative stress, impaired mitochondrial function and biogenesis, and enhanced cardiac steatosis. PMID:27106042

Chronic plus binge ethanol feeding induces myocardial oxidative stress, mitochondrial and cardiovascular dysfunction, and steatosis.

PubMed

Matyas, Csaba; Varga, Zoltan V; Mukhopadhyay, Partha; Paloczi, Janos; Lajtos, Tamas; Erdelyi, Katalin; Nemeth, Balazs T; Nan, Mintong; Hasko, Gyorgy; Gao, Bin; Pacher, Pal

2016-06-01

Alcoholic cardiomyopathy in humans develops in response to chronic excessive alcohol consumption; however, good models of alcohol-induced cardiomyopathy in mice are lacking. Herein we describe mouse models of alcoholic cardiomyopathies induced by chronic and binge ethanol (EtOH) feeding and characterize detailed hemodynamic alterations, mitochondrial function, and redox signaling in these models. Mice were fed a liquid diet containing 5% EtOH for 10, 20, and 40 days (d) combined with single or multiple EtOH binges (5 g/kg body wt). Isocalorically pair-fed mice served as controls. Left ventricular (LV) function and morphology were assessed by invasive pressure-volume conductance approach and by echocardiography. Mitochondrial complex (I, II, IV) activities, 3-nitrotyrosine (3-NT) levels, gene expression of markers of oxidative stress (gp91phox, p47phox), mitochondrial biogenesis (PGC1α, peroxisome proliferator-activated receptor α), and fibrosis were examined. Cardiac steatosis and fibrosis were investigated by histological/immunohistochemical methods. Chronic and binge EtOH feeding (already in 10 days EtOH plus single binge group) was characterized by contractile dysfunction (decreased slope of end-systolic pressure-volume relationship and preload recruitable stroke work), impaired relaxation (decreased time constant of LV pressure decay and maximal slope of systolic pressure decrement), and vascular dysfunction (impaired arterial elastance and lower total peripheral resistance). This was accompanied by enhanced myocardial oxidative/nitrative stress (3-NT; gp91phox; p47phox; angiotensin II receptor, type 1a) and deterioration of mitochondrial complex I, II, IV activities and mitochondrial biogenesis, excessive cardiac steatosis, and higher mortality. Collectively, chronic plus binge EtOH feeding in mice leads to alcohol-induced cardiomyopathies (National Institute on Alcohol Abuse and Alcoholism models) characterized by increased myocardial oxidative/nitrative stress, impaired mitochondrial function and biogenesis, and enhanced cardiac steatosis. Copyright © 2016 the American Physiological Society.

APR3 modulates oxidative stress and mitochondrial function in ARPE-19 cells.

PubMed

Li, Yuan; Zou, Xuan; Gao, Jing; Cao, Ke; Feng, Zhihui; Liu, Jiankang

2018-05-24

Impairment of retinal pigment epithelial (RPE) cells is considered a key contributor to the development of age-related macular degeneration. Apoptosis-related protein 3 (APR3) was recently discovered after treatment with all- trans retinoic acid, a pivotal molecule in RPE cells. However, the function of APR3 remains poorly understood. In the present study, we found that APR3 could interact with nuclear factor (erythroid-derived 2)-like 2, which is a regulator of phase II enzymes, and that knockdown of APR3 promoted nuclear factor (erythroid-derived 2)-like 2 nuclear translocation and activated expression of phase II enzymes, which was accompanied by improved redox status and mitochondrial activity. Overexpression of APR3 revealed its mitochondrial localization and induced a robust production of reactive oxygen species that was accompanied by impaired mitochondrial oxygen consumption, complex activity, and lower ATP content, resulting in significant changes in mitochondrial structure, which may contribute to cell apoptosis. High doses of all- trans retinoic acid treatment were found to significantly induce APR3 expression, increase reactive oxygen species levels, and decrease ATP content, which were abolished by knockdown of APR3. These results indicate that APR3 plays a vital role in regulating redox status and mitochondrial activity and thus suggest APR3 might be a potential novel target for study of treatment of age-related macular degeneration.-Li, Y., Zou, X., Gao, J., Cao, K., Feng, Z., Liu, J. APR3 modulates oxidative stress and mitochondrial function in ARPE-19 cells.

Mitochondrial threshold effects.

PubMed Central

Rossignol, Rodrigue; Faustin, Benjamin; Rocher, Christophe; Malgat, Monique; Mazat, Jean-Pierre; Letellier, Thierry

2003-01-01

The study of mitochondrial diseases has revealed dramatic variability in the phenotypic presentation of mitochondrial genetic defects. To attempt to understand this variability, different authors have studied energy metabolism in transmitochondrial cell lines carrying different proportions of various pathogenic mutations in their mitochondrial DNA. The same kinds of experiments have been performed on isolated mitochondria and on tissue biopsies taken from patients with mitochondrial diseases. The results have shown that, in most cases, phenotypic manifestation of the genetic defect occurs only when a threshold level is exceeded, and this phenomenon has been named the 'phenotypic threshold effect'. Subsequently, several authors showed that it was possible to inhibit considerably the activity of a respiratory chain complex, up to a critical value, without affecting the rate of mitochondrial respiration or ATP synthesis. This phenomenon was called the 'biochemical threshold effect'. More recently, quantitative analysis of the effects of various mutations in mitochondrial DNA on the rate of mitochondrial protein synthesis has revealed the existence of a 'translational threshold effect'. In this review these different mitochondrial threshold effects are discussed, along with their molecular bases and the roles that they play in the presentation of mitochondrial diseases. PMID:12467494

A novel MitoNEET ligand, TT01001, improves diabetes and ameliorates mitochondrial function in db/db mice.

PubMed

Takahashi, Takehiro; Yamamoto, Masashi; Amikura, Kazutoshi; Kato, Kozue; Serizawa, Takashi; Serizawa, Kanako; Akazawa, Daisuke; Aoki, Takumi; Kawai, Koji; Ogasawara, Emi; Hayashi, Jun-Ichi; Nakada, Kazuto; Kainoh, Mie

2015-02-01

The mitochondrial outer membrane protein mitoNEET is a binding protein of the insulin sensitizer pioglitazone (5-[[4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione) and is considered a novel target for the treatment of type II diabetes. Several small-molecule compounds have been identified as mitoNEET ligands using structure-based design or virtual docking studies. However, there are no reports about their therapeutic potential in animal models. Recently, we synthesized a novel small molecule, TT01001 [ethyl-4-(3-(3,5-dichlorophenyl)thioureido)piperidine-1-carboxylate], designed on the basis of pioglitazone structure. In this study, we assessed the pharmacological properties of TT01001 in both in vitro and in vivo studies. We found that TT01001 bound to mitoNEET without peroxisome proliferator-activated receptor-γ activation effect. In type II diabetes model db/db mice, TT01001 improved hyperglycemia, hyperlipidemia, and glucose intolerance, and its efficacy was equivalent to that of pioglitazone, without the pioglitazone-associated weight gain. Mitochondrial complex II + III activity of the skeletal muscle was significantly increased in db/db mice. We found that TT01001 significantly suppressed the elevated activity of the complex II + III. These results suggest that TT01001 improved type II diabetes without causing weight gain and ameliorated mitochondrial function of db/db mice. This is the first study that demonstrates the effects of a mitoNEET ligand on glucose metabolism and mitochondrial function in an animal disease model. These findings support targeting mitoNEET as a potential therapeutic approach for the treatment of type II diabetes. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Brain of Diabetic Rats.

PubMed

Ortiz-Avila, Omar; Esquivel-Martínez, Mauricio; Olmos-Orizaba, Berenice Eridani; Saavedra-Molina, Alfredo; Rodriguez-Orozco, Alain R; Cortés-Rojo, Christian

2015-01-01

Diabetic encephalopathy is a diabetic complication related to the metabolic alterations featuring diabetes. Diabetes is characterized by increased lipid peroxidation, altered glutathione redox status, exacerbated levels of ROS, and mitochondrial dysfunction. Although the pathophysiology of diabetic encephalopathy remains to be clarified, oxidative stress and mitochondrial dysfunction play a crucial role in the pathogenesis of chronic diabetic complications. Taking this into consideration, the aim of this work was to evaluate the effects of 90-day avocado oil intake in brain mitochondrial function and oxidative status in streptozotocin-induced diabetic rats (STZ rats). Avocado oil improves brain mitochondrial function in diabetic rats preventing impairment of mitochondrial respiration and mitochondrial membrane potential (ΔΨ m ), besides increasing complex III activity. Avocado oil also decreased ROS levels and lipid peroxidation and improved the GSH/GSSG ratio as well. These results demonstrate that avocado oil supplementation prevents brain mitochondrial dysfunction induced by diabetes in association with decreased oxidative stress.

A patient with congenital hyperlactataemia and Leigh syndrome: an uncommon mitochondrial variant.

PubMed

Ching, C K; Mak, Chloe M; Au, K M; Chan, K Y; Yuen, Y P; Yau, Eric K C; Ma, Louis C K; Chow, H L; Chan, Albert Y W

2013-08-01

We report an uncommon mitochondrial variant in a baby girl with congenital hyperlactataemia and Leigh syndrome. The patient presented with a single episode of generalised clonic convulsion at day 19, and was found to have isolated and persistent hyperlactataemia ranging from 3.34 to 9.26 mmol/L. She had elevated serum lactate-to-pyruvate ratios of up to 35 and high plasma alanine concentration, indicative of a respiratory chain defect. At the age of 8 months, she developed evolving neurological and imaging features compatible with Leigh syndrome. Genetic testing for common mitochondrial DNA mutations, large mitochondrial DNA deletions, and selected nuclear genes was negative. Further analysis of lymphocyte mitochondrial DNA by sequencing revealed an uncommon heteroplasmic variant, NC_012920.1(MT-ND5):m.13094T>C (p.Val253Ala), which was previously shown to reduce complex I activity. In patients in whom there was a high suspicion of mitochondrial disorder, entire mitochondrial DNA analysis may be warranted if initial screening of common mitochondrial DNA mutations is negative.

Mitochondrial gene therapy improves respiration, biogenesis, and transcription in G11778A Leber's hereditary optic neuropathy and T8993G Leigh's syndrome cells.

PubMed

Iyer, Shilpa; Bergquist, Kristen; Young, Kisha; Gnaiger, Erich; Rao, Raj R; Bennett, James P

2012-06-01

Many incurable mitochondrial disorders result from mutant mitochondrial DNA (mtDNA) and impaired respiration. Leigh's syndrome (LS) is a fatal neurodegenerative disorder of infants, and Leber's hereditary optic neuropathy (LHON) causes blindness in young adults. Treatment of LHON and LS cells harboring G11778A and T8993G mutant mtDNA, respectively, by >90%, with healthy donor mtDNA complexed with recombinant human mitochondrial transcription factor A (rhTFAM), improved mitochondrial respiration by ∼1.2-fold in LHON cells and restored >50% ATP synthase function in LS cells. Mitochondrial replication, transcription, and translation of key respiratory genes and proteins were increased in the short term. Increased NRF1, TFAMB1, and TFAMA expression alluded to the activation of mitochondrial biogenesis as a mechanism for improving mitochondrial respiration. These results represent the development of a therapeutic approach for LHON and LS patients in the near future.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy

2010-10-22

Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated withmore » reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.« less

Pig Brain Mitochondria as a Biological Model for Study of Mitochondrial Respiration.

PubMed

Fišar, Z; Hroudová, J

2016-01-01

Oxidative phosphorylation is a key process of intracellular energy transfer by which mitochondria produce ATP. Isolated mitochondria serve as a biological model for understanding the mitochondrial respiration control, effects of various biologically active substances, and pathophysiology of mitochondrial diseases. The aim of our study was to evaluate pig brain mitochondria as a proper biological model for investigation of activity of the mitochondrial electron transport chain. Oxygen consumption rates of isolated pig brain mitochondria were measured using high-resolution respirometry. Mitochondrial respiration of crude mitochondrial fraction, mitochondria purified in sucrose gradient, and mitochondria purified in Percoll gradient were assayed as a function of storage time. Oxygen flux and various mitochondrial respiratory control ratios were not changed within two days of mitochondria storage on ice. Leak respiration was found higher and Complex I-linked respiration lower in purified mitochondria compared to the crude mitochondrial fraction. Damage to both outer and inner mitochondrial membrane caused by the isolation procedure was the greatest after purification in a sucrose gradient. We confirmed that pig brain mitochondria can serve as a biological model for investigation of mitochondrial respiration. The advantage of this biological model is the stability of respiratory parameters for more than 48 h and the possibility to isolate large amounts of mitochondria from specific brain areas without the need to kill laboratory animals. We suggest the use of high-resolution respirometry of pig brain mitochondria for research of the neuroprotective effects and/or mitochondrial toxicity of new medical drugs.

miR-125a induces apoptosis, metabolism disorder and migration impairment in pancreatic cancer cells by targeting Mfn2-related mitochondrial fission

PubMed Central

Pan, Lichao; Zhou, Lin; Yin, Weijia; Bai, Jia; Liu, Rong

2018-01-01

Mitochondrial fission is important for the development and progression of pancreatic cancer (PC). However, little is known regarding its role in pancreatic cancer apoptosis, metabolism and migration. In the current study, the mechanism by which mitochondrial fission modifies the biological characteristics of PC was explored. MicroRNA-125a (miR-125a) had the ability to inhibit mitochondrial fission and contributed to cellular survival. Suppressed mitochondrial fission led to a reduction in mitochondrial debris, preserved the mitochondrial membrane potential, inhibited mitochondrial permeability transition pore opening, ablated cytochrome c leakage into the cytoplasm and reduced the pro-apoptotic protein contents, finally blocking mitochondria related apoptosis pathways. Furthermore, defective mitochondrial fission induced by miR-125a enhanced mitochondria-dependent energy metabolism by promoting activity of electron transport chain complexes. Furthermore, suppressed mitochondrial fission also contributed to PANC-1 cell migration by preserving the F-actin balance. Furthermore, mitofusin 2 (Mfn2), the key defender of mitochondrial fission, is involved in inhibition of miR125a-mediated mitochondrial fission. Low contents of miR-125a upregulated Mfn2 transcription and expression, leading to inactivation of mitochondrial fission. Ultimately, the current study determined that miR-125a and Mfn2 are regulated by hypoxia-inducible factor 1 (HIF1). Knockdown of HIF1 reversed miR-125a expression, and therefore, inhibited Mfn2 expression, leading to activation of mitochondrial fission. Collectively, the present study demonstrated mitochondrial fission as a tumor suppression process that is regulated by the HIF/miR-125a/Mfn2 pathways, acting to restrict PANC-1 cell survival, energy metabolism and migration, with potential implications for novel approaches for PC therapy. PMID:29749475

miR-125a induces apoptosis, metabolism disorder and migrationimpairment in pancreatic cancer cells by targeting Mfn2-related mitochondrial fission.

PubMed

Pan, Lichao; Zhou, Lin; Yin, Weijia; Bai, Jia; Liu, Rong

2018-07-01

Mitochondrial fission is important for the development and progression of pancreatic cancer (PC). However, little is known regarding its role in pancreatic cancer apoptosis, metabolism and migration. In the current study, the mechanism by which mitochondrial fission modifies the biological characteristics of PC was explored. MicroRNA‑125a (miR‑125a) had the ability to inhibit mitochondrial fission and contributed to cellular survival. Suppressed mitochondrial fission led to a reduction in mitochondrial debris, preserved the mitochondrial membrane potential, inhibited mitochondrial permeability transition pore opening, ablated cytochrome c leakage into the cytoplasm and reduced the pro‑apoptotic protein contents, finally blocking mitochondria related apoptosis pathways. Furthermore, defective mitochondrial fission induced by miR‑125a enhanced mitochondria‑dependent energy metabolism by promoting activity of electron transport chain complexes. Furthermore, suppressed mitochondrial fission also contributed to PANC‑1 cell migration by preserving the F‑actin balance. Furthermore, mitofusin 2 (Mfn2), the key defender of mitochondrial fission, is involved in inhibition of miR125a‑mediated mitochondrial fission. Low contents of miR‑125a upregulated Mfn2 transcription and expression, leading to inactivation of mitochondrial fission. Ultimately, the current study determined that miR‑125a and Mfn2 are regulated by hypoxia‑inducible factor 1 (HIF1). Knockdown of HIF1 reversed miR‑125a expression, and therefore, inhibited Mfn2 expression, leading to activation of mitochondrial fission. Collectively, the present study demonstrated mitochondrial fission as a tumor suppression process that is regulated by the HIF/miR‑125a/Mfn2 pathways, acting to restrict PANC‑1 cell survival, energy metabolism and migration, with potential implications for novel approaches for PC therapy.

Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex.

PubMed

Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew; Tsai, Chen-Wei; Wu, Yujiao; Willliams, Carole; Miller, Christopher

2016-04-21

Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca(2+) landscape.

Modified Metformin as a More Potent Anticancer Drug: Mitochondrial Inhibition, Redox Signaling, Antiproliferative Effects and Future EPR Studies.

PubMed

Kalyanaraman, Balaraman; Cheng, Gang; Hardy, Micael; Ouari, Olivier; Sikora, Adam; Zielonka, Jacek; Dwinell, Michael B

2017-12-01

Metformin, one of the most widely prescribed antidiabetic drugs in the world, is being repurposed as a potential drug in cancer treatment. Epidemiological studies suggest that metformin exerts anticancer effects in diabetic patients with pancreatic cancer. However, at typical antidiabetic doses the bioavailability of metformin is presumably too low to exert antitumor effects. Thus, more potent analogs of metformin are needed in order to increase its anticancer efficacy. To this end, a new class of mitochondria-targeted metformin analogs (or mito-metformins) containing a positively-charged lipophilic triphenylphosphonium group was synthesized and tested for their antitumor efficacy in pancreatic cancer cells. Results indicate that the lead compound, mito-metformin 10 , was nearly 1000-fold more potent than metformin in inhibiting mitochondrial complex I activity, inducing reactive oxygen species (superoxide and hydrogen peroxide) that stimulate redox signaling mechanisms, including the activation of adenosinemonophosphate kinase and inhibition of proliferation of pancreatic cancer cells. The potential use of the low-temperature electron paramagnetic resonance technique in assessing the role of mitochondrial complexes including complex I in tumor regression in response to metformin and mito-metformins in the in vivo setting is discussed.

A mutation screening of oncogenes, tumor suppressor gene TP53 and nuclear encoded mitochondrial complex I genes in oncocytic thyroid tumors.

PubMed

Evangelisti, Cecilia; de Biase, Dario; Kurelac, Ivana; Ceccarelli, Claudio; Prokisch, Holger; Meitinger, Thomas; Caria, Paola; Vanni, Roberta; Romeo, Giovanni; Tallini, Giovanni; Gasparre, Giuseppe; Bonora, Elena

2015-03-21

Thyroid neoplasias with oncocytic features represent a specific phenotype in non-medullary thyroid cancer, reflecting the unique biological phenomenon of mitochondrial hyperplasia in the cytoplasm. Oncocytic thyroid cells are characterized by a prominent eosinophilia (or oxyphilia) caused by mitochondrial abundance. Although disruptive mutations in the mitochondrial DNA (mtDNA) are the most significant hallmark of such tumors, oncocytomas may be envisioned as heterogeneous neoplasms, characterized by multiple nuclear and mitochondrial gene lesions. We investigated the nuclear mutational profile of oncocytic tumors to pinpoint the mutations that may trigger the early oncogenic hit. Total DNA was extracted from paraffin-embedded tissues from 45 biopsies of oncocytic tumors. High-resolution melting was used for mutation screening of mitochondrial complex I subunits genes. Specific nuclear rearrangements were investigated by RT-PCR (RET/PTC) or on isolated nuclei by interphase FISH (PAX8/PPARγ). Recurrent point mutations were analyzed by direct sequencing. In our oncocytic tumor samples, we identified rare TP53 mutations. The series of analyzed cases did not include poorly- or undifferentiated thyroid carcinomas, and none of the TP53 mutated cases had significant mitotic activity or high-grade features. Thus, the presence of disruptive TP53 mutations was completely unexpected. In addition, novel mutations in nuclear-encoded complex I genes were identified. These findings suggest that nuclear genetic lesions altering the bioenergetics competence of thyroid cells may give rise to an aberrant mitochondria-centered compensatory mechanism and ultimately to the oncocytic phenotype.

Impaired adenosine monophosphate-activated protein kinase signalling in dorsal root ganglia neurons is linked to mitochondrial dysfunction and peripheral neuropathy in diabetes.

PubMed

Roy Chowdhury, Subir K; Smith, Darrell R; Saleh, Ali; Schapansky, Jason; Marquez, Alexandra; Gomes, Suzanne; Akude, Eli; Morrow, Dwane; Calcutt, Nigel A; Fernyhough, Paul

2012-06-01

Mitochondrial dysfunction occurs in sensory neurons and may contribute to distal axonopathy in animal models of diabetic neuropathy. The adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signalling axis senses the metabolic demands of cells and regulates mitochondrial function. Studies in muscle, liver and cardiac tissues have shown that the activity of adenosine monophosphate-activated protein kinase and PGC-1α is decreased under hyperglycaemia. In this study, we tested the hypothesis that deficits in adenosine monophosphate-activated protein kinase/PGC-1α signalling in sensory neurons underlie impaired axonal plasticity, suboptimal mitochondrial function and development of neuropathy in rodent models of type 1 and type 2 diabetes. Phosphorylation and expression of adenosine monophosphate-activated protein kinase/PGC-1α and mitochondrial respiratory chain complex proteins were downregulated in dorsal root ganglia of both streptozotocin-diabetic rats and db/db mice. Adenoviral-mediated manipulation of endogenous adenosine monophosphate-activated protein kinase activity using mutant proteins modulated neurotrophin-directed neurite outgrowth in cultures of sensory neurons derived from adult rats. Addition of resveratrol to cultures of sensory neurons derived from rats after 3-5 months of streptozotocin-induced diabetes, significantly elevated adenosine monophosphate-activated protein kinase levels, enhanced neurite outgrowth and normalized mitochondrial inner membrane polarization in axons. The bioenergetics profile (maximal oxygen consumption rate, coupling efficiency, respiratory control ratio and spare respiratory capacity) was aberrant in cultured sensory neurons from streptozotocin-diabetic rats and was corrected by resveratrol treatment. Finally, resveratrol treatment for the last 2 months of a 5-month period of diabetes reversed thermal hypoalgesia and attenuated foot skin intraepidermal nerve fibre loss and reduced myelinated fibre mean axonal calibre in streptozotocin-diabetic rats. These data suggest that the development of distal axonopathy in diabetic neuropathy is linked to nutrient excess and mitochondrial dysfunction via defective signalling of the adenosine monophosphate-activated protein kinase/PGC-1α pathway.

Role of the import motor in insertion of transmembrane segments by the mitochondrial TIM23 complex.

PubMed

Popov-Čeleketić, Dušan; Waegemann, Karin; Mapa, Koyeli; Neupert, Walter; Mokranjac, Dejana

2011-06-01

The TIM23 complex mediates translocation of proteins across, and their lateral insertion into, the mitochondrial inner membrane. Translocation of proteins requires both the membrane-embedded core of the complex and its ATP-dependent import motor. Insertion of some proteins, however, occurs in the absence of ATP, questioning the need for the import motor during lateral insertion. We show here that the import motor associates with laterally inserted proteins even when its ATPase activity is not required. Furthermore, our results suggest a role for the import motor in lateral insertion. Thus, the import motor is involved in ATP-dependent translocation and ATP-independent lateral insertion.

Role of the import motor in insertion of transmembrane segments by the mitochondrial TIM23 complex

PubMed Central

Popov-Čeleketić, Dušan; Waegemann, Karin; Mapa, Koyeli; Neupert, Walter; Mokranjac, Dejana

2011-01-01

The TIM23 complex mediates translocation of proteins across, and their lateral insertion into, the mitochondrial inner membrane. Translocation of proteins requires both the membrane-embedded core of the complex and its ATP-dependent import motor. Insertion of some proteins, however, occurs in the absence of ATP, questioning the need for the import motor during lateral insertion. We show here that the import motor associates with laterally inserted proteins even when its ATPase activity is not required. Furthermore, our results suggest a role for the import motor in lateral insertion. Thus, the import motor is involved in ATP-dependent translocation and ATP-independent lateral insertion. PMID:21546912

Alda-1 Protects Against Acrolein-Induced Acute Lung Injury and Endothelial Barrier Dysfunction.

PubMed

Lu, Qing; Mundy, Miles; Chambers, Eboni; Lange, Thilo; Newton, Julie; Borgas, Diana; Yao, Hongwei; Choudhary, Gaurav; Basak, Rajshekhar; Oldham, Mahogany; Rounds, Sharon

2017-12-01

Inhalation of acrolein, a highly reactive aldehyde, causes lung edema. The underlying mechanism is poorly understood and there is no effective treatment. In this study, we demonstrated that acrolein not only dose-dependently induced lung edema but also promoted LPS-induced acute lung injury. Importantly, acrolein-induced lung injury was prevented and rescued by Alda-1, an activator of mitochondrial aldehyde dehydrogenase 2. Acrolein also dose-dependently increased monolayer permeability, disrupted adherens junctions and focal adhesion complexes, and caused intercellular gap formation in primary cultured lung microvascular endothelial cells (LMVECs). These effects were attenuated by Alda-1 and the antioxidant N-acetylcysteine, but not by the NADPH inhibitor apocynin. Furthermore, acrolein inhibited AMP-activated protein kinase (AMPK) and increased mitochondrial reactive oxygen species levels in LMVECs-effects that were associated with impaired mitochondrial respiration. AMPK total protein levels were also reduced in lung tissue of mice and LMVECs exposed to acrolein. Activation of AMPK with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside blunted an acrolein-induced increase in endothelial monolayer permeability, but not mitochondrial oxidative stress or inhibition of mitochondrial respiration. Our results suggest that acrolein-induced mitochondrial dysfunction may not contribute to endothelial barrier dysfunction. We speculate that detoxification of acrolein by Alda-1 and activation of AMPK may be novel approaches to prevent and treat acrolein-associated acute lung injury, which may occur after smoke inhalation.

The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.

PubMed

Ando, Maya; Fiesel, Fabienne C; Hudec, Roman; Caulfield, Thomas R; Ogaki, Kotaro; Górka-Skoczylas, Paulina; Koziorowski, Dariusz; Friedman, Andrzej; Chen, Li; Dawson, Valina L; Dawson, Ted M; Bu, Guojun; Ross, Owen A; Wszolek, Zbigniew K; Springer, Wolfdieter

2017-04-24

Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson's disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients' fibroblasts and in cell-based, biochemical assays. Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon mitochondrial stress and due to conformational changes in the active site does not exert kinase activity towards Ub. In patients' fibroblasts, biochemical assays and by structural analyses, we unraveled two pathomechanisms that lead to loss of function upon mutation of p.I368N and highlight potential strategies for future drug development.

Betaine is a positive regulator of mitochondrial respiration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Icksoo, E-mail: icksoolee@dankook.ac.kr

2015-01-09

Highlights: • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration. • Betaine increases mitochondrial membrane potential and cellular energy levels. • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect. - Abstract: Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro.more » Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.« less

The Paradox of Mitochondrial Dysfunction and Extended Longevity

PubMed Central

Munkácsy, Erin; Rea, Shane L.

2014-01-01

Mitochondria play numerous, essential roles in the life of eukaryotes. Disruption of mitochondrial function in humans is often pathological or even lethal. Surprisingly, in some organisms mitochondrial dysfunction can result in life extension. This paradox has been studied most extensively in the long-lived Mit mutants of the nematode Caenorhabditis elegans. In this review, we explore the major responses that are activated following mitochondrial dysfunction in these animals and how these responses potentially act to extend their life. We focus our attention on five broad areas of current research – reactive oxygen species signaling, the mitochondrial unfolded protein response, autophagy, metabolic adaptation, and the roles played by various transcription factors. Lastly, we also examine why disruption of complexes I and II differ in their ability to induce the Mit phenotype and extend lifespan. PMID:24699406

Mitochondrial Modulation by Epigallocatechin 3-Gallate Ameliorates Cisplatin Induced Renal Injury through Decreasing Oxidative/Nitrative Stress, Inflammation and NF-kB in Mice

PubMed Central

Wang, Xueping; Wang, Ping; Fu, Guanghou; Meng, Hongzhou; Wang, Yimin; Jin, Baiye

2015-01-01

Cancer chemotherapy drug cisplatin is known for its nephrotoxicity. The aim of this study is to investigate whether Epigallocatechin 3-Gallate (EGCG) can reduce cisplatin mediated side effect in kidney and to understand its mechanism of protection against tissue injury. We used a well-established 3-day cisplatin induced nephrotoxicity mice model where EGCG were administered. EGCG is a major active compound in Green Tea and have strong anti-oxidant and anti-inflammatory properties. EGCG protected against cisplatin induced renal dysfunction as measured by serum creatinine and blood urea nitrogen (BUN). EGCG improved cisplatin induced kidney structural damages such as tubular dilatation, cast formation, granulovaculoar degeneration and tubular cell necrosis as evident by PAS staining. Cisplatin induced kidney specific mitochondrial oxidative stress, impaired activities of mitochondrial electron transport chain enzyme complexes, impaired anti-oxidant defense enzyme activities such as glutathione peroxidase (GPX) and manganese superoxide dismutase (MnSOD) in mitochondria, inflammation (tumor necrosis factor α and interleukin 1β), increased accumulation of NF-κB in nuclear fraction, p53 induction, and apoptotic cell death (caspase 3 activity and DNA fragmentation). Treatment of mice with EGCG markedly attenuated cisplatin induced mitochondrial oxidative/nitrative stress, mitochondrial damages to electron transport chain activities and antioxidant defense enzyme activities in mitochondria. These mitochondrial modulations by EGCG led to protection mechanism against cisplatin induced inflammation and apoptotic cell death in mice kidney. As a result, EGCG improved renal function in cisplatin mediated kidney damage. In addition to that, EGCG attenuated cisplatin induced apoptotic cell death and mitochondrial reactive oxygen species (ROS) generation in human kidney tubular cell line HK-2. Thus, our data suggest that EGCG may represent new promising adjunct candidate for cisplatin. PMID:25875356

Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier.

PubMed

Martínez-Zamora, Ana; Meseguer, Salvador; Esteve, Juan M; Villarroya, Magda; Aguado, Carmen; Enríquez, J Antonio; Knecht, Erwin; Armengod, M-Eugenia

2015-01-01

GTPBP3 is an evolutionary conserved protein presumably involved in mitochondrial tRNA (mt-tRNA) modification. In humans, GTPBP3 mutations cause hypertrophic cardiomyopathy with lactic acidosis, and have been associated with a defect in mitochondrial translation, yet the pathomechanism remains unclear. Here we use a GTPBP3 stable-silencing model (shGTPBP3 cells) for a further characterization of the phenotype conferred by the GTPBP3 defect. We experimentally show for the first time that GTPBP3 depletion is associated with an mt-tRNA hypomodification status, as mt-tRNAs from shGTPBP3 cells were more sensitive to digestion by angiogenin than tRNAs from control cells. Despite the effect of stable silencing of GTPBP3 on global mitochondrial translation being rather mild, the steady-state levels and activity of Complex I, and cellular ATP levels were 50% of those found in the controls. Notably, the ATPase activity of Complex V increased by about 40% in GTPBP3 depleted cells suggesting that mitochondria consume ATP to maintain the membrane potential. Moreover, shGTPBP3 cells exhibited enhanced antioxidant capacity and a nearly 2-fold increase in the uncoupling protein UCP2 levels. Our data indicate that stable silencing of GTPBP3 triggers an AMPK-dependent retrograde signaling pathway that down-regulates the expression of the NDUFAF3 and NDUFAF4 Complex I assembly factors and the mitochondrial pyruvate carrier (MPC), while up-regulating the expression of UCP2. We also found that genes involved in glycolysis and oxidation of fatty acids are up-regulated. These data are compatible with a model in which high UCP2 levels, together with a reduction in pyruvate transport due to the down-regulation of MPC, promote a shift from pyruvate to fatty acid oxidation, and to an uncoupling of glycolysis and oxidative phosphorylation. These metabolic alterations, and the low ATP levels, may negatively affect heart function.

Reactive oxygen species and nitric oxide in plant mitochondria: origin and redundant regulatory systems.

PubMed

Blokhina, Olga; Fagerstedt, Kurt V

2010-04-01

Plant mitochondria differ from their mammalian counterparts in many respects, which are due to the unique and variable surroundings of plant mitochondria. In green leaves, plant mitochondria are surrounded by ample respiratory substrates and abundant molecular oxygen, both resulting from active photosynthesis, while in roots and bulky rhizomes and fruit carbohydrates may be plenty, whereas oxygen levels are falling. Several enzymatic complexes in mitochondrial electron transport chain (ETC) are capable of reactive oxygen species (ROS) formation under physiological and pathological conditions. Inherently connected parameters such as the redox state of electron carriers in the ETC, ATP synthase activity and inner mitochondrial membrane potential, when affected by external stimuli, can give rise to ROS formation via complexes I and III, and by reverse electron transport (RET) from complex II. Superoxide radicals produced are quickly scavenged by superoxide dismutase (MnSOD), and the resulting H(2)O(2) is detoxified by peroxiredoxin-thioredoxin system or by the enzymes of ascorbate-glutathione cycle, found in the mitochondrial matrix. Arginine-dependent nitric oxide (NO)-releasing activity of enzymatic origin has been detected in plant mitochondria. The molecular identity of the enzyme is not clear but the involvement of mitochondria-localized enzymes responsible for arginine catabolism, arginase and ornithine aminotransferase has been shown in the regulation of NO efflux. Besides direct control by antioxidants, mitochondrial ROS production is tightly controlled by multiple redundant systems affecting inner membrane potential: NAD(P)H-dependent dehydrogenases, alternative oxidase (AOX), uncoupling proteins, ATP-sensitive K(+) channel and a number of matrix and intermembrane enzymes capable of direct electron donation to ETC. NO removal, on the other hand, takes place either by reactions with molecular oxygen or superoxide resulting in peroxynitrite, nitrite or nitrate ions or through interaction with non-symbiotic hemoglobins or glutathione. Mitochondrial ROS and NO production is tightly controlled by multiple redundant systems providing the regulatory mechanism for redox homeostasis and specific ROS/NO signaling.

Gestational diabetes is characterized by reduced mitochondrial protein expression and altered calcium signaling proteins in skeletal muscle.

PubMed

Boyle, Kristen E; Hwang, Hyonson; Janssen, Rachel C; DeVente, James M; Barbour, Linda A; Hernandez, Teri L; Mandarino, Lawrence J; Lappas, Martha; Friedman, Jacob E

2014-01-01

The rising prevalence of gestational diabetes mellitus (GDM) affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM) and obese pregnant women with normal glucose tolerance (ONGT). Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I) subunits (NDUFS3, NDUFV2) and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, α1-syntrophin, annexin A4) in OGDM (n = 6) vs. ONGT (n = 6). Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (-60-75%) in the OGDM (n = 8) compared with ONGT (n = 10) subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum.

Effect of chronic pre-treatment with angiotensin converting enzyme inhibition on skeletal muscle mitochondrial recovery after ischemia/reperfusion.

PubMed

Thaveau, Fabien; Zoll, Joffrey; Bouitbir, Jamal; N'guessan, Benoît; Plobner, Philippe; Chakfe, Nabil; Kretz, Jean-Georges; Richard, Ruddy; Piquard, François; Geny, Bernard

2010-06-01

Impaired skeletal muscle energetic participates in peripheral arterial disease (PAD) patient's morbidity and mortality. Angiotensin converting enzyme inhibition (ACEi), cornerstone for pharmacologic risk factor management in PAD patients, might also be interesting by protecting skeletal muscle energetic. We therefore determined whether chronic ACEi might reduce ischemia-induced mitochondrial respiratory chain dysfunction in the frequent setting of hindlimb ischemia-reperfusion. Ischemic legs of rats submitted to 5 h ischemia induced by a rubber band tourniquet applied on the root of the hindlimb followed by reperfusion without (IR, n = 11) or after ACEi (n = 14; captopril 40 mg/kg per day during 28 days before surgery) were studied and compared to that of sham-operated animals (n = 11). The effect of ACEi on the non-ischemic contralateral leg was also determined in the ACEi group. Maximal oxidative capacities (V(max)) and complexes I, II and IV activities of the mitochondrial respiratory chain of the gastrocnemius muscle were determined using glutamate-malate, succinate and TMPD-ascorbate substrates. Arterial blood pressure was significantly decreased after ACEi (124 +/- 2.8 vs. 108 +/- 4.19 mmHg; P = 0.01). Ischemia-reperfusion reduced V(max) (4.4 +/- 0.4 vs. 8.7 +/- 0.5 micromol O2/min/g dry weight, -49%, P < 0.001), affecting mitochondrial complexes I, II and IV activities. ACEi failed to modulate ischemia-induced dysfunction (V(max) 5.1 +/- 0.7 micromol O2/min/g dry weight) or the non-ischemic contralateral muscle respiratory rate. Ischemia-reperfusion significantly impaired the mitochondrial respiratory chain I, II and IV complexes of skeletal muscle. Pharmacologic pre-treatment with ACEi did not prevent or increase such alterations. Further studies might be useful to improve the pharmacologic conditioning of PAD patients needing arterial revascularization.

Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Cong; Sekine, Shuichi, E-mail: ssekine@facult

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and inmore » hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.« less

The mitochondrial disulfide relay system protein GFER is mutated in autosomal-recessive myopathy with cataract and combined respiratory-chain deficiency.

PubMed

Di Fonzo, Alessio; Ronchi, Dario; Lodi, Tiziana; Fassone, Elisa; Tigano, Marco; Lamperti, Costanza; Corti, Stefania; Bordoni, Andreina; Fortunato, Francesco; Nizzardo, Monica; Napoli, Laura; Donadoni, Chiara; Salani, Sabrina; Saladino, Francesca; Moggio, Maurizio; Bresolin, Nereo; Ferrero, Iliana; Comi, Giacomo P

2009-05-01

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient's muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1(R182H) mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.

Oxidative stress in duckweed (Lemna minor L.) induced by glyphosate: Is the mitochondrial electron transport chain a target of this herbicide?

PubMed

Gomes, Marcelo Pedrosa; Juneau, Philippe

2016-11-01

We investigated the physiological responses of Lemna minor plants exposed to glyphosate. The deleterious effects of this herbicide on photosynthesis, respiration, and pigment concentrations were related to glyphosate-induced oxidative stress through hydrogen peroxide (H 2 O 2 ) accumulation. By using photosynthetic and respiratory electron transport chain (ETC) inhibitors we located the primary site of reactive oxygen species (ROS) production in plants exposed to 500 mg glyphosate l -1 . Inhibition of mitochondrial ETC Complex I by rotenone reduced H 2 O 2 concentrations in glyphosate-treated plants. Complex III activity was very sensitive to glyphosate which appears to act much like antimycin A (an inhibitor of mitochondrial ETC Complex III) by shunting electrons from semiquinone to oxygen, with resulting ROS formation. Confocal evaluations for ROS localization showed that ROS are initially produced outside of the chloroplasts upon initial glyphosate exposure. Our results indicate that in addition to interfering with the shikimate pathway, glyphosate can induce oxidative stress in plants through H 2 O 2 formation by targeting the mitochondrial ETC, which would explain its observed effects on non-target organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

RNA silencing of mitochondrial m-Nfs1 reduces Fe-S enzyme activity both in mitochondria and cytosol of mammalian cells.

PubMed

Fosset, Cédric; Chauveau, Marie-Jeanne; Guillon, Blanche; Canal, Frédéric; Drapier, Jean-Claude; Bouton, Cécile

2006-09-01

In prokaryotes and yeast, the general mechanism of biogenesis of iron-sulfur (Fe-S) clusters involves activities of several proteins among which IscS and Nfs1p provide, through cysteine desulfuration, elemental sulfide for Fe-S core formation. Although these proteins have been well characterized, the role of their mammalian homolog in Fe-S cluster biogenesis has never been evaluated. We report here the first functional study that implicates the putative cysteine desulfurase m-Nfs1 in the biogenesis of both mitochondrial and cytosolic mammalian Fe-S proteins. Depletion of m-Nfs1 in cultured fibroblasts through small interfering RNA-based gene silencing significantly inhibited the activities of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II) of the respiratory chain, as well as aconitase of the Krebs cycle, with no alteration in their protein levels. Activity of cytosolic xanthine oxidase, which holds a [2Fe-2S] cluster, was also specifically reduced, and iron-regulatory protein-1 was converted from its [4Fe-4S] aconitase form to its apo- or RNA-binding form. Reduction of Fe-S enzyme activities occurred earlier and more markedly in the cytosol than in mitochondria, suggesting that there is a mechanism that primarily dedicates m-Nfs1 to the biogenesis of mitochondrial Fe-S clusters in order to maintain cell survival. Finally, depletion of m-Nfs1, which conferred on apo-IRP-1 a high affinity for ferritin mRNA, was associated with the down-regulation of the iron storage protein ferritin.

Neuroprotective effect of curcumin as evinced by abrogation of rotenone-induced motor deficits, oxidative and mitochondrial dysfunctions in mouse model of Parkinson's disease.

PubMed

Khatri, Dharmendra K; Juvekar, Archana R

Curcumin, a natural polyphenolic compound extracted from rhizomes of Curcuma longa (turmeric), a plant in the ginger family (Zingiberaceae) has been used worldwide and extensively in Southeast Asia. Curcumin exhibited numerous biological and pharmacological activities including potent antioxidant, cardiovascular disease, anticancer, anti-inflammatory effects and neurodegenerative disorders in cell cultures and animal models. Hence, the present study was designed in order to explore the possible neuroprotective role of curcumin against rotenone induced cognitive impairment, oxidative and mitochondrial dysfunction in mice. Chronic administration of rotenone (1mg/kg i.p.) for a period of three weeks significantly impaired cognitive function (actophotometer, rotarod and open field test), oxidative defense (increased lipid peroxidation, nitrite concentration and decreased activity of superoxide dismutase, catalase and reduced glutathione level) and mitochondrial complex (II and III) enzymes activities as compared to normal control group. Three weeks of curcumin (50, 100 and 200mg/kg, p.o.) treatment significantly improved behavioral alterations, oxidative damage and mitochondrial enzyme complex activities as compared to negative control (rotenone treated) group. Curcumin treated mice also mitigated enhanced acetylcholine esterase enzyme level as compared to negative control group. We found that curcumin restored motor deficits and enhanced the activities of antioxidant enzymes suggesting its antioxidant potential in vivo. The findings of the present study conclude neuroprotective role of curcumin against rotenone induced Parkinson's in mice and offer strong justification for the therapeutic prospective of this compound in the management of PD. Copyright © 2016. Published by Elsevier Inc.

Novel piplartine-containing ruthenium complexes: synthesis, cell growth inhibition, apoptosis induction and ROS production on HCT116 cells.

PubMed

D'Sousa Costa, Cinara O; Araujo Neto, João H; Baliza, Ingrid R S; Dias, Rosane B; Valverde, Ludmila de F; Vidal, Manuela T A; Sales, Caroline B S; Rocha, Clarissa A G; Moreira, Diogo R M; Soares, Milena B P; Batista, Alzir A; Bezerra, Daniel P

2017-11-28

Piplartine (piperlongumine) is a plant-derived molecule that has been receiving intense interest due to its anticancer characteristics that target the oxidative stress. In the present paper, two novel piplartine-containing ruthenium complexes [Ru(piplartine)(dppf)(bipy)](PF 6 ) 2 (1) and [Ru(piplartine)(dppb)(bipy)](PF 6 ) 2 (2) were synthesized and investigated for their cellular and molecular responses on cancer cell lines. We found that both complexes are more potent than metal-free piplartine in a panel of cancer cell lines on monolayer cultures, as well in 3D model of cancer multicellular spheroids formed from human colon carcinoma HCT116 cells. Mechanistic studies uncovered that the complexes reduced the cell growth and caused phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase-3 activation and loss of the mitochondrial transmembrane potential on HCT116 cells. Moreover, the pre-treatment with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, reduced the complexes-induced apoptosis, indicating cell death by apoptosis through caspase-dependent and mitochondrial intrinsic pathways. Treatment with the complexes also caused a marked increase in the production of reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion and nitric oxide, and decreased reduced glutathione levels. Application of N-acetyl-cysteine, an antioxidant, reduced the ROS levels and apoptosis induced by the complexes, indicating activation of ROS-mediated apoptosis pathway. RNA transcripts of several genes, including gene related to the cell cycle, apoptosis and oxidative stress, were regulated under treatment. However, the complexes failed to induce DNA intercalation. In conclusion, the complexes are more potent than piplartine against different cancer cell lines and are able to induce caspase-dependent and mitochondrial intrinsic apoptosis on HCT116 cells by ROS-mediated pathway.

Immortalized Parkinson's disease lymphocytes have enhanced mitochondrial respiratory activity

PubMed Central

Annesley, Sarah J.; Lay, Sui T.; De Piazza, Shawn W.; Sanislav, Oana; Hammersley, Eleanor; Allan, Claire Y.; Francione, Lisa M.; Bui, Minh Q.; Chen, Zhi-Ping; Ngoei, Kevin R. W.; Tassone, Flora; Kemp, Bruce E.; Storey, Elsdon; Evans, Andrew; Loesch, Danuta Z.

2016-01-01

ABSTRACT In combination with studies of post-mortem Parkinson's disease (PD) brains, pharmacological and genetic models of PD have suggested that two fundamental interacting cellular processes are impaired – proteostasis and mitochondrial respiration. We have re-examined the role of mitochondrial dysfunction in lymphoblasts isolated from individuals with idiopathic PD and an age-matched control group. As previously reported for various PD cell types, the production of reactive oxygen species (ROS) by PD lymphoblasts was significantly elevated. However, this was not due to an impairment of mitochondrial respiration, as is often assumed. Instead, basal mitochondrial respiration and ATP synthesis are dramatically elevated in PD lymphoblasts. The mitochondrial mass, genome copy number and membrane potential were unaltered, but the expression of indicative respiratory complex proteins was also elevated. This explains the increased oxygen consumption rates by each of the respiratory complexes in experimentally uncoupled mitochondria of iPD cells. However, it was not attributable to increased activity of the stress- and energy-sensing protein kinase AMPK, a regulator of mitochondrial biogenesis and activity. The respiratory differences between iPD and control cells were sufficiently dramatic as to provide a potentially sensitive and reliable biomarker of the disease state, unaffected by disease duration (time since diagnosis) or clinical severity. Lymphoblasts from control and PD individuals thus occupy two distinct, quasi-stable steady states; a ‘normal’ and a ‘hyperactive’ state characterized by two different metabolic rates. The apparent stability of the ‘hyperactive’ state in patient-derived lymphoblasts in the face of patient ageing, ongoing disease and mounting disease severity suggests an early, permanent switch to an alternative metabolic steady state. With its associated, elevated ROS production, the ‘hyperactive’ state might not cause pathology to cells that are rapidly turned over, but brain cells might accumulate long-term damage leading ultimately to neurodegeneration and the loss of mitochondrial function observed post-mortem. Whether the ‘hyperactive’ state in lymphoblasts is a biomarker specifically of PD or more generally of neurodegenerative disease remains to be determined. PMID:27638668

Bacopa monnieri Phytochemicals Mediated Synthesis of Platinum Nanoparticles and Its Neurorescue Effect on 1-Methyl 4-Phenyl 1,2,3,6 Tetrahydropyridine-Induced Experimental Parkinsonism in Zebrafish

PubMed Central

Nellore, Jayshree; Pauline, Cynthia; Amarnath, Kanchana

2013-01-01

Current discovery demonstrates the rapid formation of platinum nanoparticles using leaf extract of a neurobeneficial plant, Bacopa monnieri (BmE). The nanoparticles (BmE-PtNPs) were stabilized and then coated with varied phytochemicals present within the leaf extract. These nanoparticles demonstrated the same activity of Complex I, as that of oxidizing NADH to NAD+ using a spectrophotometric method. This suggests that BmE-PtNPs are a potential medicinal substance for oxidative stress mediated disease with suppressed mitochondrial complex I, namely, Parkinson's disease (PD). Hence, the neuroprotective potentials of the phytochemical coated nanoparticle were explored in 1-methyl 4-phenyl 1,2,3,6 tetrahydropyridine- (MPTP-)induced experimental Parkinsonism in zebrafish model. BmE-PtNPs pretreatment significantly reversed toxic effects of MPTP by increasing the levels of dopamine, its metabolites, GSH and activities of GPx, catalase, SOD and complex I, and reducing levels of MDA along with enhanced locomotor activity. Taken together, these findings suggest that BmE-PtNPs have protective effect in MPTP-induced neurotoxicity in this model of Parkinson's disease via their dual functions as mitochondrial complex I and antioxidant activity. PMID:26317003

Mitochondrial Diabetes in Children: Seek and You Will Find It

PubMed Central

Liguori, Rosario; Ferrigno, Maddalena; Galderisi, Alfonso; Vitale, Domenico; Simonelli, Francesca; Landolfo, Paolo; Prisco, Francesco; Masullo, Mariorosario; Sacchetti, Lucia

2012-01-01

Maternally Inherited Diabetes and Deafness (MIDD) is a rare form of diabetes due to defects in mitochondrial DNA (mtDNA). 3243 A>G is the mutation most frequently associated with this condition, but other mtDNA variants have been linked with a diabetic phenotype suggestive of MIDD. From 1989 to 2009, we clinically diagnosed mitochondrial diabetes in 11 diabetic children. Diagnosis was based on the presence of one or more of the following criteria: 1) maculopathy; 2) hearing impairment; 3) maternal heritability of diabetes/impaired fasting glucose and/or hearing impairment and/or maculopathy in three consecutive generations (or in two generations if 2 or 3 members of a family were affected). We sequenced the mtDNA in the 11 probands, in their mothers and in 80 controls. We identified 33 diabetes-suspected mutations, 1/33 was 3243A>G. Most patients (91%) and their mothers had mutations in complex I and/or IV of the respiratory chain. We measured the activity of these two enzymes and found that they were less active in mutated patients and their mothers than in the healthy control pool. The prevalence of hearing loss (36% vs 75–98%) and macular dystrophy (54% vs 86%) was lower in our mitochondrial diabetic adolescents than reported in adults. Moreover, we found a hitherto unknown association between mitochondrial diabetes and celiac disease. In conclusion, mitochondrial diabetes should be considered a complex syndrome with several phenotypic variants. Moreover, deafness is not an essential component of the disease in children. The whole mtDNA should be screened because the 3243A>G variant is not as frequent in children as in adults. In fact, 91% of our patients were mutated in the complex I and/or IV genes. The enzymatic assay may be a useful tool with which to confirm the pathogenic significance of detected variants. PMID:22536343

ROS as Regulators of Mitochondrial Dynamics in Neurons.

PubMed

Cid-Castro, Carolina; Hernández-Espinosa, Diego Rolando; Morán, Julio

2018-07-01

Mitochondrial dynamics is a complex process, which involves the fission and fusion of mitochondrial outer and inner membranes. These processes organize the mitochondrial size and morphology, as well as their localization throughout the cells. In the last two decades, it has become a spotlight due to their importance in the pathophysiological processes, particularly in neurological diseases. It is known that Drp1, mitofusin 1 and 2, and Opa1 constitute the core of proteins that coordinate this intricate and dynamic process. Likewise, changes in the levels of reactive oxygen species (ROS) lead to modifications in the expression and/or activity of the proteins implicated in the mitochondrial dynamics, suggesting an involvement of these molecules in the process. In this review, we discuss the role of ROS in the regulation of fusion/fission in the nervous system, as well as the involvement of mitochondrial dynamics proteins in neurodegenerative diseases.

Fly Models of Human Diseases: Drosophila as a Model for Understanding Human Mitochondrial Mutations and Disease.

PubMed

Sen, A; Cox, R T

2017-01-01

Mitochondrial diseases are a prevalent, heterogeneous class of diseases caused by defects in oxidative phosphorylation, whose severity depends upon particular genetic mutations. These diseases can be difficult to diagnose, and current therapeutics have limited efficacy, primarily treating only symptoms. Because mitochondria play a pivotal role in numerous cellular functions, especially ATP production, their diminished activity has dramatic physiological consequences. While this in and of itself makes treating mitochondrial disease complex, these organelles contain their own DNA, mtDNA, whose products are required for ATP production, in addition to the hundreds of nucleus-encoded proteins. Drosophila offers a tractable whole-animal model to understand the mechanisms underlying loss of mitochondrial function, the subsequent cellular and tissue damage that results, and how these organelles are inherited. Human and Drosophila mtDNAs encode the same set of products, and the homologous nucleus-encoded genes required for mitochondrial function are conserved. In addition, Drosophila contain sufficiently complex organ systems to effectively recapitulate many basic symptoms of mitochondrial diseases, yet are relatively easy and fast to genetically manipulate. There are several Drosophila models for specific mitochondrial diseases, which have been recently reviewed (Foriel, Willems, Smeitink, Schenck, & Beyrath, 2015). In this review, we highlight the conservation between human and Drosophila mtDNA, the present and future techniques for creating mtDNA mutations for further study, and how Drosophila has contributed to our current understanding of mitochondrial inheritance. © 2017 Elsevier Inc. All rights reserved.

Liver mitochondrial dysfunction and electron transport chain defect induced by high dietary copper in broilers.

PubMed

Yang, Fan; Cao, Huabin; Su, Rongsheng; Guo, Jianying; Li, Chengmei; Pan, Jiaqiang; Tang, Zhaoxin

2017-09-01

Copper is an important trace mineral in the diet of poultry due to its biological activity. However, limited information is available concerning the effects of high copper on mitochondrial dysfunction. In this study, 72 broilers were used to investigate the effects of high dietary copper on liver mitochondrial dysfunction and electron transport chain defect. Birds were fed with different concentrations [11, 110, 220, and 330 mg of copper/kg dry matter (DM)] of copper from tribasic copper chloride (TBCC). The experiment lasted for 60 d. Liver tissues on d 60 were subjected to histopathological observation. Additionally, liver mitochondrial function was recorded on d 12, 36, and 60. Moreover, a site-specific defect in the electron transport chain in liver mitochondria was also identified by using various chemical inhibitors of mitochondrial respiration. The results showed different degrees of degeneration, mitochondrial swelling, and high-density electrons in hepatocytes. In addition, the respiratory control ratio (RCR) and oxidative phosphorylation rate (OPR) in liver mitochondria increased at first and then decreased in high-dose groups. Moreover, hydrogen peroxide (H2O2) generation velocity in treated groups was higher than that in control group, which were magnified by inhibiting electron transport at Complex IV. The results indicated that high dietary copper could decline liver mitochondrial function in broilers. The presence of a site-specific defect at Complex IV in liver mitochondria may be responsible for liver mitochondrial dysfunction caused by high dietary copper. © 2017 Poultry Science Association Inc.

Bim and VDAC1 are hierarchically essential for mitochondrial ATF2 mediated cell death.

PubMed

Liu, Zhaoyun; Luo, Qianfu; Guo, Chunbao

2015-01-01

ATF2 mediated cytochrome c release is the formation of a channel with some unknown factors larger than that of the individual proteins. BHS-only proteins (BH3s), such as Bim, could induce BAX and VDAC, forming a new channel. According to this facts, we can speculated that there is possible signal relationship with BH3s and ATF2, which is associated with mitochondrial-based death programs. The growth inhibitory effects of mitochondrial ATF2 were tested in cancer cell lines B16F10, A549, EG7, and LL2. Apoptosis was measured by flow cytometry. The effects of ATF2 and levels of apoptosis regulatory proteins were measured by Western blotting. The interaction of proteins were evaluated by immunoprecipitation analysis. The in vivo antitumor activity of mitochondrial ATF2 were tested in xenograft B16F10 models. Genotoxic stress enabled mitochondrial ATF2 accumulation, perturbing the HK1-VDAC1 complex, increasing mitochondrial permeability, and promoting apoptosis. ATF2 inhibition strongly reduced the conformational activation of Bim, suggesting that Bim acts downstream of ATF2. Although Bim downregulation had no effect on ATF2 activation, Bim knockdown abolished VDAC1 activation; the failure of VDAC1 activation in Bim-depleted cells could be reversed by the BH3-only protein mimic ABT-737. We also demonstrate that silencing of ATF2 in B16F10 cells increases both the incidence and prevalence of tumor xenografts in vivo, whereas stably mitochondrial ATF2 transfection inhibited B16F10 tumor xenografts growth. Altogether, these results show that ATF2 is a component of the apoptosis machinery that involves a hierarchical contribution of ATF2, Bim, and VDAC1. Our data offer new insight into the mechanism of mitochondrial ATF2 in mitochondrial apoptosis.

Growth hormone deficiency in a patient with mitochondrial disease.

PubMed

Rocha, Vera; Rocha, Dalila; Santos, Helena; Sales Marques, Jorge

2015-09-01

Mitochondrial respiratory chain (MRC) disorders, defined as primary diseases of the oxidative phosphorylation system, are a protean group of metabolic disorders, difficult to diagnose and classify. The diagnosis is complex and requires the integration of information obtained by clinical, laboratory testing, imaging and muscle biopsy. They may be associated with endocrine disorders, including hypothyroidism, diabetes mellitus, hyperinsulinemia and growth hormone (GH) deficiency. We describe a case of five years old male with polymalformative syndrome with a systemic involvement. At 6 months of age, he was sent to metabolic consultation because of facial dysmorphy and short stature. During the investigation it was diagnosed at the boy a growth hormone deficiency and because of his multisystemic involvement, muscle biopsy was carried out and showed reduced activity of complex II (38%) of the mitochondrial respiratory chain. Currently, the boy is under GH therapy with growth in the 5th percentile and coenzime Q10. Mitochondrial biology is one of the fastest growing areas in genetics and medicine. Disturbances in mitochondrial metabolism are now known to play a role not only in rare childhood diseases, but also in many common diseases of aging. In mitochondrial disorders, short stature is a common symptom, but its underlying lesion, growth hormone deficiency, is rarely investigated.

Mitochondrial-nuclear interactions and accelerated compensatory evolution: evidence from the primate cytochrome C oxidase complex.

PubMed

Osada, Naoki; Akashi, Hiroshi

2012-01-01

Accelerated rates of mitochondrial protein evolution have been proposed to reflect Darwinian coadaptation for efficient energy production for mammalian flight and brain activity. However, several features of mammalian mtDNA (absence of recombination, small effective population size, and high mutation rate) promote genome degradation through the accumulation of weakly deleterious mutations. Here, we present evidence for "compensatory" adaptive substitutions in nuclear DNA- (nDNA) encoded mitochondrial proteins to prevent fitness decline in primate mitochondrial protein complexes. We show that high mutation rate and small effective population size, key features of primate mitochondrial genomes, can accelerate compensatory adaptive evolution in nDNA-encoded genes. We combine phylogenetic information and the 3D structure of the cytochrome c oxidase (COX) complex to test for accelerated compensatory changes among interacting sites. Physical interactions among mtDNA- and nDNA-encoded components are critical in COX evolution; amino acids in close physical proximity in the 3D structure show a strong tendency for correlated evolution among lineages. Only nuclear-encoded components of COX show evidence for positive selection and adaptive nDNA-encoded changes tend to follow mtDNA-encoded amino acid changes at nearby sites in the 3D structure. This bias in the temporal order of substitutions supports compensatory weak selection as a major factor in accelerated primate COX evolution.

Insulin upregulates GRIM-19 and protects cardiac mitochondrial morphology in type 1 diabetic rats partly through PI3K/AKT signaling pathway.

PubMed

Li, Yong-Guang; Dong, Zhi-Feng; Chen, Kan-Kai; He, Ya-Ping; Dai, Xiao-Yan; Li, Shuai; Li, Jing-Bo; Zhu, Wei; Wei, Meng

2017-11-04

Insulin is involved in the development of diabetic heart disease and is important in the activities of mitochondrial complex I. However, the effect of insulin on cardiac mitochondrial nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1 subunit of retinoic-interferon-induced mortality 19 (GRIM-19) has not been characterized. The aim of this study was to investigate the effect of insulin on the mitochondrial GRIM-19 in the hearts of rats with streptozotocin (STZ)-induced type 1 diabetes. Protein changes of GRIM-19 were evaluated by western blotting and reverse transcription-quantitative polymerase chain reaction. Furthermore, the effects of insulin on mitochondrial complex I were detected in HeLa cells and H9C2 cardiac myocytes. During the development of diabetic heart disease, the cardiac function did not change within the 8 weeks, but the mitochondrial morphology was altered. The hearts from the rats with STZ-induced diabetes exhibited reduced expression of GRIM-19. Prior to the overt cardiac dilatation, mitochondrial alterations were already present. Following subcutaneous insulin injection, it was demonstrated that GRIM-19 protein was altered, as well as the mitochondrial morphology. The phosphoinositide 3-kinase inhibitor LY294002 had an effect on insulin signaling in H9C2 cardiacmyocytes, and decreased the level of GRIM-19 by half compared with that in the insulin group. The results indicate that insulin is essential for the control of cardiac mitochondrial morphology and the GRIM-19 expression partly via PI3K/AKT signaling pathways. Copyright © 2017. Published by Elsevier Inc.

Photosensitization of Intact Heart Mitochondria by the Phthalocyanine Pc 4: Correlation of Structural and Functional Deficits with Cytochrome c Release

PubMed Central

Kim, Junhwan; Fujioka, Hisashi; Oleinick, Nancy L.; Anderson, Vernon E.

2010-01-01

Singlet oxygen is produced by absorption of red light by the phthalocyanine dye, Pc 4, followed by energy transfer to dissolved triplet oxygen. Mitochondria pre-incubated with Pc 4 were illuminated by red light and the damage to mitochondrial structure and function by the generated singlet oxygen was studied. At early illumination times (3–5 min. of red light exposure), state 3 respiration was inhibited (50%) while state 4 activity increased, resulting in effectively complete uncoupling. Individual complex activities were measured and only complex IV activity was significantly reduced and exhibited a dose response while the activities of electron transport complexes I, II and III were not significantly affected. Cyt c release was an increasing function of irradiation time with 30% being released following 5 min. of illumination. Mitochondrial expansion along with changes in the structure of the cristae were observed by transmission electron microscopy following 5 min. of irradiation with an increase of large vacuoles and membrane rupture occurring following more extensive exposures. PMID:20510354

Minnelide/Triptolide Impairs Mitochondrial Function by Regulating SIRT3 in P53-Dependent Manner in Non-Small Cell Lung Cancer.

PubMed

Kumar, Ajay; Corey, Catherine; Scott, Iain; Shiva, Sruti; D'Cunha, Jonathan

2016-01-01

Minnelide/Triptolide (TL) has recently emerged as a potent anticancer drug in non-small cell lung cancer (NSCLC). However, the precise mechanism of its action remains ambiguous. In this study, we elucidated the molecular basis for TL-induced cell death in context to p53 status. Cell death was attributed to dysfunction of mitochondrial bioenergetics in p53-deficient cells, which was characterized by decreased mitochondrial respiration, steady-state ATP level and membrane potential, but augmented reactive oxygen species (ROS). Increased ROS production resulted in oxidative stress in TL-treated cells. This was exhibited by elevated nuclear levels of a redox-sensitive transcriptional factor, NF-E2-related factor-2 (NRF2), along with diminished cellular glutathione (GSH) content. We further demonstrated that in the absence of p53, TL blunted the expression of mitochondrial SIRT3 triggering increased acetylation of NDUAF9 and succinate dehydrogenase, components of complexes I and II of the electron transport chain (ETC). TL-mediated hyperacetylation of complexes I and II proteins and these complexes displayed decreased enzymatic activities. We also provide the evidence that P53 regulate steady-state level of SIRT3 through Proteasome-Pathway. Finally, forced overexpression of Sirt3, but not deacetylase-deficient mutant of Sirt3 (H243Y), restored the deleterious effect of TL on p53-deficient cells by rescuing mitochondrial bioenergetics. On contrary, Sirt3 deficiency in the background of wild-type p53 triggered TL-induced mitochondrial impairment that echoed TL effect in p53-deficeint cells. These findings illustrate a novel mechanism by which TL exerts its potent effects on mitochondrial function and ultimately the viability of NSCLC tumor.

Toxoplasma gondii Infection Is Associated with Mitochondrial Dysfunction in-Vitro

PubMed Central

Syn, Genevieve; Anderson, Denise; Blackwell, Jenefer M.; Jamieson, Sarra E.

2017-01-01

Upon invasion of host cells, the ubiquitous pathogen Toxoplasma gondii manipulates several host processes, including re-organization of host organelles, to create a replicative niche. Host mitochondrial association to T. gondii parasitophorous vacuoles is rapid and has roles in modulating host immune responses. Here gene expression profiling of T. gondii infected cells reveals enrichment of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial dysfunction 6 h post-infection. We identified 11 hub genes (HIF-1α, CASP8, FN1, POU5F1, CD44, ISG15, HNRNPA1, MDM2, RPL35, VHL, and NUPR1) and 10 predicted upstream regulators, including 4 endogenous regulators RICTOR, KDM5A, RB1, and D-glucose. We characterized a number of mitochondrial parameters in T. gondii infected human foreskin fibroblast cells over a 36 h time-course. In addition to the usual rapid recruitment and apparent enlargement of mitochondria around the parasitophorous vacuole we observed fragmented host mitochondria in infected cells, not linked to cellular apoptosis, from 24 h post-infection. An increase in mitochondrial superoxide levels in T. gondii infected cells was observed that required active parasite invasion and peaked at 30 h post-infection. Measurement of OXPHOS proteins showed decreased expression of Complex IV in infected cells at 24 h post-infection, followed by decreased expression of Complexes I and II at 36 h post-infection. No change occurred in Complex V. No difference in host mitochondrial membrane potential between infected and mock-infected cells was observed at any time. Our results show perturbation of host mitochondrial function following T. gondii infection that likely impacts on pathogenesis of disease. PMID:29312892

Muscarinic Receptor Activation Protects Cells from Apoptotic Effects of DNA Damage, Oxidative Stress, and Mitochondrial Inhibition*

PubMed Central

De Sarno, Patrizia; Shestopal, Svetlana A.; King, Taj D.; Zmijewska, Anna; Song, Ling; Jope, Richard S.

2006-01-01

The impact of muscarinic receptor stimulation was examined on apoptotic signaling induced by DNA damage, oxidative stress, and mitochondrial impairment. Exposure of human neuroblastoma SH-SY5Y cells to the DNA-damaging agent camptothecin increased p53 levels, activated caspase-3, and caused cell death. Pretreatment with oxotremorine-M, a selective agonist of muscarinic receptors that are expressed endogenously in these cells, did not affect the accumulation of p53 but greatly attenuated caspase-3 activation and protected from cell death to nearly the same extent as treatment with a general caspase inhibitor. Treatment with 50–200 μm H2O2 caused the activation of caspase-3 beginning after 2–3 h, followed by eventual cell death. Oxotremorine-M pretreatment protected cells from H2O2-inducedcaspase-3 activation and death, and this was equivalent to protection afforded by a caspase inhibitor. Muscarinic receptor stimulation also protected cells from caspase-3 activation induced by exposure to rotenone, a mitochondrial complex 1 inhibitor, but no protection was evident from staurosporine-induced caspase-3 activation. The mechanism of protection afforded by muscarinic receptor activation from camptothecin-induced apoptotic signaling involved blockade of mitochondrial cytochrome c release associated with a bolstering of mitochondrial bcl-2 levels and blockade of the translocation of Bax to mitochondria. Likely the most proximal of these events to muscarinic receptor activation, mitochondrial Bax accumulation, also was attenuated by oxotremorine-M treatment after treatment with H2O2 or rotenone. These results demonstrate that stimulation of muscarinic receptors provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. These findings suggest that neurotransmitter-induced signaling bolsters survival mechanisms, and inadequate neurotransmission may exacerbate neuronal loss. PMID:12538580

Garlic activates SIRT-3 to prevent cardiac oxidative stress and mitochondrial dysfunction in diabetes.

PubMed

Sultana, Md Razia; Bagul, Pankaj K; Katare, Parameshwar B; Anwar Mohammed, Soheb; Padiya, Raju; Banerjee, Sanjay K

2016-11-01

Cardiac complications are major contributor in the mortality of diabetic people. Mitochondrial dysfunctioning is a crucial contributor for the cardiac complications in diabetes, and SIRT-3 remains the major mitochondrial deacetylase. We hypothesized whether garlic has any role on SIRT-3 to prevent mitochondrial dysfunction in diabetic heart. Rats with developed hyperglycemia after STZ injection were divided into two groups; diabetic (Dia) and diabetic+garlic (Dia+Garl). Garlic was administered at a dose of 250mg/kg/day, orally for four weeks. An additional group was maintained to evaluate the effect of raw garlic administration on control rat heart. We have observed altered functioning of cardiac mitochondrial enzymes involved in metabolic pathways, and increased levels of cardiac ROS with decreased activity of catalase and SOD in diabetic rats. Cardiac mRNA expression of TFAM, PGC-1α, and CO1 was also altered in diabetes. In addition, reduced levels of electron transport chain complexes that observed in Dia group were normalized with garlic administration. This indicates the presence of increased oxidative stress with mitochondrial dysfunctioning in diabetic heart. We have observed reduced activity of SIRT3 and increased acetylation of MnSOD. Silencing SIRT-3 in cells also revealed the same. However, administration of garlic improved the SIRT-3 and MnSOD activity, by deacetylating MnSOD. Increased SOD activity was correlated with reduced levels of ROS in garlic-administered rat hearts. Collectively, our results provide an insight into garlic's protection to T1DM heart through activation of SIRT3-MnSOD pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

N-Methyl, N-propynyl-2-phenylethylamine (MPPE), a Selegiline Analog, Attenuates MPTP-induced Dopaminergic Toxicity with Guaranteed Behavioral Safety: Involvement of Inhibitions of Mitochondrial Oxidative Burdens and p53 Gene-elicited Pro-apoptotic Change.

PubMed

Shin, Eun-Joo; Nam, Yunsung; Lee, Ji Won; Nguyen, Phuong-Khue Thi; Yoo, Ji Eun; Tran, The-Vinh; Jeong, Ji Hoon; Jang, Choon-Gon; Oh, Young J; Youdim, Moussa B H; Lee, Phil Ho; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2016-11-01

Selegiline is a monoamine oxidase-B (MAO-B) inhibitor with anti-Parkinsonian effects, but it is metabolized to amphetamines. Since another MAO-B inhibitor N-Methyl, N-propynyl-2-phenylethylamine (MPPE) is not metabolized to amphetamines, we examined whether MPPE induces behavioral side effects and whether MPPE affects dopaminergic toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Multiple doses of MPPE (2.5 and 5 mg/kg/day) did not show any significant locomotor activity and conditioned place preference, whereas selegiline (2.5 and 5 mg/kg/day) significantly increased these behavioral side effects. Treatment with MPPE resulted in significant attenuations against decreases in mitochondrial complex I activity, mitochondrial Mn-SOD activity, and expression induced by MPTP in the striatum of mice. Consistently, MPPE significantly attenuated MPTP-induced oxidative stress and MPPE-mediated antioxidant activity appeared to be more pronounced in mitochondrial-fraction than in cytosolic-fraction. Because MPTP promoted mitochondrial p53 translocation and p53/Bcl-xL interaction, it was also examined whether mitochondrial p53 inhibitor pifithrin-μ attenuates MPTP neurotoxicity. MPPE, selegiline, or pifithrin-μ significantly attenuated mitochondrial p53/Bcl-xL interaction, impaired mitochondrial transmembrane potential, cytosolic cytochrome c release, and cleaved caspase-3 in wild-type mice. Subsequently, these compounds significantly ameliorated MPTP-induced motor impairments. Neuroprotective effects of MPPE appeared to be more prominent than those of selegiline. MPPE or selegiline did not show any additional protective effects against the attenuation by p53 gene knockout, suggesting that p53 gene is a critical target for these compounds. Our results suggest that MPPE possesses anti-Parkinsonian potentials with guaranteed behavioral safety and that the underlying mechanism of MPPE requires inhibition of mitochondrial oxidative stress, mitochondrial translocation of p53, and pro-apoptotic process.

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

PubMed Central

Madina, Bhaskara R.; Kumar, Vikas; Metz, Richard; Mooers, Blaine H.M.; Bundschuh, Ralf; Cruz-Reyes, Jorge

2014-01-01

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3′-to-5′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3′ ends and strain-specific alternative 3′ editing within 3′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs. PMID:24865612

StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation

PubMed Central

Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas

2014-01-01

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR. PMID:24422629

CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

PubMed

Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing

2016-09-01

A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p < 0.05), (ii) elevation in ΔΨm (p < 0.05), (iii) increased OCR and ATP formation (p < 0.05), (iv) increased intracellular NO levels (p < 0.05), (v) increased mitochondrial ROS production (p < 0.05), and (vi) increased susceptibility to rotenone (p < 0.05). Treatment with isradipine was able to partially rescue these negative effects of CNTF-ACM (p < 0.05). CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

PKCδ knockout mice are protected from para-methoxymethamphetamine-induced mitochondrial stress and associated neurotoxicity in the striatum of mice.

PubMed

Shin, Eun-Joo; Dang, Duy-Khanh; Tran, Hai-Quyen; Nam, Yunsung; Jeong, Ji Hoon; Lee, Young Hun; Park, Kyung Tae; Lee, Yong Sup; Jang, Choon-Gon; Hong, Jau-Shyong; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2016-11-01

Para-methoxymethamphetamine (PMMA) is a para-ring-substituted amphetamine derivative sold worldwide as an illegal psychotropic drug. Although PMMA use has been reported to lead to severe intoxication and even death, little is known about the mechanism(s) by which PMMA exerts its neurotoxic effects. Here we found that PMMA treatment resulted in phosphorylation of protein kinase Cδ (PKCδ) and subsequent mitochondrial translocation of cleaved PKCδ. PMMA-induced oxidative stress was more pronounced in mitochondria than in the cytosol. Moreover, treatment with PMMA consistently resulted in significant reductions in mitochondrial membrane potential, mitochondrial complex I activity, and mitochondrial Mn superoxide dismutase-immunoreactivity. In contrast, PMMA treatment led to a significant increase in intramitochondrial Ca 2+ level. Treatment with PMMA also significantly increased ionized calcium binding adaptor molecule 1 (Iba-1)-labeled microglial activation and upregulated tumor necrosis factor alpha (TNF-α) gene expression. PKCδ knockout attenuated these mitochondrial effects and dampened the neurotoxic effects of PMMA. Importantly, TNF-α knockout mice were significantly protected from PMMA-induced increases in phospho-PKCδ expression, mitochondrial translocation of cleaved PKCδ, and Iba-1-labeled microgliosis. Both rottlerin, a pharmacological inhibitor of PKCδ, and etanercept, a pharmacological inhibitor of TNF-α, significantly protected against PMMA-mediated induction of apoptosis, as assessed by terminal deoxynucleotidyl transferase dUDP nick end labeling (TUNEL) assays. In addition, PKCδ knockout and TNF-α knockout both resulted in decreased PMMA-mediated induction of dopaminergic loss. Therefore, our results suggest that PKCδ mediates PMMA-induced neurotoxicity by facilitating oxidative stress (mitochondria > cytosol), mitochondrial dysfunction, microglial activation, and pro-apoptotic signaling. Our results also indicate that PMMA-induced PKCδ activation requires the proinflammatory cytokine TNF-α. Copyright © 2016. Published by Elsevier Ltd.

An electrocardiographic, molecular and biochemical approach to explore the cardioprotective effect of vasopressin and milrinone against phosphide toxicity in rats.

PubMed

Jafari, Abbas; Baghaei, Amir; Solgi, Reza; Baeeri, Maryam; Chamanara, Mohsen; Hassani, Shokoufeh; Gholami, Mahdi; Ostad, Seyed Nasser; Sharifzadeh, Moahmmad; Abdollahi, Mohammad

2015-06-01

The present study was conducted to identify the protective effect of vasopressin (AVP) and milrinone on cardiovascular function, mitochondrial complex activities, cellular ATP reserve, oxidative stress, and apoptosis in rats poisoned by aluminum phosphide (AlP). Rats were divided into five groups (n = 12) including control, AlP (12.5 mg/kg), AlP + AVP (2.0 Units/kg), AlP + milrinone (0.25 mg/kg) and AlP + AVP + milrinone. After treatment, the animals were connected to an electronic cardiovascular monitoring device to monitor electrocardiographic (ECG) parameter. Finally, oxidative stress biomarkers, mitochondrial complex activities, ADP/ATP ratio and apoptosis were evaluated on the heart tissues. Results indicated that AlP administration induced ECG abnormalities along with a decline in blood pressure and heart rate. AVP and milrinone significantly ameliorated these changes in all treated groups. Considerable protective effects on oxidative stress biomarkers, complex IV activity, ADP/ATP ratio and caspase-3 and -9 activities in treated groups were also found. These findings were supported by flow cytometry assay of cardiomyocytes. In conclusion, administration of AVP and milrinone, not only improve cardiovascular functions in AlP poisoned rats in the short time, but after a long time can also restore mitochondrial function and ATP level and reduce the oxidative damage, which prevent cardiomyocytes from entering the apoptotic phase. Copyright © 2015 Elsevier Ltd. All rights reserved.

Changes in mitochondrial electron transport chain activity during insect metamorphosis.

PubMed

Chamberlin, M E

2007-02-01

The midgut of the tobacco hornworm (Manduca sexta) is a highly aerobic tissue that is destroyed by programmed cell death during larval-pupal metamorphosis. The death of the epithelium begins after commitment to pupation, and the oxygen consumption of isolated midgut mitochondria decreases soon after commitment. To assess the role of the electron transport chain in this decline in mitochondrial function, the maximal activities of complexes I-IV of the respiratory chain were measured in isolated midgut mitochondria. Whereas there were no developmental changes in the activity of complex I or III, activities of complexes II and IV [cytochrome c oxidase (COX)] were higher in mitochondria from precommitment than postcommitment larvae. This finding is consistent with a higher rate of succinate oxidation in mitochondria isolated from precommitment larvae and reveals that the metamorphic decline in mitochondrial respiration is due to the targeted destruction or inactivation of specific sites within the mitochondria, rather than the indiscriminate destruction of the organelles. The COX turnover number (e- x s(-1) x cytochrome aa3(-1)) was greater for the enzyme from precommitment than postcommitment larvae, indicating a change in the enzyme structure and/or its lipid environment during the early stages of metamorphosis. The turnover number of COX in the intact mitochondria (in organello COX) was also lower in postcommitment larvae. In addition to changes in the protein or membrane phospholipids, the metamorphic decline in this rate constant may be a result of the observed loss of endogenous cytochrome c.

Metaxins 1 and 2, two proteins of the mitochondrial protein sorting and assembly machinery, are essential for Bak activation during TNF alpha triggered apoptosis.

PubMed

Cartron, Pierre-François; Petit, Elise; Bellot, Grégory; Oliver, Lisa; Vallette, François M

2014-09-01

The proteins Bax and Bak are central in the execution phase of apoptosis; however, little is known about the partners involved in the control of this complex process. Here, we show that mitochondrial Bak is incorporated into a VDAC2/Mtx1/Mtx2 multi-protein complex in both resting and dying cells. VDAC2 is a porin that has previously been described as a partner of Bak while Mtx1 and Mtx2 are two proteins of the mitochondrial sorting and assembly machinery (SAM) that have been implicated in TNF-induced apoptosis. We show that, after the induction of apoptosis, Bak switches from its association with Mtx2 and VDAC2 to interact with Mtx1. Copyright © 2014 Elsevier Inc. All rights reserved.

Regional assessment of energy-producing metabolic activity in the endothelium of donor corneas.

PubMed

Greiner, Mark A; Burckart, Kimberlee A; Wagoner, Michael D; Schmidt, Gregory A; Reed, Cynthia R; Liaboe, Chase A; Flamme-Wiese, Miles J; Zimmerman, M Bridget; Mullins, Robert F; Kardon, Randy H; Goins, Kenneth M; Aldrich, Benjamin T

2015-05-01

We characterized mitochondrial respiration and glycolysis activity of human corneal endothelium, and compared metabolic activity between central and peripheral regions. Endothelial keratoplasty-suitable corneas were obtained from donors aged 50 to 75 years. The endothelium-Descemet membrane complex (EDM) was isolated, and 3-mm punches were obtained from central and peripheral regions. Endothelium-Descemet membrane punches were assayed for mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification) using an extracellular flux analyzer. Enzymatic (citrate synthase, glucose hexokinase) and mitochondrial density (MitoTracker) assays also were performed. Ten corneas were analyzed per assay. Metabolic activity for mitochondrial respiration and glycolysis showed expected changes to assay compounds (P < 0.01, all pairwise comparisons). Basal mitochondrial respiration and glycolysis activity did not differ between regions (P > 0.99). Similarly, central versus peripheral activity after assay compound treatment showed no significant differences (P > 0.99, all time points). The intracorneal coefficient of variation for basal readings between two and four peripheral punches was 18.5% of the mean. Although peripheral samples displayed greater enzymatic activity than central samples (P < 0.05), similar to extracellular flux results, mitochondrial density did not differ between regions (P = 0.78). Extracellular flux analysis of oxygen and pH is a valid technique for characterizing metabolic activity of human corneal endothelium. This technique demonstrates high reproducibility, allows quantification of metabolic parameters using small quantities of live cells, and permits estimation of overall metabolic output. Neither oxygen consumption nor extracellular acidification differed between central and peripheral regions of transplant suitable corneas in this series. Our results show that endothelial cell health can be quantified biochemically in transplant suitable corneas.

Evidence for inter-specific recombination among the mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex.

PubMed

Fourie, Gerda; van der Merwe, Nicolaas A; Wingfield, Brenda D; Bogale, Mesfin; Tudzynski, Bettina; Wingfield, Michael J; Steenkamp, Emma T

2013-09-08

The availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes. In the Gibberella fujikuroi species complex, the exact relationships among the so-called "African", "Asian" and "American" Clades remain largely unresolved, irrespective of the markers employed. In this study, we considered the feasibility of using mitochondrial genes to infer the phylogenetic relationships among Fusarium species in this complex. The mitochondrial genomes of representatives of the three Clades (Fusarium circinatum, F. verticillioides and F. fujikuroi) were characterized and we determined whether or not the mitochondrial genomes of these fungi have value in resolving the higher level evolutionary relationships in the complex. Overall, the mitochondrial genomes of the three species displayed a high degree of synteny, with all the genes (protein coding genes, unique ORFs, ribosomal RNA and tRNA genes) in identical order and orientation, as well as introns that share similar positions within genes. The intergenic regions and introns generally contributed significantly to the size differences and diversity observed among these genomes. Phylogenetic analysis of the concatenated protein-coding dataset separated members of the Gibberella fujikuroi complex from other Fusarium species and suggested that F. fujikuroi ("Asian" Clade) is basal in the complex. However, individual mitochondrial gene trees were largely incongruent with one another and with the concatenated gene tree, because six distinct phylogenetic trees were recovered from the various single gene datasets. The mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex are remarkably similar to those of the previously characterized Fusarium species and Sordariomycetes. Despite apparently representing a single replicative unit, all of the genes encoded on the mitochondrial genomes of these fungi do not share the same evolutionary history. This incongruence could be due to biased selection on some genes or recombination among mitochondrial genomes. The results thus suggest that the use of individual mitochondrial genes for phylogenetic inference could mask the true relationships between species in this complex.

Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

PubMed Central

Park, Song-Young; Gifford, Jayson R.; Andtbacka, Robert H. I.; Trinity, Joel D.; Hyngstrom, John R.; Garten, Ryan S.; Diakos, Nikolaos A.; Ives, Stephen J.; Dela, Flemming; Larsen, Steen; Drakos, Stavros

2014-01-01

Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s−1·mg−1, P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g−1·min−1, P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s−1·mg−1, P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production. PMID:24906913

Neuroprotective Efficacy of Mitochondrial Antioxidant MitoQ in Suppressing Peroxynitrite-Mediated Mitochondrial Dysfunction Inflicted by Lead Toxicity in the Rat Brain.

PubMed

Maiti, Arpan Kumar; Saha, Nimai Chandra; More, Sunil S; Panigrahi, Ashish Kumar; Paul, Goutam

2017-04-01

Lead (Pb) is one of the most pollutant metals that accumulate in the brain mitochondria disrupting mitochondrial structure and function. Though oxidative stress mediated by reactive oxygen species remains the most accepted mechanism of Pb neurotoxicity, some reports suggest the involvement of nitric oxide ( • NO) and reactive nitrogen species in Pb-induced neurotoxicity. But the impact of Pb neurotoxicity on mitochondrial respiratory enzyme complexes remains unknown with no relevant report highlighting the involvement of peroxynitrite (ONOO - ) in it. Herein, we investigated these effects in in vivo rat model by oral application of MitoQ, a known mitochondria-specific antioxidant with ONOO - scavenging activity. Interestingly, MitoQ efficiently alleviated ONOO - -mediated mitochondrial complexes II, III and IV inhibition, increased mitochondrial ATP production and restored mitochondrial membrane potential. MitoQ lowered enhanced caspases 3 and 9 activities upon Pb exposure and also suppressed synaptosomal lipid peroxidation and protein oxidation accompanied by diminution of nitrite production and protein-bound 3-nitrotyrosine. To ascertain our in vivo findings on mitochondrial dysfunction, we carried out similar experiments in the presence of different antioxidants and free radical scavengers in the in vitro SHSY5Y cell line model. MitoQ provided better protection compared to mercaptoethylguanidine, N-nitro-L-arginine methyl ester and superoxide dismutase suggesting the predominant involvement of ONOO - compared to • NO and O 2 •- . However, dimethylsulphoxide and catalase failed to provide protection signifying the noninvolvement of • OH and H 2 O 2 in the process. The better protection provided by MitoQ in SHSY5Y cells can be attributed to the fact that MitoQ targets mitochondria whereas mercaptoethylguanidine, N-nitro-L-arginine methyl ester and superoxide dismutase are known to target mainly cytoplasm and not mitochondria. Taken together the results from the present study clearly brings out the potential of MitoQ against ONOO - -induced toxicity upon Pb exposure indicating its therapeutic potential in metal toxicity.

Protective effects of phosphodiesterase-1 (PDE1) and ATP sensitive potassium (KATP) channel modulators against 3-nitropropionic acid induced behavioral and biochemical toxicities in experimental Huntington׳s disease.

PubMed

Gupta, Surbhi; Sharma, Bhupesh

2014-06-05

Huntington׳s disease (HD), a devastating neurodegenerative disorder, is characterized by weight loss, impairment of motor function, cognitive dysfunction, neuropsychiatric disturbances and striatal damage. Phosphodiesterase-1 (PDE1) has been implicated in various neurological diseases. Mitochondrial potassium channels in the brain take part in neuroprotection. This study has been structured to investigate the role of vinpocetine, a selective PDE1 inhibitor as well as nicorandil, selective ATP sensitive potassium (KATP) channel opener in 3-nitropropionic acid (3-NP) induced HD symptoms in rats. Systemic administration of 3-NP significantly, reduced body weight, impaired locomotion, grip strength and impaired cognition. 3-NP elicited marked oxidative stress in the brain (enhanced malondialdehyde-MDA, reduced glutathione-GSH content, superoxide dismutase-SOD and catalase-CAT), elevated brain acetylcholinesterase activity and inflammation (myeloperoxidase-MPO), with marked nitrosative stress (nitrite/nitrate) in the brain. 3-NP has also induced mitochondrial dysfunction (impaired mitochondrial NADH dehydrogenase-complex I, succinate dehydrogenase-complex II and cytochrome oxidase-complex IV) activities in the striatum of the rat. Tetrabenazine was used as a positive control. Treatment with vinpocetine, nicorandil and tetrabenazine ameliorated 3-NP induced reduction in body weight, impaired locomotion, grip strength and impaired cognition. Treatment with these drugs reduced brain striatum oxidative (MDA, GSH, SOD and CAT) and nitrosative (nitrite/nitrate) stress, acetylcholinesterase activity, inflammation and mitochondrial dysfunctions. These results indicate that vinpocetine, a selective PDE1 inhibitor and nicorandil, a KATP channel opener have attenuated 3-NP induced experimental HD. Hence, pharmacological modulation of PDE1 as well as KATP channels may be considered as potential research targets for mitigation of HD. Copyright © 2014 Elsevier B.V. All rights reserved.

Pharmacological Inhibition of Poly(ADP-Ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle

PubMed Central

Pirinen, Eija; Canto, Carles; Jo, Young-Suk; Morato, Laia; Zhang, Hongbo; Menzies, Keir; Williams, Evan G.; Mouchiroud, Laurent; Moullan, Norman; Hagberg, Carolina; Li, Wei; Timmers, Silvie; Imhof, Ralph; Verbeek, Jef; Pujol, Aurora; van Loon, Barbara; Viscomi, Carlo; Zeviani, Massimo; Schrauwen, Patrick; Sauve, Anthony; Schoonjans, Kristina; Auwerx, Johan

2014-01-01

SUMMARY We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function. PMID:24814482

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

PubMed Central

Oran, Amanda R.; Adams, Clare M.; Zhang, Xiao-yong; Gennaro, Victoria J.; Pfeiffer, Harla K.; Mellert, Hestia S.; Seidel, Hans E.; Mascioli, Kirsten; Kaplan, Jordan; Gaballa, Mahmoud R.; Shen, Chen; Rigoutsos, Isidore; King, Michael P.; Cotney, Justin L.; Arnold, Jamie J.; Sharma, Suresh D.; Martinez, Ubaldo E.; Vakoc, Christopher R.; Chodosh, Lewis A.; Thompson, James E.; Bradner, James E.; Cameron, Craig E.; Shadel, Gerald S.; Eischen, Christine M.; McMahon, Steven B.

2016-01-01

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors. PMID:27590350

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer.

PubMed

Oran, Amanda R; Adams, Clare M; Zhang, Xiao-Yong; Gennaro, Victoria J; Pfeiffer, Harla K; Mellert, Hestia S; Seidel, Hans E; Mascioli, Kirsten; Kaplan, Jordan; Gaballa, Mahmoud R; Shen, Chen; Rigoutsos, Isidore; King, Michael P; Cotney, Justin L; Arnold, Jamie J; Sharma, Suresh D; Martinez-Outschoorn, Ubaldo E; Vakoc, Christopher R; Chodosh, Lewis A; Thompson, James E; Bradner, James E; Cameron, Craig E; Shadel, Gerald S; Eischen, Christine M; McMahon, Steven B

2016-11-08

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

MTO1 mediates tissue specificity of OXPHOS defects via tRNA modification and translation optimization, which can be bypassed by dietary intervention

PubMed Central

Tischner, Christin; Hofer, Annette; Wulff, Veronika; Stepek, Joanna; Dumitru, Iulia; Becker, Lore; Haack, Tobias; Kremer, Laura; Datta, Alexandre N.; Sperl, Wolfgang; Floss, Thomas; Wurst, Wolfgang; Chrzanowska-Lightowlers, Zofia; De Angelis, Martin Hrabe; Klopstock, Thomas; Prokisch, Holger; Wenz, Tina

2015-01-01

Mitochondrial diseases often exhibit tissue-specific pathologies, but this phenomenon is poorly understood. Here we present regulation of mitochondrial translation by the Mitochondrial Translation Optimization Factor 1, MTO1, as a novel player in this scenario. We demonstrate that MTO1 mediates tRNA modification and controls mitochondrial translation rate in a highly tissue-specific manner associated with tissue-specific OXPHOS defects. Activation of mitochondrial proteases, aberrant translation products, as well as defects in OXPHOS complex assembly observed in MTO1 deficient mice further imply that MTO1 impacts translation fidelity. In our mouse model, MTO1-related OXPHOS deficiency can be bypassed by feeding a ketogenic diet. This therapeutic intervention is independent of the MTO1-mediated tRNA modification and involves balancing of mitochondrial and cellular secondary stress responses. Our results thereby establish mammalian MTO1 as a novel factor in the tissue-specific regulation of OXPHOS and fine tuning of mitochondrial translation accuracy. PMID:25552653

Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a Drosophila Model of Leigh Syndrome.

PubMed

Loewen, Carin A; Ganetzky, Barry

2018-04-01

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. Here we describe the characterization of a mutation in Drosophila ND23 , a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology, and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23 A key feature of Leigh syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. We discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23 , including a variant affecting a mitochondrially encoded component of complex I. Thus, our work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease. Copyright © 2018 by the Genetics Society of America.

GABA-BZD Receptor Modulating Mechanism of Panax quinquefolius against 72-h Sleep Deprivation Induced Anxiety like Behavior: Possible Roles of Oxidative Stress, Mitochondrial Dysfunction and Neuroinflammation

PubMed Central

Chanana, Priyanka; Kumar, Anil

2016-01-01

Rationale: Panax quinquefolius (American Ginseng) is known for its therapeutic potential against various neurological disorders, but its plausible mechanism of action still remains undeciphered. GABA (Gamma Amino Butyric Acid) plays an important role in sleep wake cycle homeostasis. Thus, there exists rationale in exploring the GABA-ergic potential of Panax quinquefolius as neuroprotective strategy in sleep deprivation induced secondary neurological problems. Objective: The present study was designed to explore the possible GABA-ergic mechanism in the neuro-protective effect of Panax quinquefolius against 72-h sleep deprivation induced anxiety like behavior, oxidative stress, mitochondrial dysfunction, HPA-axis activation and neuroinflammation. Materials and Methods: Male laca mice were sleep deprived for 72-h by using Grid suspended over water method. Panax quinquefolius (American Ginseng 50, 100, and 200 mg/kg) was administered alone and in combination with GABA modulators (GABA Cl− channel inhibitor, GABA-benzodiazepine receptor inhibitor and GABAA agonist) for 8 days, starting 5 days prior to 72-h sleep deprivation period. Various behavioral (locomotor activity, mirror chamber test), biochemical (lipid peroxidation, reduced glutathione, catalase, nitrite levels), mitochondrial complexes, neuroinflammation marker (Tumor Necrosis Factor, TNF-alpha), serum corticosterone, and histopathological sections of brains were assessed. Results: Seventy two hours sleep deprivation significantly impaired locomotor activity, caused anxiety-like behavior, conditions of oxidative stress, alterations in mitochondrial enzyme complex activities, raised serum corticosterone levels, brain TNFα levels and led to neuroinflammation like signs in discrete brain areas as compared to naive group. Panax quinquefolius (100 and 200 mg/kg) treatment restored the behavioral, biochemical, mitochondrial, molecular and histopathological alterations. Pre-treatment of GABA Cl− channel inhibitor as well as GABA-benzodiazepine receptor inhibitor, significantly reversed the protective effect of P. quinquefolius (100 mg/kg) in 72-h sleep deprived animals (P < 0.05). However, pretreatment with GABAA agonist, potentiated Panax quinquefolius's protective effect which was significant as compared to their effect per se (p < 0.05). Conclusion: GABA-ergic mechanism could be involved in the neuroprotective effect of P.quinquefolius against sleep deprivation induced anxiety-like behavior, oxidative stress, mitochondrial dysfunction, HPA axis activation and neuroinflammation. PMID:27013946

Glycolytic and mitochondrial metabolism in pancreatic islets from MSG-treated obese rats subjected to swimming training.

PubMed

Leite, Nayara de Carvalho; Ferreira, Thiago Rentz; Rickli, Sarah; Borck, Patricia Cristine; Mathias, Paulo Cezar de Freitas; Emilio, Henriette Rosa de Oliveira; Grassiolli, Sabrina

2013-01-01

Obese rats obtained by neonatal monosodium glutamate (MSG) administration present insulin hypersecretion. The metabolic mechanism by which glucose catabolism is coupled to insulin secretion in the pancreatic β-cells from MSG-treated rats is understood. The purpose of this study was to evaluate glucose metabolism in pancreatic islets from MSG-treated rats subjected to swimming training. MSG-treated and control (CON) rats swam for 30 minutes (3 times/week) over a period of 10 weeks. Pancreatic islets were isolated and incubated with glucose in the presence of glycolytic or mitochondrial inhibitors. Swimming training attenuated fat pad accumulation, avoiding changes in the plasma levels of lipids, glucose and insulin in MSG-treated rats. Adipocyte and islet hypertrophy observed in MSG-treated rats were attenuated by exercise. Pancreatic islets from MSG-treated obese rats also showed insulin hypersecretion, greater glucose transporter 2 (GLUT2) expression, increased glycolytic flux and reduced mitochondrial complex III activity. Swimming training attenuated islet hypertrophy and normalised GLUT2 expression, contributing to a reduction in the glucose responsiveness of pancreatic islets from MSG-treated rats without altering glycolytic flux. However, physical training increased the activity of mitochondrial complex III in pancreatic islets from MSG-treated rats without a subsequent increase in glucose-induced insulin secretion. Copyright © 2013 S. Karger AG, Basel.

Berberine attenuates oxidative stress and hepatocytes apoptosis via protecting mitochondria in blunt snout bream Megalobrama amblycephala fed high-fat diets.

PubMed

Lu, Kang-Le; Wang, Li-Na; Zhang, Ding-Dong; Liu, Wen-Bin; Xu, Wei-Na

2017-02-01

High-fat diets may have favorable effects on growth and cost, but high-fat diets often induce excessive fat deposition, resulting in liver damage. This study aimed to identify the hepatoprotective of a Chinese herb (berberine) for blunt snout bream (Megalobrama amblycephala). Fish were fed with a normal diet (LFD, 5 % fat), high-fat diet (HFD, 15 % fat) or berberine-supplemented diets (BSD, 15 % fat with berberine 50 or 100 mg/kg level) for 8 weeks. After the feeding, histology, oxidative status and mitochondrial function of liver were assessed. The results showed that HFD caused fat accumulation, oxidative stress and apoptosis in hepatocytes of fish. Hepatocytes in HFD group appeared to be hypertrophied, with larger liver cells diameter than these of LFD group. Berberine-supplemented diets could attenuate oxidative stress and hepatocytes apoptosis. HFD induced the decreasing mitochondrial complexes activities and bulk density and surface area density. Berberine improved function of mitochondrial respiratory chain via increasing the complex activities. Moreover, the histological results showed that berberine has the potential to repair mitochondrial ultrastructural damage and elevate the density in cells. In conclusion, our study demonstrated that berberine has attenuated liver damage induced by the high fat mainly via the protection for mitochondria.

Low abundance of the matrix arm of complex I in mitochondria predicts longevity in mice

PubMed Central

Miwa, Satomi; Jow, Howsun; Baty, Karen; Johnson, Amy; Czapiewski, Rafal; Saretzki, Gabriele; Treumann, Achim; von Zglinicki, Thomas

2014-01-01

Mitochondrial function is an important determinant of the ageing process; however, the mitochondrial properties that enable longevity are not well understood. Here we show that optimal assembly of mitochondrial complex I predicts longevity in mice. Using an unbiased high-coverage high-confidence approach, we demonstrate that electron transport chain proteins, especially the matrix arm subunits of complex I, are decreased in young long-living mice, which is associated with improved complex I assembly, higher complex I-linked state 3 oxygen consumption rates and decreased superoxide production, whereas the opposite is seen in old mice. Disruption of complex I assembly reduces oxidative metabolism with concomitant increase in mitochondrial superoxide production. This is rescued by knockdown of the mitochondrial chaperone, prohibitin. Disrupted complex I assembly causes premature senescence in primary cells. We propose that lower abundance of free catalytic complex I components supports complex I assembly, efficacy of substrate utilization and minimal ROS production, enabling enhanced longevity. PMID:24815183

Human neuronal coenzyme Q10 deficiency results in global loss of mitochondrial respiratory chain activity, increased mitochondrial oxidative stress and reversal of ATP synthase activity: implications for pathogenesis and treatment.

PubMed

Duberley, Kate E C; Abramov, Andrey Y; Chalasani, Annapurna; Heales, Simon J; Rahman, Shamima; Hargreaves, Iain P

2013-01-01

Disorders of coenzyme Q(10) (CoQ(10)) biosynthesis represent the most treatable subgroup of mitochondrial diseases. Neurological involvement is frequently observed in CoQ(10) deficiency, typically presenting as cerebellar ataxia and/or seizures. The aetiology of the neurological presentation of CoQ(10) deficiency has yet to be fully elucidated and therefore in order to investigate these phenomena we have established a neuronal cell model of CoQ(10) deficiency by treatment of neuronal SH-SY5Y cell line with para-aminobenzoic acid (PABA). PABA is a competitive inhibitor of the CoQ(10) biosynthetic pathway enzyme, COQ2. PABA treatment (1 mM) resulted in a 54 % decrease (46 % residual CoQ(10)) decrease in neuronal CoQ(10) status (p < 0.01). Reduction of neuronal CoQ(10) status was accompanied by a progressive decrease in mitochondrial respiratory chain enzyme activities, with a 67.5 % decrease in cellular ATP production at 46 % residual CoQ(10). Mitochondrial oxidative stress increased four-fold at 77 % and 46 % residual CoQ(10). A 40 % increase in mitochondrial membrane potential was detected at 46 % residual CoQ(10) with depolarisation following oligomycin treatment suggesting a reversal of complex V activity. This neuronal cell model provides insights into the effects of CoQ(10) deficiency on neuronal mitochondrial function and oxidative stress, and will be an important tool to evaluate candidate therapies for neurological conditions associated with CoQ(10) deficiency.

Transcriptional upregulation of mitochondrial uncoupling protein 2 protects against oxidative stress-associated neurogenic hypertension.

PubMed

Chan, Samuel H H; Wu, Chiung-Ai; Wu, Kay L H; Ho, Ying-Hao; Chang, Alice Y W; Chan, Julie Y H

2009-10-23

Mitochondrial uncoupling proteins (UCPs) belong to a superfamily of mitochondrial anion transporters that uncouple ATP synthesis from oxidative phosphorylation and mitigates mitochondrial reactive oxygen species production. We assessed the hypothesis that UCP2 participates in central cardiovascular regulation by maintaining reactive oxygen species homeostasis in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons that maintain vasomotor tone located. We also elucidated the molecular mechanisms that underlie transcriptional upregulation of UCP2 in response to oxidative stress in RVLM. In Sprague-Dawley rats, transcriptional upregulation of UCP2 in RVLM by rosiglitazone, an activator of its transcription factor peroxisome proliferator-activated receptor (PPAR)gamma, reduced mitochondrial hydrogen peroxide level in RVLM and systemic arterial pressure. Oxidative stress induced by microinjection of angiotensin II into RVLM augmented UCP2 mRNA or protein expression in RVLM, which was antagonized by comicroinjection of NADPH oxidase inhibitor (diphenyleneiodonium chloride), superoxide dismutase mimetic (tempol), or p38 mitogen-activated protein kinase inhibitor (SB203580) but not by extracellular signal-regulated kinase 1/2 inhibitor (U0126). Angiotensin II also induced phosphorylation of the PPARgamma coactivator, PPARgamma coactivator (PGC)-1alpha, and an increase in formation of PGC-1alpha/PPARgamma complexes in a p38 mitogen-activated protein kinase-dependent manner. Intracerebroventricular infusion of angiotensin II promoted an increase in mitochondrial hydrogen peroxide production in RVLM and chronic pressor response, which was potentiated by gene knockdown of UCP2 but blunted by rosiglitazone. These results suggest that transcriptional upregulation of mitochondrial UCP2 in response to an elevation in superoxide plays an active role in feedback regulation of reactive oxygen species production in RVLM and neurogenic hypertension associated with chronic oxidative stress.

Titanium dioxide nanoparticle-induced cytotoxicity and the underlying mechanism in mouse myocardial cells

NASA Astrophysics Data System (ADS)

Zhou, Yingjun; Hong, Fashui; Wang, Ling

2017-11-01

Exposure to fine particulate matter (PM) is known to cause cardiovascular disease. While extensive research has focused on the risk of atmospheric PM to public health, particularly heart disease, limited studies to date have attempted to clarify the molecular mechanisms underlying myocardial cell damage caused by exposure to titanium dioxide nanoparticles (TiO2 NPs). Data from the current investigation showed that TiO2 NPs are deposited in myocardial mitochondria via the blood circulation accompanied by obvious ultrastructural changes and impairment of mitochondrial structure and function in mouse myocardial cells, including reduction in mitochondrial membrane potential and ATP production, aggravation of oxidative stress along with increased levels of reactive oxygen species, malondialdehyde and protein carbonyl, and decreased glutathione content and enzymatic activities, including superoxide dismutase and glutathione peroxidase. Furthermore, TiO2 NPs induced a significant decrease in the activities of complex I, complex II, complex III, complex IV, succinate dehydrogenase, NADH oxidase, Ca2+-ATPase, Na+/K+-ATPase, and Ca2+/Mg2+-ATPase, and upregulation of cytokine expression (including cytochrome c, caspase-3, and p-JNK) in mitochondria-mediated apoptosis while downregulating Bcl-2 expression in mouse myocardial cells. Our results collectively indicate that chronic exposure to TiO2 NPs induces damage in mitochondrial structure and function as well as mitochondria-mediated apoptosis in mouse myocardial cells, which may be closely associated with heart disease in animals and humans.

Low-Level Laser Irradiation Improves Depression-Like Behaviors in Mice.

PubMed

Xu, Zhiqiang; Guo, Xiaobo; Yang, Yong; Tucker, Donovan; Lu, Yujiao; Xin, Ning; Zhang, Gaocai; Yang, Lingli; Li, Jizhen; Du, Xiangdong; Zhang, Quanguang; Xu, Xingshun

2017-08-01

Major depressive disorder (MDD) is one of the leading forms of psychiatric disorders, characterized by aversion to mobility, neurotransmitter deficiency, and energy metabolic decline. Low-level laser therapy (LLLT) has been investigated in a variety of neurodegenerative disorders associated with mitochondrial dysfunction and functional impairments. The goal of this study was to examine the effect of LLLT on depression-like behaviors and to explore the potential mechanism by detecting mitochondrial function following LLLT. Depression models in space restriction mice and Abelson helper integration site-1 (Ahi1) knockout (KO) mice were employed in this work. Our results revealed that LLLT effectively improved depression-like behaviors, in the two depression mice models, by decreasing immobility duration in behavioral despair tests. In addition, ATP biosynthesis and the level of mitochondrial complex IV expression and activity were significantly elevated in prefrontal cortex (PFC) following LLLT. Intriguingly, LLLT has no effects on ATP content and mitochondrial complex I-IV levels in other tested brain regions, hippocampus and hypothalamus. As a whole, these findings shed light on a novel strategy of transcranial LLLT on depression improvement by ameliorating neurotransmitter abnormalities and promoting mitochondrial function in PFC. The present work provides concrete groundwork for further investigation of LLLT for depression treatment.

UCP1 deficiency causes brown fat respiratory chain depletion and sensitizes mitochondria to calcium overload-induced dysfunction.

PubMed

Kazak, Lawrence; Chouchani, Edward T; Stavrovskaya, Irina G; Lu, Gina Z; Jedrychowski, Mark P; Egan, Daniel F; Kumari, Manju; Kong, Xingxing; Erickson, Brian K; Szpyt, John; Rosen, Evan D; Murphy, Michael P; Kristal, Bruce S; Gygi, Steven P; Spiegelman, Bruce M

2017-07-25

Brown adipose tissue (BAT) mitochondria exhibit high oxidative capacity and abundant expression of both electron transport chain components and uncoupling protein 1 (UCP1). UCP1 dissipates the mitochondrial proton motive force (Δp) generated by the respiratory chain and increases thermogenesis. Here we find that in mice genetically lacking UCP1, cold-induced activation of metabolism triggers innate immune signaling and markers of cell death in BAT. Moreover, global proteomic analysis reveals that this cascade induced by UCP1 deletion is associated with a dramatic reduction in electron transport chain abundance. UCP1-deficient BAT mitochondria exhibit reduced mitochondrial calcium buffering capacity and are highly sensitive to mitochondrial permeability transition induced by reactive oxygen species (ROS) and calcium overload. This dysfunction depends on ROS production by reverse electron transport through mitochondrial complex I, and can be rescued by inhibition of electron transfer through complex I or pharmacologic depletion of ROS levels. Our findings indicate that the interscapular BAT of Ucp1 knockout mice exhibits mitochondrial disruptions that extend well beyond the deletion of UCP1 itself. This finding should be carefully considered when using this mouse model to examine the role of UCP1 in physiology.

Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin.

PubMed

Teixeira, Pedro F; Masuyer, Geoffrey; Pinho, Catarina M; Branca, Rui M M; Kmiec, Beata; Wallin, Cecilia; Wärmländer, Sebastian K T S; Berntsson, Ronnie P-A; Ankarcrona, Maria; Gräslund, Astrid; Lehtiö, Janne; Stenmark, Pål; Glaser, Elzbieta

2018-02-02

Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLN E475Q in complex with the products of neurotensin cleavage at 2.7Å revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-β peptide, Aβ1-40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aβ35-40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cholesterol contributes to dopamine-neuronal loss in MPTP mouse model of Parkinson's disease: Involvement of mitochondrial dysfunctions and oxidative stress.

PubMed

Paul, Rajib; Choudhury, Amarendranath; Kumar, Sanjeev; Giri, Anirudha; Sandhir, Rajat; Borah, Anupom

2017-01-01

Hypercholesterolemia is a known contributor to the pathogenesis of Alzheimer's disease while its role in the occurrence of Parkinson's disease (PD) is only conjecture and far from conclusive. Altered antioxidant homeostasis and mitochondrial functions are the key mechanisms in loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain in PD. Hypercholesterolemia is reported to cause oxidative stress and mitochondrial dysfunctions in the cortex and hippocampus regions of the brain in rodents. However, the impact of hypercholesterolemia on the midbrain dopaminergic neurons in animal models of PD remains elusive. We tested the hypothesis that hypercholesterolemia in MPTP model of PD would potentiate dopaminergic neuron loss in SN by disrupting mitochondrial functions and antioxidant homeostasis. It is evident from the present study that hypercholesterolemia in naïve animals caused dopamine neuronal loss in SN with subsequent reduction in striatal dopamine levels producing motor impairment. Moreover, in the MPTP model of PD, hypercholesterolemia exacerbated MPTP-induced reduction of striatal dopamine as well as dopaminergic neurons in SN with motor behavioral depreciation. Activity of mitochondrial complexes, mainly complex-I and III, was impaired severely in the nigrostriatal pathway of hypercholesterolemic animals treated with MPTP. Hypercholesterolemia caused oxidative stress in the nigrostriatal pathway with increased generation of hydroxyl radicals and enhanced activity of antioxidant enzymes, which were further aggravated in the hypercholesterolemic mice with Parkinsonism. In conclusion, our findings provide evidence of increased vulnerability of the midbrain dopaminergic neurons in PD with hypercholesterolemia.

A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Junghwa; Jung, Hye Jin; Jeong, Seung Hun

2014-12-12

Highlights: • We constructed mitochondrial protein UQCRB mutant stable cell lines on the basis of a human case report. • These mutant cell lines exhibit pro-angiogenic activity with enhanced VEGF expression. • Proliferation of mutant cell lines was regulated by UQCRB inhibitors. • UQCRB may have a functional role in angiogenesis. - Abstract: Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identificationmore » and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders.« less

The pleiotropic effects of decanoic acid treatment on mitochondrial function in fibroblasts from patients with complex I deficient Leigh syndrome.

PubMed

Kanabus, Marta; Fassone, Elisa; Hughes, Sean David; Bilooei, Sara Farahi; Rutherford, Tricia; Donnell, Maura O'; Heales, Simon J R; Rahman, Shamima

2016-05-01

There is growing interest in the use of the ketogenic diet (KD) to treat inherited metabolic diseases including mitochondrial disorders. However, neither the mechanism whereby the diet may be working, nor if it could benefit all patients with mitochondrial disease, is known. This study focusses on decanoic acid (C10), a component of the medium chain triglyceride KD, and a ligand for the nuclear receptor PPAR-γ known to be involved in mitochondrial biogenesis. The effects of C10 were investigated in primary fibroblasts from a cohort of patients with Leigh syndrome (LS) caused by nuclear-encoded defects of respiratory chain complex I, using mitochondrial respiratory chain enzyme assays, gene expression microarray, qPCR and flow cytometry. Treatment with C10 increased citrate synthase activity, a marker of cellular mitochondrial content, in 50 % of fibroblasts obtained from individuals diagnosed with LS in a PPAR-γ-mediated manner. Gene expression analysis and qPCR studies suggested that treating cells with C10 supports fatty acid metabolism, through increasing ACADVL and CPT1 expression, whilst downregulating genes involved in glucose metabolism (PDK3, PDK4). PCK2, involved in blocking glucose metabolism, was upregulated, as was CAT, encoding catalase. Moreover, treatment with C10 also decreased oxidative stress in complex I deficient (rotenone treated) cells. However, since not all cells from subjects with LS appeared to respond to C10, prior cellular testing in vitro could be employed as a means for selecting individuals for subsequent clinical studies involving C10 preparations.

A new mutation in MT-ND1 m.3928G>C p.V208L causes Leigh disease with infantile spasms.

PubMed

Wray, Carter D; Friederich, Marisa W; du Sart, Desiree; Pantaleo, Sarah; Smet, Joél; Kucera, Cathlin; Fenton, Laura; Scharer, Gunter; Van Coster, Rudy; Van Hove, Johan L K

2013-11-01

New mutations in mitochondrial DNA encoded genes of complex I are rarely reported. An infant developed Leigh disease with infantile spasms. Complex I enzyme activity was deficient and response to increasing coenzyme Q concentrations was reduced. Complex I assembly was intact. A new mutation in MT-ND1 m.3928G>C p.V208L, affecting a conserved amino acid in a critical domain, part of the coenzyme Q binding pocket, was present at high heteroplasmy. The unaffected mother did not carry measurable mutant mitochondrial DNA, but concern remained for gonadal mosaicism. Prenatal testing was possible for a subsequent sibling. The ND1 p.V208L mutation causes Leigh disease. © 2013.

Enhanced Respiratory Chain Supercomplex Formation in Response to Exercise in Human Skeletal Muscle.

PubMed

Greggio, Chiara; Jha, Pooja; Kulkarni, Sameer S; Lagarrigue, Sylviane; Broskey, Nicholas T; Boutant, Marie; Wang, Xu; Conde Alonso, Sonia; Ofori, Emmanuel; Auwerx, Johan; Cantó, Carles; Amati, Francesca

2017-02-07

Mitochondrial dysfunction is a hallmark of multiple metabolic complications. Physical activity is known to increase mitochondrial content in skeletal muscle, counteracting age-related decline in muscle function and protecting against metabolic and cardiovascular complications. Here, we investigated the effect of 4 months of exercise training on skeletal muscle mitochondria electron transport chain complexes and supercomplexes in 26 healthy, sedentary older adults. Exercise differentially modulated respiratory complexes. Complex I was the most upregulated complex and not stoichiometrically associated to the other complexes. In contrast to the other complexes, complex I was almost exclusively found assembled in supercomplexes in muscle mitochondria. Overall, supercomplex content was increased after exercise. In particular, complexes I, III, and IV were redistributed to supercomplexes in the form of I+III 2 +IV. Taken together, our results provide the first evidence that exercise affects the stoichiometry of supercomplex formation in humans and thus reveal a novel adaptive mechanism for increased energy demand. Copyright © 2017 Elsevier Inc. All rights reserved.

Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis.

PubMed

Han, Jie; Goldstein, Leslie A; Gastman, Brian R; Rabinowich, Hannah

2006-04-14

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1. Direct degradation of Mcl-1 by TRAIL-activated caspase-8 or caspase-3 produces Mcl-1-free Bim that mediates a Bax-dependent apoptotic cascade. Using Mcl-1 or Bim RNAi, we demonstrate that a loss in Mcl-1 expression significantly enhances the mitochondrial apoptotic response to TRAIL that is now mediated by freed Bim. Whereas overexpression of Mcl-1 contributes to the preservation of the mitochondrial membrane potential, Mcl-1 knockdown facilitates the Bim-mediated dissipation of this potential. Loss of Mcl-1 contributes to an increased level of caspase activity downstream of the mitochondrial response to TRAIL. Furthermore, the Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome c, Smac, and HtrA2 in response to Bim. These are the first findings to demonstrate the involvement of Bim in the TRAIL-mediated mitochondrial cascade. They also suggest that Mcl-1 may serve as a direct substrate for TRAIL-activated caspases implying the existence of a novel TRAIL/caspase-8/Mcl-1/Bim communication mechanism between the extrinsic and the intrinsic apoptotic pathways.

Melatonin enhances neural stem cell differentiation and engraftment by increasing mitochondrial function.

PubMed

Mendivil-Perez, Miguel; Soto-Mercado, Viviana; Guerra-Librero, Ana; Fernandez-Gil, Beatriz I; Florido, Javier; Shen, Ying-Qiang; Tejada, Miguel A; Capilla-Gonzalez, Vivian; Rusanova, Iryna; Garcia-Verdugo, José M; Acuña-Castroviejo, Darío; López, Luis Carlos; Velez-Pardo, Carlos; Jimenez-Del-Rio, Marlene; Ferrer, José M; Escames, Germaine

2017-09-01

Neural stem cells (NSCs) are regarded as a promising therapeutic approach to protecting and restoring damaged neurons in neurodegenerative diseases (NDs) such as Parkinson's disease and Alzheimer's disease (PD and AD, respectively). However, new research suggests that NSC differentiation is required to make this strategy effective. Several studies have demonstrated that melatonin increases mature neuronal markers, which reflects NSC differentiation into neurons. Nevertheless, the possible involvement of mitochondria in the effects of melatonin during NSC differentiation has not yet been fully established. We therefore tested the impact of melatonin on NSC proliferation and differentiation in an attempt to determine whether these actions depend on modulating mitochondrial activity. We measured proliferation and differentiation markers, mitochondrial structural and functional parameters as well as oxidative stress indicators and also evaluated cell transplant engraftment. This enabled us to show that melatonin (25 μM) induces NSC differentiation into oligodendrocytes and neurons. These effects depend on increased mitochondrial mass/DNA/complexes, mitochondrial respiration, and membrane potential as well as ATP synthesis in NSCs. It is also interesting to note that melatonin prevented oxidative stress caused by high levels of mitochondrial activity. Finally, we found that melatonin enriches NSC engraftment in the ND mouse model following transplantation. We concluded that a combined therapy involving transplantation of NSCs pretreated with pharmacological doses of melatonin could efficiently restore neuronal cell populations in PD and AD mouse models depending on mitochondrial activity promotion. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Rapid identification of a novel complex I MT-ND3 m.10134C>A mutation in a Leigh syndrome patient.

PubMed

Miller, David K; Menezes, Minal J; Simons, Cas; Riley, Lisa G; Cooper, Sandra T; Grimmond, Sean M; Thorburn, David R; Christodoulou, John; Taft, Ryan J

2014-01-01

Leigh syndrome (LS) is a rare progressive multi-system neurodegenerative disorder, the genetics of which is frequently difficult to resolve. Rapid determination of the genetic etiology of LS in a 5-year-old girl facilitated inclusion in Edison Pharmaceutical's phase 2B clinical trial of EPI-743. SNP-arrays and high-coverage whole exome sequencing were performed on the proband, both parents and three unaffected siblings. Subsequent multi-tissue targeted high-depth mitochondrial sequencing was performed using custom long-range PCR amplicons. Tissue-specific mutant load was also assessed by qPCR. Complex I was interrogated by spectrophotometric enzyme assays and Western Blot. No putatively causal mutations were identified in nuclear-encoded genes. Analysis of low-coverage off-target mitochondrial reads revealed a previously unreported mitochondrial mutation in the proband in MT-ND3 (m.10134C>A, p.Q26K), a Complex I mitochondrial gene previously associated with LS. Targeted investigations demonstrated that this mutation was 1% heteroplasmic in the mother's blood and homoplasmic in the proband's blood, fibroblasts, liver and muscle. Enzyme assays revealed decreased Complex I activity. The identification of this novel LS MT-ND3 variant, the genomics of which was accomplished in less than 3.5 weeks, indicates that rapid genomic approaches may prove useful in time-sensitive cases with an unresolved genetic diagnosis.

Elevated extracellular [K+] inhibits death-receptor- and chemical-mediated apoptosis prior to caspase activation and cytochrome c release.

PubMed Central

Thompson, G J; Langlais, C; Cain, K; Conley, E C; Cohen, G M

2001-01-01

Efflux of intracellular K(+) and cell shrinkage are features of apoptosis in many experimental systems, and a regulatory role has been proposed for cytoplasmic [K(+)] in initiating apoptosis. We have investigated this in both death-receptor-mediated and chemical-induced apoptosis. Using Jurkat T cells pre-loaded with the K(+) ion surrogate (86)Rb(+), we have demonstrated an efflux of intracellular K(+) during apoptosis that was concomitant with, but did not precede, other apoptotic changes, including phosphatidylserine externalization, mitochondrial depolarization and cell shrinkage. To further clarify the role of K(+) ions in apoptosis, cytoprotection by elevated extracellular [K(+)] was studied. Induction of apoptosis by diverse death-receptor and chemical stimuli in two cell lines was inhibited prior to phosphatidylserine externalization, mitochondrial depolarization, cytochrome c release and caspase activation. Using a cell-free system, we have demonstrated a novel mechanism by which increasing [K(+)] inhibited caspase activation. In control dATP-activated lysates, Apaf-1 oligomerized to a biologically active caspase processing approximately 700 kDa complex and an inactive approximately 1.4 MDa complex. Increasing [K(+)] inhibited caspase activation by preventing formation of the approximately 700 kDa complex, but not of the inactive complex. Thus intracellular and extracellular [K(+)] markedly affect caspase activation and the initiation of apoptosis induced by both death-receptor ligation and chemical stress. PMID:11415444

Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes*

PubMed Central

Im, Ilkyun; Jang, Mi-jin; Park, Seung Ju; Lee, Sang-Hee; Choi, Jin-Ho; Yoo, Han-Wook; Kim, Seyun; Han, Yong-Mahn

2015-01-01

A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD+/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver. PMID:26491018

AMPK activity regulates trafficking of mitochondria to the leading edge during cell migration and matrix invasion

PubMed Central

Cunniff, Brian; McKenzie, Andrew J.; Heintz, Nicholas H.; Howe, Alan K.

2016-01-01

Cell migration is a complex behavior involving many energy-expensive biochemical events that iteratively alter cell shape and location. Mitochondria, the principal producers of cellular ATP, are dynamic organelles that fuse, divide, and relocate to respond to cellular metabolic demands. Using ovarian cancer cells as a model, we show that mitochondria actively infiltrate leading edge lamellipodia, thereby increasing local mitochondrial mass and relative ATP concentration and supporting a localized reversal of the Warburg shift toward aerobic glycolysis. This correlates with increased pseudopodial activity of the AMP-activated protein kinase (AMPK), a critically important cellular energy sensor and metabolic regulator. Furthermore, localized pharmacological activation of AMPK increases leading edge mitochondrial flux, ATP content, and cytoskeletal dynamics, whereas optogenetic inhibition of AMPK halts mitochondrial trafficking during both migration and the invasion of three-dimensional extracellular matrix. These observations indicate that AMPK couples local energy demands to subcellular targeting of mitochondria during cell migration and invasion. PMID:27385336

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. In thismore » paper, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q 6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Finally, given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.« less

Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

PubMed

Trotta, Andrew P; Gelles, Jesse D; Serasinghe, Madhavika N; Loi, Patrick; Arbiser, Jack L; Chipuk, Jerry E

2017-07-14

The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAF V600E ), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAF V600E is pharmacologically inhibited by targeted therapies ( e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAF V600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G 1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

Metabolically induced heteroplasmy shifting and L-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

PubMed Central

Desquiret-Dumas, Valerie; Gueguen, Naig; Barth, Magalie; Chevrollier, Arnaud; Hancock, Saege; Wallace, Douglas C; Amati-Bonneau, Patrizia; Henrion, Daniel; Bonneau, Dominique; Reynier, Pascal; Procaccio, Vincent

2012-01-01

The m.3243A>G variant in the mitochondrial tRNALeu (UUR) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood. We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of L-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of L-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and L-arginine therapy may constitute promising therapeutic strategies against MELAS. PMID:22306605

Osthole attenuates spinal cord ischemia-reperfusion injury through mitochondrial biogenesis-independent inhibition of mitochondrial dysfunction in rats.

PubMed

Zhou, Yue-fei; Li, Liang; Feng, Feng; Yuan, Hua; Gao, Da-kuan; Fu, Luo-an; Fei, Zhou

2013-12-01

Osthole, the main bioactive compounds isolated from the traditional Chinese medical herb broad Cnidium monnieri (L.) cusson, has been shown to exert spectrum of pharmacologic activities. The aim of this study was to investigate the potential neuroprotective effects of osthole against spinal cord ischemia-reperfusion injury in rats. Osthole was administrated at the concentration of 0.1, 1, 10, 50, or 200 mg/kg (intraperitoneally) 1 h before spinal cord ischemia. The effects on spinal cord injury were measured by spinal cord water content, infarct volume, hematoxylin and eosin staining, and neurologic assessment. Mitochondria were purified from injured spinal cord tissue to determine mitochondrial function. We found that treatment with osthole (10 and 50 mg/kg) significantly decreased spinal cord water content and infarct volume, preserved normal motor neurons, and improved neurologic functions. These protective effects can be also observed even if the treatment was delayed to 4 h after reperfusion. Osthole treatment preserved mitochondrial membrane potential level, reduced reactive oxygen species production, increased adenosine triphosphate generation, and inhibited cytochrome c release in mitochondrial samples. Moreover, osthole increased mitochondria respiratory chain complex activities in spinal cord tissue, with no effect on mitochondrial DNA content and the expression of mitochondrial-specific transcription factors. All these findings demonstrate the neuroprotective effect of osthole in spinal cord ischemia-reperfusion injury model and suggest that oshtole-induced neuroprotection was mediated by mitochondrial biogenesis-independent inhibition of mitochondrial dysfunction. Copyright © 2013 Elsevier Inc. All rights reserved.

Tissue- and cell-type–specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model

PubMed Central

Hämäläinen, Riikka H.; Manninen, Tuula; Koivumäki, Hanna; Kislin, Mikhail; Otonkoski, Timo; Suomalainen, Anu

2013-01-01

Mitochondrial DNA (mtDNA) mutations manifest with vast clinical heterogeneity. The molecular basis of this variability is mostly unknown because the lack of model systems has hampered mechanistic studies. We generated induced pluripotent stem cells from patients carrying the most common human disease mutation in mtDNA, m.3243A>G, underlying mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. During reprogramming, heteroplasmic mtDNA showed bimodal segregation toward homoplasmy, with concomitant changes in mtDNA organization, mimicking mtDNA bottleneck during epiblast specification. Induced pluripotent stem cell–derived neurons and various tissues derived from teratomas manifested cell-type specific respiratory chain (RC) deficiency patterns. Similar to MELAS patient tissues, complex I defect predominated. Upon neuronal differentiation, complex I specifically was sequestered in perinuclear PTEN-induced putative kinase 1 (PINK1) and Parkin-positive autophagosomes, suggesting active degradation through mitophagy. Other RC enzymes showed normal mitochondrial network distribution. Our data show that cellular context actively modifies RC deficiency manifestation in MELAS and that autophagy is a significant component of neuronal MELAS pathogenesis. PMID:24003133

Mitochondrial Ca2+ and Regulation of the Permeability Transition Pore

PubMed Central

Hurst, Stephen; Hoek, Jan; Sheu, Shey-Shing

2017-01-01

The mitochondrial permeability transition pore was originally described in the 1970’s as a Ca2+ activated pore and has since been attributed to the pathogenesis of many diseases. Here we evaluate how each of the current models of the pore complex fit to what is known about how Ca2+ regulates the pore, and any insight that provides into the molecular identity of the pore complex. We also discuss the central role of Ca2+ in modulating the pore’s open probability by directly regulating processes, such as ATP/ADP balance through the tricarboxylic acid cycle, electron transport chain, and mitochondrial membrane potential. We review how Ca2+ influences second messengers such as reactive oxygen/nitrogen species production and polyphosphate formation. We discuss the evidence for how Ca2+ regulates post-translational modification of cyclophilin D including phosphorylation by glycogen synthase kinase 3 beta, deacetylation by sirtuins, and oxidation/nitrosylation of key residues. Lastly we introduce a novel view into how Ca2+ activated proteolysis through calpains in the mitochondria may be a driver of sustained pore opening during pathologies such as ischemia reperfusion injury. PMID:27497945

The effect of artichoke (Cynara scolymus L.) extract on respiratory chain system activity in rat liver mitochondria.

PubMed

Juzyszyn, Z; Czerny, B; Myśliwiec, Z; Pawlik, A; Droździk, M

2010-06-01

The effect of artichoke extract on mitochondrial respiratory chain (MRC) activity in isolated rat liver mitochondria (including reaction kinetics) was studied. The effect of the extract on the activity of isolated cytochrome oxidase was also studied. Extract in the range of 0.68-2.72 microg/ml demonstrated potent and concentration-dependent inhibitory activity. Concentrations > or =5.4 microg/ml entirely inhibited MRC activity. The succinate oxidase system (MRC complexes II-IV) was the most potently inhibited, its activity at an extract concentration of 1.36 microg/ml being reduced by 63.3% compared with the control (p < 0.05). The results suggest a complex inhibitory mechanism of the extract. Inhibition of the succinate oxidase system was competitive (K(i) = 0.23 microg/ml), whereas isolated cytochrome oxidase was inhibited noncompetitively (K(i) = 126 microg/ml). The results of this study suggest that the salubrious effects of artichoke extracts may rely in part on the effects of their active compounds on the activity of the mitochondrial respiratory chain system.

A Krebs Cycle Component Limits Caspase Activation Rate through Mitochondrial Surface Restriction of CRL Activation.

PubMed

Aram, Lior; Braun, Tslil; Braverman, Carmel; Kaplan, Yosef; Ravid, Liat; Levin-Zaidman, Smadar; Arama, Eli

2016-04-04

How cells avoid excessive caspase activity and unwanted cell death during apoptotic caspase-mediated removal of large cellular structures is poorly understood. We investigate caspase-mediated extrusion of spermatid cytoplasmic contents in Drosophila during spermatid individualization. We show that a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase β subunit (A-Sβ), binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. In vitro and in vivo evidence suggests that this interaction occurs on the mitochondrial surface, thereby limiting the source of CRL3 complex activation to the vicinity of this organelle and reducing the potential rate of caspase activation by at least 60%. Domain swapping between A-Sβ and the GTP-specific SCSβ (G-Sβ), which functions redundantly in the Krebs cycle, show that the metabolic and structural roles of A-Sβ in spermatids can be uncoupled, highlighting a moonlighting function of this Krebs cycle component in CRL activation. Copyright © 2016 Elsevier Inc. All rights reserved.

Gem1 and ERMES Do Not Directly Affect Phosphatidylserine Transport from ER to Mitochondria or Mitochondrial Inheritance

PubMed Central

Nguyen, Tammy T; Lewandowska, Agnieszka; Choi, Jae-Yeon; Markgraf, Daniel F; Junker, Mirco; Bilgin, Mesut; Ejsing, Christer S; Voelker, Dennis R; Rapoport, Tom A; Shaw, Janet M

2012-01-01

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology. PMID:22409400

Clinical manifestations and enzymatic activities of mitochondrial respiratory chain complexes in Pearson marrow-pancreas syndrome with 3-methylglutaconic aciduria: a case report and literature review.

PubMed

Sato, Takeshi; Muroya, Koji; Hanakawa, Junko; Iwano, Reiko; Asakura, Yumi; Tanaka, Yukichi; Murayama, Kei; Ohtake, Akira; Hasegawa, Tomonobu; Adachi, Masanori

2015-12-01

Pearson marrow-pancreas syndrome (PS) is a rare mitochondrial disorder. Impaired mitochondrial respiratory chain complexes (MRCC) differ among individuals and organs, which accounts for variable clinical pictures. A subset of PS patients develop 3-methylglutaconic aciduria (3-MGA-uria), but the characteristic symptoms and impaired MRCC remain unknown. Our patient, a girl, developed pancytopenia, hyperlactatemia, steatorrhea, insulin-dependent diabetes mellitus, liver dysfunction, Fanconi syndrome, and 3-MGA-uria. She died from cerebral hemorrhage at 3 years of age. We identified a novel 5.4-kbp deletion of mitochondrial DNA. The enzymatic activities of MRCC I and IV were markedly reduced in the liver and muscle and mildly reduced in skin fibroblasts and the heart. To date, urine organic acid analysis has been performed on 29 PS patients, including our case. Eight patients had 3-MGA-uria, while only one patient did not. The remaining 20 patients were not reported to have 3-MGA-uria. In this paper, we included these 20 patients as PS patients without 3-MGA-uria. PS patients with and without 3-MGA-uria have similar manifestations. Only a few studies have examined the enzymatic activities of MRCC. No clinical characteristics distinguish between PS patients with and without 3-MGA-uria. The correlation between 3-MGA-uria and the enzymatic activities of MRCC remains to be elucidated. • The clinical characteristics of patients with Pearson marrow-pancreas syndrome and 3-methylglutaconic aciduria remain unknown. • No clinical characteristics distinguish between Pearson marrow-pancreas syndrome patients with and without 3-methylglutaconic aciduria.

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression.

PubMed

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Kandimalla, Ramesh J L; Bal, Amanjit; Gill, Kiran Dip

2013-12-01

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10mg/kgb.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits-NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. © 2013.

Adult cases of mitochondrial DNA depletion due to TK2 defect: an expanding spectrum.

PubMed

Béhin, A; Jardel, C; Claeys, K G; Fagart, J; Louha, M; Romero, N B; Laforêt, P; Eymard, B; Lombès, A

2012-02-28

In this study we aim to demonstrate the occurrence of adult forms of TK2 mutations causing progressive mitochondrial myopathy with significant muscle mitochondrial DNA (mtDNA) depletion. Patients' investigations included serum creatine kinase, blood lactate, electromyographic, echocardiographic, and functional respiratory analyses as well as TK2 gene sequencing and TK2 activity measurement. Mitochondrial activities and mtDNA were analyzed in the patients' muscle biopsy. The 3 adult patients with TK2 mutations presented with slowly progressive myopathy compatible with a fairly normal life during decades. Apart from its much slower progression, these patients' phenotype closely resembled that of pediatric cases including early onset, absence of CNS symptoms, generalized muscle weakness predominating on axial and proximal muscles but affecting facial, ocular, and respiratory muscles, typical mitochondrial myopathy with a mosaic pattern of COX-negative and ragged-red fibers, combined mtDNA-dependent respiratory complexes deficiency and mtDNA depletion. In accordance with the disease's relatively slow progression, the residual mtDNA content was higher than that observed in pediatric cases. That difference was not explained by the type of the TK2 mutations or by the residual TK2 activity. TK2 mutations can cause mitochondrial myopathy with a slow progression. Comparison of patients with similar mutations but different disease progression might address potential mechanisms of mtDNA maintenance modulation.

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria.

PubMed

Jesina, P; Kholová, D; Bolehovská, R; Cervinková, Z; Drahota, Z; Houstek, J

2004-01-01

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.

Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress: Involving of mitochondria and energy metabolism.

PubMed

He, Weiliang; Cui, Lili; Zhang, Cong; Zhang, Xiangjian; He, Junna; Xie, Yanzhao; Chen, Yanxia

2017-01-01

Oxidative stress has been demonstrated to be involved in the etiology of several neurobiological disorders. Sonic hedgehog (Shh), a secreted glycoprotein factor, has been implicated in promoting several aspects of brain remodeling process. Mitochondria may play an important role in controlling fundamental processes in neuroplasticity. However, little evidence is available about the effect and the potential mechanism of Shh on neurite outgrowth in primary cortical neurons under oxidative stress. Here, we revealed that Shh treatment significantly increased the viability of cortical neurons in a dose-dependent manner, which was damaged by hydrogen peroxide (H 2 O 2 ). Shh alleviated the apoptosis rate of H 2 O 2 -induced neurons. Shh also increased neuritogenesis injuried by H 2 O 2 in primary cortical neurons. Moreover, Shh reduced the generation of reactive oxygen species (ROS), increased the activities of SOD and and decreased the productions of MDA. In addition, Shh protected mitochondrial functions, elevated the cellular ATP levels and amelioratesd the impairment of mitochondrial complex II activities of cortical neurons induced by H 2 O 2 . In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H 2 O 2 -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

The Mitochondrial Disulfide Relay System Protein GFER Is Mutated in Autosomal-Recessive Myopathy with Cataract and Combined Respiratory-Chain Deficiency

PubMed Central

Di Fonzo, Alessio; Ronchi, Dario; Lodi, Tiziana; Fassone, Elisa; Tigano, Marco; Lamperti, Costanza; Corti, Stefania; Bordoni, Andreina; Fortunato, Francesco; Nizzardo, Monica; Napoli, Laura; Donadoni, Chiara; Salani, Sabrina; Saladino, Francesca; Moggio, Maurizio; Bresolin, Nereo; Ferrero, Iliana; Comi, Giacomo P.

2009-01-01

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient's muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1R182H mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease. PMID:19409522

Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate.

PubMed

Miyayama, Takamitsu; Arai, Yuta; Suzuki, Noriyuki; Hirano, Seishiro

2013-03-08

Silver (Ag) possesses antibacterial activity and has been used in wound dressings and deodorant powders worldwide. However, the metabolic behavior and biological roles of Ag in mammals have not been well characterized. In the present study, we exposed human bronchial epithelial cells (BEAS-2B) to AgNO3 and investigated uptake and intracellular distribution of Ag, expression of metallothionein (MT), generation of reactive oxygen species (ROS), and changes in mitochondrial respiration. The culture medium concentration of Ag decreased with time and stabilized at 12h. The concentration of both Ag and MT in the soluble cellular fraction increased up to 3h and then decreased, indicating that cytosolic Ag relocated to the insoluble fraction of the cells. The levels of mRNAs for the major human MT isoforms MT-I and MT-II paralleled with the protein levels of Ag-MT. The intensity of fluorescence derived from ROS was elevated in the mitochondrial region at 24h. Ag decreased mitochondrial oxygen consumption in a dose-dependent manner and the activity of mitochondrial complex I-IV enzymes was significantly inhibited following exposure to Ag. In a separate experiment, we found that hydrogen peroxide (H2O2) at concentrations as low as 0.001% (equivalent to the concentration of H2O2 in Ag-exposed cells) removed Ag from MT. These results suggest MT was decomposed by cytosolic H2O2, and then Ag released from MT relocated to insoluble cellular fractions and inhibited electron chain transfer of mitochondrial complexes, which eventually led to cell damage. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

Mitochondrial network complexity emerges from fission/fusion dynamics.

PubMed

Zamponi, Nahuel; Zamponi, Emiliano; Cannas, Sergio A; Billoni, Orlando V; Helguera, Pablo R; Chialvo, Dante R

2018-01-10

Mitochondrial networks exhibit a variety of complex behaviors, including coordinated cell-wide oscillations of energy states as well as a phase transition (depolarization) in response to oxidative stress. Since functional and structural properties are often interwinded, here we characterized the structure of mitochondrial networks in mouse embryonic fibroblasts using network tools and percolation theory. Subsequently we perturbed the system either by promoting the fusion of mitochondrial segments or by inducing mitochondrial fission. Quantitative analysis of mitochondrial clusters revealed that structural parameters of healthy mitochondria laid in between the extremes of highly fragmented and completely fusioned networks. We confirmed our results by contrasting our empirical findings with the predictions of a recently described computational model of mitochondrial network emergence based on fission-fusion kinetics. Altogether these results offer not only an objective methodology to parametrize the complexity of this organelle but also support the idea that mitochondrial networks behave as critical systems and undergo structural phase transitions.

Mitochondrial NADH Fluorescence is Enhanced by Complex I Binding

PubMed Central

Blinova, Ksenia; Levine, Rodney L.; Boja, Emily S.; Griffiths, Gary L.; Shi, Zhen-Dan; Ruddy, Brian; Balaban, Robert S.

2012-01-01

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10 fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state. PMID:18702505

Ginsenoside Rg3 improves cardiac mitochondrial population quality: Mimetic exercise training

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Mengwei; Huang, Chenglin; Wang, Cheng

Highlights: •Rg3 is an ergogenic aid. •Rg3 improves mitochondrial antioxidant capacity. •Rg3 regulates mitochondria dynamic remodeling. •Rg3 alone matches some the benefits of aerobic exercise. -- Abstract: Emerging evidence indicates exercise training could mediate mitochondrial quality control through the improvement of mitochondrial dynamics. Ginsenoside Rg3 (Rg3), one of the active ingredients in Panax ginseng, is well known in herbal medicine as a tonic and restorative agent. However, the molecular mechanism underlying the beneficial effects of Rg3 has been elusive. In the present study, we compared the effects of Rg3 administration with aerobic exercise on mitochondrial adaptation in cardiac muscle tissuemore » of Sprague–Dawley (SD) rats. Three groups of SD rats were studied: (1) sedentary control, (2) Rg3-treated and (3) aerobic exercise trained. Both aerobic exercise training and Rg3 supplementation enhanced peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α) and nuclear factor-E2-related factor 2 (Nrf2) protein levels in cardiac muscle. The activation of PGC-1α led to increased mRNA levels of mitochondrial transcription factor A (Tfam) and nuclear related factor 1(Nrf1), these changes were accompanied by increases in mitochondrial DNA copy number and complex protein levels, while activation of Nrf2 increased levels of phase II detoxifying enzymes, including nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1(NQO1), superoxide dismutase (MnSOD) and catalase. Aerobic exercise also enhanced mitochondrial autophagy pathway activity, including increased conversion of LC3-I to LC3-II and greater expression of beclin1 and autophagy-related protein 7 (ATG7), these effects of aerobic exercise are comparable to that of Rg3. These results demonstrate that Rg3 mimics improved cardiac adaptations to exercise by regulating mitochondria dynamic remodeling and enhancing the quantity and quality of mitochondria.« less

Mitochondrial dysfunction in autism.

PubMed

Giulivi, Cecilia; Zhang, Yi-Fan; Omanska-Klusek, Alicja; Ross-Inta, Catherine; Wong, Sarah; Hertz-Picciotto, Irva; Tassone, Flora; Pessah, Isaac N

2010-12-01

Impaired mitochondrial function may influence processes highly dependent on energy, such as neurodevelopment, and contribute to autism. No studies have evaluated mitochondrial dysfunction and mitochondrial DNA (mtDNA) abnormalities in a well-defined population of children with autism. To evaluate mitochondrial defects in children with autism. Observational study using data collected from patients aged 2 to 5 years who were a subset of children participating in the Childhood Autism Risk From Genes and Environment study in California, which is a population-based, case-control investigation with confirmed autism cases and age-matched, genetically unrelated, typically developing controls, that was launched in 2003 and is still ongoing. Mitochondrial dysfunction and mtDNA abnormalities were evaluated in lymphocytes from 10 children with autism and 10 controls. Oxidative phosphorylation capacity, mtDNA copy number and deletions, mitochondrial rate of hydrogen peroxide production, and plasma lactate and pyruvate. The reduced nicotinamide adenine dinucleotide (NADH) oxidase activity (normalized to citrate synthase activity) in lymphocytic mitochondria from children with autism was significantly lower compared with controls (mean, 4.4 [95% confidence interval {CI}, 2.8-6.0] vs 12 [95% CI, 8-16], respectively; P = .001). The majority of children with autism (6 of 10) had complex I activity below control range values. Higher plasma pyruvate levels were found in children with autism compared with controls (0.23 mM [95% CI, 0.15-0.31 mM] vs 0.08 mM [95% CI, 0.04-0.12 mM], respectively; P = .02). Eight of 10 cases had higher pyruvate levels but only 2 cases had higher lactate levels compared with controls. These results were consistent with the lower pyruvate dehydrogenase activity observed in children with autism compared with controls (1.0 [95% CI, 0.6-1.4] nmol × [min × mg protein](-1) vs 2.3 [95% CI, 1.7-2.9] nmol × [min × mg protein](-1), respectively; P = .01). Children with autism had higher mitochondrial rates of hydrogen peroxide production compared with controls (0.34 [95% CI, 0.26-0.42] nmol × [min × mg of protein](-1) vs 0.16 [95% CI, 0.12-0.20] nmol × [min × mg protein](-1) by complex III; P = .02). Mitochondrial DNA overreplication was found in 5 cases (mean ratio of mtDNA to nuclear DNA: 239 [95% CI, 217-239] vs 179 [95% CI, 165-193] in controls; P = 10(-4)). Deletions at the segment of cytochrome b were observed in 2 cases (ratio of cytochrome b to ND1: 0.80 [95% CI, 0.68-0.92] vs 0.99 [95% CI, 0.93-1.05] for controls; P = .01). In this exploratory study, children with autism were more likely to have mitochondrial dysfunction, mtDNA overreplication, and mtDNA deletions than typically developing children.

Effect of palladium α-lipoic acid complex on energy in the brain mitochondria of aged rats.

PubMed

Ajith, Thekkuttuparambil Ananthanarayanan; Nima, Nalin; Veena, Ravindran Kalathil; Janardhanan, Kainoor Krishnankutty; Antonawich, Francis

2014-01-01

According to the mitochondrial mutation theory of aging, the impairment of mitochondrial functions and decline of cellular bioenergetics are induced by highly reactive oxygen species (ROS). Supplementation with antioxidants may protect mitochondria against respiration-linked oxidative stress and reduce decay by preserving genomic and structural integrity. Several clinical studies have reported beneficial effects of α-lipoic acid (LA) administration in individuals with Alzheimer's disease, particularly improving their spatial orientation; however, no studies have been reported on the effects of palladium α-lipoic acid (Pd-LA). The current study examined the effects of the Pd-LA complex on mitochondrial energy status in the brains of aged rats. The study used male Wistar rats, some that were older than 24 mo and weighed approximately 350 ± 50 g and some that were younger than 24 mo and weighed approximately 175 ± 25 g. The research team divided the rats into 5 groups of 6 rats. The study was conducted at the Amala Cancer Research Centre in Amala Nagar, Thrissur, Kerala, India. Three groups of rats were controls: (1) young controls administered no solution, (2) aged controls administered 1 mL/kg of a 0.25% solution (PO) of sodium hydroxide (NaOH), and (3) positive aged controls treated with LA (7.6 mg/kg, PO) dissolved in an alkaline saline (0.25% NaOH, w/v). Two groups were intervention groups: (1) aged rats treated with 1.2 mg/kg of Pd-LA (PO) and (2) aged rats treated with 23.5 mg/kg of Pd-LA (PO). The research team administered the solutions once daily for 30 d. After 30 d, all animals were sacrificed. The research team evaluated serum transaminases, lactate dehydrogenase (LDH), serum urea, and creatinine. The activities of superoxide dismutase (SOD), catalase (CAT), and the levels of reduced glutathione (GSH) were determined in the blood samples. Krebs cycle dehydrogenases were evaluated in the brain mitochondria. Furthermore, the activities of the respiratory chain complexes I, III and IV as well as adenosine triphosphate (ATP) levels were estimated in the mitochondrial fraction. The study found that Pd-LA elevated the mitochondrial ATP levels in the brains of aged rats by enhancing the activity of not only the Krebs cycle dehydrogenases but also complexes I and IV. Furthermore, Pd-LA improved the body weight and blood antioxidant status of aged rats without affecting the functions of liver or renal cells. The results of the current study demonstrate that Pd-LA improves mitochondrial energy status in the brains of aged rats. The effects can be attributed to the enhancing effect on the Krebs cycle dehydrogenase and the activities of complexes I, III, and IV. The results further support the possible use of Pd-LA as an adjuvant treatment, together with the standard cholinesterase inhibitors, in individuals with mild or moderate dementia caused by Alzheimer's disease (AD).

Role of membrane contact sites in protein import into mitochondria

PubMed Central

Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

2015-01-01

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. PMID:25514890

Statins Affect Skeletal Muscle Performance: Evidence for Disturbances in Energy Metabolism.

PubMed

Allard, Neeltje A E; Schirris, Tom J J; Verheggen, Rebecca J; Russel, Frans G M; Rodenburg, Richard J; Smeitink, Jan A M; Thompson, Paul D; Hopman, Maria T E; Timmers, Silvie

2018-01-01

Statin myopathy is linked to disturbances in mitochondrial function and exercise intolerance. To determine whether differences exist in exercise performance, muscle function, and muscle mitochondrial oxidative capacity and content between symptomatic and asymptomatic statin users, and control subjects. Cross-sectional study. Department of Physiology, Radboud University Medical Center. Long-term symptomatic and asymptomatic statin users, and control subjects (n = 10 per group). Maximal incremental cycling tests, involuntary electrically stimulated isometric quadriceps-muscle contractions, and biopsy of vastus lateralis muscle. Maximal exercise capacity, substrate use during exercise, muscle function, and mitochondrial energy metabolism. Peak oxygen uptake, maximal work load, and ventilatory efficiency were comparable between groups, but both statin groups had a depressed anaerobic threshold compared with the control group (P = 0.01). Muscle relaxation time was prolonged in both statin groups compared with the control group and rate of maximal force rise was decreased (Ptime×group < 0.001 for both measures). Mitochondrial activity of complexes II and IV was lower in symptomatic statin users than control subjects and tended to be lower for complex (C) III (CII: P = 0.03; CIII: P = 0.05; CIV: P = 0.04). Mitochondrial content tended to be lower in both statin groups than in control subjects. Statin use attenuated substrate use during maximal exercise performance, induced muscle fatigue during repeated muscle contractions, and decreased muscle mitochondrial oxidative capacity. This suggests disturbances in mitochondrial oxidative capacity occur with statin use even in patients without statin-induced muscle complaints. Copyright © 2017 Endocrine Society

Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dykens, James A.; Jamieson, Joseph; Marroquin, Lisa

2008-12-01

As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanidemore » toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.« less

Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro.

PubMed

Dykens, James A; Jamieson, Joseph; Marroquin, Lisa; Nadanaciva, Sashi; Billis, Puja A; Will, Yvonne

2008-12-01

As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies.

PubMed

Feichtinger, René G; Oláhová, Monika; Kishita, Yoshihito; Garone, Caterina; Kremer, Laura S; Yagi, Mikako; Uchiumi, Takeshi; Jourdain, Alexis A; Thompson, Kyle; D'Souza, Aaron R; Kopajtich, Robert; Alston, Charlotte L; Koch, Johannes; Sperl, Wolfgang; Mastantuono, Elisa; Strom, Tim M; Wortmann, Saskia B; Meitinger, Thomas; Pierre, Germaine; Chinnery, Patrick F; Chrzanowska-Lightowlers, Zofia M; Lightowlers, Robert N; DiMauro, Salvatore; Calvo, Sarah E; Mootha, Vamsi K; Moggio, Maurizio; Sciacco, Monica; Comi, Giacomo P; Ronchi, Dario; Murayama, Kei; Ohtake, Akira; Rebelo-Guiomar, Pedro; Kohda, Masakazu; Kang, Dongchon; Mayr, Johannes A; Taylor, Robert W; Okazaki, Yasushi; Minczuk, Michal; Prokisch, Holger

2017-10-05

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp -/- mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp -/- MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A novel mutation in NDUFS4 causes Leigh syndrome in an Ashkenazi Jewish family.

PubMed

Anderson, S L; Chung, W K; Frezzo, J; Papp, J C; Ekstein, J; DiMauro, S; Rubin, B Y

2008-12-01

Leigh syndrome is a neurodegenerative disorder of infancy or childhood generally due to mutations in nuclear or mitochondrial genes involved in mitochondrial energy metabolism. We performed linkage analysis in an Ashkenazi Jewish (AJ) family without consanguinity with three affected children. Linkage to microsatellite markers D5S1969 and D5S407 led to evaluation of the complex I gene NDUFS4, in which we identified a novel homozygous c.462delA mutation that disrupts the reading frame. The resulting protein lacks a cAMP-dependent protein kinase phosphorylation site required for activation of mitochondrial respiratory chain complex I. In a random sample of 5000 healthy AJ individuals, the carrier frequency of the NDUFS4 mutation c.462delA was 1 in 1000, suggesting that it should be considered in all AJ patients with Leigh syndrome.

Mitochondrial Ca2+ influx targets cardiolipin to disintegrate respiratory chain complex II for cell death induction

PubMed Central

Hwang, M-S; Schwall, C T; Pazarentzos, E; Datler, C; Alder, N N; Grimm, S

2014-01-01

Massive Ca2+ influx into mitochondria is critically involved in cell death induction but it is unknown how this activates the organelle for cell destruction. Using multiple approaches including subcellular fractionation, FRET in intact cells, and in vitro reconstitutions, we show that mitochondrial Ca2+ influx prompts complex II of the respiratory chain to disintegrate, thereby releasing an enzymatically competent sub-complex that generates excessive reactive oxygen species (ROS) for cell death induction. This Ca2+-dependent dissociation of complex II is also observed in model membrane systems, but not when cardiolipin is replaced with a lipid devoid of Ca2+ binding. Cardiolipin is known to associate with complex II and upon Ca2+ binding coalesces into separate homotypic clusters. When complex II is deprived of this lipid, it disintegrates for ROS formation and cell death. Our results reveal Ca2+ binding to cardiolipin for complex II disintegration as a pivotal step for oxidative stress and cell death induction. PMID:24948011

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions.

PubMed

Deegan, Shane; Saveljeva, Svetlana; Logue, Susan E; Pakos-Zebrucka, Karolina; Gupta, Sanjeev; Vandenabeele, Peter; Bertrand, Mathieu J M; Samali, Afshin

2014-01-01

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.

Inactivation of brain mitochondrial Lon protease by peroxynitrite precedes electron transport chain dysfunction.

PubMed

Stanyer, Lee; Jorgensen, Wenche; Hori, Osamu; Clark, John B; Heales, Simon J R

2008-09-01

The accumulation of oxidatively modified proteins has been shown to be a characteristic feature of many neurodegenerative disorders and its regulation requires efficient proteolytic processing. One component of the mitochondrial proteolytic system is Lon, an ATP-dependent protease that has been shown to degrade oxidatively modified aconitase in vitro and may thus play a role in defending against the accumulation of oxidized matrix proteins in mitochondria. Using an assay system that allowed us to distinguish between basal and ATP-stimulated Lon protease activity, we have shown in isolated non-synaptic rat brain mitochondria that Lon protease is highly susceptible to oxidative inactivation by peroxynitrite (ONOO(-)). This susceptibility was more pronounced with regard to ATP-stimulated activity, which was inhibited by 75% in the presence of a bolus addition of 1mM ONOO(-), whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10-1000 microM) revealed that a decline in Lon protease activity preceded electron transport chain (ETC) dysfunction (complex I, II-III and IV) and that ATP-stimulated activity was approximately fivefold more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO(-) exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex. Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress.

Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response.

PubMed

Zhang, Conggang; Liu, Zeyu; Bunker, Eric; Ramirez, Adrian; Lee, Schuyler; Peng, Yinghua; Tan, Aik-Choon; Eckhardt, S Gail; Chapnick, Douglas A; Liu, Xuedong

2017-09-08

Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m -chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Kinetic Modeling of the Mitochondrial Energy Metabolism of Neuronal Cells: The Impact of Reduced α-Ketoglutarate Dehydrogenase Activities on ATP Production and Generation of Reactive Oxygen Species

PubMed Central

Berndt, Nikolaus; Bulik, Sascha; Holzhütter, Hermann-Georg

2012-01-01

Reduced activity of brain α-ketoglutarate dehydrogenase complex (KGDHC) occurs in a number of neurodegenerative diseases like Parkinson's disease and Alzheimer's disease. In order to quantify the relation between diminished KGDHC activity and the mitochondrial ATP generation, redox state, transmembrane potential, and generation of reactive oxygen species (ROS) by the respiratory chain (RC), we developed a detailed kinetic model. Model simulations revealed a threshold-like decline of the ATP production rate at about 60% inhibition of KGDHC accompanied by a significant increase of the mitochondrial membrane potential. By contrast, progressive inhibition of the enzyme aconitase had only little impact on these mitochondrial parameters. As KGDHC is susceptible to ROS-dependent inactivation, we also investigated the reduction state of those sites of the RC proposed to be involved in ROS production. The reduction state of all sites except one decreased with increasing degree of KGDHC inhibition suggesting an ROS-reducing effect of KGDHC inhibition. Our model underpins the important role of reduced KGDHC activity in the energetic breakdown of neuronal cells during development of neurodegenerative diseases. PMID:22719765

Mitochondrial peptides modulate mitochondrial function during cellular senescence.

PubMed

Kim, Su-Jeong; Mehta, Hemal H; Wan, Junxiang; Kuehnemann, Chisaka; Chen, Jingcheng; Hu, Ji-Fan; Hoffman, Andrew R; Cohen, Pinchas

2018-06-10

Cellular senescence is a complex cell fate response that is thought to underlie several age-related pathologies. Despite a loss of proliferative potential, senescent cells are metabolically active and produce energy-consuming effectors, including senescence-associated secretory phenotypes (SASPs). Mitochondria play crucial roles in energy production and cellular signaling, but the key features of mitochondrial physiology and particularly of mitochondria-derived peptides (MDPs), remain underexplored in senescence responses. Here, we used primary human fibroblasts made senescent by replicative exhaustion, doxorubicin or hydrogen peroxide treatment, and examined the number of mitochondria and the levels of mitochondrial respiration, mitochondrial DNA methylation and the mitochondria-encoded peptides humanin, MOTS-c, SHLP2 and SHLP6. Senescent cells showed increased numbers of mitochondria and higher levels of mitochondrial respiration, variable changes in mitochondrial DNA methylation, and elevated levels of humanin and MOTS-c. Humanin and MOTS-c administration modestly increased mitochondrial respiration and selected components of the SASP in doxorubicin-induced senescent cells partially via JAK pathway. Targeting metabolism in senescence cells is an important strategy to reduce SASP production for eliminating the deleterious effects of senescence. These results provide insight into the role of MDPs in mitochondrial energetics and the production of SASP components by senescent cells.

Disruption of hepatic mitochondrial bioenergetics is not a primary mechanism for the toxicity of methoprene - relevance for toxicological assessment.

PubMed

Monteiro, J P; Oliveira, P J; Moreno, A J M; Jurado, A S

2008-07-01

Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) is an insect growth regulator generally used to control insect populations by preventing insect maturation. So far, the effects of the insecticide on mitochondrial bioenergetics were not investigated. In the present work, liver mitochondria from Wistar rats were isolated and features of mitochondrial physiology were characterized in the presence of methoprene. High concentrations of methoprene, in the range of 40-100 nmol/mg of protein could decrease the transmembrane electric potential (Delta Psi) developed by mitochondria and, at the highest concentration, methoprene prevented complete Delta Psi repolarization after ADP addition. The effect was more evident using succinate than with ascorbate+TMPD as substrate. State 3 respiration was approximately 60% inhibited by 80 nmol of methoprene/mg of protein, while state 4 respiration, within the same range of methoprene concentrations, showed a slight increase, when both glutamate-malate and succinate were used as substrates. Additionally, FCCP-stimulated respiration was inhibited to an extent comparable to the effect on state 3, which suggests an interaction of methoprene with the respiratory chain, more evident with glutamate/malate as substrate. The activity of complex I (NADH-ubiquinone oxidorreductase) and that of the segment comprehending complexes II and III (succinate-cytochrome c reductase) were decreased in the presence of methoprene (approximately 60% and 85% of inhibition, respectively, with 300 nmol of methoprene/mg of protein), while the activities of cytochrome c oxidase and ATPase do not seem to be affected. Furthermore, the action of methoprene on the mitochondrial permeability transition was also studied, showing that the insecticide (in the range of 30-80 nmol mg(-1) of protein) decreases the susceptibility of liver mitochondria to the opening of the transition pore, even in non-energized mitochondria. These results lead to the conclusion that methoprene interference with hepatic mitochondrial function occurs only for high concentrations, which implies that the noxious effects of the insecticide reported for a number of non-target organisms are not fully attributable to mitochondrial effects. Therefore, it seems that mitochondrial activity does not represent the primary target for methoprene toxic action.

Impaired adenosine monophosphate-activated protein kinase signalling in dorsal root ganglia neurons is linked to mitochondrial dysfunction and peripheral neuropathy in diabetes

PubMed Central

Smith, Darrell R.; Saleh, Ali; Schapansky, Jason; Marquez, Alexandra; Gomes, Suzanne; Akude, Eli; Morrow, Dwane; Calcutt, Nigel A.; Fernyhough, Paul

2012-01-01

Mitochondrial dysfunction occurs in sensory neurons and may contribute to distal axonopathy in animal models of diabetic neuropathy. The adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signalling axis senses the metabolic demands of cells and regulates mitochondrial function. Studies in muscle, liver and cardiac tissues have shown that the activity of adenosine monophosphate-activated protein kinase and PGC-1α is decreased under hyperglycaemia. In this study, we tested the hypothesis that deficits in adenosine monophosphate-activated protein kinase/PGC-1α signalling in sensory neurons underlie impaired axonal plasticity, suboptimal mitochondrial function and development of neuropathy in rodent models of type 1 and type 2 diabetes. Phosphorylation and expression of adenosine monophosphate-activated protein kinase/PGC-1α and mitochondrial respiratory chain complex proteins were downregulated in dorsal root ganglia of both streptozotocin-diabetic rats and db/db mice. Adenoviral-mediated manipulation of endogenous adenosine monophosphate-activated protein kinase activity using mutant proteins modulated neurotrophin-directed neurite outgrowth in cultures of sensory neurons derived from adult rats. Addition of resveratrol to cultures of sensory neurons derived from rats after 3–5 months of streptozotocin-induced diabetes, significantly elevated adenosine monophosphate-activated protein kinase levels, enhanced neurite outgrowth and normalized mitochondrial inner membrane polarization in axons. The bioenergetics profile (maximal oxygen consumption rate, coupling efficiency, respiratory control ratio and spare respiratory capacity) was aberrant in cultured sensory neurons from streptozotocin-diabetic rats and was corrected by resveratrol treatment. Finally, resveratrol treatment for the last 2 months of a 5-month period of diabetes reversed thermal hypoalgesia and attenuated foot skin intraepidermal nerve fibre loss and reduced myelinated fibre mean axonal calibre in streptozotocin-diabetic rats. These data suggest that the development of distal axonopathy in diabetic neuropathy is linked to nutrient excess and mitochondrial dysfunction via defective signalling of the adenosine monophosphate-activated protein kinase/PGC-1α pathway. PMID:22561641

The life of plant mitochondrial complex I.

PubMed

Braun, Hans-Peter; Binder, Stefan; Brennicke, Axel; Eubel, Holger; Fernie, Alisdair R; Finkemeier, Iris; Klodmann, Jennifer; König, Ann-Christine; Kühn, Kristina; Meyer, Etienne; Obata, Toshihiro; Schwarzländer, Markus; Takenaka, Mizuki; Zehrmann, Anja

2014-11-01

The mitochondrial NADH dehydrogenase complex (complex I) of the respiratory chain has several remarkable features in plants: (i) particularly many of its subunits are encoded by the mitochondrial genome, (ii) its mitochondrial transcripts undergo extensive maturation processes (e.g. RNA editing, trans-splicing), (iii) its assembly follows unique routes, (iv) it includes an additional functional domain which contains carbonic anhydrases and (v) it is, indirectly, involved in photosynthesis. Comprising about 50 distinct protein subunits, complex I of plants is very large. However, an even larger number of proteins are required to synthesize these subunits and assemble the enzyme complex. This review aims to follow the complete "life cycle" of plant complex I from various molecular perspectives. We provide arguments that complex I represents an ideal model system for studying the interplay of respiration and photosynthesis, the cooperation of mitochondria and the nucleus during organelle biogenesis and the evolution of the mitochondrial oxidative phosphorylation system. Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Seokheon; Kim, Joo Yeon; Hwang, Joohyun

Highlights: •Heptachlor inhibited mitochondrial electron transport chain complex III activity. •Heptachlor promoted generation of reactive oxygen species. •Heptachlor induced Bax activation. •Heptachlor induced mitochondria-mediated and caspase-dependent apoptosis. -- Abstract: Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complexmore » III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.« less

Mitochondrial hepato-encephalopathy due to deficiency of QIL1/MIC13 (C19orf70), a MICOS complex subunit.

PubMed

Zeharia, Avraham; Friedman, Jonathan R; Tobar, Ana; Saada, Ann; Konen, Osnat; Fellig, Yacov; Shaag, Avraham; Nunnari, Jodi; Elpeleg, Orly

2016-12-01

The mitochondrial inner membrane possesses distinct subdomains including cristae, which are lamellar structures invaginated into the mitochondrial matrix and contain the respiratory complexes. Generation of inner membrane domains requires the complex interplay between the respiratory complexes, mitochondrial lipids and the recently identified mitochondrial contact site and cristae organizing system (MICOS) complex. Proper organization of the mitochondrial inner membrane has recently been shown to be important for respiratory function in yeast. Here we aimed at a molecular diagnosis in a brother and sister from a consanguineous family who presented with a neurodegenerative disorder accompanied by hyperlactatemia, 3-methylglutaconic aciduria, disturbed hepatocellular function with abnormal cristae morphology in liver and cerebellar and vermis atrophy, which suggest mitochondrial dysfunction. Using homozygosity mapping and exome sequencing the patients were found to be homozygous for the p.(Gly15Glufs*75) variant in the QIL1/MIC13 (C19orf70) gene. QIL1/MIC13 is a constituent of MICOS, a six subunit complex that helps to form and/or stabilize cristae junctions and determine the placement, distribution and number of cristae within mitochondria. In patient fibroblasts both MICOS subunits QIL1/MIC13 and MIC10 were absent whereas MIC60 was present in a comparable abundance to that of the control. We conclude that QIL1/MIC13 deficiency in human, is associated with disassembly of the MICOS complex, with the associated aberration of cristae morphology and mitochondrial respiratory dysfunction. 3-Methylglutaconic aciduria is associated with variants in genes encoding mitochondrial inner membrane organizing determinants, including TAZ, DNAJC19, SERAC1 and QIL1/MIC13.

Leigh disease presenting in utero due to a novel missense mutation in the mitochondrial DNA-ND3.

PubMed

Leshinsky-Silver, Esther; Lev, Dorit; Malinger, Gustavo; Shapira, Daniel; Cohen, Sarit; Lerman-Sagie, Tally; Saada, Ann

2010-05-01

Leigh syndrome can be caused by defects in both nuclear and mitochondrial genes involved in energy metabolism. Recently, an increasing number of mutations in mitochondrial DNA encoding regions, especially in NADH dehydrogenase (respiratory chain complex I) subunits, have been reported as causative of early onset Leigh syndrome. We describe a patient whose fetal brain ultrasound demonstrated periventricular pseudocyst suggestive of a possible mitochondrial disorder who presented postnatally with Leigh syndrome. A muscle biopsy demonstrated a partial decrease in complex I and pyruvate dehydrogenase (PDH-E1 alpha) activity. Sequencing of the PDH-E1 alpha gene did not reveal any mutation. Sequencing of the mtDNA revealed a novel heteroplasmic G10254A (D66N) mutation in the ND3 gene. This change results in a substitution of aspartic acid to asparagine in a highly conserved domain of the ND3 subunit. The mutation could not be detected in the mother's blood or urine sediment. Blue native gel electrophoresis of muscle mitochondria revealed a normal size, albeit a decreased level of complex I. The G10254A substitution in the mtDNA-ND3 gene is another cause of maternally inherited Leigh syndrome. This case demonstrates that periventricular pseudocysts may be the initial in utero presentation in patients with mitochondrial disorders. We emphasize the importance of screening the mtDNA in pediatric patients as the first step in molecular diagnosis of Leigh syndrome. (c) 2010 Elsevier Inc. All rights reserved.

Characterizing the mechanism of thiazolidinedione-induced hepatotoxicity: An in vitro model in mitochondria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Dan; Wu, Chun-qi; Li, Ze-jun

Objective: To characterize the mechanism of action of thiazolidinedione (TZD)-induced liver mitochondrial toxicity caused by troglitazone, rosiglitazone, and pioglitazone in HepaRG cells. Methods: Human hepatoma cells (HepaRG) were treated with troglitazone, rosiglitazone, or pioglitazone (12.5, 25, and 50 μM) for 48 h. The Seahorse Biosciences XF24 Flux Analyzer was used to measure mitochondrial oxygen consumption. The effect of TZDs on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry. The mitochondrial ultrastructure of HepaRG cells was observed under a transmission electrical microscope (TEM). mtDNA content was evaluated by real-time PCR, and ATP content and mitochondrialmore » respiratory chain (MRC) complex I, II, III, IV activity were measured via chemiluminescence. Results were considered statistically significant at p < 0.05. Results: Among the three drugs, troglitazone exhibited the highest potency, followed by rosiglitazone, and then pioglitazone. The TZDs caused varying degrees of mitochondrial respiratory function disorders including decreases in oxygen consumption, MRC activity, and ATP level, and an elevation in ROS level. TZD treatment resulted in mtDNA content decline, reduction in MMP, and alterations of mitochondrial structure. Conclusion: All investigated TZDs show a certain degree of mitochondrial toxicity, with troglitazone exhibiting the highest potency. The underlying mechanism of TZD-induced hepatotoxicity may be associated with alterations in mitochondrial respiratory function disorders, oxidative stress, and changes in membrane permeability. These parameters may be used early in drug development to further optimize risk:benefit profiles. - Highlights: • We compared three TZD mitochondrial toxicity characteristics in HepaRG cells. • TZD induced respiratory disorders and mitochondrial structural damage. • Mitochondrial toxicity evaluation presents guidance value for hepatotoxicity.« less

Nitric Oxide and Mitochondrial Function in Neurological Diseases.

PubMed

Ghasemi, Mehdi; Mayasi, Yunis; Hannoun, Anas; Eslami, Seyed Majid; Carandang, Raphael

2018-04-15

Mitochondria are key cellular organelles that play crucial roles in the energy production and regulation of cellular metabolism. Accumulating evidence suggests that mitochondrial activity can be modulated by nitric oxide (NO). As a key neurotransmitter in biologic systems, NO mediates the majority of its function through activation of the cyclic guanylyl cyclase (cGC) signaling pathway and S-nitrosylation of a variety of proteins involved in cellular functioning including those involved in mitochondrial biology. Moreover, excess NO or the formation of reactive NO species (RNS), e.g., peroxynitrite (ONOO - ), impairs mitochondrial functioning and this, in conjunction with nuclear events, eventually affects neuronal cell metabolism and survival, contributing to the pathogenesis of several neurodegenerative diseases. In this review we highlight the possible mechanisms underlying the noxious effects of excess NO and RNS on mitochondrial function including (i) negative effects on electron transport chain (ETC); (ii) ONOO - -mediated alteration in mitochondrial permeability transition; (iii) enhanced mitochondrial fragmentation and autophagy through S-nitrosylation of key proteins involved in this process such as dynamin-related protein 1 (DRP-1) and Parkin/PINK1 (protein phosphatase and tensin homolog-induced kinase 1) complex; (iv) alterations in the mitochondrial metabolic pathways including Krebs cycle, glycolysis, fatty acid metabolism, and urea cycle; and finally (v) mitochondrial ONOO - -induced nuclear toxicity and subsequent release of apoptosis-inducing factor (AIF) from mitochondria, causing neuronal cell death. These proposed mechanisms highlight the multidimensional nature of NO and its signaling in the mitochondrial function. Understanding the mechanisms by which NO mediates mitochondrial (dys)function can provide new insights into the treatment of neurodegenerative diseases. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Mitochondrial dysfunction precedes depression of AMPK/AKT signaling in insulin resistance induced by high glucose in primary cortical neurons.

PubMed

Peng, Yunhua; Liu, Jing; Shi, Le; Tang, Ying; Gao, Dan; Long, Jiangang; Liu, Jiankang

2016-06-01

Recent studies have demonstrated brain insulin signaling impairment and mitochondrial dysfunction in diabetes. Hyperinsulinemia and hyperlipidemia arising from diabetes have been linked to neuronal insulin resistance, and hyperglycemia induces peripheral sensory neuronal impairment and mitochondrial dysfunction. However, how brain glucose at diabetic conditions elicits cortical neuronal insulin signaling impairment and mitochondrial dysfunction remains unknown. In the present study, we cultured primary cortical neurons with high glucose levels and investigated the neuronal mitochondrial function and insulin response. We found that mitochondrial function was declined in presence of 10 mmol/L glucose, prior to the depression of AKT signaling in primary cortical neurons. We further demonstrated that the cerebral cortex of db/db mice exhibited both insulin resistance and loss of mitochondrial complex components. Moreover, we found that adenosine monophosphate-activated protein kinase (AMPK) inactivation is involved in high glucose-induced mitochondrial dysfunction and insulin resistance in primary cortical neurons and neuroblastoma cells, as well as in cerebral cortex of db/db mice, and all these impairments can be rescued by mitochondrial activator, resveratrol. Taken together, our results extend the finding that high glucose (≥10 mmol/L) comparable to diabetic brain extracellular glucose level leads to neuronal mitochondrial dysfunction and resultant insulin resistance, and targeting mitochondria-AMPK signaling might be a promising strategy to protect against diabetes-related neuronal impairment in central nerves system. We found that high glucose (≥10 mmol/L), comparable to diabetic brain extracellular glucose level, leads to neuronal mitochondrial dysfunction and resultant insulin resistance in an AMPK-dependent manner, and targeting mitochondria-AMPK signaling might be a promising strategy to protect against diabetes-related neuronal impairment in central nerves system. © 2016 International Society for Neurochemistry.

Dopamine transporter SPECT in patients with mitochondrial disorders

PubMed Central

Minnerop, M; Kornblum, C; Joe, A; Tatsch, K; Kunz, W; Klockgether, T; Wullner, U; Reinhardt, M

2005-01-01

Objective : To investigate the dopaminergic system in patients with known mitochondrial disorders and complex I deficiency. Methods: Dopamine transporter density was studied in 10 female patients with mitochondrial complex I deficiency by 123I-FP-CIT (N-ß-fluoropropyl-2ß-carbomethyl-3ß-(4-iodophenyl)-nortropane) SPECT. Results: No differences in 123I-FP-CIT striatal binding ratios were observed and no correlation of the degree of complex I deficiency and striatal binding ratios could be detected. Conclusions: These data argue against the possibility that mitochondrial complex I deficiency by itself is sufficient to elicit dopaminergic cell loss. PMID:15608010

A fluorescence assay for peptide translocation into mitochondria.

PubMed

Martinez-Caballero, Sonia; Peixoto, Pablo M V; Kinnally, Kathleen W; Campo, María Luisa

2007-03-01

Translocation of the presequence is an early event in import of preproteins across the mitochondrial inner membrane by the TIM23 complex. Import of signal peptides, whose sequences mimic mitochondrial import presequences, was measured using a novel, qualitative, fluorescence assay in about 1h. This peptide assay was used in conjunction with classical protein import analyses and electrophysiological approaches to examine the mechanisms underlying the functional effects of depleting two TIM23 complex components. Tim23p forms, at least in part, the pore of this complex while Tim44p forms part of the translocation motor. Depletion of Tim23p eliminates TIM23 channel activity, which interferes with both peptide and preprotein translocation. In contrast, depletion of Tim44p disrupts preprotein but not peptide translocation, which has no effect on TIM23 channel activity. Two conclusions were made. First, this fluorescence peptide assay was validated as two different mutants were accurately identified. Hence, this assay could provide a rapid means of screening mutants to identify those that fail an initial step in import, i.e., translocation of the presequence. Second, translocation of signal peptides required normal channel activity and disruption of the presequence translocase-associated motor complex did not modify TIM23 channel activity nor prevent presequence translocation.

MicroRNA-210 regulates mitochondrial free radical response to hypoxia and krebs cycle in cancer cells by targeting iron sulfur cluster protein ISCU.

PubMed

Favaro, Elena; Ramachandran, Anassuya; McCormick, Robert; Gee, Harriet; Blancher, Christine; Crosby, Meredith; Devlin, Cecilia; Blick, Christopher; Buffa, Francesca; Li, Ji-Liang; Vojnovic, Borivoj; Pires das Neves, Ricardo; Glazer, Peter; Iborra, Francisco; Ivan, Mircea; Ragoussis, Jiannis; Harris, Adrian L

2010-04-26

Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis. In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

Inhibition of neuroinflammation and mitochondrial dysfunctions by carbenoxolone in the rotenone model of Parkinson's disease.

PubMed

Thakur, Poonam; Nehru, Bimla

2015-02-01

α-Synuclein aggregation contributes to the Parkinson's disease (PD) pathology in multiple ways-the two most important being the activation of neuroinflammation and mitochondrial dysfunction. Our recent studies have shown the beneficial effects of a heat shock protein (HSP) inducer, carbenoxolone (Cbx), in reducing the aggregation of α-synuclein in a rotenone-based rat model of PD. The present study was designed to explore its ability to attenuate the α-synuclein-mediated alterations in neuroinflammation and mitochondrial functions. The PD model was generated by the rotenone administration (2 mg/kg b.wt.) to the male SD rats for a period of 5 weeks. Cbx (20 mg/kg b.wt.) co-administration was seen to reduce the activation of astrocytes incited by rotenone. Subsequently, the release of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β was inhibited. Further, the expression level of various inflammatory mediators such as COX-2, iNOS, and NF-κB was also reduced following Cbx co-treatment. Cbx was also shown to reduce the rotenone-induced decline in activity of mitochondrial complexes-I, -II, and -IV. Protection of mitochondrial functions and reduction in neuroinflammation lead to the lesser production of ROS and subsequently reduced oxidative stress. This was reflected by the increase in both the cytosolic and mitochondrial GSH levels as well as SOD activity during Cbx co-treatment. Thus, Cbx reduces the inflammatory response and improves the mitochondrial dysfunctions by reducing α-synuclein aggregation. In addition, it also reduces the associated oxidative stress. Due to its ability to target the multiple pathways implicated in the PD, Cbx can serve as a highly beneficial prophylactic agent.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

PubMed

Hao, Enkui; Mukhopadhyay, Partha; Cao, Zongxian; Erdélyi, Katalin; Holovac, Eileen; Liaudet, Lucas; Lee, Wen-Shin; Haskó, György; Mechoulam, Raphael; Pacher, Pál

2015-01-06

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX's cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

PubMed Central

Hao, Enkui; Mukhopadhyay, Partha; Cao, Zongxian; Erdélyi, Katalin; Holovac, Eileen; Liaudet, Lucas; Lee, Wen-Shin; Haskó, György; Mechoulam, Raphael; Pacher, Pál

2015-01-01

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX’s cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury. PMID:25569804

Novel Blood Biomarkers Are Associated with White Matter Lesions in Fragile X- Associated Tremor/Ataxia Syndrome.

PubMed

Loesch, Danuta Z; Annesley, Sarah J; Trost, Nicholas; Bui, Minh Q; Lay, Sui T; Storey, Elsdon; De Piazza, Shawn W; Sanislav, Oana; Francione, Lisa M; Hammersley, Eleanor M; Tassone, Flora; Francis, David; Fisher, Paul R

2017-01-01

The need for accessible cellular biomarkers of neurodegeneration in carriers of the fragile X mental retardation 1 (FMR1) premutation (PM) alleles. To assess the mitochondrial status and respiration in blood lymphoblasts from PM carriers manifesting the fragile X-associated tremor/ataxia syndrome (FXTAS) and non-FXTAS carriers, and their relationship with the brain white matter lesions. Oxygen consumption rates (OCR) and ATP synthesis using a Seahorse XFe24 Extracellular Flux Analyser, and steady-state parameters of mitochondrial function were assessed in cultured lymphoblasts from 16 PM males (including 11 FXTAS patients) and 9 matched controls. The regional white matter hyperintensity (WMH) scores were obtained from MRI. Mitochondrial respiratory activity was significantly elevated in lymphoblasts from PM carriers compared with controls, with a 2- to 3-fold increase in basal and maximum OCR attributable to complex I activity, and ATP synthesis, accompanied by unaltered mitochondrial mass and membrane potential. The changes, which were more advanced in FXTAS patients, were significantly associated with the WMH scores in the supratentorial regions. The dramatic increase in mitochondrial activity in lymphoblasts from PM carriers may represent either the early stages of disease (specific alterations in short-lived blood cells) or an activation of the lymphocytes under pathological situations. These changes may provide early, convenient blood biomarkers of clinical involvements. © 2016 S. Karger AG, Basel.

Mitochondrial transfer from Wharton's jelly-derived mesenchymal stem cells to mitochondria-defective cells recaptures impaired mitochondrial function.

PubMed

Lin, Hung-Yu; Liou, Chia-Wei; Chen, Shang-Der; Hsu, Te-Yao; Chuang, Jiin-Haur; Wang, Pei-Wen; Huang, Sheng-Teng; Tiao, Mao-Meng; Chen, Jin-Bor; Lin, Tsu-Kung; Chuang, Yao-Chung

2015-05-01

Adult mesenchymal stem cell (MSC)-conducted mitochondrial transfer has been recently shown to rescue cellular bioenergetics and prevent cell death caused by mitochondrial dysfunction. Wharton's jelly-derived MSCs (WJMSCs) harvested from postpartum umbilical cords are an accessible and abundant source of stem cells. This study aimed to determine the capability of WJMSCs to transfer their own mitochondria and rescue impaired oxidative phosphorylation (OXPHOS) and bioenergetics caused by mitochondrial DNA defects. To do this, WJMSCs were co-cultured with mitochondrial DNA (mtDNA)-depleted ρ(0) cells and the recapture of mitochondrial function was evaluated. WJMSCs were shown to be capable of transferring their own mitochondria into ρ(0) cells and underwent interorganellar mixture within these cells. Permissive culture media (BrdU-containing and pyruvate- and uridine-free) sieved out a survival cell population from the co-cultured WJMSCs (BrdU-sensitive) and ρ(0) cells (pyruvate/uridine-free). The survival cells had mtDNA identical to that of WJMSCs, whereas they expressed cellular markers identical to that of ρ(0) cells. Importantly, these ρ(0)-plus -WJMSC-mtDNA (ρ(+W)) cells recovered the expression of mtDNA-encoded proteins and exhibited functional oxygen consumption and respiratory control, as well as the activity of electron transport chain (ETC) complexes I, II, III and IV. In addition, ETC complex V-inhibitor-sensitive ATP production and metabolic shifting were also recovered. Furthermore, cellular behaviors including attachment-free proliferation, aerobic viability and OXPHOS-reliant cellular motility were also regained after mitochondrial transfer by WJMSCs. The therapeutic effect of WJMSCs-derived mitochondrial transfer was able to stably sustain for at least 45 passages. In conclusion, this study suggests that WJMSCs may serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction through the donation of healthy mitochondria to cells with genetic mitochondrial defects. Copyright © 2015 Elsevier B.V. All rights reserved.

Genetic ablation of calcium-independent phospholipase A2gamma leads to alterations in mitochondrial lipid metabolism and function resulting in a deficient mitochondrial bioenergetic phenotype.

PubMed

Mancuso, David J; Sims, Harold F; Han, Xianlin; Jenkins, Christopher M; Guan, Shao Ping; Yang, Kui; Moon, Sung Ho; Pietka, Terri; Abumrad, Nada A; Schlesinger, Paul H; Gross, Richard W

2007-11-30

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A(2) gamma (iPLA(2)gamma), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA(2)gamma in cellular bioenergetics, we generated mice null for the iPLA(2)gamma gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA(2)gamma display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA(2)gamma is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

The Mitochondrial Calcium Uniporter: Mice can live and die without it

PubMed Central

Harrington, Josephine L; Murphy, Elizabeth

2014-01-01

Calcium is of critical importance to mitochondrial and cell function, and calcium signaling is highly localized in the cell. When stimulated, mitochondria are capable of rapidly taking up calcium, affecting both matrix energetics within mitochondria and shaping the amplitude and frequency of cytosolic calcium “waves”. During pathological conditions a large increase in mitochondrial calcium levels is thought to activate the mitochondrial permeability transition pore, resulting in cell death. The protein responsible for mitochondrial calcium uptake, the mitochondrial calcium uniporter (MCU), was identified in 2011 and its molecular elucidation has stimulated and invigorated research in this area. MCU knockout mice have been created, a variety of other regulators have been identified, and a disease phenotype in humans has been attributed to the loss of a uniporter regulator. In the three years since its molecular elucidation, further research into the MCU has revealed a complex uniporter, and raised many questions about its physiologic and pathologic cell roles. PMID:25451167

Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease

PubMed Central

Lood, Christian; Blanco, Luz P.; Purmalek, Monica M.; Carmona-Rivera, Carmelo; De Ravin, Suk S.; Smith, Carolyne K.; Malech, Harry L.; Ledbetter, Jeffrey A.; Elkon, Keith B.; Kaplan, Mariana J.

2015-01-01

Neutrophil extracellular traps (NETs) are implicated in autoimmunity but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes, inducers of NETosis, require mitochondrial ROS for maximal NET stimulation. During this process, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro and, when injected into mice, stimulates type-I interferon (IFN) signaling through a pathway dependent on the DNA sensor, STING. Mitochondrial ROS is also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus (SLE). This was also observed in individuals with chronic granulomatous disease (CGD), which lack NADPH-oxidase activity, but still develop autoimmunity and type I-IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type-I IFN responses in a mouse model of lupus. These findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases. PMID:26779811

Mitochondrial function as a therapeutic target in heart failure

PubMed Central

Brown, David A.; Perry, Justin B.; Allen, Mitchell E.; Sabbah, Hani N.; Stauffer, Brian L.; Shaikh, Saame Raza; Cleland, John G. F.; Colucci, Wilson S.; Butler, Javed; Voors, Adriaan A.; Anker, Stefan D.; Pitt, Bertram; Pieske, Burkert; Filippatos, Gerasimos; Greene, Stephen J.; Gheorghiade, Mihai

2017-01-01

Heart failure is a pressing worldwide public-health problem with millions of patients having worsening heart failure. Despite all the available therapies, the condition carries a very poor prognosis. Existing therapies provide symptomatic and clinical benefit, but do not fully address molecular abnormalities that occur in cardiomyocytes. This shortcoming is particularly important given that most patients with heart failure have viable dysfunctional myocardium, in which an improvement or normalization of function might be possible. Although the pathophysiology of heart failure is complex, mitochondrial dysfunction seems to be an important target for therapy to improve cardiac function directly. Mitochondrial abnormalities include impaired mitochondrial electron transport chain activity, increased formation of reactive oxygen species, shifted metabolic substrate utilization, aberrant mitochondrial dynamics, and altered ion homeostasis. In this Consensus Statement, insights into the mechanisms of mitochondrial dysfunction in heart failure are presented, along with an overview of emerging treatments with the potential to improve the function of the failing heart by targeting mitochondria. PMID:28004807

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.

PubMed

Zhang, Yuxun; Bharathi, Sivakama S; Rardin, Matthew J; Lu, Jie; Maringer, Katherine V; Sims-Lucas, Sunder; Prochownik, Edward V; Gibson, Bradford W; Goetzman, Eric S

2017-06-16

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro , succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5 -/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5 -/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5 -/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Short Communication: Transplacental Nucleoside Analogue Exposure and Mitochondrial Parameters in HIV-Uninfected Children

PubMed Central

Brogly, Susan B.; DiMauro, Salvatore; Van Dyke, Russell B.; Williams, Paige L.; Naini, Ali; Libutti, Daniel E.; Choi, Julia; Chung, Michelle

2011-01-01

Abstract Transplacental nucleoside analogue exposure can affect infant mitochondrial DNA (mtDNA). We evaluated mitochondria in peripheral blood mononuclear cells of children with and without clinical signs of mitochondrial dysfunction (MD) and antiretroviral (ARV) exposure. We previously identified 20 children with signs of MD (cases) among 1037 HIV-uninfected children born to HIV-infected women. We measured mtDNA copies/cell and oxidative phosphorylation (OXPHOS) NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV) protein levels and enzyme activities, determined mtDNA haplogroups and deletions in 18 of 20 cases with stored samples and in sex- and age-matched HIV-uninfected children, both ARV exposed and unexposed, (1) within 18 months of birth and (2) at the time of presentation of signs of MD. In specimens drawn within 18 months of birth, mtDNA levels were higher and OXPHOS protein levels and enzyme activities lower in cases than controls. In contrast, at the time of MD presentation, cases and ARV-exposed controls had lower mtDNA levels, 214 and 215 copies/cell, respectively, than ARV-unexposed controls, 254 copies/cell. OXPHOS protein levels and enzyme activities were lower in cases than exposed controls, and higher in cases than unexposed controls, except for complex IV activity, which was higher in cases. Haplotype H was less frequent among cases (6%) than controls (31%). No deletions were found. The long-term significance of these small but potentially important alterations should continue to be studied as these children enter adolescence and adulthood. PMID:21142587

Selective muscle fiber loss and molecular compensation in mitochondrial myopathy due to TK2 deficiency.

PubMed

Vilà, Maya R; Villarroya, Joan; García-Arumí, Elena; Castellote, Amparo; Meseguer, Anna; Hirano, Michio; Roig, Manuel

2008-04-15

A 12-year-old patient with mitochondrial DNA (mtDNA) depletion syndrome due to TK2 gene mutations has been evaluated serially over the last 10 years. We observed progressive muscle atrophy with selective loss of type 2 muscle fibers and, despite severe depletion of mtDNA, normal activities of respiratory chain (RC) complexes and levels of COX II mitochondrial protein in the remaining muscle fibers. These results indicate that compensatory mechanisms account for the slow progression of the disease. Identification of factors that ameliorate mtDNA depletion may reveal new therapeutic targets for these devastating disorders.

Aconitase couples metabolic regulation to mitochondrial DNA maintenance.

PubMed

Chen, Xin Jie; Wang, Xiaowen; Kaufman, Brett A; Butow, Ronald A

2005-02-04

Mitochondrial DNA (mtDNA) is essential for cells to maintain respiratory competency and is inherited as a protein-DNA complex called the nucleoid. We have identified 22 mtDNA-associated proteins in yeast, among which is mitochondrial aconitase (Aco1p). We show that this Krebs-cycle enzyme is essential for mtDNA maintenance independent of its catalytic activity. Regulation of ACO1 expression by the HAP and retrograde metabolic signaling pathways directly affects mtDNA maintenance. When constitutively expressed, Aco1p can replace the mtDNA packaging function of the high-mobility-group protein Abf2p. Thus, Aco1p may integrate metabolic signals and mtDNA maintenance.

Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans

PubMed Central

Dingley, Stephen D.; Polyak, Erzsebet; Ostrovsky, Julian; Srinivasan, Satish; Lee, Icksoo; Rosenfeld, Amy B.; Tsukikawa, Mai; Xiao, Rui; Selak, Mary A.; Coon, Joshua J.; Hebert, Alexander S.; Grimsrud, Paul A.; Kwon, Young Joon; Pagliarini, David J.; Gai, Xiaowu; Schurr, Theodore G.; Hüttemann, Maik; Nakamaru-Ogiso, Eiko; Falk, Marni J.

2014-01-01

Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20 °C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25 °C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856 > N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856 > N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear– mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism. PMID:24534730

Rat liver mitochondrial dysfunction by addition of copper(II) or iron(III) ions.

PubMed

Saporito-Magriñá, Christian; Musacco-Sebio, Rosario; Acosta, Juan M; Bajicoff, Sofía; Paredes-Fleitas, Paola; Boveris, Alberto; Repetto, Marisa G

2017-01-01

Increased copper (Cu) and iron (Fe) levels in liver and brain are associated to oxidative stress and damage with increased phospholipid oxidation process. The aim of this work was to assess the toxic effects of Cu 2+ and Fe 3+ addition to rat liver mitochondria by determining mitochondrial respiration in states 3 (active respiration) and 4 (resting respiration), and phospholipid peroxidation. Both, Cu 2+ and Fe 3+ produced decreases in O 2 consumption in a concentration-dependent manner in active state 3: both ions by 42% with malate-glutamate as complex I substrate (concentration for half maximal response (C 50 ) 60μM Cu 2+ and 1.25mM Fe 3+ ), and with succinate as complex II substrate: 64-69% with C 50 of 50μM Cu 2+ and with C 50 of 1.25mM of Fe 3+ . Respiratory control decreased with Cu 2+ (C 50 50μM) and Fe 3+ (C 50 1.25-1-75mM) with both substrates. Cu 2+ produced a 2-fold increase and Fe 3+ a 5-fold increase of thiobarbituric acid-reactive substances (TBARS) content from 25μM Cu 2+ (C 50 40μM) and from 100μM Fe 3+ (C 50 1.75mM). Supplementations with Cu 2+ and Fe 3+ ions induce mitochondrial dysfunction with phospholipid peroxidation in rat liver mitochondria. Although is proved that a Fenton/Haber Weiss mechanism of oxidative damage occurs in metal-ion induced mitochondrial toxicity, slightly different responses to the metal ions suggest some differences in the mechanism of intracellular toxicity. The decreased rates of mitochondrial respiration and the alteration of mitochondrial function by phospholipid and protein oxidations lead to mitochondrial dysfunction, cellular dyshomeostasis and cell death. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of alpha-melanocyte-stimulating hormone on mitochondrial energy metabolism in rats of different age-groups.

PubMed

Feichtinger, René G; Pétervári, Erika; Zopf, Michaela; Vidali, Silvia; Aminzadeh-Gohari, Sepideh; Mayr, Johannes A; Kofler, Barbara; Balaskó, Márta

2017-08-01

Hypothalamic alpha-melanocyte-stimulating hormone (α-MSH) is a key catabolic mediator of energy homeostasis. Its anorexigenic and hypermetabolic effects show characteristic age-related alterations that may be part of the mechanism of middle-aged obesity and geriatric anorexia/cachexia seen in humans and other mammals. We aimed to investigate the role of α-MSH in mitochondrial energy metabolism during the course of aging in a rodent model. To determine the role of α-MSH in mitochondrial energy metabolism in muscle, we administered intracerebroventricular (ICV) infusions of α-MSH for 7-days to different age-groups of male Wistar rats. The activities of oxidative phosphorylation complexes I to V and citrate synthase were determined and compared to those of age-matched controls. We also quantified mitochondrial DNA (mtDNA) copy number and measured the expression of the master regulators of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor gamma (PPARγ). The peptide reduced weight gain in juvenile rats to one fifth of that of controls and increased the weight loss in older animals by about five fold. Mitochondrial DNA copy number inversely correlated with changes in body weight in controls, but not in α-MSH-treated animals. The strong increase in body weight in young rats was associated with a low mtDNA copy number and high PPARγ mRNA levels in controls. Expression of PGC-1α and PPARγ declined with age, whereas OXPHOS and citrate synthase enzyme activities were unchanged. In contrast, α-MSH treatment suppressed OXPHOS enzyme and citrate synthase activity. In conclusion, our results showed age-related differences in the metabolic effects of α-MSH. In addition, administration of α-MSH suppressed citrate synthase and OXPHOS activities independent of age. These findings suggest that α-MSH exposure may inhibit mitochondrial biogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation*

PubMed Central

Letts, James A.; Degliesposti, Gianluca; Fiedorczuk, Karol; Skehel, Mark; Sazanov, Leonid A.

2016-01-01

NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies. PMID:27672209

Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: benefits of Alda-1

PubMed Central

Gomes, Katia M.S.; Bechara, Luiz R.G.; Lima, Vanessa M.; Ribeiro, Márcio A.C.; Campos, Juliane C.; Dourado, Paulo M.; Kowaltowski, Alicia J.; Mochly-Rosen, Daria; Ferreira, Julio C.B.

2015-01-01

Background/Objectives We previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy is unknown and should be established to determine the optimal time window for drug treatment. Methods Here we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H2O2 release during the 4 weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats. Results We observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H2O2 release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24hrs after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats. Conclusion Taken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction cardiomyopathy. PMID:25464432

Late Administration of a Palladium Lipoic Acid Complex (POLY-MVA) Modifies Cardiac Mitochondria but Not Functional or Structural Manifestations of Radiation-Induced Heart Disease in a Rat Model

PubMed Central

Sridharan, Vijayalakshmi; Seawright, John W.; Antonawich, Francis J.; Garnett, Merrill; Cao, Maohua; Singh, Preeti; Boerma, Marjan

2017-01-01

Exposure of the heart to ionizing radiation can cause adverse myocardial remodeling. In small animal models, local heart irradiation causes persistent alterations in cardiac mitochondrial function and swelling. POLY-MVA is a dietary supplement that contains a palladium lipoic acid complex that targets mitochondrial complex I and has been demonstrated to have greater redox potential than lipoic acid alone. POLY-MVA improves mitochondrial function and anti-oxidant enzyme activity in the aged rat heart. In this study, we tested whether POLY-MVA can mitigate cardiac effects of ionizing radiation. Adult male rats were exposed to local heart X rays with a daily dose of 9 Gy for 5 consecutive days. Eighteen weeks after irradiation, POLY-MVA was administered orally at 1 ml/kg bodyweight per day during weekdays, for 6 weeks. Alterations in cardiac function as measured with echocardiography coincided with enhanced mitochondrial swelling, a reduction in mitochondrial expression of complex II, manifestations of adverse remodeling such as a reduction in myocardial microvessel density and an increase in collagen deposition and mast cell numbers. POLY-MVA enhanced left ventricular expression of superoxide dismutase 2, but only in sham-irradiated animals. In irradiated animals, POLY-MVA caused a reduction in markers of inflammatory infiltration, CD2 and CD68. Moreover, POLY-MVA mitigated the effects of radiation on mitochondria. Nonetheless, POLY-MVA did not mitigate adverse cardiac remodeling, suggesting that this tissue remodeling may not be alleviated by altering cardiac mitochondria alone. However, we cannot exclude the possibility that an earlier onset of POLY-MVA administration may have more profound effects on radiation-induced cardiac remodeling. PMID:28231026

O-GlcNAcase deficiency suppresses skeletal myogenesis and insulin sensitivity in mice through the modulation of mitochondrial homeostasis.

PubMed

Wang, Xun; Feng, Zhihui; Wang, Xueqiang; Yang, Liang; Han, Shujun; Cao, Ke; Xu, Jie; Zhao, Lin; Zhang, Yong; Liu, Jiankang

2016-06-01

O-GlcNAcylation is implicated in modulating mitochondrial function, which is closely involved in regulating muscle metabolism. The presence of O-GlcNAcase (OGA), the enzyme involved in the removal of O-GlcNAc, in mitochondria was recently confirmed in rats. In the present study, we investigated the regulation of myogenesis and muscle insulin sensitivity to OGA in mice, with a focus on mitochondria. C57BL/6J mice fed a high-fat diet for 4 months were used to observe mitochondrial density, activity and O-GlcNAcylation in muscle. Small interfering RNA and overexpression vectors were used to modulate protein content in vitro. High-fat feeding decreased the OGA level and largely increased mitochondrial O-GlcNAcylation in mouse skeletal muscle that was accompanied by decreased levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), decreased mitochondrial density and disrupted mitochondrial complex activities. Knockdown of OGA in C2C12 myoblasts promoted PGC-1α degradation, resulting in the suppression of mitochondrial biogenesis and myogenesis, whereas neither knockdown of O-GlcNAc transferase nor overexpression of OGA had significant effects on myogenesis. Mitochondrial dysfunction as evidenced by decreased ATP content and increased reactive oxygen species production, and increased lipid and protein oxidation was observed in both myoblasts and myotubes after OGA knockdown. Meanwhile, elevated O-GlcNAcylation through either OGA knockdown or treatment with the OGA inhibitor PUGNAc and the O-GlcNAc transferase substrate D-GlcNAc suppressed myotube insulin signalling transduction and glucose uptake. OGA overexpression had no significant effect on insulin sensitivity but sufficiently improved the insulin resistance induced by D-GlcNAc treatment. These data suggest that OGA can modulate mitochondrial density via PGC-1α and mitochondrial function via protein O-GlcNAcylation. In this manner, OGA appears to play a key role in myogenesis and the development of muscle insulin resistance.

Magnolol protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity through activation of mitochondrial function.

PubMed

Choi, Eun Mi

2012-06-01

Antimycin A treatment of cells blocks the mitochondrial electron transport chain and leads to elevated ROS generation. In the present study, we investigated the protective effects of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, on antimycin A-induced toxicity in osteoblastic MC3T3-E1 cells. Osteoblastic MC3T3-E1 cells were pre-incubated with magnolol before treatment with antimycin A. Cell viability and mineralization of osteoblasts were assessed by MTT assay and Alizarin Red staining, respectively. Mitochondrial dysfunction in cells was measured by mitochondrial membrane potential (MMP), complex IV activity, and ATP level. The cellular antioxidant effect of magnolol in osteoblastic MC3T3-E1 cells was assessed by measuring cardiolipin oxidation, mitochondrial superoxide levels, and nitrotyrosine content. Phosphorylated cAMP-response element-binding protein (CREB ) was evaluated using ELISA assay. Pretreatment with magnolol prior to antimycin A exposure significantly reduced antimycin A-induced osteoblast dysfunction by preventing MMP dissipation, ATP loss, and CREB inactivation. Magnolol also reduced cardiolipin peroxidation, mitochondrial superoxide, and nitrotyrosine production induced by antimycin A. These results suggest that magnolol has a protective effect against antimycin A-induced cell damage by its antioxidant effects and the attenuation of mitochondrial dysfunction. All these data indicate that magnolol may reduce or prevent osteoblast degeneration in osteoporosis or other degenerative disorders.

Protosappanin B protects PC12 cells against oxygen-glucose deprivation-induced neuronal death by maintaining mitochondrial homeostasis via induction of ubiquitin-dependent p53 protein degradation.

PubMed

Zeng, Ke-Wu; Liao, Li-Xi; Zhao, Ming-Bo; Song, Fang-Jiao; Yu, Qian; Jiang, Yong; Tu, Peng-Fei

2015-03-15

Protosappanin B (PTB) is a bioactive dibenzoxocin derivative isolated from Caesalpinia sappan L. Here, we investigated the neuroprotective effects and the potential mechanisms of PTB on oxygen-glucose deprivation (OGD)-injured PC12 cells. Results showed that PTB significantly increased cell viability, inhibited cell apoptosis and up-regulated the expression of growth-associated protein 43 (a marker of neural outgrowth). Moreover, our study revealed that PTB effectively maintained mitochondrial homeostasis by up-regulation of mitochondrial membrane potential (MMP), inhibition of cytochrome c release from mitochondria and inactivation of mitochondrial caspase-9/3 apoptosis pathway. Further study showed that PTB significantly promoted cytoplasmic component degradation of p53 protein, a key negative regulator for mitochondrial function, resulting in a release of Bcl-2 from p53-Bcl-2 complex and an enhancing translocation of Bcl-2 to mitochondrial outer membrane. Finally, we found the degradation of p53 protein was induced by PTB via activation of a MDM2-dependent ubiquitination process. Taken together, our findings provided a new viewpoint of neuronal protection strategy for anoxia and ischemic injury with natural small molecular dibenzoxocin derivative by activating ubiquitin-dependent p53 protein degradation as well as increasing mitochondrial function. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel C12orf65 mutations in patients with axonal neuropathy and optic atrophy.

PubMed

Tucci, Arianna; Liu, Yo-Tsen; Preza, Elisabeth; Pitceathly, Robert D S; Chalasani, Annapurna; Plagnol, Vincent; Land, John M; Trabzuni, Daniah; Ryten, Mina; Jaunmuktane, Zane; Reilly, Mary M; Brandner, Sebastian; Hargreaves, Iain; Hardy, John; Singleton, Andrew B; Abramov, Andrey Y; Houlden, Henry

2014-05-01

Charcot-Marie Tooth disease (CMT) forms a clinically and genetically heterogeneous group of disorders. Although a number of disease genes have been identified for CMT, the gene discovery for some complex form of CMT has lagged behind. The association of neuropathy and optic atrophy (also known as CMT type 6) has been described with autosomaldominant, recessive and X-linked modes of inheritance. Mutations in Mitofusin 2 have been found to cause dominant forms of CMT6. Phosphoribosylpyrophosphate synthetase-I mutations cause X-linked CMT6, but until now, mutations in the recessive forms of disease have never been identified. We here describe a family with three affected individuals who inherited in an autosomal recessive fashion a childhood onset neuropathy and optic atrophy. Using homozygosity mapping in the family and exome sequencing in two affected individuals we identified a novel protein-truncating mutation in the C12orf65 gene, which encodes for a protein involved in mitochondrial translation. Using a variety of methods we investigated the possibility of mitochondrial impairment in the patients cell lines. We described a large consanguineous family with neuropathy and optic atrophy carrying a loss of function mutation in the C12orf65 gene. We report mitochondrial impairment in the patients cell lines, followed by multiple lines of evidence which include decrease of complex V activity and stability (blue native gel assay), decrease in mitochondrial respiration rate and reduction of mitochondrial membrane potential. This work describes a mutation in the C12orf65 gene that causes recessive form of CMT6 and confirms the role of mitochondrial dysfunction in this complex axonal neuropathy.

Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction.

PubMed

Varela, Ana T; Gomes, Ana P; Simões, Anabela M; Teodoro, João S; Duarte, Filipe V; Rolo, Anabela P; Palmeira, Carlos M

2008-12-01

Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10microM, significantly depresses the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25microM stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.

Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varela, Ana T.; Gomes, Ana P.; Simoes, Anabela M.

2008-12-01

Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10{mu}M, significantly depressesmore » the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25{mu}M stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.« less

Adenoviral Gene Transfer of Hepatic Stimulator Substance Confers Resistance Against Hepatic Ischemia–Reperfusion Injury by Improving Mitochondrial Function

PubMed Central

Jiang, Shu-Jun; Li, Wen

2013-01-01

Abstract Hepatic stimulator substance (HSS) has been suggested to protect liver cells from various toxins. However, the precise role of HSS in hepatic ischemia–reperfusion (I/R) injury remains unknown. This study aims to elucidate whether overexpression of HSS could attenuate hepatic ischemia–reperfusion injury and its possible mechanisms. Both in vivo hepatic I/R injury in mice and in vitro hypoxia–reoxygenation (H/R) in a cell model were used to evaluate the effect of HSS protection after adenoviral gene transfer. Moreover, a possible mitochondrial mechanism of HSS protection was investigated. Efficient transfer of the HSS gene into liver inhibited hepatic I/R injury in mice, as evidenced by improvement in liver function tests, the preservation of hepatic morphology, and a reduction in hepatocyte apoptosis. HSS overexpression also inhibited H/R-induced cell death, as detected by cell viability and cell apoptosis assays. The underlying mechanism of this hepatic protection might involve the attenuation of mitochondrial dysfunction and mitochondrial-dependent cell apoptosis, as shown by the good preservation of mitochondrial ultrastructure, mitochondrial membrane potential, and the inhibition of cytochrome c leakage and caspase activity. Moreover, the suppression of H/R-induced mitochondrial ROS production and the maintenance of mitochondrial respiratory chain complex activities may participate in this mechanism. This new function of HSS expands the possibility of its application for the prevention of I/R injury, such as hepatic resection and liver transplantation in clinical practice. PMID:23461564

Voluntary exercise promotes beneficial anti-aging mechanisms in SAMP8 female brain.

PubMed

Bayod, Sergi; Guzmán-Brambila, Carolina; Sanchez-Roige, Sandra; Lalanza, Jaume F; Kaliman, Perla; Ortuño-Sahagun, Daniel; Escorihuela, Rosa M; Pallàs, Mercè

2015-02-01

Regular physical exercise mediates health and longevity promotion involving Sirtuin 1 (SIRT1)-regulated pathways. The anti-aging activity of SIRT1 is achieved, at least in part, by means of fine-tuning the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway by preventing the transition of an originally pro-survival program into a pro-aging mechanism. Additionally, SIRT1 promotes mitochondrial function and reduces the production of reactive oxygen species (ROS) through regulating peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), the master controller of mitochondrial biogenesis. Here, by using senescence-accelerated mice prone 8 (SAMP8) as a model for aging, we determined the effect of wheel-running as a paradigm for long-term voluntary exercise on SIRT1-AMPK pathway and mitochondrial functionality measured by oxidative phosphorylation (OXPHOS) complex content in the hippocampus and cortex. We found differential activation of SIRT1 in both tissues and hippocampal-specific activation of AMPK. These findings correlated well with significant changes in OXPHOS in the hippocampal, but not in the cerebral cortex, area. Collectively, the results revealed greater benefits of the exercise in the wheel-running intervention in a murine model of senescence, which was directly related with mitochondrial function and which was mediated through the modulation of SIRT1 and AMPK pathways.

Correlation of mitochondrial protein expression in complexes I to V with natural and induced forms of canine idiopathic dilated cardiomyopathy.

PubMed

Lopes, Rosana; Solter, Philip F; Sisson, D David; Oyama, Mark A; Prosek, Robert

2006-06-01

To identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with natural or induced dilated cardiomyopathy (DCM). Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with DCM induced by rapid right ventricular pacing. Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by 2-fold or greater between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. A total of 22 altered mitochondrial proteins were identified in complexes I to V. Ten and 12 were found in complex I and complexes II to V, respectively. Five were mitochondrial encoded, and 17 were nuclear encoded. Most altered mitochondrial proteins in tissue specimens from dogs with naturally occurring DCM were associated with complexes I and V, whereas in tissue specimens from dogs subjected to rapid ventricular pacing, complexes I and IV were more affected. In the experimentally induced form of DCM, only nuclear-encoded subunits were changed in complex I. In both disease groups, the 22-kd subunit was downregulated. Natural and induced forms of DCM resulted in altered mitochondrial protein expression in complexes I to V. However, subcellular differences between the experimental and naturally occurring forms of DCM may exist.

Lack of β, β-carotene -9’, 10’-oxygenase 2 leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice

PubMed Central

Wu, Lei; Guo, Xin; Hartson, Steven D.; Davis, Mary Abby; He, Hui; Medeiros, Denis M.; Wang, Weiqun; Clarke, Stephen L.; Lucas, Edralin; Smith, Brenda J.; von Lintig, Johannes; Lin, Dingbo

2017-01-01

Scope β,β-carotene-9’,10’-dioxygenase 2 (BCO2) is a carotenoid cleavage enzyme localized to the inner mitochondrial membrane in mammals. This study was aimed to assess the impact of genetic ablation of BCO2 on hepatic oxidative stress through mitochondrial function in mice. Methods and Results Liver samples from 6 week old male BCO2−/− knockout (KO) and isogenic wild-type (WT) mice were subjected to proteomics and functional activity assays. Compared to the WT, KO mice consumed more food (by 18 %) yet displayed significantly lower body weight (by 12 %). Mitochondrial proteomic results demonstrated that loss of BCO2 was associated with quantitative changes of the mitochondrial proteome mainly shown by suppressed expression of enzymes and/or proteins involved in fatty acid β–oxidation, the tricarboxylic acid cycle, and the electron transport chain (ETC). The mitochondrial basal respiratory rate, proton leak, and ETC complex II capacity were significantly elevated in the livers of KO compared to WT mice. Moreover, elevated reactive oxygen species and increased mitochondrial protein carbonylation were also demonstrated in liver of KO mice. Conclusions Loss of BCO2 induces mitochondrial hyperactivation, mitochondrial stress and changes of the mitochondrial proteome, leading to mitochondrial energy insufficiency. BCO2 appears to be critical for proper hepatic mitochondrial function. PMID:27991717

Lycopene Prevents Amyloid [Beta]-Induced Mitochondrial Oxidative Stress and Dysfunctions in Cultured Rat Cortical Neurons.

PubMed

Qu, Mingyue; Jiang, Zheng; Liao, Yuanxiang; Song, Zhenyao; Nan, Xinzhong

2016-06-01

Brains affected by Alzheimer's disease (AD) show a large spectrum of mitochondrial alterations at both morphological and genetic level. The causal link between β-amyloid (Aβ) and mitochondrial dysfunction has been established in cellular models of AD. We observed previously that lycopene, a member of the carotenoid family of phytochemicals, could counteract neuronal apoptosis and cell damage induced by Aβ and other neurotoxic substances, and that this neuroprotective action somehow involved the mitochondria. The present study aims to investigate the effects of lycopene on mitochondria in cultured rat cortical neurons exposed to Aβ. It was found that lycopene attenuated Aβ-induced oxidative stress, as evidenced by the decreased intracellular reactive oxygen species generation and mitochondria-derived superoxide production. Additionally, lycopene ameliorated Aβ-induced mitochondrial morphological alteration, opening of the mitochondrial permeability transition pores and the consequent cytochrome c release. Lycopene also improved mitochondrial complex activities and restored ATP levels in Aβ-treated neuron. Furthermore, lycopene prevented mitochondrial DNA damages and improved the protein level of mitochondrial transcription factor A in mitochondria. Those results indicate that lycopene protects mitochondria against Aβ-induced damages, at least in part by inhibiting mitochondrial oxidative stress and improving mitochondrial function. These beneficial effects of lycopene may account for its protection against Aβ-induced neurotoxicity.

Impaired mitochondrial oxidative phosphorylation and supercomplex assembly in rectus abdominis muscle of diabetic obese individuals.

PubMed

Antoun, Ghadi; McMurray, Fiona; Thrush, A Brianne; Patten, David A; Peixoto, Alyssa C; Slack, Ruth S; McPherson, Ruth; Dent, Robert; Harper, Mary-Ellen

2015-12-01

Skeletal muscle mitochondrial dysfunction has been documented in patients with type 2 diabetes mellitus; however, specific respiratory defects and their mechanisms are poorly understood. The aim of the current study was to examine oxidative phosphorylation and electron transport chain (ETC) supercomplex assembly in rectus abdominis muscles of 10 obese diabetic and 10 obese non-diabetic individuals. Twenty obese women undergoing Roux-en-Y gastric bypass surgery were recruited for this study. Muscle samples were obtained intraoperatively and subdivided for multiple analyses, including high-resolution respirometry and assessment of supercomplex assembly. Clinical data obtained from referring physicians were correlated with laboratory findings. Participants in both groups were of a similar age, weight and BMI. Mitochondrial respiration rates were markedly reduced in diabetic vs non-diabetic patients. This defect was observed during maximal ADP-stimulated respiration in the presence of complex I-linked substrates and complex I- and II-linked substrates, and during maximal uncoupled respiration. There were no differences in fatty acid (octanoyl carnitine) supported respiration, leak respiration or isolated activity of cytochrome c oxidase. Intriguingly, significant correlations were found between glycated haemoglobin (HbA1c) levels and maximal respiration or respiration supported by complex I, complex I and II or fatty acid. In the muscle of diabetic patients, blue native gel electrophoresis revealed a striking decrease in complex I, III and IV containing ETC supercomplexes. These findings support the hypothesis that ETC supercomplex assembly may be an important underlying mechanism of muscle mitochondrial dysfunction in type 2 diabetes mellitus.

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

PubMed

Danylovych, H V

2016-01-01

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

Cholesterol contributes to dopamine-neuronal loss in MPTP mouse model of Parkinson’s disease: Involvement of mitochondrial dysfunctions and oxidative stress

PubMed Central

Kumar, Sanjeev; Giri, Anirudha; Sandhir, Rajat

2017-01-01

Hypercholesterolemia is a known contributor to the pathogenesis of Alzheimer’s disease while its role in the occurrence of Parkinson’s disease (PD) is only conjecture and far from conclusive. Altered antioxidant homeostasis and mitochondrial functions are the key mechanisms in loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain in PD. Hypercholesterolemia is reported to cause oxidative stress and mitochondrial dysfunctions in the cortex and hippocampus regions of the brain in rodents. However, the impact of hypercholesterolemia on the midbrain dopaminergic neurons in animal models of PD remains elusive. We tested the hypothesis that hypercholesterolemia in MPTP model of PD would potentiate dopaminergic neuron loss in SN by disrupting mitochondrial functions and antioxidant homeostasis. It is evident from the present study that hypercholesterolemia in naïve animals caused dopamine neuronal loss in SN with subsequent reduction in striatal dopamine levels producing motor impairment. Moreover, in the MPTP model of PD, hypercholesterolemia exacerbated MPTP-induced reduction of striatal dopamine as well as dopaminergic neurons in SN with motor behavioral depreciation. Activity of mitochondrial complexes, mainly complex-I and III, was impaired severely in the nigrostriatal pathway of hypercholesterolemic animals treated with MPTP. Hypercholesterolemia caused oxidative stress in the nigrostriatal pathway with increased generation of hydroxyl radicals and enhanced activity of antioxidant enzymes, which were further aggravated in the hypercholesterolemic mice with Parkinsonism. In conclusion, our findings provide evidence of increased vulnerability of the midbrain dopaminergic neurons in PD with hypercholesterolemia. PMID:28170429

Mitochondrial impairments contribute to Spinocerebellar ataxia type 1 progression and can be ameliorated by the mitochondria-targeted antioxidant MitoQ.

PubMed

Stucki, David M; Ruegsegger, Céline; Steiner, Silvio; Radecke, Julika; Murphy, Michael P; Zuber, Benoît; Saxena, Smita

2016-08-01

Spinocerebellar ataxia type 1 (SCA1), due to an unstable polyglutamine expansion within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), decreasing motor coordination and causing death within 10-15 years of diagnosis. Currently, there are no therapies available to slow down disease progression. As secondary cellular impairments contributing to SCA1 progression are poorly understood, here, we focused on identifying those processes by performing a PC specific proteome profiling of Sca1(154Q/2Q) mice at a symptomatic stage. Mass spectrometry analysis revealed prominent alterations in mitochondrial proteins. Immunohistochemical and serial block-face scanning electron microscopy analyses confirmed that PCs underwent age-dependent alterations in mitochondrial morphology. Moreover, colorimetric assays demonstrated impairment of the electron transport chain complexes (ETC) and decrease in ATPase activity. Subsequently, we examined whether the mitochondria-targeted antioxidant MitoQ could restore mitochondrial dysfunction and prevent SCA1-associated pathology in Sca1(154Q/2Q) mice. MitoQ treatment both presymptomatically and when symptoms were evident ameliorated mitochondrial morphology and restored the activities of the ETC complexes. Notably, MitoQ slowed down the appearance of SCA1-linked neuropathology such as lack of motor coordination as well as prevented oxidative stress-induced DNA damage and PC loss. Our work identifies a central role for mitochondria in PC degeneration in SCA1 and provides evidence for the supportive use of mitochondria-targeted therapeutics in slowing down disease progression. Copyright © 2016 Elsevier Inc. All rights reserved.

Glia Maturation Factor Dependent Inhibition of Mitochondrial PGC-1α Triggers Oxidative Stress-Mediated Apoptosis in N27 Rat Dopaminergic Neuronal Cells.

PubMed

Selvakumar, Govindhasamy Pushpavathi; Iyer, Shankar S; Kempuraj, Duraisamy; Raju, Murugesan; Thangavel, Ramasamy; Saeed, Daniyal; Ahmed, Mohammad Ejaz; Zahoor, Harris; Raikwar, Sudhanshu P; Zaheer, Smita; Zaheer, Asgar

2018-01-30

Parkinson's disease (PD) is a progressive neurodegenerative disease affecting over five million individuals worldwide. The exact molecular events underlying PD pathogenesis are still not clearly known. Glia maturation factor (GMF), a neuroinflammatory protein in the brain plays an important role in the pathogenesis of PD. Mitochondrial dysfunctions and oxidative stress trigger apoptosis leading to dopaminergic neuronal degeneration in PD. Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α or PPARGC-α) acts as a transcriptional co-regulator of mitochondrial biogenesis and energy metabolism by controlling oxidative phosphorylation, antioxidant activity, and autophagy. In this study, we found that incubation of immortalized rat dopaminergic (N27) neurons with GMF influences the expression of peroxisome PGC-1α and increases oxidative stress, mitochondrial dysfunction, and apoptotic cell death. We show that incubation with GMF reduces the expression of PGC-1α with concomitant decreases in the mitochondrial complexes. Besides, there is increased oxidative stress and depolarization of mitochondrial membrane potential (MMP) in these cells. Further, GMF reduces tyrosine hydroxylase (TH) expression and shifts Bax/Bcl-2 expression resulting in release of cytochrome-c and increased activations of effector caspase expressions. Transmission electron microscopy analyses revealed alteration in the mitochondrial architecture. Our results show that GMF acts as an important upstream regulator of PGC-1α in promoting dopaminergic neuronal death through its effect on oxidative stress-mediated apoptosis. Our current data suggest that GMF is a critical risk factor for PD and suggest that it could be explored as a potential therapeutic target to inhibit PD progression.

DNA interaction, antioxidant activity, and bioactivity studies of two ruthenium(II) complexes

NASA Astrophysics Data System (ADS)

Han, Bing-Jie; Jiang, Guang-Bin; Yao, Jun-Hua; Li, Wei; Wang, Ji; Huang, Hong-Liang; Liu, Yun-Jun

2015-01-01

Two new ruthenium(II) polypyridyl complexes [Ru(dmb)2(dcdppz)](ClO4)2 (1) and [Ru(bpy)2(dcdppz)](ClO4)2 (2) were prepared and characterized. The crystal structure of the complex 2 was solved by single crystal X-ray diffraction. The complex crystallizes in the monoclinic system, space group P21/n with a = 12.9622(14) Å, b = 17.1619(19) Å, c = 22.7210(3) Å, β = 100.930(2)°, R = 0.0536, Rω = 0.1111. The DNA-binding constants for complexes 1 and 2 were determined to be 1.92 × 105 (s = 1.72) and 2.24 × 105 (s = 1.86) M-1, respectively. The DNA-binding behaviors showed that complexes 1 and 2 interact with DNA by intercalative mode. The antioxidant activities of the ligand and the complexes were performed. Ligand, dcdppz, has no cytotoxicity against the selected cell lines. Complex 1 shows higher cytotoxicity than complex 2, but lower than cisplatin toward selected cell lines. The apoptosis and cell cycle arrest were investigated, and the apoptotic mechanism of BEL-7402 cells was studied by reactive oxygen species (ROS), mitochondrial membrane potential and western blot analysis. Complex 1 induces apoptosis in BEL-7402 cells through ROS-mediated mitochondrial dysfunction pathway and by regulating the expression of Bcl-2 family proteins.

Mitochondrial activity and dynamics changes regarding metabolism in ageing and obesity.

PubMed

López-Lluch, Guillermo

2017-03-01

Mitochondria play an essential role in ageing and longevity. During ageing, a general deregulation of metabolism occurs, affecting molecular, cellular and physiological activities in the organism. Dysfunction of mitochondria has been associated with ageing and age-related diseases indicating their importance in the maintenance of cell homeostasis. Three major nutritional sensors, mTOR, AMPK and Sirtuins are involved in the control of mitochondrial physiology. These nutritional sensors control mitochondrial biogenesis, dynamics by regulating fusion and fission processes, and turnover through mito- and autophagy. Apart of the known factors involved in fusion, OPA1 and mitofusins, and fission, DRP1 and FIS1, emerging factors such as prohibitins and sestrins can play important functions in mitochondrial dynamics regulation. Mitochondria is also affected by sexual hormones that suffer drastic changes during ageing. The recent literature demonstrates the complex interaction between nutritional sensors and mitochondrial homeostasis in the physiology of adipose tissue and in the accumulation of fat in other organs such as muscle and liver. In this article, the role of mitochondrial homeostasis in ageing and age-dependent fat accumulation is revised. This review highlights the importance of mitochondria in the accumulation of fat during ageing and related diseases such as obesity, metabolic syndrome or type 2 diabetes mellitus. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Adipocyte Fatty Acid-Binding Protein Promotes Palmitate-Induced Mitochondrial Dysfunction and Apoptosis in Macrophages

PubMed Central

Li, Hui; Xiao, Yang; Tang, Lin; Zhong, Feng; Huang, Gan; Xu, Jun-Mei; Xu, Ai-Min; Dai, Ru-Ping; Zhou, Zhi-Guang

2018-01-01

A high level of circulating free fatty acids (FFAs) is known to be an important trigger for macrophage apoptosis during the development of atherosclerosis. However, the underlying mechanism by which FFAs result in macrophage apoptosis is not well understood. In cultured human macrophage Thp-1 cells, we showed that palmitate (PA), the most abundant FFA in circulation, induced excessive reactive oxidative substance production, increased malondialdehyde concentration, and decreased adenosine triphosphate levels. Furthermore, PA treatment also led to mitochondrial dysfunction, including the decrease of mitochondrial number, the impairment of respiratory complex IV and succinate dehydrogenase activity, and the reduction of mitochondrial membrane potential. Mitochondrial apoptosis was also detected after PA treatment, indicated by a decrease in cytochrome c release, downregulation of Bcl-2, upregulation of Bax, and increased caspase-3 activity. PA treatment upregulated the expression of adipocyte fatty acid-binding protein (A-FABP), a critical regulator of fatty acid trafficking and lipid metabolism. Inhibition of A-FABP with BMS309403, a small-molecule A-FABP inhibitor, almost reversed all of these indexes. Thus, this study suggested that PA-mediated macrophage apoptosis through A-FABP upregulation, which subsequently resulted in mitochondrial dysfunction and reactive oxidative stress. Inhibition of A-FABP may be a potential therapeutic target for macrophage apoptosis and to delay the progress of atherosclerosis. PMID:29441065

Apoptotic effect of novel Schiff based CdCl₂(C₁₄H₂₁N₃O₂) complex is mediated via activation of the mitochondrial pathway in colon cancer cells.

PubMed

Hajrezaie, Maryam; Paydar, Mohammadjavad; Looi, Chung Yeng; Moghadamtousi, Soheil Zorofchian; Hassandarvish, Pouya; Salga, Muhammad Saleh; Karimian, Hamed; Shams, Keivan; Zahedifard, Maryam; Majid, Nazia Abdul; Ali, Hapipah Mohd; Abdulla, Mahmood Ameen

2015-03-13

The development of metal-based agents has had a tremendous role in the present progress in cancer chemotherapy. One well-known example of metal-based agents is Schiff based metal complexes, which hold great promise for cancer therapy. Based on the potential of Schiff based complexes for the induction of apoptosis, this study aimed to examine the cytotoxic and apoptotic activity of a CdCl2(C14H21N3O2) complex on HT-29 cells. The complex exerted a potent suppressive effect on HT-29 cells with an IC50 value of 2.57 ± 0.39 after 72 h of treatment. The collapse of the mitochondrial membrane potential and the elevated release of cytochrome c from the mitochondria to the cytosol indicate the involvement of the intrinsic pathway in the induction of apoptosis. The role of the mitochondria-dependent apoptotic pathway was further proved by the significant activation of the initiator caspase-9 and the executioner caspases-3 and -7. In addition, the activation of caspase-8, which is associated with the suppression of NF-κB translocation to the nucleus, also revealed the involvement of the extrinsic pathway in the induced apoptosis. The results suggest that the CdCl2(C14H21N3O2) complex is able to induce the apoptosis of colon cancer cells and is a potential candidate for future cancer studies.

Deletion of protein kinase C-ε attenuates mitochondrial dysfunction and ameliorates ischemic renal injury.

PubMed

Nowak, Grazyna; Takacsova-Bakajsova, Diana; Megyesi, Judit

2017-01-01

Previously, we documented that activation of protein kinase C-ε (PKC-ε) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of PKC-ε decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC-ε levels in mitochondria of ischemic kidneys increased 24 h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44 and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC-ε-deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO but not in WT animals. PKC-ε deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC-ε deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC-ε activation in hypoxic RPTC in primary culture exacerbated, whereas PKC-ε inhibition reduced, decreases in: 1) complex I- and complex II-coupled state 3 respirations and 2) activities of complexes I, III, and IV. We conclude that PKC-ε activation mediates 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia. Copyright © 2017 the American Physiological Society.

Deletion of protein kinase C-ε attenuates mitochondrial dysfunction and ameliorates ischemic renal injury

PubMed Central

Takacsova-Bakajsova, Diana; Megyesi, Judit

2016-01-01

Previously, we documented that activation of protein kinase C-ε (PKC-ε) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of PKC-ε decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC-ε levels in mitochondria of ischemic kidneys increased 24 h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44 and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC-ε-deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO but not in WT animals. PKC-ε deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC-ε deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC-ε activation in hypoxic RPTC in primary culture exacerbated, whereas PKC-ε inhibition reduced, decreases in: 1) complex I- and complex II-coupled state 3 respirations and 2) activities of complexes I, III, and IV. We conclude that PKC-ε activation mediates 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia. PMID:27760765

Energy metabolism determines the sensitivity of human hepatocellular carcinoma cells to mitochondrial inhibitors and biguanide drugs.

PubMed

Hsu, Chia-Chi; Wu, Ling-Chia; Hsia, Cheng-Yuan; Yin, Pen-Hui; Chi, Chin-Wen; Yeh, Tien-Shun; Lee, Hsin-Chen

2015-09-01

Human hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide particularly in Asia. Deregulation of cellular energetics was recently included as one of the cancer hallmarks. Compounds that target the mitochondria in cancer cells were proposed to have therapeutic potential. Biguanide drugs which inhibit mitochondrial complex I and repress mTOR signaling are clinically used to treat type 2 diabetes mellitus patients (T2DM) and were recently found to reduce the risk of HCC in T2DM patients. However, whether alteration of energy metabolism is involved in regulating the sensitivity of HCC to biguanide drugs is still unclear. In the present study, we treated four HCC cell lines with mitochondrial inhibitors (rotenone and oligomycin) and biguanide drugs (metformin and phenformin), and found that the HCC cells which had a higher mitochondrial respiration rate were more sensitive to these treatments; whereas the HCC cells which exhibited higher glycolysis were more resistant. When glucose was replaced by galactose in the medium, the altered energy metabolism from glycolysis to mitochondrial respiration in the HCC cells enhanced the cellular sensitivity to mitochondrial inhibitors and biguanides. The energy metabolism change enhanced AMP-activated protein kinase (AMPK) activation, mTOR repression and downregulation of cyclin D1 and Mcl-1 in response to the mitochondrial inhibitors and biguanides. In conclusion, our results suggest that increased mitochondrial oxidative metabolism upregulates the sensitivity of HCC to biguanide drugs. Enhancing the mitochondrial oxidative metabolism in combination with biguanide drugs may be a therapeutic strategy for HCC.

SIMILAR PATTERNS OF MITOCHONDRIAL VULNERABILITY AND RESCUE INDUCED BY GENETIC MODIFICATION OF α-SYNUCLEIN, PARKIN AND DJ-1 IN C. ELEGANS*

PubMed Central

Westlund, Beth; Perier, Celine; Burnam, Lucinda; Sluder, Anne; Hoener, Marius; Rodrigues, Cecilia MP; Alfonso, Aixa; Steer, Clifford; Liu, Leo; Przedborski, Serge; Wolozin, Benjamin

2014-01-01

How genetic and environmental factors interact in Parkinson’s disease is poorly understood. We have now compared the patterns of vulnerability and rescue of C. elegans with genetic modifications of three different genetic factors implicated in PD. We observed that expressing α-synuclein, deleting parkin (K08E3.7) or knocking down DJ-1 (B0432.2) or parkin, produces similar patterns of pharmacological vulnerability and rescue. C. elegans lines with these genetic changes were more vulnerable than non-transgenic nematodes to mitochondrial complex I inhibitors, including rotenone, fenperoximate, pyridaben or stigmatellin. In contrast, the genetic manipulations did not increase sensitivity to paraquat, sodium azide, divalent metal ions (FeII or CuII) or etoposide compared to non-transgenic nematodes. Each of the PD-related lines was also partially rescued by the anti-oxidant probucol, the mitochondrial complex II activator, D-β-hydroxybutyrate (DβHB) or the anti-apoptotic bile acid tauroursodeoxycholic acid (TUDCA). Complete protection in all lines was achieved by combining DβHB with TUDCA but not with probucol. These results show that diverse PD-related genetic modifications disrupt mitochondrial function in C. elegans, and they raise the possibility that mitochondrial disruption is a pathway shared in common by many types of familial PD. PMID:16239214

UCP1 deficiency causes brown fat respiratory chain depletion and sensitizes mitochondria to calcium overload-induced dysfunction

PubMed Central

Kazak, Lawrence; Chouchani, Edward T.; Stavrovskaya, Irina G.; Lu, Gina Z.; Jedrychowski, Mark P.; Egan, Daniel F.; Kumari, Manju; Kong, Xingxing; Erickson, Brian K.; Szpyt, John; Rosen, Evan D.; Murphy, Michael P.; Kristal, Bruce S.; Gygi, Steven P.; Spiegelman, Bruce M.

2017-01-01

Brown adipose tissue (BAT) mitochondria exhibit high oxidative capacity and abundant expression of both electron transport chain components and uncoupling protein 1 (UCP1). UCP1 dissipates the mitochondrial proton motive force (Δp) generated by the respiratory chain and increases thermogenesis. Here we find that in mice genetically lacking UCP1, cold-induced activation of metabolism triggers innate immune signaling and markers of cell death in BAT. Moreover, global proteomic analysis reveals that this cascade induced by UCP1 deletion is associated with a dramatic reduction in electron transport chain abundance. UCP1-deficient BAT mitochondria exhibit reduced mitochondrial calcium buffering capacity and are highly sensitive to mitochondrial permeability transition induced by reactive oxygen species (ROS) and calcium overload. This dysfunction depends on ROS production by reverse electron transport through mitochondrial complex I, and can be rescued by inhibition of electron transfer through complex I or pharmacologic depletion of ROS levels. Our findings indicate that the interscapular BAT of Ucp1 knockout mice exhibits mitochondrial disruptions that extend well beyond the deletion of UCP1 itself. This finding should be carefully considered when using this mouse model to examine the role of UCP1 in physiology. PMID:28630339

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

PubMed

Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

2014-01-01

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

Mitochondrial Bioenergetics and Dysfunction in Failing Heart.

PubMed

Sheeran, Freya L; Pepe, Salvatore

2017-01-01

Energy insufficiency has been recognized as a key feature of systolic heart failure. Although mitochondria have long been known to sustain myocardial work energy supply, the capacity to therapeutically target mitochondrial bioenergetics dysfunction is hampered by a complex interplay of multiple perturbations that progressively compound causing myocardial failure and collapse. Compared to non-failing human donor hearts, activity rates of complexes I and IV, nicotinamide nucleotide transhydrogenase (NADPH-transhydrogenase, Nnt) and the Krebs cycle enzymes isocitrate dehydrogenase, malate dehydrogenase and aconitase are markedly decreased in end-stage heart failure. Diminished REDOX capacity with lower total glutathione and coenzyme Q 10 levels are also a feature of chronic left ventricular failure. Decreased enzyme activities in part relate to abundant and highly specific oxidative, nitrosylative, and hyperacetylation modifications. In this brief review we highlight that energy deficiency in end-stage failing human left ventricle predominantly involves concomitantly impaired activities of key electron transport chain and Krebs cycle enzymes rather than altered expression of respective genes or proteins. Augmented oxidative modification of these enzyme subunit structures, and the formation of highly reactive secondary metabolites, implicates dysfunction due to diminished capacity for management of mitochondrial reactive oxygen species, which contribute further to progressive decreases in bioenergetic capacity and contractile function in human heart failure.

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) andmore » Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded subunits. • It decreases the mtDNA copy number and mitochondrial content in rat brain. • It down-regulates the mRNA and protein levels of PGC-1α, NRF-1, NRF-2 and Tfam. • It also disturbs the mitochondrial or nuclear architecture of neurons. • Finally it also decreases mitochondrial number in HC and CS regions of rat brain.« less

Depleted energy charge and increased pulmonary endothelial permeability induced by mitochondrial complex I inhibition are mitigated by coenzyme Q1 in the isolated perfused rat lung.

PubMed

Bongard, Robert D; Yan, Ke; Hoffmann, Raymond G; Audi, Said H; Zhang, Xiao; Lindemer, Brian J; Townsley, Mary I; Merker, Marilyn P

2013-12-01

Mitochondrial dysfunction is associated with various forms of lung injury and disease that also involve alterations in pulmonary endothelial permeability, but the relationship, if any, between the two is not well understood. This question was addressed by perfusing isolated intact rat lung with a buffered physiological saline solution in the absence or presence of the mitochondrial complex I inhibitor rotenone (20 μM). Compared to control, rotenone depressed whole lung tissue ATP from 5.66 ± 0.46 (SEM) to 2.34 ± 0.15 µmol · g(-1) dry lung, with concomitant increases in the ADP:ATP and AMP:ATP ratios. Rotenone also increased lung perfusate lactate (from 12.36 ± 1.64 to 38.62 ± 3.14 µmol · 15 min(-1) perfusion · g(-1) dry lung) and the lactate:pyruvate ratio, but had no detectable impact on lung tissue GSH:GSSG redox status. The amphipathic quinone coenzyme Q1 (CoQ1; 50 μM) mitigated the impact of rotenone on the adenine nucleotide balance, wherein mitigation was blocked by NAD(P)H-quinone oxidoreductase 1 or mitochondrial complex III inhibitors. In separate studies, rotenone increased the pulmonary vascular endothelial filtration coefficient (Kf) from 0.043 ± 0.010 to 0.156 ± 0.037 ml · min(-1) · cm H2O(-1) · g(-1) dry lung, and CoQ1 protected against the effect of rotenone on Kf. A second complex I inhibitor, piericidin A, qualitatively reproduced the impact of rotenone on Kf and the lactate:pyruvate ratio. Taken together, the observations imply that pulmonary endothelial barrier integrity depends on mitochondrial bioenergetics as reflected in lung tissue ATP levels and that compensatory activation of whole lung glycolysis cannot protect against pulmonary endothelial hyperpermeability in response to mitochondrial blockade. The study further suggests that low-molecular-weight amphipathic quinones may have therapeutic utility in protecting lung barrier function in mitochondrial insufficiency. Published by Elsevier Inc.