Effect of Nanosecond RF Pulses on Mitochondrial Membranes

NASA Astrophysics Data System (ADS)

Zharkova, L. P.; Romanchenko, I. V.; Bol'shakov, M. A.; Rostov, V. V.

2017-12-01

Effect of nanosecond RF pulses on the state of isolated mitochondria and their membranes is investigated. Mitochondrial suspensions are exposed to periodic RF pulses with durations from 4 to 25 ns, frequencies from 0.6 to 1.0 GHz, amplitudes from 0.1 to 36 kV/cm, and pulse repetition frequencies 8-25 Hz. The integrity of the mitochondrial membranes is estimated from their resistance to electric current. The possibility of opening of protein pores with nonspecific permeability is determined from a change in the mitochondrial volume by registration of optical density of organelle suspension.

Role of Pterocarpus santalinus against mitochondrial dysfunction and membrane lipid changes induced by ulcerogens in rat gastric mucosa.

PubMed

Narayan, Shoba; Devi, R S; Devi, C S Shyamala

2007-11-20

Free radicals produced by ulcerogenic agents affect the TCA cycle enzymes located in the outer membrane of the mitochondria. Upon induction with ulcerogens, peroxidation of membrane lipids bring about alterations in the mitochondrial enzyme activity. This indicates an increase in the permeability levels of the mitochondrial membrane. The ability of PSE to scavenge the reactive oxygen species results in restoration of activities of TCA cycle enzymes. NSAIDs interfere with the mitochondrial beta-oxidation of fatty acids in vitro and in vivo, resulting in uncoupling of mitochondrial oxidative phosphorylation process. This usually results in diminished cellular ATP production. The recovery of gastric mucosal barrier function through maintenance of energy metabolism results in maintenance of ATP levels, as observed in this study upon treatment with PSE. Membrane integrity altered by peroxidation is known to have a modified fatty acid composition, a disruption of permeability, a decrease in electrical resistance, and increase in flip-flopping between monolayers and inactivated cross-linked proteins. The severe depletion of arachidonic acid in ulcer induced groups was prevented upon treatment with PSE. The acid inhibitory property of the herbal extract enables the maintenance of GL activity upon treatment with PSE. The ability to prevent membrane peroxidation has been traced to the presence of active constituents in the PSE. In essence, PSE has been found to prevent mitochondrial dysfunction, provide mitochondrial cell integrity, through the maintenance of lipid bilayer by its ability to provide a hydrophobic character to the gastric mucosa, further indicating its ability to reverse the action of NSAIDs and mast cell degranulators in gastric mucosa.

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

Gradual changes in permeability of inner mitochondrial membrane precede the mitochondrial permeability transition.

PubMed

Balakirev, M Y; Zimmer, G

1998-08-01

Some compounds are known to induce solute-nonselective permeability of the inner mitochondrial membrane (IMM) in Ca2+-loaded mitochondria. Existing data suggest that this process, following the opening of a mitochondrial permeability transition pore, is preceded by different solute-selective permeable states of IMM. At pH 7, for instance, the K0.5 for Ca2+-induced pore opening is 16 microM, a value 80-fold above a therapeutically relevant shift of intracellular Ca2+ during ischemia in vivo. The present work shows that in the absence of Ca2+, phenylarsine oxide and tetraalkyl thiuram disulfides (TDs) are able to induce a complex sequence of IMM permeability changes. At first, these agents activated an electrogenic K+ influx into the mitochondria. This K+-specific pathway had K0.5 = 35 mM for K+ and was inhibited by bromsulfalein with Ki = 2.5 microM. The inhibitors of mitochondrial KATP channel, ATP and glibenclamide, did not inhibit K+ transport via this pathway. Moreover, 50 microM glibenclamide induced by itself K+ influx into the mitochondria. After the increase in K+ permeability of IMM, mitochondria become increasingly permeable to protons. Mechanisms of H+ leak and nonselective permeability increase could also be different depending on the type of mitochondrial permeability transition (MPT) inducer. Thus, permeabilization of mitochondria induced by phenylarsine oxide was fully prevented by ADP and/or cyclosporin A, whereas TD-induced membrane alterations were insensitive toward these inhibitors. It is suggested that MPT in vivo leading to irreversible apoptosis is irrelevant in reversible ischemia/reperfusion injury. Copyright 1998 Academic Press.

Phytol shows anti-angiogenic activity and induces apoptosis in A549 cells by depolarizing the mitochondrial membrane potential.

PubMed

Sakthivel, Ravi; Malar, Dicson Sheeja; Devi, Kasi Pandima

2018-06-13

In the present study, the antiproliferative activity of phytol and its mechanism of action against human lung adenocarcinoma cell line A549 were studied in detail. Results showed that phytol exhibited potent antiproliferative activity against A549 cells in a dose and time-dependent manner with an IC 50 value of 70.81 ± 0.32 μM and 60.7 ± 0.47 μM at 24 and 48 h, respectively. Phytol showed no adverse toxic effect in normal human lung cells (L-132), but mild toxic effect was observed when treated with maximum dose (67 and 84 μM). No membrane-damaging effect was evidenced by PI staining and SEM analysis. The results of mitochondrial membrane potential analysis, cell cycle analysis, FT-IR and Western blotting analysis clearly demonstrated the molecular mechanism of phytol as induction of apoptosis in A549 cells, as evidenced by formation of shrinked cell morphology with membrane blebbing, depolarization of mitochondrial membrane potential, increased cell population in the sub-G0 phase, band variation in the DNA and lipid region, downregulation of Bcl-2, upregulation of Bax and the activation of caspase-9 and -3. In addition, phytol inhibited the CAM vascular growth as evidenced by CAM assay, which positively suggests that phytol has anti-angiogenic potential. Taken together, these findings clearly demonstrate the mode of action by which phytol induces cell death in A549 lung adenocarcinoma cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

[The effects of electromagnetic pulse on fluidity and lipid peroxidation of mitochondrial membrane].

PubMed

Wang, Changzhen; Cong, Jianbo; Xian, Hong; Cao, Xiaozhe; Sun, Cunpu; Wu, Ke

2002-08-01

To study the effects of intense electromagnetic pulse(EMP) on the biological effects of mitochondrial membrane. Rat liver mitochondrial suspension was exposed to EMP at 60 kV/m level. The changes of membrane lipid fluidity and membrane protein mobility were detected by ESR and spin label technique. Malondialdehyde(MDA) was detected by spectrophotometer. The mobility of membrane protein decreased significantly(P < 0.05). Correlation time (tau c) of control group was (0.501 +/- 0.077) x 10(-9)s, and tau c of EMP group was (0.594 +/- 0.049) x 10(-9)s, indicating that the mobility of protein was restricted. The fluidity of mitochondrial membrane increased significantly(P < 0.05) at the same time. Order parameter(S) of mitochondrial membrane lipid in control group was 0.63 +/- 0.01, while S of EMP group was 0.61 +/- 0.01(P < 0.05). MDA decreased significantly. The mobility and lipid peroxidation of mitochondrial membrane may be disturbed after EMP exposure.

NITRIC OXIDE, MITOCHONDRIAL HYPERPOLARIZATION AND T-CELL ACTIVATION

PubMed Central

Nagy, Gyorgy; Koncz, Agnes; Fernandez, David; Perl, Andras

2007-01-01

T lymphocyte activation is associated with nitric oxide (NO) production that plays an essential role in multiple T cell functions. NO acts as a messenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulates mitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many cell types, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both T lymphocyte activation and apoptosis, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cell signaling pathways. Overproduction of NO in systemic lupus erythematosus (SLE) induces mitochondrial biogenesis and alters Ca2+ signaling. Thus, while NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to the pathogenesis of autoimmunity. PMID:17462531

Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

PubMed

Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

2016-05-01

Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.

Sonic hedgehog pathway activation increases mitochondrial abundance and activity in hippocampal neurons

PubMed Central

Yao, Pamela J.; Manor, Uri; Petralia, Ronald S.; Brose, Rebecca D.; Wu, Ryan T. Y.; Ott, Carolyn; Wang, Ya-Xian; Charnoff, Ari; Lippincott-Schwartz, Jennifer; Mattson, Mark P.

2017-01-01

Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. We present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway affects multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense, as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons. PMID:27932496

Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes

PubMed Central

Hoogerheide, David P.; Noskov, Sergei Y.; Jacobs, Daniel; Bergdoll, Lucie; Silin, Vitalii; Worcester, David L.; Abramson, Jeff; Nanda, Hirsh; Rostovtseva, Tatiana K.; Bezrukov, Sergey M.

2017-01-01

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques—surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations—suggest that α-tubulin’s amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic “mitochondrial” membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents. PMID:28420794

Impaired Bioenergetics in Mutant Mitochondrial DNA Determines Cell Fate During Seizure-Like Activity.

PubMed

Kovac, Stjepana; Preza, Elisavet; Houlden, Henry; Walker, Matthew C; Abramov, Andrey Y

2018-04-27

Mutations in genes affecting mitochondrial proteins are increasingly recognised in patients with epilepsy, but the factors determining cell fate during seizure activity in these mutations remain unknown. Fluorescent dye imaging techniques were applied to fibroblast cell lines from patients suffering from common mitochondrial mutations and to age-matched controls. Using live cell imaging techniques in fibroblasts, we show that fibroblasts with mutations in the mitochondrial genome had reduced mitochondrial membrane potential and NADH pools and higher redox indices, indicative of respiratory chain dysfunction. Increasing concentrations of ferutinin, a Ca 2+ ionophore, led to oscillatory Ca 2+ signals in fibroblasts resembling dynamic Ca 2+ changes that occur during seizure-like activity. Co-monitoring of mitochondrial membrane potential (ΔΨ m ) changes induced by ferutinin showed accelerated membrane depolarisation and cell collapse in fibroblasts with mutations in the mitochondrial genome when compared to controls. Ca 2+ flash photolysis using caged Ca 2+ confirmed impaired Ca 2+ handling in fibroblasts with mitochondrial mutations. Findings indicate that intracellular Ca 2+ levels cannot be compensated during periods of hyperexcitability, leading to Ca 2+ overload and subsequent cell death in mitochondrial diseases.

Calcium transport across the inner mitochondrial membrane: molecular mechanisms and pharmacology

PubMed Central

Csordás, György; Várnai, Peter; Golenár, Tünde; Sheu, Shey-Shing; Hajnóczky, György

2011-01-01

Growing evidence supports that mitochondrial calcium uptake is important for cell metabolism, signaling and survival. However, both the molecular nature of the mitochondrial Ca2+ transport sites and the calcium signals they respond to remained elusive. Recent RNA interference studies have identified new candidate proteins for Ca2+ uptake across the inner mitochondrial membrane, including LETM1, MCU, MICU1 and NCLX. The sensitivity of these factors to several drugs has been tested and in parallel, some new inhibitors of mitochondrial Ca2+ uptake have been described. This paper provides an update on the pharmacological aspects of the molecular mechanisms of the inner mitochondrial membrane Ca2+ transport. PMID:22123069

Mitochondrial calcium ion and membrane potential transients follow the pattern of epileptiform discharges in hippocampal slice cultures.

PubMed

Kovács, Richard; Kardos, Julianna; Heinemann, Uwe; Kann, Oliver

2005-04-27

Emerging evidence suggests that mitochondrial dysfunction contributes to the pathophysiology of epilepsy. Recurrent mitochondrial Ca2+ ion load during seizures might act on mitochondrial membrane potential (DeltaPsim) and proton motive force. By using electrophysiology and confocal laser-scanning microscopy, we investigated the effects of epileptiform activity, as induced by low-Mg2+ ion perfusion in hippocampal slice cultures, on changes in DeltaPsim and in mitochondrial Ca2+ ion concentration ([Ca2+]m). The mitochondrial compartment was identified by monitoring DeltaPsim in the soma and dendrites of patched CA3 pyramidal cells using the mitochondria-specific voltage-sensitive dye rhodamine-123 (Rh-123). Interictal activity was accompanied by localized mitochondrial depolarization that was restricted to a few mitochondria in small dendrites. In contrast, robust Rh-123 release into the cytosol was observed during seizure-like events (SLEs), indicating simultaneous depolarization of mitochondria. This was critically dependent on Ca2+ ion uptake and extrusion, because inhibition of the mitochondrial Ca2+ ion uniporter by Ru360 and the mitochondrial Na+/Ca2+ ion exchanger by 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one but not the inhibitor of mitochondrial permeability transition pore, cyclosporin A, decreased the SLE-associated mitochondrial depolarization. The Ca2+ ion dependence of simultaneous mitochondrial depolarization suggested enhanced Ca2+ ion cycling across mitochondrial membranes during epileptiform activity. Indeed, [Ca2+]m fluctuated during interictal activity in single dendrites, and these fluctuations spread over the entire mitochondrial compartment during SLEs, as revealed using mitochondria-specific dyes (rhod-2 and rhod-ff) and spatial frequency-based image analysis. These findings strengthen the hypothesis that epileptic activity results in Ca2+ ion-dependent changes in mitochondrial function that might contribute to the

The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

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

Serasinghe, Madhavika N.; Mitochondrial Research and Innovation Group, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; Yoon, Yisang

2008-11-15

Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six {alpha}-helices ({alpha}1-{alpha}6) out of which {alpha}2-{alpha}5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found thatmore » hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the {alpha}1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that {alpha}1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the {alpha}5 helix and the linker between {alpha}3 and {alpha}4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by {alpha}1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.« less

Mechanisms Underlying the Essential Role of Mitochondrial Membrane Lipids in Yeast Chronological Aging

PubMed Central

Medkour, Younes; Dakik, Paméla; McAuley, Mélissa; Mohammad, Karamat; Mitrofanova, Darya

2017-01-01

The functional state of mitochondria is vital to cellular and organismal aging in eukaryotes across phyla. Studies in the yeast Saccharomyces cerevisiae have provided evidence that age-related changes in some aspects of mitochondrial functionality can create certain molecular signals. These signals can then define the rate of cellular aging by altering unidirectional and bidirectional communications between mitochondria and other organelles. Several aspects of mitochondrial functionality are known to impact the replicative and/or chronological modes of yeast aging. They include mitochondrial electron transport, membrane potential, reactive oxygen species, and protein synthesis and proteostasis, as well as mitochondrial synthesis of iron-sulfur clusters, amino acids, and NADPH. Our recent findings have revealed that the composition of mitochondrial membrane lipids is one of the key aspects of mitochondrial functionality affecting yeast chronological aging. We demonstrated that exogenously added lithocholic bile acid can delay chronological aging in yeast because it elicits specific changes in mitochondrial membrane lipids. These changes allow mitochondria to operate as signaling platforms that delay yeast chronological aging by orchestrating an institution and maintenance of a distinct cellular pattern. In this review, we discuss molecular and cellular mechanisms underlying the essential role of mitochondrial membrane lipids in yeast chronological aging. PMID:28593023

Structural Changes and Proapoptotic Peroxidase Activity of Cardiolipin-Bound Mitochondrial Cytochrome c

PubMed Central

Mandal, Abhishek; Hoop, Cody L.; DeLucia, Maria; Kodali, Ravindra; Kagan, Valerian E.; Ahn, Jinwoo; van der Wel, Patrick C.A.

2015-01-01

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly 13C,15N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the

Induced mitochondrial membrane potential for modeling solitonic conduction of electrotonic signals

PubMed Central

Poznanski, R. R.; Cacha, L. A.; Ali, J.; Rizvi, Z. H.; Yupapin, P.; Salleh, S. H.; Bandyopadhyay, A.

2017-01-01

A cable model that includes polarization-induced capacitive current is derived for modeling the solitonic conduction of electrotonic potentials in neuronal branchlets with microstructure containing endoplasmic membranes. A solution of the nonlinear cable equation modified for fissured intracellular medium with a source term representing charge ‘soakage’ is used to show how intracellular capacitive effects of bound electrical charges within mitochondrial membranes can influence electrotonic signals expressed as solitary waves. The elastic collision resulting from a head-on collision of two solitary waves results in localized and non-dispersing electrical solitons created by the nonlinearity of the source term. It has been shown that solitons in neurons with mitochondrial membrane and quasi-electrostatic interactions of charges held by the microstructure (i.e., charge ‘soakage’) have a slower velocity of propagation compared with solitons in neurons with microstructure, but without endoplasmic membranes. When the equilibrium potential is a small deviation from rest, the nonohmic conductance acts as a leaky channel and the solitons are small compared when the equilibrium potential is large and the outer mitochondrial membrane acts as an amplifier, boosting the amplitude of the endogenously generated solitons. These findings demonstrate a functional role of quasi-electrostatic interactions of bound electrical charges held by microstructure for sustaining solitons with robust self-regulation in their amplitude through changes in the mitochondrial membrane equilibrium potential. The implication of our results indicate that a phenomenological description of ionic current can be successfully modeled with displacement current in Maxwell’s equations as a conduction process involving quasi-electrostatic interactions without the inclusion of diffusive current. This is the first study in which solitonic conduction of electrotonic potentials are generated by

Induced mitochondrial membrane potential for modeling solitonic conduction of electrotonic signals.

PubMed

Poznanski, R R; Cacha, L A; Ali, J; Rizvi, Z H; Yupapin, P; Salleh, S H; Bandyopadhyay, A

2017-01-01

A cable model that includes polarization-induced capacitive current is derived for modeling the solitonic conduction of electrotonic potentials in neuronal branchlets with microstructure containing endoplasmic membranes. A solution of the nonlinear cable equation modified for fissured intracellular medium with a source term representing charge 'soakage' is used to show how intracellular capacitive effects of bound electrical charges within mitochondrial membranes can influence electrotonic signals expressed as solitary waves. The elastic collision resulting from a head-on collision of two solitary waves results in localized and non-dispersing electrical solitons created by the nonlinearity of the source term. It has been shown that solitons in neurons with mitochondrial membrane and quasi-electrostatic interactions of charges held by the microstructure (i.e., charge 'soakage') have a slower velocity of propagation compared with solitons in neurons with microstructure, but without endoplasmic membranes. When the equilibrium potential is a small deviation from rest, the nonohmic conductance acts as a leaky channel and the solitons are small compared when the equilibrium potential is large and the outer mitochondrial membrane acts as an amplifier, boosting the amplitude of the endogenously generated solitons. These findings demonstrate a functional role of quasi-electrostatic interactions of bound electrical charges held by microstructure for sustaining solitons with robust self-regulation in their amplitude through changes in the mitochondrial membrane equilibrium potential. The implication of our results indicate that a phenomenological description of ionic current can be successfully modeled with displacement current in Maxwell's equations as a conduction process involving quasi-electrostatic interactions without the inclusion of diffusive current. This is the first study in which solitonic conduction of electrotonic potentials are generated by polarization

Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease.

PubMed

Pera, Marta; Larrea, Delfina; Guardia-Laguarta, Cristina; Montesinos, Jorge; Velasco, Kevin R; Agrawal, Rishi R; Xu, Yimeng; Chan, Robin B; Di Paolo, Gilbert; Mehler, Mark F; Perumal, Geoffrey S; Macaluso, Frank P; Freyberg, Zachary Z; Acin-Perez, Rebeca; Enriquez, Jose Antonio; Schon, Eric A; Area-Gomez, Estela

2017-11-15

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by γ-secretase to generate the β-amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

NASA Astrophysics Data System (ADS)

Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

2010-03-01

The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

Mitochondrial Ca2+ and membrane potential, an alternative pathway for Interleukin 6 to regulate CD4 cell effector function

PubMed Central

Yang, Rui; Lirussi, Dario; Thornton, Tina M; Jelley-Gibbs, Dawn M; Diehl, Sean A; Case, Laure K; Madesh, Muniswamy; Taatjes, Douglas J; Teuscher, Cory; Haynes, Laura; Rincón, Mercedes

2015-01-01

IL-6 plays an important role in determining the fate of effector CD4 cells and the cytokines that these cells produce. Here we identify a novel molecular mechanism by which IL-6 regulates CD4 cell effector function. We show that IL-6-dependent signal facilitates the formation of mitochondrial respiratory chain supercomplexes to sustain high mitochondrial membrane potential late during activation of CD4 cells. Mitochondrial hyperpolarization caused by IL-6 is uncoupled from the production of ATP by oxidative phosphorylation. However, it is a mechanism to raise the levels of mitochondrial Ca2+ late during activation of CD4 cells. Increased levels of mitochondrial Ca2+ in the presence of IL-6 are used to prolong Il4 and Il21 expression in effector CD4 cells. Thus, the effect of IL-6 on mitochondrial membrane potential and mitochondrial Ca2+ is an alternative pathway by which IL-6 regulates effector function of CD4 cells and it could contribute to the pathogenesis of inflammatory diseases. DOI: http://dx.doi.org/10.7554/eLife.06376.001 PMID:25974216

High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice

PubMed Central

Kahle, M.; Schäfer, A.; Seelig, A.; Schultheiß, J.; Wu, M.; Aichler, M.; Leonhardt, J.; Rathkolb, B.; Rozman, J.; Sarioglu, H.; Hauck, S.M.; Ueffing, M.; Wolf, E.; Kastenmueller, G.; Adamski, J.; Walch, A.; Hrabé de Angelis, M.; Neschen, S.

2014-01-01

Objective Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. Methods We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. Results Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. Conclusions We assume HF-induced modifications in membrane lipid

Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

PubMed

Raza Shaikh, Saame; Brown, David A

2013-01-01

Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

Role of mitochondrial permeability transition pores in mitochondrial autophagy.

PubMed

Rodriguez-Enriquez, Sara; He, Lihua; Lemasters, John J

2004-12-01

During autophagy, cells rid themselves of damaged and superfluous mitochondria, as well as other organelles. This activation of mitochondrial turnover could be the result of changes in the physiological state of mitochondria. Confocal microscopy and fluorescence techniques indicate that onset of mitochondrial permeability transition is one such change. The mitochondrial permeability transition is a reversible phenomenon whereby the mitochondrial inner membrane becomes freely permeable to solutes of less than 1500 Da. At onset of the mitochondrial permeability transition, mitochondria depolarize, uncouple, and undergo large amplitude swelling due to opening of permeability transition pores, which may form by aggregation of damaged, misfolded membrane proteins. When injurious cellular stresses occur, cells may protect themselves using autophagy to remove damaged mitochondria and mutated mitochondrial DNA. Ca(2+) overloading, reactive oxygen and nitrogen species, decreased mitochondrial membrane potential, and oxidation of pyridine nucleotides and glutathione all promote mitochondrial damage and onset of the mitochondrial permeability transition. The mitochondrial permeability transition is also associated with necrosis and apoptosis after a variety of stimuli. This review emphasizes the role of the mitochondrial permeability transition as a key event in mitochondrial autophagy.

Withaferin-A Induces Apoptosis in Osteosarcoma U2OS Cell Line via Generation of ROS and Disruption of Mitochondrial Membrane Potential.

PubMed

Zhang, Hui-Liang; Zhang, Hong

2017-01-01

Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania sominifera . It has tremendous pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis inducing drug-like molecules. Osteosarcoma is a rare type of osteocancer, affecting human. The present study therefore focused on the evaluation of antitumor potential of WF-A against several osteosarcoma cell lines. MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC 50 . DAPI staining was used to confirm the apoptosis inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. The results revealed that that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC 50 ranging from 0.32 to 7.6 μM. The lowest IC 50 (0.32 μM) was observed against U2OS cell line and therefore it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced ROS-mediated reduction in mitochondrial membrane potential ΔΨm) in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. We propose that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential. WF-A exhibits strong anticancer activity against osteosarcoma cell linesAntiproliferative activity of WF-A is via induction of apoptosisWF-A induced ROS-mediated reduction in mitochondrial membrane potentialWF-A induced expression of caspase-3 in osteosarcoma cells. Abbreviations used: WA: Withaferin A; ROS: Reactive oxygen species; OS: Osteosarcoma; MMP: Mitochondrial membrane potential.

Mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia.

PubMed

Ali, Shimaa E; Thoen, Even; Evensen, Øystein; Wiik-Nielsen, Jannicke; Gamil, Amr A A; Skaar, Ida

2014-01-01

There has been a significant increase in the incidence of Saprolegnia infections over the past decades, especially after the banning of malachite green. Very often these infections are associated with high economic losses in salmonid farms and hatcheries. The use of boric acid to control the disease has been investigated recently both under in vitro and in vivo conditions, however its possible mode of action against fish pathogenic Saprolegnia is not known. In this study, we have explored the transformation in Saprolegnia spores/hyphae after exposure to boric acid (1 g/L) over a period 4-24 h post treatment. Using transmission electron microscopy (TEM), early changes in Saprolegnia spores were detected. Mitochondrial degeneration was the most obvious sign observed following 4 h treatment in about 20% of randomly selected spores. We also investigated the effect of the treatment on nuclear division, mitochondrial activity and function using confocal laser scanning microscopy (CLSM). Fluorescence microscopy was also used to test the effect of treatment on mitochondrial membrane potential and formation of reactive oxygen species. Additionally, the viability and proliferation of treated spores that correlated to mitochondrial enzymatic activity were tested using an MTS assay. All obtained data pointed towards changes in the mitochondrial structure, membrane potential and enzymatic activity following treatment. We have found that boric acid has no effect on the integrity of membranes of Saprolegnia spores at concentrations tested. It is therefore likely that mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia spp.

Mitochondrial Dysfunction Is Involved in the Toxic Activity of Boric Acid against Saprolegnia

PubMed Central

Ali, Shimaa E.; Thoen, Even; Evensen, Øystein; Wiik-Nielsen, Jannicke; Gamil, Amr A. A.; Skaar, Ida

2014-01-01

There has been a significant increase in the incidence of Saprolegnia infections over the past decades, especially after the banning of malachite green. Very often these infections are associated with high economic losses in salmonid farms and hatcheries. The use of boric acid to control the disease has been investigated recently both under in vitro and in vivo conditions, however its possible mode of action against fish pathogenic Saprolegnia is not known. In this study, we have explored the transformation in Saprolegnia spores/hyphae after exposure to boric acid (1 g/L) over a period 4–24 h post treatment. Using transmission electron microscopy (TEM), early changes in Saprolegnia spores were detected. Mitochondrial degeneration was the most obvious sign observed following 4 h treatment in about 20% of randomly selected spores. We also investigated the effect of the treatment on nuclear division, mitochondrial activity and function using confocal laser scanning microscopy (CLSM). Fluorescence microscopy was also used to test the effect of treatment on mitochondrial membrane potential and formation of reactive oxygen species. Additionally, the viability and proliferation of treated spores that correlated to mitochondrial enzymatic activity were tested using an MTS assay. All obtained data pointed towards changes in the mitochondrial structure, membrane potential and enzymatic activity following treatment. We have found that boric acid has no effect on the integrity of membranes of Saprolegnia spores at concentrations tested. It is therefore likely that mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia spp. PMID:25354209

Reconstitution of the protein insertion machinery of the mitochondrial inner membrane.

PubMed Central

Haucke, V; Schatz, G

1997-01-01

We have reconstituted the protein insertion machinery of the yeast mitochondrial inner membrane into proteoliposomes. The reconstituted proteoliposomes have a distinct morphology and protein composition and correctly insert the ADP/ATP carrier (AAC) and Tim23p, two multi-spanning integral proteins of the mitochondrial inner membrane. The reconstituted system requires a membrane potential, but not Tim44p or mhsp70, both of which are required for the ATP-driven translocation of proteins into the matrix. The protein insertion machinery can thus operate independently of the energy-transducing Tim44p-mhsp70 complex. PMID:9303300

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

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

Pardo-Andreu, Gilberto L., E-mail: gilbertopardo@infomed.sld.cu; Departamento de Fisica e Quimica, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. Cafe s/n, 14040-903 Ribeirao Preto, SP; Nunez-Figueredo, Yanier

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 {mu}M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca{sup 2+} efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. Allmore » effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP{sup +} transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. - Graphical abstract: Guttiferone-A permeabilizes mitochondrial membrane and induces cancer cell death Display Omitted Highlights: > We addressed the involvement of mitochondria in guttiferone (GA) toxicity toward cancer cells. > GA promoted membrane permeabilization, membrane potential dissipation, NAD(P)H depletion, ROS accumulation and ATP depletion. > These

Metabolic Remodeling Precedes Mitochondrial Outer Membrane Permeabilization in Human Glioma Xenograft Cells

PubMed Central

Ponnala, Shivani; Chetty, Chandramu; Veeravalli, Krishna Kumar; Dinh, Dzung H.; Klopfenstein, Jeffrey D.; Rao, Jasti S.

2011-01-01

Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch glioma cells glycolytic metabolism to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated glycolysis to OXPHOS switch. OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to change in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from glycolytic to OXPHOS pathway through an inhibitory effect on Akt, ROS induction, and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for treatment of glioma. PMID:22076676

Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

PubMed

Ponnala, Shivani; Chetty, Chandramu; Veeravalli, Krishna Kumar; Dinh, Dzung H; Klopfenstein, Jeffrey D; Rao, Jasti S

2012-02-01

Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch the glycolytic metabolism of glioma cells to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated OXPHOS to glycolysis switch. The OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to changes in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from the glycolytic to the OXPHOS pathway through an inhibitory effect on Akt, ROS induction and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for the treatment of glioma.

Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands.

PubMed Central

Kinnally, K W; Zorov, D B; Antonenko, Y N; Snyder, S H; McEnery, M W; Tedeschi, H

1993-01-01

The mitochrondrial benzodiazepine receptor (mBzR) binds a subset of benzodiazepines and isoquinoline carboxamides with nanomolar affinity and consists of the voltage-dependent anion channel, the adenine nucleotide translocator, and an 18-kDa protein. The effect of ligands of the mBzR on two inner mitochondrial membrane channel activities was determined with patch-clamp techniques. The relative inhibitory potencies of the drugs resemble their binding affinities for the mBzR. Ro5-4864 and protoporphyrin IX inhibit activity of the multiple conductance channel (MCC) and the mitochondrial centum-picosiemen (mCtS) channel activities at nanomolar concentrations. PK11195 inhibits mCtS activity at similar levels. Higher concentrations of protoporphyrin IX induce MCC but possibly not mCtS activity. Clonazepam, which has low affinity for mBzR, is at least 500 times less potent at both channel activities. Ro15-1788, which also has a low mBzR affinity, inhibits MCC at very high concentrations (16 microM). The findings indicate an association of these two channel activities with the proteins forming the mBzR complex and are consistent with an interaction of inner and outer membrane channels. PMID:7679505

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress

PubMed Central

Lee, Chun Pong; Maksaev, Grigory; Jensen, Gregory S.; Murcha, Monika W.; Wilson, Margaret E.; Fricker, Mark; Hell, Ruediger; Haswell, Elizabeth S.; Millar, A. Harvey; Sweetlove, Lee

2016-01-01

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in E. coli, MSL1 forms a stretch-activated ion channel with a slight preference for anions and provides protection against hypo-osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo-osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1-1 mutants under moderate heat- and heavy-metal-stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing but is complementary and appears to be important under similar conditions. PMID:27505616

A Mitochondrial Membrane Exopolyphosphatase Is Modulated by, and Plays a Role in, the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus) microplus Embryos

PubMed Central

Campos, Eldo; Façanha, Arnoldo R.; Costa, Evenilton P.; Fraga, Amanda; Moraes, Jorge; da Silva Vaz, Itabajara; Masuda, Aoi; Logullo, Carlos

2011-01-01

The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (Pi) and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 μg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5′-triphosphate synthesis in hard tick embryos. PMID:21747692

Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

PubMed

Anand, Sanjeev K; Gaba, Amit; Singh, Jaswant; Tikoo, Suresh K

2014-02-01

Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

PubMed

Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Radiation inactivation method provides evidence that membrane-bound mitochondrial creatine kinase is an oligomer

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

Quemeneur, E.; Eichenberger, D.; Goldschmidt, D.

1988-06-30

Lyophilized suspensions of rabbit heart mitochondria have been irradiated with varying doses of gamma rays. Mitochondrial creatine kinase activity was inactivated exponentially with a radiation inactivation size of 352 or 377 kDa depending upon the initial medium. These values are in good agreement with the molecular mass previously deduced from by permeation experiments: 357 kDa. This is the first direct evidence showing that the native form of mitochondrial creatine kinase is associated to the inner membrane as an oligomer, very likely an octamer.

Mitofusins and the mitochondrial permeability transition: the potential downside of mitochondrial fusion

PubMed Central

Papanicolaou, Kyriakos N.; Phillippo, Matthew M.

2012-01-01

Mitofusins (Mfn-1 and Mfn-2) are transmembrane proteins that bind and hydrolyze guanosine 5′-triphosphate to bring about the merging of adjacent mitochondrial membranes. This event is necessary for mitochondrial fusion, a biological process that is critical for organelle function. The broad effects of mitochondrial fusion on cell bioenergetics have been extensively studied, whereas the local effects of mitofusin activity on the structure and integrity of the fusing mitochondrial membranes have received relatively little attention. From the study of fusogenic proteins, theoretical models, and simulations, it has been noted that the fusion of biological membranes is associated with local perturbations on the integrity of the membrane that present in the form of lipidic holes which open on the opposing bilayers. These lipidic holes represent obligate intermediates that make the fusion process thermodynamically more favorable and at the same time induce leakage to the fusing membranes. In this perspectives article we present the relevant evidence selected from a spectrum of membrane fusion/leakage models and attempt to couple this information with observations conducted with cardiac myocytes or mitochondria deficient in Mfn-1 and Mfn-2. More specifically, we argue in favor of a situation whereby mitochondrial fusion in cardiac myocytes is coupled with outer mitochondrial membrane destabilization that is opportunistically employed during the process of mitochondrial permeability transition. We hope that these insights will initiate research on this new hypothesis of mitochondrial permeability transition regulation, a poorly understood mitochondrial function with significant consequences on myocyte survival. PMID:22636681

Altered Mitochondrial Membrane Potential, Mass, and Morphology in the Mononuclear Cells of Humans with Type 2 Diabetes

PubMed Central

Widlansky, Michael E.; Wang, Jingli; Shenouda, Sherene M.; Hagen, Tory M.; Smith, Anthony R.; Kizhakekuttu, Tinoy J.; Kluge, Matthew A.; Weihrauch, Dorothee; Gutterman, David D.; Vita, Joseph A.

2010-01-01

Mitochondrial membrane hyperpolarization and morphological changes are important in inflammatory cell activation. Despite the pathophysiological relevance, no valid and reproducible method for measuring mitochondrial homeostasis in human inflammatory cells is currently available. This study's purpose was to define and validate reproducible methods for measuring relevant mitochondrial perturbations and to determine whether these methods could discern mitochondrial perturbations in type 2 diabetes mellitus (T2DM), a condition associated with altered mitochondrial homeostasis. We employed 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzamidazol-carboncyanine (JC-1) to estimate mitochondrial membrane potential (ψm) and acridine orange 10-nonyl bromide (NAO) to assess mitochondrial mass in human mononuclear cells isolated from blood. Both assays were reproducible. We validated our findings by electron microscopy and pharmacological manipulation of ψm. We measured JC-1 and NAO fluorescence in the mononuclear cells of 27 T2DM patients and 32 controls. Mitochondria were more polarized (P=0.02) and mitochondrial mass was lower in T2DM (P=0.008). Electron microscopy demonstrated diabetic mitochondria were smaller, more spherical, and occupied less cellular area in T2DM. Mitochondrial superoxide production was higher in T2DM (P=0.01). Valid and reproducible measurements of mitochondrial homeostasis can be made in human mononuclear cells using these fluorophores. Further, potential clinically relevant perturbations in mitochondrial homeostasis in T2DM human mononuclear cells can be detected. PMID:20621033

Reconstitution of proapoptotic BAK function in liposomes reveals a dual role for mitochondrial lipids in the BAK-driven membrane permeabilization process.

PubMed

Landeta, Olatz; Landajuela, Ane; Gil, David; Taneva, Stefka; Di Primo, Carmelo; Sot, Begoña; Valle, Mikel; Frolov, Vadim A; Basañez, Gorka

2011-03-11

BAK is a key effector of mitochondrial outer membrane permeabilization (MOMP) whose molecular mechanism of action remains to be fully dissected in intact cells, mainly due to the inherent complexity of the intracellular apoptotic machinery. Here we show that the core features of the BAK-driven MOMP pathway can be reproduced in a highly simplified in vitro system consisting of recombinant human BAK lacking the carboxyl-terminal 21 residues (BAKΔC) and tBID in combination with liposomes bearing an appropriate lipid environment. Using this minimalist reconstituted system we established that tBID suffices to trigger BAKΔC membrane insertion, oligomerization, and pore formation. Furthermore, we demonstrate that tBID-activated BAKΔC permeabilizes the membrane by forming structurally dynamic pores rather than a large proteinaceous channel of fixed size. We also identified two distinct roles played by mitochondrial lipids along the molecular pathway of BAKΔC-induced membrane permeabilization. First, using several independent approaches, we showed that cardiolipin directly interacts with BAKΔC, leading to a localized structural rearrangement in the protein that "primes" BAKΔC for interaction with tBID. Second, we provide evidence that selected curvature-inducing lipids present in mitochondrial membranes specifically modulate the energetic expenditure required to create the BAKΔC pore. Collectively, our results support the notion that BAK functions as a direct effector of MOMP akin to BAX and also adds significantly to the growing evidence indicating that mitochondrial membrane lipids are actively implicated in BCL-2 protein family function.

Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

PubMed

Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

2012-01-01

Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress.

PubMed

Lee, Chun Pong; Maksaev, Grigory; Jensen, Gregory S; Murcha, Monika W; Wilson, Margaret E; Fricker, Mark; Hell, Ruediger; Haswell, Elizabeth S; Millar, A Harvey; Sweetlove, Lee J

2016-12-01

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch-activated ion channel with a slight preference for anions and provides protection against hypo-osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo-osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1-1 mutants under moderate heat- and heavy-metal-stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Dissecting Stop Transfer versus Conservative Sorting Pathways for Mitochondrial Inner Membrane Proteins in Vivo*

PubMed Central

Park, Kwangjin; Botelho, Salomé Calado; Hong, Joonki; Österberg, Marie; Kim, Hyun

2013-01-01

Mitochondrial inner membrane proteins that carry an N-terminal presequence are sorted by one of two pathways: stop transfer or conservative sorting. However, the sorting pathway is known for only a small number of proteins, in part due to the lack of robust experimental tools with which to study. Here we present an approach that facilitates determination of inner membrane protein sorting pathways in vivo by fusing a mitochondrial inner membrane protein to the C-terminal part of Mgm1p containing the rhomboid cleavage region. We validated the Mgm1 fusion approach using a set of proteins for which the sorting pathway is known, and determined sorting pathways of inner membrane proteins for which the sorting mode was previously uncharacterized. For Sdh4p, a multispanning membrane protein, our results suggest that both conservative sorting and stop transfer mechanisms are required for insertion. Furthermore, the sorting process of Mgm1 fusion proteins was analyzed under different growth conditions and yeast mutant strains that were defective in the import motor or the m-AAA protease function. Our results show that the sorting of mitochondrial proteins carrying moderately hydrophobic transmembrane segments is sensitive to cellular conditions, implying that mitochondrial import and membrane sorting in the physiological environment may be dynamically tuned. PMID:23184936

Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes.

PubMed

Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

2014-01-01

Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO.

Dimethyl Sulfoxide Damages Mitochondrial Integrity and Membrane Potential in Cultured Astrocytes

PubMed Central

Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

2014-01-01

Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO. PMID:25238609

Activated mitofusin 2 signals mitochondrial fusion, interferes with Bax activation, and reduces susceptibility to radical induced depolarization.

PubMed

Neuspiel, Margaret; Zunino, Rodolfo; Gangaraju, Sandhya; Rippstein, Peter; McBride, Heidi

2005-07-01

Mitochondrial fusion in higher eukaryotes requires at least two essential GTPases, Mitofusin 1 and Mitofusin 2 (Mfn2). We have created an activated mutant of Mfn2, which shows increased rates of nucleotide exchange and decreased rates of hydrolysis relative to wild type Mfn2. Mitochondrial fusion is stimulated dramatically within heterokaryons expressing this mutant, demonstrating that hydrolysis is not requisite for the fusion event, and supporting a role for Mfn2 as a signaling GTPase. Although steady-state mitochondrial fusion required the conserved intermembrane space tryptophan residue, this requirement was overcome within the context of the hydrolysis-deficient mutant. Furthermore, the punctate localization of Mfn2 is lost in the dominant active mutants, indicating that these sites are functionally controlled by changes in the nucleotide state of Mfn2. Upon staurosporine-stimulated cell death, activated Bax is recruited to the Mfn2-containing puncta; however, Bax activation and cytochrome c release are inhibited in the presence of the dominant active mutants of Mfn2. The dominant active form of Mfn2 also protected the mitochondria against free radical-induced permeability transition. In contrast to staurosporine-induced outer membrane permeability transition, pore opening induced through the introduction of free radicals was dependent upon the conserved intermembrane space residue. This is the first evidence that Mfn2 is a signaling GTPase regulating mitochondrial fusion and that the nucleotide-dependent activation of Mfn2 concomitantly protects the organelle from permeability transition. The data provide new insights into the critical relationship between mitochondrial membrane dynamics and programmed cell death.

[Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

PubMed

Zhao, H; Teng, X M; Li, Y F

2017-11-25

Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all P< 0.05]. The copy number of mtDNA and the mitochondrial membrane potential in classⅠ frozen embryo group were significantly higher than those in classⅡ frozen embryo group (both P< 0.05). Conclusion: The mtDNA copy number and the mitochondrial membrane potential of embryos of the better quality embryo are higher.

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane.

PubMed

Wenz, Lena-Sophie; Opaliński, Lukasz; Schuler, Max-Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; van der Laan, Martin; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

2014-06-01

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane. © 2014 The Authors.

Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

NASA Astrophysics Data System (ADS)

Shiva, Sruti; Brookes, Paul S.; Patel, Rakesh P.; Anderson, Peter G.; Darley-Usmar, Victor M.

2001-06-01

An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (<20 μM). In this study, we present evidence for a consumption of NO in mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia

PubMed Central

Cooper, Helen M.; Yang, Yang; Ylikallio, Emil; Khairullin, Rafil; Woldegebriel, Rosa; Lin, Kai-Lan; Euro, Liliya; Palin, Eino; Wolf, Alexander; Trokovic, Ras; Isohanni, Pirjo; Kaakkola, Seppo; Auranen, Mari; Lönnqvist, Tuula; Wanrooij, Sjoerd

2017-01-01

Abstract De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity. PMID:28158749

Erythropoietin activates SIRT1 to protect human cardiomyocytes against doxorubicin-induced mitochondrial dysfunction and toxicity.

PubMed

Cui, Lan; Guo, Jiabin; Zhang, Qiang; Yin, Jian; Li, Jin; Zhou, Wei; Zhang, Tingfen; Yuan, Haitao; Zhao, Jun; Zhang, Li; Carmichael, Paul L; Peng, Shuangqing

2017-06-05

The hormone erythropoietin (EPO) has been demonstrated to protect against chemotherapy drug doxorubicin (DOX)-induced cardiotoxicity, but the underlying mechanism remains obscure. We hypothesized that silent mating type information regulation 2 homolog 1 (SIRT1), an NAD + -dependent protein deacetylase that activates peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), plays a crucial role in regulating mitochondrial function and mediating the beneficial effect of EPO. Our study in human cardiomyocyte AC16 cells showed that DOX-induced cytotoxicity and mitochondrial dysfunction, as manifested by decreased mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential, and increased mitochondrial superoxide accumulation, can be mitigated by EPO pretreatment. EPO was found to upregulate SIRT1 activity and protein expression to reverse DOX-induced acetylation of PGC-1α and suppression of a suite of PGC-1α-activated genes involved in mitochondrial function and biogenesis, such as nuclear respiratory factor-1 (NRF1), mitochondrial transcription factor A (TFAM), citrate synthase (CS), superoxide dismutase 2 (SOD2), cytochrome c oxidase IV (COXIV), and voltage-dependent anion channel (VDAC). Silencing of SIRT1 via small RNA interference sensitized AC16 cells to DOX-induced cytotoxicity and reduction in mtDNA copy number. Although with SIRT1 silenced, EPO could reverse to some extent DOX-induced mitochondrial superoxide accumulation, loss of mitochondrial membrane potential and ATP depletion, it failed to normalize protein expression of PGC-1α and its downstream genes. Taken together, our results indicated that EPO may activate SIRT1 to enhance mitochondrial function and protect against DOX-induced cardiotoxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Nobiletin attenuates neurotoxic mitochondrial calcium overload through K+ influx and ΔΨm across mitochondrial inner membrane.

PubMed

Lee, Ji Hyung; Amarsanaa, Khulan; Wu, Jinji; Jeon, Sang-Chan; Cui, Yanji; Jung, Sung-Cherl; Park, Deok-Bae; Kim, Se-Jae; Han, Sang-Heon; Kim, Hyun-Wook; Rhyu, Im Joo; Eun, Su-Yong

2018-05-01

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (ΔΨ m ). Therefore, pharmacological manipulation of ΔΨ m can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ΔΨ m against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity (100 µM, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 µM)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca 2+ (5 µM). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ΔΨ m were completely abolished in K + -free medium on pure isolated mitochondria. Taken together, results demonstrate that K + influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K + influx is probably mediated, at least in part, by activation of mitochondrial K + channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

Direct Membrane Association Drives Mitochondrial Fission by the Parkinson Disease-associated Protein α-Synuclein*♦

PubMed Central

Nakamura, Ken; Nemani, Venu M.; Azarbal, Farnaz; Skibinski, Gaia; Levy, Jon M.; Egami, Kiyoshi; Munishkina, Larissa; Zhang, Jue; Gardner, Brooke; Wakabayashi, Junko; Sesaki, Hiromi; Cheng, Yifan; Finkbeiner, Steven; Nussbaum, Robert L.; Masliah, Eliezer; Edwards, Robert H.

2011-01-01

The protein α-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of α-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by α- than β- or γ-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. α-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease. PMID:21489994

Evidence of proteolipid domain formation in an inner mitochondrial membrane mimicking model.

PubMed

Cheniour, Mouhedine; Brewer, Jonathan; Bagatolli, Luis; Marcillat, Olivier; Granjon, Thierry

2017-05-01

Mitochondrial creatine kinase (mtCK) is highly abundant in mitochondria; its quantity is equimolecular to the Adenylic Nucleotide Translocator and represents 1% of the mitochondrial proteins. It is a multitask protein localized in the mitochondria intermembrane space where it binds to the specific cardiolipin (CL) phospholipid. If mtCK was initially thought to be exclusively implicated in energy transfer between mitochondria and cytosol through a mechanism referred to as the phosphocreatine shuttle, several recent studies suggested an additional role in maintaining mitochondria membrane structure. To further characterized mtCK binding process we used multiphoton excitation fluorescence microscopy coupled with Giant Unilamellar Vesicles (GUV) and laurdan as fluorescence probe. We gathered structural and dynamical information on the molecular events occurring during the binding of mtCK to the mitochondria inner membrane. We present the first visualization of mtCK-induced CL segregation on a bilayer model forming micrometer-size proteolipid domains at the surface of the GUV. Those microdomains, which only occurred when CL is included in the lipid mixture, were accompanied by the formation of protein multimolecular assembly, vesicle clamping, and changes in both vesicle curvature and membrane fluidity CONCLUSION: Those results highlighted the importance of the highly abundant mtCK in the lateral organization of the mitochondrial inner membrane. Microdomains were induced in mitochondria-mimicking membranes composed of natural phospholipids without cholesterol and/or sphingolipids differing from the proposed cytoplasmic membrane rafts. Those findings as well as membrane curvature modification were discussed in relation with protein-membrane interaction and protein cluster involvement in membrane morphology. Copyright © 2017 Elsevier B.V. All rights reserved.

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

AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function

PubMed Central

Taniane, Caitlin; Farrell, Geoffrey; Arias, Irwin M.; Lippincott-Schwartz, Jennifer; Fu, Dong

2016-01-01

Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury. PMID:27792760

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

Glycogen synthase kinase 3-mediated voltage-dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation.

PubMed

Martel, Cecile; Allouche, Maya; Esposti, Davide Degli; Fanelli, Elena; Boursier, Céline; Henry, Céline; Chopineau, Joel; Calamita, Giuseppe; Kroemer, Guido; Lemoine, Antoinette; Brenner, Catherine

2013-01-01

Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early mitochondrial impairment and late hepatocyte cell death. However, mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high-fat diet, and in vitro models of lipotoxicity, we show that outer mitochondrial membrane permeability is altered and identified a posttranslational modification of voltage-dependent anion channel (VDAC), a membrane channel and NADH oxidase, as a cause of early mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. VDAC acts as an early sensor of lipid toxicity and its GSK3-mediated phosphorylation status controls outer mitochondrial membrane permeabilization in hepatosteatosis. Copyright © 2012 American Association for the Study of Liver Diseases.

Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

USDA-ARS?s Scientific Manuscript database

Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>500) known and predicted TA proteins in Arabidopsis for those annotated, based on Gene Ontology, to possess mitochondrial...

Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis*

PubMed Central

Huang, Kai; Zhang, Jingjing; O'Neill, Katelyn L.; Gurumurthy, Channabasavaiah B.; Quadros, Rolen M.; Tu, Yaping; Luo, Xu

2016-01-01

The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo. In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant BidD60E and a BH3 defective mutant BidG94E failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis. PMID:27053107

A nontoxic, photostable and high signal-to-noise ratio mitochondrial probe with mitochondrial membrane potential and viscosity detectivity

NASA Astrophysics Data System (ADS)

Chen, Yanan; Qi, Jianguo; Huang, Jing; Zhou, Xiaomin; Niu, Linqiang; Yan, Zhijie; Wang, Jianhong

2018-01-01

Herein, we reported a yellow emission probe 1-methyl-4-(6-morpholino-1, 3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl) pyridin-1-ium iodide which could specifically stain mitochondria in living immortalized and normal cells. In comparison to the common mitochondria tracker (Mitotracker Deep Red, MTDR), this probe was nontoxic, photostable and ultrahigh signal-to-noise ratio, which could real-time monitor mitochondria for a long time. Moreover, this probe also showed high sensitivity towards mitochondrial membrane potential and intramitochondrial viscosity change. Consequently, this probe was used for imaging mitochondria, detecting changes in mitochondrial membrane potential and intramitochondrial viscosity in physiological and pathological processes.

Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

PubMed

Ge, Yan; Yoshiie, Kiyotaka; Kuribayashi, Futoshi; Lin, Mingqun; Rikihisa, Yasuko

2005-01-01

The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A. phagocytophilum. Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

PubMed Central

Merkwirth, Carsten; Morbin, Michela; Brönneke, Hella S.; Jordan, Sabine D.; Rugarli, Elena I.; Langer, Thomas

2012-01-01

Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies

Loss of Drp1 function alters OPA1 processing and changes mitochondrial membrane organization

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

Moepert, Kristin; Hajek, Petr; Frank, Stephan

2009-08-01

RNAi mediated loss of Drp1 function changes mitochondrial morphology in cultured HeLa and HUVEC cells by shifting the balance of mitochondrial fission and fusion towards unopposed fusion. Over time, inhibition of Drp1 expression results in the formation of a highly branched mitochondrial network along with 'bulge'-like structures. These changes in mitochondrial morphology are accompanied by a reduction in levels of Mitofusin 1 (Mfn1) and 2 (Mfn2) and a modified proteolytic processing of OPA1 isoforms, resulting in the inhibition of cell proliferation. In addition, our data imply that bulge formation is driven by Mfn1 action along with particular proteolytic short-OPA1 (s-OPA1)more » variants: Loss of Mfn2 in the absence of Drp1 results in an increase of Mfn1 levels along with processed s-OPA1-isoforms, thereby enhancing continuous 'fusion' and bulge formation. Moreover, bulge formation might reflect s-OPA1 mitochondrial membrane remodeling activity, resulting in the compartmentalization of cytochrome c deposits. The proteins Yme1L and PHB2 appeared not associated with the observed enhanced OPA1 proteolysis upon RNAi of Drp1, suggesting the existence of other OPA1 processing controlling proteins. Taken together, Drp1 appears to affect the activity of the mitochondrial fusion machinery by unbalancing the protein levels of mitofusins and OPA1.« less

Mitochondrial cardiolipin/phospholipid trafficking: the role of membrane contact site complexes and lipid transfer proteins.

PubMed

Schlattner, Uwe; Tokarska-Schlattner, Malgorzata; Rousseau, Denis; Boissan, Mathieu; Mannella, Carmen; Epand, Richard; Lacombe, Marie-Lise

2014-04-01

Historically, cellular trafficking of lipids has received much less attention than protein trafficking, mostly because its biological importance was underestimated, involved sorting and translocation mechanisms were not known, and analytical tools were limiting. This has changed during the last decade, and we discuss here some progress made in respect to mitochondria and the trafficking of phospholipids, in particular cardiolipin. Different membrane contact site or junction complexes and putative lipid transfer proteins for intra- and intermembrane lipid translocation have been described, involving mitochondrial inner and outer membrane, and the adjacent membranes of the endoplasmic reticulum. An image emerges how cardiolipin precursors, remodeling intermediates, mature cardiolipin and its oxidation products could migrate between membranes, and how this trafficking is involved in cardiolipin biosynthesis and cell signaling events. Particular emphasis in this review is given to mitochondrial nucleoside diphosphate kinase D and mitochondrial creatine kinases, which emerge to have roles in both, membrane junction formation and lipid transfer. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

PubMed Central

Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

2014-01-01

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster.

PubMed

Wiley, Sandra E; Paddock, Mark L; Abresch, Edward C; Gross, Larry; van der Geer, Peter; Nechushtai, Rachel; Murphy, Anne N; Jennings, Patricia A; Dixon, Jack E

2007-08-17

The outer mitochondrial membrane protein mitoNEET was discovered as a binding target of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class used to treat type 2 diabetes (Colca, J. R., McDonald, W. G., Waldon, D. J., Leone, J. W., Lull, J. M., Bannow, C. A., Lund, E. T., and Mathews, W. R. (2004) Am. J. Physiol. 286, E252-E260). We have shown that mitoNEET is a member of a small family of proteins containing a 39-amino-acid CDGSH domain. Although the CDGSH domain is annotated as a zinc finger motif, mitoNEET was shown to contain iron (Wiley, S. E., Murphy, A. N., Ross, S. A., van der Geer, P., and Dixon, J. E. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 5318-5323). Optical and electron paramagnetic resonance spectroscopy showed that it contained a redox-active pH-labile Fe-S cluster. Mass spectrometry showed the loss of 2Fe and 2S upon cofactor extrusion. Spectroscopic studies of recombinant proteins showed that the 2Fe-2S cluster was coordinated by Cys-3 and His-1. The His ligand was shown to be involved in the observed pH lability of the cluster, indicating that loss of this ligand via protonation triggered release of the cluster. mitoNEET is the first identified 2Fe-2S-containing protein located in the outer mitochondrial membrane. Based on the biophysical data and domain fusion analysis, mitoNEET may function in Fe-S cluster shuttling and/or in redox reactions.

Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes

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

Yasuzaki, Yukari; Yamada, Yuma; Harashima, Hideyoshi, E-mail: harasima@pharm.hokudai.ac.jp

2010-06-25

Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we providemore » a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.« less

Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

PubMed Central

Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

2011-01-01

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

Phylogenetic Analysis of Mitochondrial Outer Membrane β-Barrel Channels

PubMed Central

Wojtkowska, Małgorzata; Jąkalski, Marcin; Pieńkowska, Joanna R.; Stobienia, Olgierd; Karachitos, Andonis; Przytycka, Teresa M.; Weiner, January; Kmita, Hanna; Makałowski, Wojciech

2012-01-01

Transport of molecules across mitochondrial outer membrane is pivotal for a proper function of mitochondria. The transport pathways across the membrane are formed by ion channels that participate in metabolite exchange between mitochondria and cytoplasm (voltage-dependent anion-selective channel, VDAC) as well as in import of proteins encoded by nuclear genes (Tom40 and Sam50/Tob55). VDAC, Tom40, and Sam50/Tob55 are present in all eukaryotic organisms, encoded in the nuclear genome, and have β-barrel topology. We have compiled data sets of these protein sequences and studied their phylogenetic relationships with a special focus on the position of Amoebozoa. Additionally, we identified these protein-coding genes in Acanthamoeba castellanii and Dictyostelium discoideum to complement our data set and verify the phylogenetic position of these model organisms. Our analysis show that mitochondrial β-barrel channels from Archaeplastida (plants) and Opisthokonta (animals and fungi) experienced many duplication events that resulted in multiple paralogous isoforms and form well-defined monophyletic clades that match the current model of eukaryotic evolution. However, in representatives of Amoebozoa, Chromalveolata, and Excavata (former Protista), they do not form clearly distinguishable clades, although they locate basally to the plant and algae branches. In most cases, they do not posses paralogs and their sequences appear to have evolved quickly or degenerated. Consequently, the obtained phylogenies of mitochondrial outer membrane β-channels do not entirely reflect the recent eukaryotic classification system involving the six supergroups: Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta. PMID:22155732

Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis.

PubMed

Huang, Kai; Zhang, Jingjing; O'Neill, Katelyn L; Gurumurthy, Channabasavaiah B; Quadros, Rolen M; Tu, Yaping; Luo, Xu

2016-05-27

The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid(D60E) and a BH3 defective mutant Bid(G94E) failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Profiling of the Tox21 Chemical Collection for Mitochondrial Function to Identify Compounds that Acutely Decrease Mitochondrial Membrane Potential

PubMed Central

Attene-Ramos, Matias S.; Huang, Ruili; Michael, Sam; Witt, Kristine L.; Richard, Ann; Tice, Raymond R.; Simeonov, Anton; Austin, Christopher P.

2014-01-01

Background: Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding whether different environmental chemicals and druglike molecules impact mitochondrial function represents an initial step in predicting exposure-related toxicity and defining a possible role for such compounds in the onset of various diseases. Objectives: We sought to identify individual chemicals and general structural features associated with changes in mitochondrial membrane potential (MMP). Methods: We used a multiplexed [two end points in one screen; MMP and adenosine triphosphate (ATP) content] quantitative high throughput screening (qHTS) approach combined with informatics tools to screen the Tox21 library of 10,000 compounds (~ 8,300 unique chemicals) at 15 concentrations each in triplicate to identify chemicals and structural features that are associated with changes in MMP in HepG2 cells. Results: Approximately 11% of the compounds (913 unique compounds) decreased MMP after 1 hr of treatment without affecting cell viability (ATP content). In addition, 309 compounds decreased MMP over a concentration range that also produced measurable cytotoxicity [half maximal inhibitory concentration (IC50) in MMP assay/IC50 in viability assay ≤ 3; p < 0.05]. More than 11% of the structural clusters that constitute the Tox21 library (76 of 651 clusters) were significantly enriched for compounds that decreased the MMP. Conclusions: Our multiplexed qHTS approach allowed us to generate a robust and reliable data set to evaluate the ability of thousands of drugs and environmental compounds to decrease MMP. The use of structure-based clustering analysis allowed us to identify molecular features that are likely responsible for the observed activity. Citation: Attene-Ramos MS, Huang R, Michael S, Witt KL, Richard A, Tice RR, Simeonov A, Austin CP, Xia M. 2015. Profiling of the Tox

Mitochondrial targeted curcumin exhibits anticancer effects through disruption of mitochondrial redox and modulation of TrxR2 activity.

PubMed

Jayakumar, Sundarraj; Patwardhan, Raghavendra S; Pal, Debojyoti; Singh, Babita; Sharma, Deepak; Kutala, Vijay Kumar; Sandur, Santosh Kumar

2017-12-01

Mitocurcumin is a derivative of curcumin, which has been shown to selectively enter mitochondria. Here we describe the anti-tumor efficacy of mitocurcumin in lung cancer cells and its mechanism of action. Mitocurcumin, showed 25-50 fold higher efficacy in killing lung cancer cells as compared to curcumin as demonstrated by clonogenic assay, flow cytometry and high throughput screening assay. Treatment of lung cancer cells with mitocurcumin significantly decreased the frequency of cancer stem cells. Mitocurcumin increased the mitochondrial reactive oxygen species (ROS), decreased the mitochondrial glutathione levels and induced strand breaks in the mitochondrial DNA. As a result, we observed increased BAX to BCL-2 ratio, cytochrome C release into the cytosol, loss of mitochondrial membrane potential and increased caspase-3 activity suggesting that mitocurcumin activates the intrinsic apoptotic pathway. Docking studies using mitocurcumin revealed that it binds to the active site of the mitochondrial thioredoxin reductase (TrxR2) with high affinity. In corroboration with the above finding, mitocurcumin decreased TrxR activity in cell free as well as the cellular system. The anti-cancer activity of mitocurcumin measured in terms of apoptotic cell death and the decrease in cancer stem cell frequency was accentuated by TrxR2 overexpression. This was due to modulation of TrxR2 activity to NADPH oxidase like activity by mitocurcumin, resulting in higher ROS accumulation and cell death. Thus, our findings reveal mitocurcumin as a potent anticancer agent with better efficacy than curcumin. This study also demonstrates the role of TrxR2 and mitochondrial DNA damage in mitocurcumin mediated killing of cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Polyhydroxybutyrate targets mammalian mitochondria and increases permeability of plasmalemmal and mitochondrial membranes.

PubMed

Elustondo, Pia A; Angelova, Plamena R; Kawalec, Michał; Michalak, Michał; Kurcok, Piotr; Abramov, Andrey Y; Pavlov, Evgeny V

2013-01-01

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle.

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

Multifunctional Mitochondrial AAA Proteases

PubMed Central

Glynn, Steven E.

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle. PMID:28589125

Multifunctional Mitochondrial AAA Proteases.

PubMed

Glynn, Steven E

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

PubMed

Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

2014-01-01

Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

Mitochondrial membrane permeabilization and cell death during myocardial infarction: roles of calcium and reactive oxygen species

PubMed Central

Webster, Keith A

2013-01-01

Excess generation of reactive oxygen species (ROS) and cytosolic calcium accumulation play major roles in the initiation of programmed cell death during acute myocardial infarction. Cell death may include necrosis, apoptosis and autophagy, and combinations thereof. During ischemia, calcium handling between the sarcoplasmic reticulum and myofilament is disrupted and calcium is diverted to the mitochondria causing swelling. Reperfusion, while essential for survival, reactivates energy transduction and contractility and causes the release of ROS and additional ionic imbalance. During acute ischemia–reperfusion, the principal death pathways are programmed necrosis and apoptosis through the intrinsic pathway, initiated by the opening of the mitochondrial permeability transition pore and outer mitochondrial membrane permeabilization, respectively. Despite intense investigation, the mechanisms of action and modes of regulation of mitochondrial membrane permeabilization are incompletely understood. Extrinsic apoptosis, necroptosis and autophagy may also contribute to ischemia–reperfusion injury. In this review, the roles of dysregulated calcium and ROS and the contributions of Bcl-2 proteins, as well as mitochondrial morphology in promoting mitochondrial membrane permeability change and the ensuing cell death during myocardial infarction are discussed. PMID:23176689

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity.

PubMed

Stelmashook, E V; Lozier, E R; Goryacheva, E S; Mergenthaler, P; Novikova, S V; Zorov, D B; Isaev, N K

2010-09-27

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca(2+)](i)) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca(2+)](i)). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca(2+)](i) increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca(2+)](i). Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca(2+)](i), demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca(2+)](i) and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca(2+)](i) and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Toxins in Botanical Dietary Supplements: Blue Cohosh Components Disrupt Cellular Respiration and Mitochondrial Membrane Potential

PubMed Central

Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B.; Khan, Ikhlas A.; Nagle, Dale G.; Zhou, Yu-Dong

2014-01-01

Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA “Black Box” warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3) exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage. PMID:24328138

The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis*

PubMed Central

Barnwal, Bhaskar; Karlberg, Helen; Mirazimi, Ali; Tan, Yee-Joo

2016-01-01

Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93–140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells. PMID:26574543

Polyhydroxybutyrate Targets Mammalian Mitochondria and Increases Permeability of Plasmalemmal and Mitochondrial Membranes

PubMed Central

Elustondo, Pia A.; Angelova, Plamena R.; Kawalec, Michał; Michalak, Michał; Kurcok, Piotr; Abramov, Andrey Y.; Pavlov, Evgeny V.

2013-01-01

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle. PMID:24086638

Interleukin-15 Modulates Adipose Tissue by Altering Mitochondrial Mass and Activity

PubMed Central

Barra, Nicole G.; Palanivel, Rengasamy; Denou, Emmanuel; Chew, Marianne V.; Gillgrass, Amy; Walker, Tina D.; Kong, Josh; Richards, Carl D.; Jordana, Manel; Collins, Stephen M.; Trigatti, Bernardo L.; Holloway, Alison C.; Raha, Sandeep; Steinberg, Gregory R.; Ashkar, Ali A.

2014-01-01

Interleukin-15 (IL-15) is an immunomodulatory cytokine that affects body mass regulation independent of lymphocytes; however, the underlying mechanism(s) involved remains unknown. In an effort to investigate these mechanisms, we performed metabolic cage studies, assessed intestinal bacterial diversity and macronutrient absorption, and examined adipose mitochondrial activity in cultured adipocytes and in lean IL-15 transgenic (IL-15tg), overweight IL-15 deficient (IL-15−/−), and control C57Bl/6 (B6) mice. Here we show that differences in body weight are not the result of differential activity level, food intake, or respiratory exchange ratio. Although intestinal microbiota differences between obese and lean individuals are known to impact macronutrient absorption, differing gut bacteria profiles in these murine strains does not translate to differences in body weight in colonized germ free animals and macronutrient absorption. Due to its contribution to body weight variation, we examined mitochondrial factors and found that IL-15 treatment in cultured adipocytes resulted in increased mitochondrial membrane potential and decreased lipid deposition. Lastly, IL-15tg mice have significantly elevated mitochondrial activity and mass in adipose tissue compared to B6 and IL-15−/− mice. Altogether, these results suggest that IL-15 is involved in adipose tissue regulation and linked to altered mitochondrial function. PMID:25517731

Crystal structure of mitochondrial respiratory membrane protein complex II.

PubMed

Sun, Fei; Huo, Xia; Zhai, Yujia; Wang, Aojin; Xu, Jianxing; Su, Dan; Bartlam, Mark; Rao, Zihe

2005-07-01

The mitochondrial respiratory Complex II or succinate:ubiquinone oxidoreductase (SQR) is an integral membrane protein complex in both the tricarboxylic acid cycle and aerobic respiration. Here we report the first crystal structure of Complex II from porcine heart at 2.4 A resolution and its complex structure with inhibitors 3-nitropropionate and 2-thenoyltrifluoroacetone (TTFA) at 3.5 A resolution. Complex II is comprised of two hydrophilic proteins, flavoprotein (Fp) and iron-sulfur protein (Ip), and two transmembrane proteins (CybL and CybS), as well as prosthetic groups required for electron transfer from succinate to ubiquinone. The structure correlates the protein environments around prosthetic groups with their unique midpoint redox potentials. Two ubiquinone binding sites are discussed and elucidated by TTFA binding. The Complex II structure provides a bona fide model for study of the mitochondrial respiratory system and human mitochondrial diseases related to mutations in this complex.

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.

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

[Change in the lipid composition of the inner mitochondrial membranes in rat organs during adaptation to heat].

PubMed

Zubareva, E V; Seferova, R I; Denisova, N A

1991-01-01

Under conditions of adaptation to heating lipid composition in mitochondrial membranes of rat inner tissues was altered as follows: an increase in relative concentration of plasmalogenous forms of phospholipids (kidney, heart) and in content of saturated fatty acids (liver tissue), a decrease in the index of fatty acids unsaturation and in the ratio of fatty acids omega-3/omega-6. The alterations observed enabled the membranes to keep sufficient amount of liquidity essential for functional activity of mitochondria in heating.

Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter.

PubMed

Nagampalli, Raghavendra Sashi Krishna; Quesñay, José Edwin Neciosup; Adamoski, Douglas; Islam, Zeyaul; Birch, James; Sebinelli, Heitor Gobbi; Girard, Richard Marcel Bruno Moreira; Ascenção, Carolline Fernanda Rodrigues; Fala, Angela Maria; Pauletti, Bianca Alves; Consonni, Sílvio Roberto; de Oliveira, Juliana Ferreira; Silva, Amanda Cristina Teixeira; Franchini, Kleber Gomes; Leme, Adriana Franco Paes; Silber, Ariel Mariano; Ciancaglini, Pietro; Moraes, Isabel; Dias, Sandra Martha Gomes; Ambrosio, Andre Luis Berteli

2018-02-22

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

Molecular insights into the m-AAA protease-mediated dislocation of transmembrane helices in the mitochondrial inner membrane.

PubMed

Lee, Seoeun; Lee, Hunsang; Yoo, Suji; Kim, Hyun

2017-12-08

Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane; thus, proper regulation of these proteins is essential for cell viability. The m -AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments, but the molecular mechanism involved in these processes remains elusive. This study investigated the role of the TM domains in the m -AAA protease by systematic replacement of one TM domain at a time in yeast. Our data indicated that replacement of the Yta10 TM2 domain abolishes membrane dislocation for only a subset of substrates, whereas replacement of the Yta12 TM2 domain impairs membrane dislocation for all tested substrates, suggesting different roles of the TM domains in each m -AAA protease subunit. Furthermore, m -AAA protease-mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate. This finding suggested that the m -AAA protease cannot dislocate large hydrophilic domains across the membrane, indicating that the membrane dislocation probably occurs in a lipid environment. In summary, this study highlights previously underappreciated biological roles of TM domains of the m -AAA proteases in mediating the recognition and dislocation of membrane-embedded substrates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial lipids in neurodegeneration.

PubMed

Aufschnaiter, Andreas; Kohler, Verena; Diessl, Jutta; Peselj, Carlotta; Carmona-Gutierrez, Didac; Keller, Walter; Büttner, Sabrina

2017-01-01

Mitochondrial dysfunction is a common feature of many neurodegenerative diseases, including proteinopathies such as Alzheimer's or Parkinson's disease, which are characterized by the deposition of aggregated proteins in the form of insoluble fibrils or plaques. The distinct molecular processes that eventually result in mitochondrial dysfunction during neurodegeneration are well studied but still not fully understood. However, defects in mitochondrial fission and fusion, mitophagy, oxidative phosphorylation and mitochondrial bioenergetics have been linked to cellular demise. These processes are influenced by the lipid environment within mitochondrial membranes as, besides membrane structure and curvature, recruitment and activity of different proteins also largely depend on the respective lipid composition. Hence, the interaction of neurotoxic proteins with certain lipids and the modification of lipid composition in different cell compartments, in particular mitochondria, decisively impact cell death associated with neurodegeneration. Here, we discuss the relevance of mitochondrial lipids in the pathological alterations that result in neuronal demise, focussing on proteinopathies.

The Involvement of Mitochondrial Membrane Potential in Cross-Resistance Between Radiation and Docetaxel

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

Kuwahara, Yoshikazu; Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai; Roudkenar, Mehryar Habibi

2016-11-01

Purpose: To understand the molecular mechanisms underlying cancer cell radioresistance, clinically relevant radioresistant (CRR) cells that continue to proliferate during exposure to 2 Gy/day X-rays for more than 30 days were established. A modified high-density survival assay for anticancer drug screening revealed that CRR cells were resistant to an antimicrotubule agent, docetaxel (DTX). The involvement of reactive oxygen species (ROS) from mitochondria (mtROS) in the cross-resistance to X-rays and DTX was studied. Methods and Materials: Sensitivity to anticancer agents was determined by a modified high-density cell survival or water-soluble tetrazolium salt assay. DTX-induced mtROS generation was determined by MitoSOX redmore » staining. JC-1 staining was used to visualize mitochondrial membrane potential. DTX-induced DNA double-strand breaks were determined by γ-H2AX staining. To obtain mitochondrial DNA-lacking (ρ{sup 0}) cells, the cells were cultured for 3 to 4 weeks in medium containing ethidium bromide. Results: Treatment with DTX increased mtROS in parental cells but not in CRR cells. DTX induced DNA double-strand breaks in parental cells. The mitochondrial membrane potential of CRR cells was lower in CRR cells than in parental cells. Depletion of mtDNA induced DTX resistance in parental cells. Treatment with dimethyl sulfoxide also induced DTX resistance in parental cells. Conclusions: The mitochondrial dysfunction observed in CRR cells contributes to X-ray and DTX cross-resistance. The activation of oxidative phosphorylation in CRR cells may represent an effective approach to overcome radioresistant cancers. In general, the overexpression of β-tubulin or multidrug efflux pumps is thought to be involved in DTX resistance. In the present study, we discovered another DTX resistant mechanism by investigating CRR cells.« less

The mitochondrial-targeted antioxidant, MitoQ, increases liver mitochondrial cardiolipin content in obesogenic diet-fed rats.

PubMed

Fouret, Gilles; Tolika, Evanthia; Lecomte, Jérôme; Bonafos, Béatrice; Aoun, Manar; Murphy, Michael P; Ferreri, Carla; Chatgilialoglu, Chryssostomos; Dubreucq, Eric; Coudray, Charles; Feillet-Coudray, Christine

2015-10-01

Cardiolipin (CL), a unique mitochondrial phospholipid, plays a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics. The present study was designed to determine the effect of MitoQ, a mitochondrial-targeted antioxidant, on the content of liver mitochondrial membrane phospholipids, in particular CL, and its fatty acid composition in obesogenic diet-fed rats. To do this, twenty-four 6week old male Sprague Dawley rats were randomized into three groups of 8 animals and fed for 8weeks with either a control diet, a high fat diet (HF), or a HF diet with MitoQ (HF+MitoQ). Phospholipid classes and fatty acid composition were assayed by chromatographic methods in liver and liver mitochondria. Mitochondrial bioenergetic function was also evaluated. While MitoQ had no or slight effects on total liver fatty acid composition and phospholipid classes and their fatty acid composition, it had major effects on liver mitochondrial phospholipids and mitochondrial function. Indeed, MitoQ both increased CL synthase gene expression and CL content of liver mitochondria and increased 18:2n-6 (linoleic acid) content of mitochondrial phospholipids by comparison to the HF diet. Moreover, mitochondrial CL content was positively correlated to mitochondrial membrane fluidity, membrane potential and respiration, as well as to ATP synthase activity, while it was negatively correlated to mitochondrial ROS production. These findings suggest that MitoQ may decrease pathogenic alterations to CL content and profiles, thereby preserving mitochondrial function and attenuating the development of some of the features of metabolic syndrome in obesogenic diet-fed rats. Copyright © 2015 Elsevier B.V. All rights reserved.

Mitochondrial-dependent Autoimmunity in Membranous Nephropathy of IgG4-related Disease

PubMed Central

Buelli, Simona; Perico, Luca; Galbusera, Miriam; Abbate, Mauro; Morigi, Marina; Novelli, Rubina; Gagliardini, Elena; Tentori, Chiara; Rottoli, Daniela; Sabadini, Ettore; Saito, Takao; Kawano, Mitsuhiro; Saeki, Takako; Zoja, Carlamaria; Remuzzi, Giuseppe; Benigni, Ariela

2015-01-01

The pathophysiology of glomerular lesions of membranous nephropathy (MN), including seldom-reported IgG4-related disease, is still elusive. Unlike in idiopathic MN where IgG4 prevails, in this patient IgG3 was predominant in glomerular deposits in the absence of circulating anti-phospholipase A2 receptor antibodies, suggesting a distinct pathologic process. Here we documented that IgG4 retrieved from the serum of our propositus reacted against carbonic anhydrase II (CAII) at the podocyte surface. In patient's biopsy, glomerular CAII staining increased and co-localized with subepithelial IgG4 deposits along the capillary walls. Patient's IgG4 caused a drop in cell pH followed by mitochondrial dysfunction, excessive ROS production and cytoskeletal reorganization in cultured podocytes. These events promoted mitochondrial superoxide-dismutase-2 (SOD2) externalization on the plasma membrane, becoming recognizable by complement-binding IgG3 anti-SOD2. Among patients with IgG4-related disease only sera of those with IgG4 anti-CAII antibodies caused low intracellular pH and mitochondrial alterations underlying SOD2 externalization. Circulating IgG4 anti-CAII can cause podocyte injury through processes of intracellular acidification, mitochondrial oxidative stress and neoantigen induction in patients with IgG4 related disease. The onset of MN in a subset of patients could be due to IgG4 antibodies recognizing CAII with consequent exposure of mitochondrial neoantigen in the context of multifactorial pathogenesis of disease. PMID:26137589

Melatonin: A Mitochondrial Targeting Molecule Involving Mitochondrial Protection and Dynamics

PubMed Central

Tan, Dun-Xian; Manchester, Lucien C.; Qin, Lilan; Reiter, Russel J.

2016-01-01

Melatonin has been speculated to be mainly synthesized by mitochondria. This speculation is supported by the recent discovery that aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT) is localized in mitochondria of oocytes and the isolated mitochondria generate melatonin. We have also speculated that melatonin is a mitochondria-targeted antioxidant. It accumulates in mitochondria with high concentration against a concentration gradient. This is probably achieved by an active transportation via mitochondrial melatonin transporter(s). Melatonin protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting the mitochondrial permeability transition pore (MPTP), and activating uncoupling proteins (UCPs). Thus, melatonin maintains the optimal mitochondrial membrane potential and preserves mitochondrial functions. In addition, mitochondrial biogenesis and dynamics is also regulated by melatonin. In most cases, melatonin reduces mitochondrial fission and elevates their fusion. Mitochondrial dynamics exhibit an oscillatory pattern which matches the melatonin circadian secretory rhythm in pinealeocytes and probably in other cells. Recently, melatonin has been found to promote mitophagy and improve homeostasis of mitochondria. PMID:27999288

Habitual physical activity in mitochondrial disease.

PubMed

Apabhai, Shehnaz; Gorman, Grainne S; Sutton, Laura; Elson, Joanna L; Plötz, Thomas; Turnbull, Douglass M; Trenell, Michael I

2011-01-01

Mitochondrial disease is the most common neuromuscular disease and has a profound impact upon daily life, disease and longevity. Exercise therapy has been shown to improve mitochondrial function in patients with mitochondrial disease. However, no information exists about the level of habitual physical activity of people with mitochondrial disease and its relationship with clinical phenotype. Habitual physical activity, genotype and clinical presentations were assessed in 100 patients with mitochondrial disease. Comparisons were made with a control group individually matched by age, gender and BMI. Patients with mitochondrial disease had significantly lower levels of physical activity in comparison to matched people without mitochondrial disease (steps/day; 6883±3944 vs. 9924±4088, p = 0.001). 78% of the mitochondrial disease cohort did not achieve 10,000 steps per day and 48% were classified as overweight or obese. Mitochondrial disease was associated with less breaks in sedentary activity (Sedentary to Active Transitions, % per day; 13±0.03 vs. 14±0.03, p = 0.001) and an increase in sedentary bout duration (bout lengths/fraction of total sedentary time; 0.206±0.044 vs. 0.187±0.026, p = 0.001). After adjusting for covariates, higher physical activity was moderately associated with lower clinical disease burden (steps/day; r(s) = -0.49; 95% CI -0.33, -0.63, P<0.01). There were no systematic differences in physical activity between different genotypes mitochondrial disease. These results demonstrate for the first time that low levels of physical activity are prominent in mitochondrial disease. Combined with a high prevalence of obesity, physical activity may constitute a significant and potentially modifiable risk factor in mitochondrial disease.

Regulation of Mitochondrial Dynamics and Autophagy by the Mitochondria-Associated Membrane.

PubMed

Tagaya, Mitsuo; Arasaki, Kohei

2017-01-01

Mitochondria are powerhouses and central to metabolism in cells. They are highly dynamic organelles that continuously fuse, divide, and move along the cytoskeleton to form the mitochondrial network. The fusion and fission are catalyzed by four dynamin-related GTPases in mammals that are controlled by a variety of protein-protein interactions and posttranslational modifications. Mitochondrial dynamics and metabolism are linked and regulate each other. Starvation induces mitochondrial elongation, which enables the mitochondria to produce energy more efficiently and to escape from autophagic degradation. Damaged portions of mitochondria are removed from the healthy parts by division, and subsequently degraded via a specific mode of autophagy termed mitophagy. Recent studies shed light on the contribution of the endoplasmic reticulum to mitochondrial dynamics and the cooperation of the two organelles for the progression of autophagy including mitophagy. A subdomain of the endoplasmic reticulum apposed to mitochondria is called the mitochondria-associated membrane (MAM), which comprises a unique set of proteins that interact with mitochondrial proteins. Here we review our current understanding of the molecular mechanisms of mitochondrial dynamics and mitochondria-related processes in the context of the interaction with the endoplasmic reticulum.

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

Dietary Tocotrienol/γ-Cyclodextrin Complex Increases Mitochondrial Membrane Potential and ATP Concentrations in the Brains of Aged Mice

PubMed Central

Schloesser, Anke; Esatbeyoglu, Tuba; Piegholdt, Stefanie; Dose, Janina; Ikuta, Naoko; Okamoto, Hinako; Ishida, Yoshiyuki; Terao, Keiji; Matsugo, Seiichi; Rimbach, Gerald

2015-01-01

Brain aging is accompanied by a decrease in mitochondrial function. In vitro studies suggest that tocotrienols, including γ- and δ-tocotrienol (T3), may exhibit neuroprotective properties. However, little is known about the effect of dietary T3 on mitochondrial function in vivo. In this study, we monitored the effect of a dietary T3/γ-cyclodextrin complex (T3CD) on mitochondrial membrane potential and ATP levels in the brain of 21-month-old mice. Mice were fed either a control diet or a diet enriched with T3CD providing 100 mg T3 per kg diet for 6 months. Dietary T3CD significantly increased mitochondrial membrane potential and ATP levels compared to those of controls. The increase in MMP and ATP due to dietary T3CD was accompanied by an increase in the protein levels of the mitochondrial transcription factor A (TFAM). Furthermore, dietary T3CD slightly increased the mRNA levels of superoxide dismutase, γ-glutamyl cysteinyl synthetase, and heme oxygenase 1 in the brain. Overall, the present data suggest that T3CD increases TFAM, mitochondrial membrane potential, and ATP synthesis in the brains of aged mice. PMID:26301044

Lipopolysaccharide-induced mitochondrial dysfunction in boar sperm is mediated by activation of oxidative phosphorylation.

PubMed

He, Bin; Guo, Huiduo; Gong, Yabin; Zhao, Ruqian

2017-01-01

Lipopolysaccharide (LPS) has been reported to exert detrimental effects on boar sperm viability. In the present study, LPS was detected in boar semen samples at an average level of 0.62 ± 0.14 μg/mL. We treated boar sperm with 1 μg/mL LPS for 6 hours and examined alterations in sperm motility and apoptosis, together with mitochondrial functionality and mitochondrial reactive oxygen species generation. The expression and the location of toll-like receptor 4 (TLR4) and mitochondrial transcription factor A (TFAM) were determined to reveal possible mechanisms. LPS-treated sperm showed significant reduction in motility (P < 0.05) and viability (P < 0.05). LPS induced sperm mitochondrial damage via oxidative stress which is indicated by marked ultrastructural changes in the mitochondria including swelling, disorientation and vacuole, a decrease of mitochondrial membrane potential (ΔΨm; P < 0.05), as well as an increase of malondialdehyde levels (P < 0.01). Moreover, the production of mitochondrial reactive oxygen species through oxidative phosphorylation (OXPHOS) was significantly (P < 0.05) increased, which leads to oxidative stress. The copy number of mitochondrial DNA was significantly (P < 0.05) higher in LPS-treated sperm. Moreover, cytochrome c oxidase subunit IV (COXIV), an important subunit in mitochondrial electron transport chain and OXPHOS, was significantly (P < 0.05) upregulated after LPS treatment. TFAM, the key transcription factor that activates mitochondrial DNA replication and transcription, was translocated from the head to the midpiece of sperm where mitochondria are distributed in LPS-treated sperm. Taken together, these results indicate that LPS-induced decrease of motility and viability in boar sperm is mediated by abnormal activation of OXPHOS and mitochondrial membrane lipid peroxidation. These findings may provide new insights in understanding the mechanisms underlying the bacterial infection-induced sperm damage

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed Central

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-01-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria. PMID:10816421

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-06-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

Withaferin-A induces apoptosis in osteosarcoma U2OS cell line via generation of ROS and disruption of mitochondrial membrane potential.

PubMed

Li, A-X; Sun, M; Li, X

2017-03-01

Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania somnifera. It has marked pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis-inducing drug-like molecules. Osteosarcoma is a rare type of bone cancer affecting humans. The objective of the present study was therefore to evaluate the antitumor potential of WF-A against several osteosarcoma cell lines. MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC50. DAPI staining was used to confirm the apoptosis-inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. The results of the present study revealed that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC50 ranging from 0.32 to 7.6 µM. The lowest IC50 (0.32 µM) was observed against U2OS cell line and, therefore, it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced a ROS-mediated reduction in mitochondrial membrane potential in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. We suggest that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential.

[Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase].

PubMed

Danylovych, H V; Danylovych, Iu V; Kolomiiets', O V; Kosterin, S O; Rodik, R V; Cherenok, S O; Kal'chenko, V I; Chunikhin, O Iu; Horchev, V F; Karakhim, S O

2012-01-01

The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

Mitochondrial decay in hepatocytes from old rats: Membrane potential declines, heterogeneity and oxidants increase

PubMed Central

Hagen, Tory M.; Yowe, David L.; Bartholomew, James C.; Wehr, Carol M.; Do, Katherine L.; Park, Jin-Y.; Ames, Bruce N.

1997-01-01

Mitochondrial function during aging was assessed in isolated rat hepatocytes to avoid the problem of differential lysis when old, fragile mitochondria are isolated. Rhodamine 123, a fluorescent dye that accumulates in mitochondria on the basis of their membrane potential, was used as a probe to determine whether this key function is affected by aging. A marked fluorescent heterogeneity was observed in hepatocytes from old (20–28 months) but not young (3–5 months) rats, suggesting age-associated alterations in mitochondrial membrane potential, the driving force for ATP synthesis. Three distinct cell subpopulations were separated by centrifugal elutriation; each exhibited a unique rhodamine 123 fluorescence pattern, with the largest population from old rats having significantly lower fluorescence than that seen in young rats. This apparent age-associated alteration in mitochondrial membrane potential was confirmed by measurements with radioactive tetraphenylphosphonium bromide. Cells from young rats had a calculated membrane potential of −154 mV, in contrast to that of the three subpopulations from old rats of −70 mV (the largest population), −93 mV, and −154 mV. Production of oxidants was examined using 2′,7′dichlorofluorescin, a dye that forms a fluorescent product upon oxidation. The largest cell subpopulation and a minor one from old animals produced significantly more oxidants than cells from young rats. To investigate the molecular cause(s) for the heterogeneity, we determined the levels of an age-associated mtDNA deletion. No significant differences were seen in the three subpopulations, indicating that the mitochondrial decay is due to other mutations, epigenetic changes, or both. PMID:9096346

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

Mitochondrial modulators in experimental Huntington's disease: reversal of mitochondrial dysfunctions and cognitive deficits.

PubMed

Mehrotra, Arpit; Kanwal, Abhinav; Banerjee, Sanjay Kumar; Sandhir, Rajat

2015-06-01

Huntington's disease (HD) is a chronic neurodegenerative condition involving impaired mitochondrial functions. The present study evaluates the therapeutic potential of combined administration of mitochondrial modulators: alpha-lipoic acid and acetyl-l-carnitine on mitochondrial dysfunctions in 3-NP-induced HD. Our results reveal 3-NP administration resulted in compromise of mitochondrial functions in terms of: (1) impaired activity of mitochondrial respiratory chain enzymes, altered cytochrome levels, reduced histochemical staining of complex-II and IV, reduced in-gel activity of complex-I to V, and reduced mRNA expression of respiratory chain complexes; (2) enhanced mitochondrial oxidative stress indicated by increased malondialdehyde, protein carbonyls, reactive oxygen species and nitrite levels, along with decreased Mn-superoxide dismutase and catalase activity; (3) mitochondrial structural changes measured by mitochondrial swelling, reduced mitochondrial membrane potential and ultra-structure changes; (4) increased cytosolic cytochrome c levels, caspase-3 and -9 activity along with altered expression of apoptotic proteins (AIF, Bim, Bad, and Bax); and (5) impaired cognitive functions assessed using Morris water maze and Y-maze. Combination of mitochondrial modulators (alpha-lipoic acid + acetyl-l-carnitine) on the other hand ameliorated 3-NP-induced mitochondrial dysfunctions, oxidative stress, histologic alterations, and behavioral deficits, suggesting their therapeutic efficacy in the management of HD. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of Insecticides on the Fluidity of Mitochondrial Membranes of the Diamondback Moth, Plutella xylostella, Resistant and Susceptible to Avermectin

PubMed Central

Hu, J.; Liang, P.; Shi, X.; Gao, X.

2008-01-01

The effects of various insecticides on the fluidity of mitochondrial membranes and cross-resistance were investigated in the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) using strains that were both resistant and susceptible to avermectin. The resistant strain of P. xylostella, AV-R, developed 1078-fold resistance to avermetins with a high level of cross-resistance to the analogs of avermectins, ivermectin and emamectin benzoate. It had more than 1000 times greater resistance when compared with the avermectin-susceptible strain, XH-S. Mitochondrial membrane fluidity was measured by detecting fluorescence polarization using DPH (1,6-Diphenyl -1,3,5-hexatriene) as the fluorescence probe. Abamectin, emamectin benzoate, ivermectin, cypermethrin and fenvalerate decreased the fluidity of mitochondrial membranes in the XH-S strain at 25°C. However, fipronil and acephate did not change the fluidity of mitochondrial membrane when the concentration of these insecticides was 1×10-4 mol/L. Membrane fluidity increased as the temperature increased. The thermotropic effect on the polarization value of DPH increased as the insecticide concentration was increased. There was a significant difference of mitochondrial membrane fluidity between both XH-S and AV-R when temperature was less than 25°C and no difference was observed when the temperature was more than 25°C. The low-dose abamectin (0.11 mg/L) in vivo treatment caused a significant change of membrane fluidity in the XH-S strain and no change in the AV-R strain. However, a high-dose abamectin (11.86 mg/L) resulted in 100% mortality of the XH-S strain. In vivo treatment may cause a significant change of membrane fluidity in the AV-R strain PMID:20345311

Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation

PubMed Central

Hung, Victoria; Lam, Stephanie S; Udeshi, Namrata D; Svinkina, Tanya; Guzman, Gaelen; Mootha, Vamsi K; Carr, Steven A; Ting, Alice Y

2017-01-01

The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP’s ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes. DOI: http://dx.doi.org/10.7554/eLife.24463.001 PMID:28441135

Mitochondrial metabolic regulation by GRP78

PubMed Central

Prasad, Manoj; Pawlak, Kevin J.; Burak, William E.; Perry, Elizabeth E.; Marshall, Brendan; Whittal, Randy M.; Bose, Himangshu S.

2017-01-01

Steroids, essential for mammalian survival, are initiated by cholesterol transport by steroidogenic acute regulatory protein (StAR). Appropriate protein folding is an essential requirement of activity. Endoplasmic reticulum (ER) chaperones assist in folding of cytoplasmic proteins, whereas mitochondrial chaperones fold only mitochondrial proteins. We show that glucose regulatory protein 78 (GRP78), a master ER chaperone, is also present at the mitochondria-associated ER membrane (MAM), where it folds StAR for delivery to the outer mitochondrial membrane. StAR expression and activity are drastically reduced following GRP78 knockdown. StAR folding starts at the MAM region; thus, its cholesterol fostering capacity is regulated by GRP78 long before StAR reaches the mitochondria. In summary, GRP78 is an acute regulator of steroidogenesis at the MAM, regulating the intermediate folding of StAR that is crucial for its activity. PMID:28275724

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

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

Effects of mitochondrial poisons on glutathione redox potential and carotid body chemoreceptor activity.

PubMed

Gomez-Niño, A; Agapito, M T; Obeso, A; Gonzalez, C

2009-01-01

Low oxygen sensing in chemoreceptor cells involves the inhibition of specific plasma membrane K(+) channels, suggesting that mitochondria-derived reactive oxygen species (ROS) link hypoxia to K(+) channel inhibition, subsequent cell depolarization and activation of neurotransmitter release. We have used several mitochondrial poisons, alone and in combination with the antioxidant N-acetylcysteine (NAC), and quantify their capacity to alter GSH/GSSG levels and glutathione redox potential (E(GSH)) in rat diaphragm. Selected concentrations of mitochondrial poisons with or without NAC were tested for their capacity to activate neurotransmitter release in chemoreceptor cells and to alter ATP levels in intact rat carotid body (CB). We found that rotenone (1 microM), antimycin A (0.2 microg/ml) and sodium azide (5mM) decreased E(GSH); NAC restored E(GSH) to control values. At those concentrations mitochondrial poisons activated neurotransmitter release from CB chemoreceptor cells and decreased CB ATP levels, NAC being ineffective to modify these responses. Additional experiments with 3-nitroprionate (5mM), lower concentrations of rotenone and dinitrophenol revealed variable relationships between E(GSH) and chemoreceptor cell neurotransmitter release responses and ATP levels. These findings indicate a lack of correlation between mitochondrial-generated modifications of E(GSH) and chemoreceptor cells activity. This lack of correlation renders unlikely that alteration of mitochondrial production of ROS is the physiological pathway chemoreceptor cells use to signal hypoxia.

Dihydroartemisinin Induces Apoptosis in Human Bladder Cancer Cell Lines Through Reactive Oxygen Species, Mitochondrial Membrane Potential, and Cytochrome C Pathway

PubMed Central

Poupel, Farhad; Aghaei, Mahmoud; Movahedian, Ahmad; Jafari, Seyyed Mehdi; Shahrestanaki, Mohammad Keyvanloo

2017-01-01

Background: Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin and has antiproliferative effect. However, such effects of DHA have not yet been revealed for bladder cancer cells. Methods: We used as bladder cancer cell lines to examine the effect of DHA on the cell viability, cell apoptosis, and monitoring of mitochondrial membrane potential (ΔΨm) changes. Furthermore, the effect of DHA on the reactive oxygen species (ROS) production and cytochrome c release were also detected. We employed MTT assay to investigate the cell proliferation effect of DHA on the EJ-138 and HTB-9 human bladder cancer cells. Annexin/PI staining, caspase-3 activity assay, Bcl-2/Bax protein expression, mitochondrial membrane potential assay, cytochrome c release, and ROS analysis were used for apoptosis detection. Results: DHA significantly reduced cell viability in a dose-dependent manner. Cytotoxicity of DHA was suppressed by N-acetylcysteine. The growth inhibition effect of DHA was related to the induction of cell apoptosis, which were manifested by annexin V-FITC staining, activation of caspase-3. DHA also increased ROS generation, cytochrome c release, and loss of mitochondrial transmembrane potential (ΔΨm) in cells. In addition, the downregulation of regulatory protein Bcl-2 and upregulation of Bax protein by DHA were also observed. Conclusions: These findings demonstrated that DHA induces apoptosis through mitochondrial signaling pathway. These suggest that DHA may be a potential agent for induction of apoptosis in human bladder cancer cells. PMID:29114376

Copper effects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at different salinities.

PubMed

Lauer, Mariana Machado; de Oliveira, Camila Bento; Yano, Natalia Lie Inocencio; Bianchini, Adalto

2012-11-01

The estuarine crab Neohelice granulata was exposed (96 h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2 ppt salinity, 1 mg Cu/L; isosmotic crabs: 30 ppt salinity, 5 mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2 ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, increased lactate dehydrogenase activity, and reduced the mitochondrial membrane potential. In posterior gills, copper inhibited hexokinase and pyruvate kinase activity, and increased citrate synthase activity. In anterior gills of crabs acclimated to 30 ppt salinity, copper exposure inhibited phosphofructokinase and citrate synthase activity, and increased hexokinase activity. In posterior gills, copper inhibited phosphofructokinase and pyruvate kinase activity, and increased hexokinase and lactate dehydrogenase activity. Copper did not affect cytochrome c oxidase activity in either anterior or posterior gills of crabs acclimated to 2 and 30 ppt salinity. These findings indicate that exposure to a sublethal copper concentration affects the activity of enzymes involved in glycolysis and Krebs cycle, especially in anterior (respiratory) gills of hyperosmoregulating crabs. Changes observed indicate a switch from aerobic to anaerobic metabolism, characterizing a situation of functional hypoxia. In this case, reduced mitochondrial membrane potential would suggest a decrease in ATP production. Although gills of isosmotic crabs were also affected by copper exposure, changes

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

VDAC electronics: 1. VDAC-hexo(gluco)kinase generator of the mitochondrial outer membrane potential.

PubMed

Lemeshko, Victor V

2014-05-01

The simplest mechanism of the generation of the mitochondrial outer membrane potential (OMP) by the VDAC (voltage-dependent anion channel)-hexokinase complex (VHC), suggested earlier, and by the VDAC-glucokinase complex (VGC), was computationally analyzed. Even at less than 4% of VDACs bound to hexokinase, the calculated OMP is high enough to trigger the electrical closure of VDACs beyond the complexes at threshold concentrations of glucose. These results confirmed our previous hypothesis that the Warburg effect is caused by the electrical closure of VDACs, leading to global restriction of the outer membrane permeability coupled to aerobic glycolysis. The model showed that the inhibition of the conductance and/or an increase in the voltage sensitivity of a relatively small fraction of VDACs by factors like tubulin potentiate the electrical closure of the remaining free VDACs. The extrusion of calcium ions from the mitochondrial intermembrane space by the generated OMP, positive inside, might increase cancer cell resistance to death. Within the VGC model, the known effect of induction of ATP release from mitochondria by accumulated glucose-6-phosphate in pancreatic beta cells might result not only of the known effect of GK dissociation from the VDAC-GK complex, but also of a decrease in the free energy of glucokinase reaction, leading to the OMP decrease and VDAC opening. We suggest that the VDAC-mediated electrical control of the mitochondrial outer membrane permeability, dependent on metabolic conditions, is a fundamental physiological mechanism of global regulation of mitochondrial functions and of cell death. Copyright © 2014 Elsevier B.V. All rights reserved.

Architecture Mapping of the Inner Mitochondrial Membrane Proteome by Chemical Tools in Live Cells.

PubMed

Lee, Song-Yi; Kang, Myeong-Gyun; Shin, Sanghee; Kwak, Chulhwan; Kwon, Taejoon; Seo, Jeong Kon; Kim, Jong-Seo; Rhee, Hyun-Woo

2017-03-15

The inner mitochondrial membrane (IMM) proteome plays a central role in maintaining mitochondrial physiology and cellular metabolism. Various important biochemical reactions such as oxidative phosphorylation, metabolite production, and mitochondrial biogenesis are conducted by the IMM proteome, and mitochondria-targeted therapeutics have been developed for IMM proteins, which is deeply related for various human metabolic diseases including cancer and neurodegenerative diseases. However, the membrane topology of the IMM proteome remains largely unclear because of the lack of methods to evaluate it in live cells in a high-throughput manner. In this article, we reveal the in vivo topological direction of 135 IMM proteins, using an in situ-generated radical probe with genetically targeted peroxidase (APEX). Owing to the short lifetime of phenoxyl radicals generated in situ by submitochondrial targeted APEX and the impermeability of the IMM to small molecules, the solvent-exposed tyrosine residues of both the matrix and intermembrane space (IMS) sides of IMM proteins were exclusively labeled with the radical probe in live cells by Matrix-APEX and IMS-APEX, respectively and identified by mass spectrometry. From this analysis, we confirmed 58 IMM protein topologies and we could determine the topological direction of 77 IMM proteins whose topology at the IMM has not been fully characterized. We also found several IMM proteins (e.g., LETM1 and OXA1) whose topological information should be revised on the basis of our results. Overall, our identification of structural information on the mitochondrial inner-membrane proteome can provide valuable insights for the architecture and connectome of the IMM proteome in live cells.

Cytosolic Calcium Coordinates Mitochondrial Energy Metabolism with Presynaptic Activity

PubMed Central

Chouhan, Amit K.; Ivannikov, Maxim V.; Lu, Zhongmin; Sugimori, Mutsuyuki; Llinas, Rodolfo R.; Macleod, Gregory T.

2012-01-01

Most neurons fire in bursts, imposing episodic energy demands, but how these demands are coordinated with oxidative phosphorylation is still unknown. Here, using fluorescence imaging techniques on presynaptic termini of Drosophila motor neurons (MNs), we show that mitochondrial matrix pH (pHm), inner membrane potential (Δψm), and NAD(P)H levels ([NAD(P)H]m) increase within seconds of nerve stimulation. The elevations of pHm, Δψm, and [NAD(P)H]m indicate an increased capacity for ATP production. Elevations in pHm were blocked by manipulations which blocked mitochondrial Ca2+ uptake, including replacement of extracellular Ca2+ with Sr2+, and application of either tetraphenylphosphonium chloride or KB-R7943, indicating that it is Ca2+ that stimulates presynaptic mitochondrial energy metabolism. To place this phenomenon within the context of endogenous neuronal activity, the firing rates of a number of individually identified MNs were determined during fictive locomotion. Surprisingly, although endogenous firing rates are significantly different, there was little difference in presynaptic cytosolic Ca2+ levels ([Ca2+]c) between MNs when each fires at its endogenous rate. The average [Ca2+]c level (329±11nM) was slightly above the average Ca2+ affinity of the mitochondria (281±13nM). In summary, we show that when MNs fire at endogenous rates [Ca2+]c is driven into a range where mitochondria rapidly acquire Ca2+. As we also show that Ca2+ stimulates presynaptic mitochondrial energy metabolism, we conclude that [Ca2+]c levels play an integral role in coordinating mitochondrial energy metabolism with presynaptic activity in Drosophila MNs. PMID:22279208

Proteolytic cleavage by the inner membrane peptidase (IMP) complex or Oct1 peptidase controls the localization of the yeast peroxiredoxin Prx1 to distinct mitochondrial compartments.

PubMed

Gomes, Fernando; Palma, Flávio Romero; Barros, Mario H; Tsuchida, Eduardo T; Turano, Helena G; Alegria, Thiago G P; Demasi, Marilene; Netto, Luis E S

2017-10-13

Yeast Prx1 is a mitochondrial 1-Cys peroxiredoxin that catalyzes the reduction of endogenously generated H 2 O 2 Prx1 is synthesized on cytosolic ribosomes as a preprotein with a cleavable N-terminal presequence that is the mitochondrial targeting signal, but the mechanisms underlying Prx1 distribution to distinct mitochondrial subcompartments are unknown. Here, we provide direct evidence of the following dual mitochondrial localization of Prx1: a soluble form in the intermembrane space and a form in the matrix weakly associated with the inner mitochondrial membrane. We show that Prx1 sorting into the intermembrane space likely involves the release of the protein precursor within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase. We also found that during its import into the matrix compartment, Prx1 is sequentially cleaved by mitochondrial processing peptidase and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 cleaved eight amino acid residues from the N-terminal region of Prx1 inside the matrix, without interfering with its peroxidase activity in vitro Remarkably, the processing of peroxiredoxin (Prx) proteins by Oct1 appears to be an evolutionarily conserved process because yeast Oct1 could cleave the human mitochondrial peroxiredoxin Prx3 when expressed in Saccharomyces cerevisiae Altogether, the processing of peroxiredoxins by Imp2 or Oct1 likely represents systems that control the localization of Prxs into distinct compartments and thereby contribute to various mitochondrial redox processes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Study on the effect of reactive oxygen species-mediated oxidative stress on the activation of mitochondrial apoptosis and the tenderness of yak meat.

PubMed

Wang, Lin-Lin; Yu, Qun-Li; Han, Ling; Ma, Xiu-Li; Song, Ren-De; Zhao, Suo-Nan; Zhang, Wen-Hua

2018-04-01

This study investigated the effect of reactive oxygen species-mediated oxidative stress on activation of mitochondrial apoptosis and tenderness of yak meat during postmortem ageing. Oxidative stress degree, Ca 2+ levels, membrane permeability transition pore opening, mitochondrial membrane potential, apoptotic factors and the shear force were examined. Results showed that the ROS generated by H 2 O 2 significantly increased mitochondrial oxidative stress by decreasing the activities of superoxide dismutase, catalase and glutathione peroxidase, and increasing lipid peroxidation. Furthermore, oxidative stress enhanced Ca 2+ production and cytochrome c release, changed the levels of Bcl-2 family proteins and activated caspase-9 and -3 activities. Ultimately, oxidative stress increased the apoptosis rate and tenderness of yak meat. These observations confirmed that ROS-mediated oxidative stress participates in the activation of the apoptotic cascade reaction involving Ca 2+ and Bcl-2 family proteins. The results further suggested that ROS-mediated oxidative stress plays a significant role in meat tenderization through the mitochondrial apoptotic pathway. Copyright © 2017. Published by Elsevier Ltd.

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.

Modulation of mitochondrial ion transport by inorganic polyphosphate - essential role in mitochondrial permeability transition pore.

PubMed

Baev, Artyom Y; Negoda, Alexander; Abramov, Andrey Y

2017-02-01

Inorganic polyphosphate (polyP) is a biopolymer of phosphoanhydride-linked orthophosphate residues. PolyP is involved in multiple cellular processes including mitochondrial metabolism and cell death. We used artificial membranes and isolated mitochondria to investigate the role of the polyP in mitochondrial ion transport and in activation of PTP. Here, we found that polyP can modify ion permeability of de-energised mitochondrial membranes but not artificial membranes. This permeability was selective for Ba 2+ and Ca 2+ but not for other monovalent and bivalent cations and can be blocked by inhibitors of the permeability transition pore - cyclosporine A or ADP. Lower concentrations of polyP modulate calcium dependent permeability transition pore opening. Increase in polyP concentrations and elongation chain length of the polymer causes calcium independent swelling in energized conditions. Physiologically relevant concentrations of inorganic polyP can regulate calcium dependent as well calcium independent mitochondrial permeability transition pore opening. This raises the possibility that cytoplasmic polyP can be an important contributor towards regulation of the cell death.

Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes

PubMed Central

Gillies, Laura A; Du, Han; Peters, Bjoern; Knudson, C. Michael; Newmeyer, Donald D.; Kuwana, Tomomi

2015-01-01

Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼100–160 nm in diameter after 60–90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10–500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress. PMID:25411335

The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM)

PubMed Central

Frank, Daniel O.; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

2015-01-01

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated. PMID:25875815

The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

PubMed

Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

2015-01-01

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

Cisplatin impairs rat liver mitochondrial functions by inducing changes on membrane ion permeability: prevention by thiol group protecting agents.

PubMed

Custódio, José B A; Cardoso, Carla M P; Santos, Maria S; Almeida, Leonor M; Vicente, Joaquim A F; Fernandes, Maria A S

2009-05-02

Cisplatin (CisPt) is the most important platinum anticancer drug widely used in the treatment of head, neck, ovarian and testicular cancers. However, the mechanisms by which CisPt induces cytotoxicity, namely hepatotoxicity, are not completely understood. The goal of this study was to investigate the influence of CisPt on rat liver mitochondrial functions (Ca(2+)-induced mitochondrial permeability transition (MPT), mitochondrial bioenergetics, and mitochondrial oxidative stress) to better understand the mechanism underlying its hepatotoxicity. The effect of thiol group protecting agents and some antioxidants against CisPt-induced mitochondrial damage was also investigated. Treatment of rat liver mitochondria with CisPt (20nmol/mg protein) induced Ca(2+)-dependent mitochondrial swelling, depolarization of membrane potential (DeltaPsi), Ca(2+) release, and NAD(P)H fluorescence intensity decay. These effects were prevented by cyclosporine A (CyA), a potent and specific inhibitor of the MPT. In the concentration range of up to 40nmol/mg protein, CisPt slightly inhibited state 3 and stimulated state 2 and state 4 respiration rates using succinate as respiratory substrate. The respiratory indexes, respiratory control ratio (RCR) and ADP/O ratios, the DeltaPsi, and the ADP phosphorylation rate were also depressed. CisPt induced mitochondrial inner membrane permeabilization to protons (proton leak) but did not induce significant changes on mitochondrial H(2)O(2) generation. All the effects induced by CisPt on rat liver mitochondria were prevented by thiol group protecting agents namely, glutathione (GSH), dithiothreitol (DTT), N-acetyl-L-cysteine (NAC) and cysteine (CYS), whereas superoxide-dismutase (SOD), catalase (CAT) and ascorbate (ASC) were without effect. In conclusion, the anticancer drug CisPt: (1) increases the sensitivity of mitochondria to Ca(2+)-induced MPT; (2) interferes with mitochondrial bioenergetics by increasing mitochondrial inner membrane

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

PubMed Central

Hönlinger, A; Bömer, U; Alconada, A; Eckerskorn, C; Lottspeich, F; Dietmeier, K; Pfanner, N

1996-01-01

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase. Images PMID:8641278

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

PubMed

He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

2012-07-01

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

Molecular Dynamics Simulations of Creatine Kinase and Adenine Nucleotide Translocase in Mitochondrial Membrane Patch*

PubMed Central

Karo, Jaanus; Peterson, Pearu; Vendelin, Marko

2012-01-01

Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies. PMID:22241474

Mitochondrial uncoupling proteins in unicellular eukaryotes.

PubMed

Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Antos-Krzeminska, Nina; Sluse, Francis E

2010-01-01

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species. Copyright © 2009 Elsevier B.V. All rights reserved.

Ebselen protects mitochondrial function and oxidative stress while inhibiting the mitochondrial apoptosis pathway after acute spinal cord injury.

PubMed

Jia, Zhi-Qiang; Li, San-Qiang; Qiao, Wei-Qiang; Xu, Wen-Zhong; Xing, Jian-Wu; Liu, Jian-Tao; Song, Hui; Gao, Zhong-Yang; Xing, Bing-Wen; He, Xi-Jing

2018-05-04

Ebselen is a fat-soluble small molecule and organic selenium compound that regulates the activity of glutathione peroxidase to alleviate mitochondrial oxidative stress and improve mitochondrial function. In the present study, we aimed to investigate the effects of ebselen on mitochondrial oxidative stress response, mitochondrial apotosis, and motor behaviors after spinal cord injury (SCI). We found that ebselen significantly increased the BBB score in motor behavior, thus suggesting a rescue effect of ebselen on motor function after SCI in rats. Meanwhile, we revealed that ebselen can increase glutathione (GSH) content as well as superoxide dismutase (SOD) and catalase (CAT) activities after SCI-this suggests ebselen has an antioxidant effect. Furthermore, the ATP content and Na + -K + -ATPase activity in mitochondria were increased by ebselen after SCI, while the mitochondrial membrane potential (MMP) was decreased by ebselen. The Cytochrome C and Smac release from mitochondria were reduced by ebselen after SCI, thus indicating improved membrane permeability by ebselen. Moreover, the alterations in caspase-3, Bax and Bcl-2 protein expression, as well as the proportion of cell apoptosis were improved by ebselen treatment, which together suggested that ebselen has an inhibitory effect on mitochondrial apotosis pathways after SCI. Taken together, our results suggest that ebselen can inhibit secondary damage caused by spinal cord injury. Indeed it plays a neuroprotective role in spinal cord injury perhaps by improving mitochondrial function and inhibiting the mitochondrial apoptosis pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

USDA-ARS?s Scientific Manuscript database

Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>600) known and predicted TA proteins in Arabidopsis thaliana for those annotated, based on Gene Ontology, to possess mitoc...

The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy

PubMed Central

Vincent, Amy E.; Ng, Yi Shiau; White, Kathryn; Davey, Tracey; Mannella, Carmen; Falkous, Gavin; Feeney, Catherine; Schaefer, Andrew M.; McFarland, Robert; Gorman, Grainne S.; Taylor, Robert W.; Turnbull, Doug M.; Picard, Martin

2016-01-01

Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease. PMID:27506553

Constitutive Activation of PINK1 Protein Leads to Proteasome-mediated and Non-apoptotic Cell Death Independently of Mitochondrial Autophagy*

PubMed Central

Akabane, Shiori; Matsuzaki, Kohei; Yamashita, Shun-ichi; Arai, Kana; Okatsu, Kei; Kanki, Tomotake; Matsuda, Noriyuki; Oka, Toshihiko

2016-01-01

Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), a Ser/Thr kinase, and PARKIN, a ubiquitin ligase, are causal genes for autosomal recessive early-onset parkinsonism. Multiple lines of evidence indicate that PINK1 and PARKIN cooperatively control the quality of the mitochondrial population via selective degradation of damaged mitochondria by autophagy. Here, we report that PINK1 and PARKIN induce cell death with a 12-h delay after mitochondrial depolarization, which differs from the time profile of selective autophagy of mitochondria. This type of cell death exhibited definite morphologic features such as plasma membrane rupture, was insensitive to a pan-caspase inhibitor, and did not involve mitochondrial permeability transition. Expression of a constitutively active form of PINK1 caused cell death in the presence of a pan-caspase inhibitor, irrespective of the mitochondrial membrane potential. PINK1-mediated cell death depended on the activities of PARKIN and proteasomes, but it was not affected by disruption of the genes required for autophagy. Furthermore, fluorescence and electron microscopic analyses revealed that mitochondria were still retained in the dead cells, indicating that PINK1-mediated cell death is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play critical roles in selective cell death in which damaged mitochondria are retained, independent of mitochondrial autophagy. PMID:27302064

Ca2+ and mitochondrial ROS: Both hero and villain in membrane repair.

PubMed

Cooper, Sandra T

2017-09-05

Membrane repair is a symphony of signaling, conducted principally by the steep influx of Ca 2+ through an injured membrane. In this issue of Science Signaling , Horn et al reveal unique interplay between Ca 2+ influx and mitochondrially generated reactive oxygen species (mtROS) to enhance actin-mediated wound closure for survival of injured mammalian muscle and nonmuscle cells. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

PubMed

Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

2016-01-01

The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A

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

Siu, W.P.; Pun, Pamela Boon Li; Latchoumycandane, Calivarathan

2008-03-15

Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (> 500 {mu}M) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 {mu}M) fully inhibited diclofenac-induced cell injury, suggesting thatmore » mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca{sup 2+} chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca{sup 2+}-Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.« less

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.

Metabolic activation of hepatotoxic drug (benzbromarone) induced mitochondrial membrane permeability transition

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

Shirakawa, Maho; Sekine, Shuichi; Tanaka, Ayaka

The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assaymore » system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1′,6-(OH){sub 2} BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100 μM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites. - Highlights: • We constructed a sequential assay system for toxic metabolite induced MPT in one pot. • 14 drugs (e.g. benzbromarone (BBR)) induced toxic metabolite dependent MPT. • Both the production of toxic metabolite and MPT could be inhibited by CYP inhibitors. • This system could evaluate the comprehensive MPT without purification of metabolites.« less

Increases in Intracellular Zinc Enhance Proliferative Signaling as well as Mitochondrial and Endolysosomal Activity in Human Melanocytes.

PubMed

Rudolf, Emil; Rudolf, Kamil

2017-01-01

Zinc (Zn) is an important microelement required by skin cells for a variety of biological processes. The role of Zn in melanocyte proliferation and homeostasis has to date not been investigated. Human dermal melanocytes were isolated from patients and their proliferative activity determined along with both total and labile Zn content. Subsequently, changes in proliferation as well as in Zn content were determined upon exposure of the dermal melanocytes to external Zn. Further in-depth analyses were undertaken aimed at measuring the expression of proliferation-related proteins (determined by immunoblotting and densitometry), as well as changes in mitochondrial biogenesis and membrane potential (assessed by fluorescence-based cellometry) along with endolysosomal activity (determined by spectrofluorimetrically-measured elevation in fluorescence of lysosomal-aimed non-fuorescent substrate). Human skin melanocytes accumulate externally added Zn, a process which dose-dependently enhances their injury or proliferative activity. Enhanced proliferation is accompanied by an increased expression of the proteins AKT3, ERK1/2, c-MYC and CYCD. In addition, Zn-enriched melanocytes exhibit enhanced mitochondrial biogenesis, with individual mitochondria possessing stabilized mitochondrial membrane potential as well as showing elevated ATP and superoxide levels. Moreover, upon external exposure, Zn enters lysosomes/melanosomes, the activity of which is stimulated along with the process of autophagy. The determination of the unique Zn-dependent stimulation of melanocytes and in particular the enhancement of the cells' mitochondrial as well as lysosomal/melanosomal activities may prove important in tracing the sequence of steps in the process of melanomagenesis. © 2017 The Author(s). Published by S. Karger AG, Basel.

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.

Piracetam improves mitochondrial dysfunction following oxidative stress

PubMed Central

Keil, Uta; Scherping, Isabel; Hauptmann, Susanne; Schuessel, Katin; Eckert, Anne; Müller, Walter E

2005-01-01

Mitochondrial dysfunction including decrease of mitochondrial membrane potential and reduced ATP production represents a common final pathway of many conditions associated with oxidative stress, for example, hypoxia, hypoglycemia, and aging. Since the cognition-improving effects of the standard nootropic piracetam are usually more pronounced under such pathological conditions and young healthy animals usually benefit little by piracetam, the effect of piracetam on mitochondrial dysfunction following oxidative stress was investigated using PC12 cells and dissociated brain cells of animals treated with piracetam. Piracetam treatment at concentrations between 100 and 1000 μM improved mitochondrial membrane potential and ATP production of PC12 cells following oxidative stress induced by sodium nitroprusside (SNP) and serum deprivation. Under conditions of mild serum deprivation, piracetam (500 μM) induced a nearly complete recovery of mitochondrial membrane potential and ATP levels. Piracetam also reduced caspase 9 activity after SNP treatment. Piracetam treatment (100–500 mg kg−1 daily) of mice was also associated with improved mitochondrial function in dissociated brain cells. Significant improvement was mainly seen in aged animals and only less in young animals. Moreover, the same treatment reduced antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in aged mouse brain only, which are elevated as an adaptive response to the increased oxidative stress with aging. In conclusion, therapeutically relevant in vitro and in vivo concentrations of piracetam are able to improve mitochondrial dysfunction associated with oxidative stress and/or aging. Mitochondrial stabilization and protection might be an important mechanism to explain many of piracetam's beneficial effects in elderly patients. PMID:16284628

Analysis of functional domains of rat mitochondrial Fis1, the mitochondrial fission-stimulating protein

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

Jofuku, Akihiro; Ishihara, Naotada; Mihara, Katsuyoshi

2005-07-29

In yeast, mitochondrial-fission is regulated by the cytosolic dynamin-like GTPase (Dnm1p) in conjunction with a peripheral protein, Mdv1p, and a C-tail-anchored outer membrane protein, Fis1p. In mammals, a dynamin-related protein (Drp1) and Fis1 are involved in the mitochondrial-fission reaction as Dnm1 and Fis1 orthologues, respectively. The involvement of other component(s), such as the Mdv1 homologue, and the mechanisms regulating mitochondrial-fission remain unclear. Here, we identified rat Fis1 (rFis1) and analyzed its structure-function relationship. Blue-native-polyacrylamide gel electrophoresis revealed that rFis1 formed a {approx}200-kDa complex in the outer mitochondrial membrane. Its expression in HeLa cells promoted extensive mitochondrial fragmentation, and gene knock-downmore » by RNAi induced extension of the mitochondrial networks. Taking advantage of these properties, we analyzed functional domains of rFis1. These experiments revealed that the N-terminal and C-terminal segments are both essential for oligomeric rFis1 interaction, and the middle TPR-like domains regulate proper oligomer assembly. Any mutations that disturb the proper oligomeric assembly compromise mitochondrial division-stimulating activity of rFis1.« less

Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes.

PubMed

Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa; Peterson, Darrell L; Berrueta, Lisbeth; Salmen, Siham

2016-05-01

Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins.

PubMed

Imai, Kenichiro; Fujita, Naoya; Gromiha, M Michael; Horton, Paul

2011-01-28

The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs) often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs) have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal) for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism.

Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

PubMed Central

2011-01-01

Background The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs) often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs) have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal) for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. Results We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Conclusions Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism. PMID:21272379

Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets.

PubMed

Choo, Hyo-Jung; Kholmukhamedov, Andaleb; Zhou, ChengZing; Jobe, Shawn

2017-08-01

Phosphatidylserine exposure mediates platelet procoagulant function and regulates platelet life span. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet phosphatidylserine exposure. Here, we investigate (1) the role of mitochondrial events in platelet phosphatidylserine exposure initiated by these distinct stimuli and (2) the cellular interactions of the procoagulant platelet in vitro and in vivo. Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets, phosphatidylserine externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D-dependent IMM disruption that coincided with phosphatidylserine exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with phosphatidylserine exposure. A nonmitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel phosphatidylserine-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet phosphatidylserine externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus. Platelet phosphatidylserine externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in phosphatidylserine-externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the

Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation.

PubMed

Davila, M Plaza; Muñoz, P Martin; Bolaños, J M Gallardo; Stout, T A E; Gadella, B M; Tapia, J A; da Silva, C Balao; Ferrusola, C Ortega; Peña, F J

2016-12-01

To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also significantly reduced mitochondrial membrane potential. Sperm motility almost completely ceased after the inhibition of mitochondrial respiration and both percentage of motile sperm and sperm velocity were reduced in the presence of mitochondrial uncouplers. Inhibition of ATP synthesis resulted in the loss of sperm membrane integrity and increased the production of reactive oxygen species by degenerating sperm. Inhibition of glycolysis by deoxyglucose led to reduced sperm velocities and reduced ATP content, but not to loss of membrane integrity. These results suggest that, in contrast to many other mammalian species, stallion spermatozoa rely primarily on oxidative phosphorylation to generate the energy required for instance to maintain a functional Na + /K + gradient, which is dependent on an Na + -K + antiporter ATPase, which relates directly to the noted membrane integrity loss. Under aerobic conditions, however, glycolysis also provides the energy required for sperm motility. © 2016 Society for Reproduction and Fertility.

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

Bacterial Origin of a Mitochondrial Outer Membrane Protein Translocase

PubMed Central

Harsman, Anke; Niemann, Moritz; Pusnik, Mascha; Schmidt, Oliver; Burmann, Björn M.; Hiller, Sebastian; Meisinger, Chris; Schneider, André; Wagner, Richard

2012-01-01

Mitochondria are of bacterial ancestry and have to import most of their proteins from the cytosol. This process is mediated by Tom40, an essential protein that forms the protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic translocase of the outer mitochondrial membrane (ATOM), a protein that shows similarities to both eukaryotic Tom40 and bacterial protein translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other protein translocases reveals that ATOM closely resembles bacterial-type protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes. PMID:22778261

The acylphloroglucinols hyperforin and myrtucommulone A cause mitochondrial dysfunctions in leukemic cells by direct interference with mitochondria.

PubMed

Wiechmann, Katja; Müller, Hans; Fischer, Dagmar; Jauch, Johann; Werz, Oliver

2015-11-01

The acylphloroglucinols hyperforin (Hypf) and myrtucommulone A (MC A) induce death of cancer cells by triggering the intrinsic/mitochondrial pathway of apoptosis, accompanied by a loss of the mitochondrial membrane potential and release of cytochrome c. However, the upstream targets and mechanisms leading to these mitochondrial events in cancer cells remain elusive. Here we show that Hypf and MC A directly act on mitochondria derived from human leukemic HL-60 cells and thus, disrupt mitochondrial functions. In isolated mitochondria, Hypf and MC A efficiently impaired mitochondrial viability (EC50 = 0.2 and 0.9 µM, respectively), caused loss of the mitochondrial membrane potential (at 0.03 and 0.1 µM, respectively), and suppressed mitochondrial ATP synthesis (IC50 = 0.2 and 0.5 µM, respectively). Consequently, the compounds activated the adenosine monophosphate-activated protein kinase (AMPK) in HL-60 cells, a cellular energy sensor involved in apoptosis of cancer cells. Side by side comparison with the protonophore CCCP and the ATP synthase inhibitor oligomycin suggest that Hypf and MC A act as protonophores that primarily dissipate the mitochondrial membrane potential by direct interaction with the mitochondrial membrane. Together, Hypf and MC A abolish the mitochondrial proton motive force that on one hand impairs mitochondrial viability and on the other cause activation of AMPK due to lowered ATP levels which may further facilitate the intrinsic mitochondrial pathway of apoptosis.

Soluble guanylate cyclase activation during ischemic injury in mice protects against postischemic inflammation at the mitochondrial level.

PubMed

Wang, Derek Z; Jones, Allan W; Wang, Walter Z; Wang, Meifang; Korthuis, Ronald J

2016-05-01

The aim was to determine whether treatment with BAY 60-2770, a selective activator of oxidized soluble guanylate cyclase (sGC), near the end of an ischemic event would prevent postischemic inflammation and mitochondrial dysfunction in wild-type (WT) and heme oxygenase-1 KO (HO-1(-/-)) mice. This protocol prevented increases in leukocyte rolling (LR) and adhesion (LA) to intestinal venules along with elevated TNFα and circulating neutrophil levels that accompany ischemia-reperfusion (I/R) in both animal models. We further hypothesized that a component of BAY 60-2770 treatment involves maintenance of mitochondrial membrane integrity during I/R. Measurements on isolated enterocytes of calcein fluorescence (mitochondrial permeability) and JC-1 fluorescence ratio (mitochondrial membrane potential) were reduced by I/R, indicating formation of mitochondrial permeability transition pores (mPTP). These effects were abrogated by BAY 60-2770 as well as cyclosporin A and SB-216763, which prevented mPTP opening and inhibited glycogen synthase kinase-3β (GSK-3β), respectively. Western blots of WT and HO-1(-/-) enterocytes indicated that GSK-3β phosphorylation on Ser(9) (inhibitory site) was reduced by half following I/R alone (increased GSK-3β activity) and increased by one-third (reduced GSK-3β activity) following BAY 60-2770. Other investigators have associated phosphorylation of the GSK-3β substrate cyclophilin D (pCyPD) with mPTP formation. We observed a 60% increase in pCyPD after I/R, whereas BAY 60-2770 treatment of sham and I/R groups reduced pCyPD by about 20%. In conclusion, selective activation of oxidized sGC of WT and HO-1(-/-) during ischemia protects against I/R-induced inflammation and preserves mucosal integrity in part by reducing pCyPD production and mPTP formation. Copyright © 2016 the American Physiological Society.

Phosphorylation of Mitochondrial Polyubiquitin by PINK1 Promotes Parkin Mitochondrial Tethering

PubMed Central

Shiba-Fukushima, Kahori; Arano, Taku; Matsumoto, Gen; Inoshita, Tsuyoshi; Yoshida, Shigeharu; Ishihama, Yasushi; Ryu, Kwon-Yul; Nukina, Nobuyuki; Hattori, Nobutaka; Imai, Yuzuru

2014-01-01

The kinase PINK1 and the E3 ubiquitin (Ub) ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin's ubiquitin-like (UBl) domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR) domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved. PMID:25474007

Substrate-dependent changes in mitochondrial function, intracellular free calcium concentration and membrane channels in pancreatic beta-cells.

PubMed

Duchen, M R; Smith, P A; Ashcroft, F M

1993-08-15

Microfluorimetric and patch-clamp techniques have been combined to determine the relationship between changes in mitochondrial metabolism, the activity of KATP channels and changes in intracellular free calcium concentration ([Ca2+]i) in isolated pancreatic beta-cells in response to glucose, ketoisocaproic acid (KIC) and the electron donor couple tetramethyl p-phenylenediamine (TMPD) and ascorbate. Exposure of cells to 20 mM glucose raised NAD(P)H autofluorescence after a delay of 28 +/- 1 s (mean +/- S.E.M., n = 30). The mitochondrial inner membrane potential, delta psi m (monitored using rhodamine 123 fluorescence), hyperpolarized with a latency of 49 +/- 6 s (n = 17), and the [Ca2+]i rose after 129 +/- 13 s (n = 5). The amplitudes of the metabolic changes were graded appropriately with glucose concentration over the range 2.5-20 mM. All variables responded to KIC with shorter latencies: NAD(P)H autofluorescence rose after a delay of 20 +/- 3 s (n = 5) and rhodamine 123 changed after 21 +/- 3 s (n = 6). The electron donor couple, TMPD with ascorbate, rapidly hyperpolarized delta psi m and raised [Ca2+]i. When [Ca2+]i was raised by sustained exposure to 20 mM glucose, TMPD had no further effect. TMPD also decreased whole-cell KATP currents and depolarized the cell membrane, measured with the perforated patch configuration. These data are consistent with a central role for mitochondrial oxidative phosphorylation in coupling changes in glucose concentration with the secretion of insulin.

CaMKII determines mitochondrial stress responses in heart

PubMed Central

Joiner, Mei-ling A.; Koval, Olha M.; Jingdong, Li; He, B. Julie; Allamargot, Chantal; Gao, Zhan; Luczak, Elizabeth D.; Hall, Duane D.; Fink, Brian D.; Chen, Biyi; Yang, Jinying; Moore, Steven A.; Scholz, Thomas D.; Strack, Stefan; Mohler, Peter J.; Sivitz, William I.; Song, Long-Sheng; Anderson, Mark E.

2012-01-01

Myocardial cell death is initiated by excessive mitochondrial Ca2+ entry, causing Ca2+ overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm)1,2. However, the signaling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU)3–5 are not known. The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is activated in ischemia reperfusion (I/R), myocardial infarction (MI) and neurohumoral injury, common causes of myocardial death and heart failure, suggesting CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (IMCU). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A (CsA), an mPTP antagonist with clinical efficacy in I/R injury6, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to I/R injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition are resistant to I/R injury, MI and neurohumoral injury, suggesting pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry and suggest mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure dysfunction in response to common experimental forms of pathophysiological stress. PMID:23051746

Elevated hydrostatic pressures induce apoptosis and oxidative stress through mitochondrial membrane depolarization in PC12 neuronal cells: A cell culture model of glaucoma.

PubMed

Tök, Levent; Nazıroğlu, Mustafa; Uğuz, Abdülhadi Cihangir; Tök, Ozlem

2014-10-01

Despite the importance of oxidative stress and apoptosis through mitochondrial depolarization in neurodegenerative diseases, their roles in etiology of glaucoma are poorly understood. We aimed to investigate whether oxidative stress and apoptosis formation are altered in rat pheochromocytoma-derived cell line-12 (PC12) neuronal cell cultures exposed to elevated different hydrostatic pressures as a cell culture model of glaucoma. Cultured PC12 cells were subjected to 0, 15 and 70 mmHg hydrostatic pressure for 1 and 24 h. Then, the following values were analyzed: (a) cell viability; (b) lipid peroxidation and intracellular reactive oxygen species production; (c) mitochondrial membrane depolarization; (d) cell apoptosis; (e) caspase-3 and caspase-9 activities; (f) reduced glutathione (GSH) and glutathione peroxidase (GSH-Px). The hydrostatic pressures (15 and 70 mmHg) increased oxidative cell damage through a decrease of GSH and GSH-Px values, and increasing mitochondrial membrane potential. Additionally, 70 mmHg hydrostatic pressure for 24 h indicated highest apoptotic effects, as demonstrated by plate reader analyses of apoptosis, caspase-3 and -9 values. The present data indicated oxidative stress, apoptosis and mitochondrial changes in PC12 cell line during different hydrostatic pressure as a cell culture model of glaucoma. This findings support the view that mitochondrial oxidative injury contributes early to glaucomatous optic neuropathy.

Accelerated recovery of mitochondrial membrane potential by GSK-3β inactivation affords cardiomyocytes protection from oxidant-induced necrosis.

PubMed

Sunaga, Daisuke; Tanno, Masaya; Kuno, Atsushi; Ishikawa, Satoko; Ogasawara, Makoto; Yano, Toshiyuki; Miki, Takayuki; Miura, Tetsuji

2014-01-01

Loss of mitochondrial membrane potential (ΔΨm) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨm recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨm contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K+ (mKATP) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨm of H9c2 cells was determined by tetramethylrhodamine ethyl ester (TMRE) before or after 1-h exposure to antimycin A (AA), an inducer of reactive oxygen species (ROS) production at complex III. Opening of the mitochondrial permeability transition pore (mPTP) was determined by mitochondrial loading of calcein. AA reduced ΔΨm to 15 ± 1% of the baseline and induced calcein leak from mitochondria. ΔΨm was recovered to 51 ± 3% of the baseline and calcein-loadable mitochondria was 6 ± 1% of the control at 1 h after washout of AA. mKATP channel openers improved the ΔΨm recovery and mitochondrial calcein to 73 ± 2% and 30 ± 7%, respectively, without change in ΔΨm during AA treatment. Activation of the mKATP channel induced inhibitory phosphorylation of GSK-3β and suppressed ROS production, LDH release and apoptosis after AA washout. Knockdown of GSK-3β and pharmacological inhibition of GSK-3β mimicked the effects of mKATP channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨm. These results indicate that inactivation of GSK-3β directly or indirectly by mKATP channel activation facilitates recovery of ΔΨm by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.

[Effect of DNA polymerase beta on apoptosis and mitochondrial membrane potential induced by hydroquinone, a metabolite of benzene].

PubMed

Chen, Chen; Yang, Mo; Zhang, Zun-zhen; Wu, Mei; Deng, Wen-wen

2011-12-01

To explore the effect and mechanism of DNA polymerase β expression level on cell apoptosis and mitochondrial membrane potential induced by hydroquinone. Polβ wild-type cells (polβ+/+), polβ overexpressed cells (polβ oe) and polβ null cells (polβ-/-) were applied as a model cell system, The effect of cell apoptosis and mitochondrial membrane potential induced by different doses of hydroquinone were analyzed by flow cytometry. The ROS and ·OH assay kit were used to examine the cellular ROS and ·OH level. The activity of cellular SOD and GSH-Px were tested by Chemiluminescence method after exposed to different concentrations of hydroquinone. With the dose of hydroquinone increased, the rate of apoptosis and falling of mitochondrial membrane potential (ΔΨm) in cells were increased compared with the control. When compared with polβ+/+ cells, the rate of apoptosis in polβ-/- cells exposed to 20.00, 40.00, 80.00 µmol/L hydroquinone increased and the rate of apoptosis in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone decreased (P < 0.05). Compared with polβ+/+ cells (20.60% ± 0.57%, 37.95% ± 0.64%, 44.50% ± 1.27%, 57.55% ± 1.06%), the rate of cell which undergone mitochondrial depolarization in polβ-/- cells treated with 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (33.60% ± 1.55%, 46.05% ± 1.77%, 52.75% ± 2.05%, 75.20% ± 0.56%) increased. The rate of cell which undergone mitochondrial depolarization in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (16.05% ± 1.20%, 29.80% ± 1.21%, 35.15% ± 1.06%, 53.80% ± 0.85%) decreased (P < 0.05). When compared with polβ+/+ cells, fluorescent intensity of polβ-/- cells treated with different dosages of hydroquinone increased, while which of polβ oe cells decreased (P < 0.05). Compared with polβ+/+ cells, ·OH level of polβ-/- cells treated with 20.00, 40.00 µmol/L hydroquinone significantly enhanced, while which of polβ oe cells decreased

Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins.

PubMed

Mellgren, Ronald L

2008-04-24

HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside GM1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A-C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein

Mitochondrial antibodies in primary biliary cirrhosis

PubMed Central

Berg, P. A.; Roitt, I. M.; Doniach, D.; Cooper, H. M.

1969-01-01

The effect on the mitochondrial antigen of different agents known to influence the integrity and structure of membranes has been studied using quantitative complement fixation with autoantibodies from the serum of a patient with primary biliary cirrhosis. The susceptibility to proteolytic enzymes suggests that the antigen is a protein. Activity depends upon an association with phospholipids. Addition of phospholipids prevents loss of antigen during artificial ageing of mitochondria at 37°. Activity is lost after treatment with phospholipases or solvents which extract phospholipids. Antigen is also destroyed by surface active agents which dissociate the link with phospholipid but those which weaken bonds between phospholipids and hydrophobic molecules yield fragments of antigen-containing membrane structures which, nonetheless, still react with the mitochondrial autoantibody. ImagesFIG. 2FIG. 4 PMID:5804537

Modulation of mitochondrial activity in HaCaT keratinocytes by the cell penetrating peptide Z-Gly-RGD(DPhe)-mitoparan.

PubMed

Richardson, Adam; Muir, Lewis; Mousdell, Sasha; Sexton, Darren; Jones, Sarah; Howl, John; Ross, Kehinde

2018-01-30

Biologically active cell penetrating peptides (CPPs) are an emerging class of therapeutic agent. The wasp venom peptide mastoparan is an established CPP that modulates mitochondrial activity and triggers caspase-dependent apoptosis in cancer cells, as does the mastoparan analogue mitoparan (mitP). Mitochondrial depolarisation and activation of the caspase cascade also underpins the action of dithranol, a topical agent for treatment of psoriasis. The effects of a potent mitP analogue on mitochondrial activity were therefore examined to assess its potential as a novel approach for targeting mitochondria for the treatment of psoriasis. In HaCaT keratinocytes treated with the mitP analogue Z-Gly-RGD(DPhe)-mitP for 24 h, a dose-dependent loss of mitochondrial activity was observed using the methyl-thiazolyl-tetrazolium (MTT) assay. At 10 μmol L -1 , MTT activity was less than 30% that observed in untreated cells. Staining with the cationic dye JC-1 suggested that Z-Gly-RGD(DPhe)-mitP also dissipated the mitochondrial membrane potential, with a threefold increase in mitochondrial depolarisation levels. However, caspase activity appeared to be reduced by 24 h exposure to Z-Gly-RGD(DPhe)-mitP treatment. Furthermore, Z-Gly-RGD(DPhe)-mitP treatment had little effect on overall cell viability. Our findings suggest Z-Gly-RGD(DPhe)-mitP promotes the loss of mitochondrial activity but does not appear to evoke apoptosis in HaCaT keratinocytes.

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.

The reaction pathway of membrane-bound rat liver mitochondrial monoamine oxidase

PubMed Central

Houslay, Miles D.; Tipton, Keith F.

1973-01-01

1. A preparation of a partly purified mitochondrial outer-membrane fraction suitable for kinetic investigations of monoamine oxidase is described. 2. An apparatus suitable for varying the O2 concentration in a spectrophotometer cuvette is described. 3. The reaction catalysed by the membrane-bound enzyme is shown to proceed by a double-displacement (Ping Pong) mechanism, and a formal mechanism is proposed. 4. KCN, NaN3, benzyl cyanide and 4-cyanophenol are shown to be reversible inhibitors of the enzyme. 5. The non-linear reciprocal plot obtained with impure preparations of benzylamine, which is typical of high substrate inhibition, is shown to be due to aldehyde contamination of the substrate. PMID:4778271

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane.

PubMed

Zhang, Liang; Trushin, Sergey; Christensen, Trace A; Tripathi, Utkarsh; Hong, Courtney; Geroux, Rachel E; Howell, Kyle G; Poduslo, Joseph F; Trushina, Eugenia

2018-06-01

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype. Copyright © 2018 The Authors. Published by

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane

PubMed Central

Zhang, Liang; Trushin, Sergey; Christensen, Trace A.; Tripathi, Utkarsh; Hong, Courtney; Geroux, Rachel E.; Howell, Kyle G.; Poduslo, Joseph F.; Trushina, Eugenia

2018-01-01

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer’s Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype. PMID:29477640

Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition.

PubMed

Petit, P X; Goubern, M; Diolez, P; Susin, S A; Zamzami, N; Kroemer, G

1998-04-10

Upon induction of permeability transition with different agents (Ca2+, tert-butyl hydroperoxide, atractyloside), mouse hepatocyte mitochondria manifest a disruption of outer membrane integrity leading to the release of cytochrome c and apoptosis-inducing factor (AIF), two proteins which are involved in programmed cell death (apoptosis). Chelation of Ca2+ shortly (within 2 min) after its addition to isolated mitochondria reestablished the mitochondrial transmembrane potential (deltapsi(m)), prevented induction of large amplitude swelling and release of both cytochrome c and AIF. In contrast, late Ca2+ chelation (10 min after addition of Ca2+) failed to affect these parameters. Cytochrome c appears to be released through a mechanically damaged outer mitochondrial membrane rather than via a specific release mechanism. These findings clarify the mechanisms through which irreversible permeability transition occurs with subsequent large amplitude swelling culminating in the release of intermembrane proteins from mitochondria. Moreover, they confirm the hypothesis formulated by Skulachev [FEBS Lett. 397 (1996) 7-10 and Q. Rev. Biophys. 29 (1996) 169-2021 linking permeability transition to activation of the apoptogenic catabolic enzymes.

The force exerted by the membrane potential during protein import into the mitochondrial matrix

NASA Technical Reports Server (NTRS)

Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

2004-01-01

The force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated on the basis of continuum electrostatics. The force is found to vary from 3.0 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 6.5 to 12 A, its measured range. In the present model, the decrease in force with increasing pore width arises from the shielding effect of water. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a purely membranous pore without water gives a force of 3.2 pN per unit charge, which should represent an upper limit. When applied to mitochondrial import experiments on the protein barnase, these results imply that forces between 11 +/- 2 pN and 13.5 +/- 2.5 pN catalyze the unfolding of barnase in those experiments. A comparison of these results with unfolding forces measured using atomic force microscopy is made.

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.

Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V.

PubMed

Shimada, Satoru; Maeda, Shintaro; Hikita, Masahide; Mieda-Higa, Kaoru; Uene, Shigefumi; Nariai, Yukiko; Shinzawa-Itoh, Kyoko

2018-04-24

Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46.0% (w/v) or 31.5% (w/v) based on weight. After 0.7% (w/v) deoxycholic acid, 0.4% (w/v) decyl maltoside, and 7.2% (w/v) potassium chloride were added to the mitochondrial membranes, those membranes were solubilized. At a membrane concentration of 46%, complex V was selectively solubilized, whereas at a concentration of 31.5% (w/v), complexes I and III were solubilized. Two steps-sucrose density gradient centrifugation and anion-exchange chromatography on a POROS HQ 20 μm column-enabled selective purification of samples that retained their structure and functions. These two steps enabled complexes I, III, and V to be purified in two days with a high yield. Complexes I, III, and V were stabilized with n-decyl-β-D-maltoside. A total of 200 mg-300 mg of those complexes from one bovine heart (1.1 kg muscle) was purified with good reproducibility, and the complexes retained the same functions they possessed while in mitochondrial membranes. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Ocean acidification impacts on sperm mitochondrial membrane potential bring sperm swimming behaviour near its tipping point.

PubMed

Schlegel, Peter; Binet, Monique T; Havenhand, Jonathan N; Doyle, Christopher J; Williamson, Jane E

2015-04-01

Broadcast spawning marine invertebrates are susceptible to environmental stressors such as climate change, as their reproduction depends on the successful meeting and fertilization of gametes in the water column. Under near-future scenarios of ocean acidification, the swimming behaviour of marine invertebrate sperm is altered. We tested whether this was due to changes in sperm mitochondrial activity by investigating the effects of ocean acidification on sperm metabolism and swimming behaviour in the sea urchin Centrostephanus rodgersii. We used a fluorescent molecular probe (JC-1) and flow cytometry to visualize mitochondrial activity (measured as change in mitochondrial membrane potential, MMP). Sperm MMP was significantly reduced in ΔpH -0.3 (35% reduction) and ΔpH -0.5 (48% reduction) treatments, whereas sperm swimming behaviour was less sensitive with only slight changes (up to 11% decrease) observed overall. There was significant inter-individual variability in responses of sperm swimming behaviour and MMP to acidified seawater. We suggest it is likely that sperm exposed to these changes in pH are close to their tipping point in terms of physiological tolerance to acidity. Importantly, substantial inter-individual variation in responses of sperm swimming to ocean acidification may increase the scope for selection of resilient phenotypes, which, if heritable, could provide a basis for adaptation to future ocean acidification. © 2015. Published by The Company of Biologists Ltd.

The role of mitochondrial fusion and StAR phosphorylation in the regulation of StAR activity and steroidogenesis.

PubMed

Castillo, Ana F; Orlando, Ulises; Helfenberger, Katia E; Poderoso, Cecilia; Podesta, Ernesto J

2015-06-15

The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroidogenesis, i.e. the delivery of cholesterol from the outer (OMM) to the inner (IMM) mitochondrial membrane. StAR is a 37-kDa protein with an N-terminal mitochondrial targeting sequence that is cleaved off during mitochondrial import to yield 30-kDa intramitochondrial StAR. StAR acts exclusively on the OMM and its activity is proportional to how long it remains on the OMM. However, the precise fashion and the molecular mechanism in which StAR remains on the OMM have not been elucidated yet. In this work we will discuss the role of mitochondrial fusion and StAR phosphorylation by the extracellular signal-regulated kinases 1/2 (ERK1/2) as part of the mechanism that regulates StAR retention on the OMM and activity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Lowered iPLA2γ activity causes increased mitochondrial lipid peroxidation and mitochondrial dysfunction in a rotenone-induced model of Parkinson's disease.

PubMed

Chao, Honglu; Liu, Yinlong; Fu, Xian; Xu, Xiupeng; Bao, Zhongyuan; Lin, Chao; Li, Zheng; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

2018-02-01

iPLA 2 γ, calcium-independent phospholipase A 2 γ, discerningly hydrolyses glycerophospholipids to liberate free fatty acids. iPLA 2 γ-deficiency has been associated with abnormal mitochondrial function. More importantly, the iPLA 2 family is causative proteins in mitochondrial neurodegenerative disorders such as parkinsonian disorders. However, the mechanisms by which iPLA 2 γ affects Parkinson's disease (PD) remain unknown. Mitochondrion stress has a key part in rotenone-induced dopaminergic neuronal degeneration. The present evaluation revealed that lowered iPLA 2 γ function provokes the parkinsonian phenotype and leads to the reduction of dopamine and its metabolites, lowered survival, locomotor deficiencies, and organismal hypersensitivity to rotenone-induced oxidative stress. In addition, lowered iPLA 2 γ function escalated the amount of mitochondrial irregularities, including mitochondrial reactive oxygen species (ROS) regeneration, reduced ATP synthesis, reduced glutathione levels, and abnormal mitochondrial morphology. Further, lowered iPLA 2 γ function was tightly linked with strengthened lipid peroxidation and mitochondrial membrane flaws following rotenone treatment, which can cause cytochrome c release and eventually apoptosis. These results confirmed the important role of iPLA 2 γ, whereby decreasing iPLA 2 γ activity aggravates mitochondrial degeneration to induce neurodegenerative disorders in a rotenone rat model of Parkinson's disease. These findings may be useful in the design of rational approaches for the prevention and treatment of PD-associated symptoms. Copyright © 2017 Elsevier Inc. All rights reserved.

Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction.

PubMed

Resseguie, Emily A; Staversky, Rhonda J; Brookes, Paul S; O'Reilly, Michael A

2015-08-01

High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM. Because ATM maintains mitochondrial homeostasis, we hypothesized that hyperoxia induces both mitochondrial dysfunction and ROS that activate ATM. In A549 lung epithelial cells, hyperoxia decreased mitochondrial respiratory reserve capacity at 12h and basal respiration by 48 h. ROS were significantly increased at 24h, yet mitochondrial DNA double strand breaks were not detected. ATM was not required for activating p53 when mitochondrial respiration was inhibited by chronic exposure to antimycin A. Also, ATM was not further activated by mitochondrial ROS, which were enhanced by depleting manganese superoxide dismutase (SOD2). In contrast, ATM dampened the accumulation of mitochondrial ROS during exposure to hyperoxia. Our findings suggest that hyperoxia-induced mitochondrial dysfunction and ROS do not activate ATM. ATM more likely carries out its canonical response to nuclear DNA damage and may function to attenuate mitochondrial ROS that contribute to oxygen toxicity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Three-dimensional organization of the endoplasmic reticulum membrane around the mitochondrial constriction site in mammalian cells revealed by using focused-ion beam tomography.

PubMed

Ohta, Keisuke; Okayama, Satoko; Togo, Akinobu; Nakamura, Kei-Ichiro

2014-11-01

The endoplasmic reticulum (ER) and mitochondria associate at multiple contact sites to form specific domains known as mitochondria-ER associated membranes (MAMs) that play a role in the regulation of various cellular processes such as Ca2+ transfer, autophagy, and inflammation. Recently, it has been suggested that MAMs are also involved in mitochondrial dynamics, especially fission events. Cytological analysis showed that ER tubules were frequently located close to each other in mitochondrial fission sites that accumulate fission-related proteins. Three-dimensional (3D) imaging of ER-mitochondrial contacts in yeast mitochondria by using cryo-electron tomography also showed that ER tubules were attached near the constriction site, which is considered to be a fission site1). MAMs have been suggested to play a role in the initiation of mitochondrial fission, although the molecular relationships between MAMs and the mitochondrial fission process have not been established. Although an ER-mitochondrial membrane association has also been observed at the fission site in mammalian mitochondria, the detailed organization of MAMs around mammalian mitochondria remains to be established. To visualize the 3D distribution of the ER-mitochondrial contacts around the mitochondria, especially around the constriction site in mammalian cells, we attempted 3D structural analysis of the mammalian cytoplasm using high-resolution focused ion-beam scanning electron microscopy (FIB-SEM) tomography, and observed the distribution pattern of ER contacts around the mammalian mitochondrial constriction site.Rat hepatocytes and HeLa cells were used. Liver tissue was obtained from male rats (Wistar, 6W) fixed by transcardial perfusion of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) under deep anesthesia. HeLa cells were fixed with the same fixative. The specimens were then stained en bloc to enhance membrane contrast and embedded in epoxy resin2). The surface of

Mitochondrial-targeted DNA delivery using a DF-MITO-Porter, an innovative nano carrier with cytoplasmic and mitochondrial fusogenic envelopes

NASA Astrophysics Data System (ADS)

Yamada, Yuma; Kawamura, Eriko; Harashima, Hideyoshi

2012-08-01

Mitochondrial gene therapy has the potential for curing a variety of diseases that are associated with mitochondrial DNA mutations and/or defects. To achieve this, it will be necessary to deliver therapeutic agents into the mitochondria in diseased cells. A number of mitochondrial drug delivery systems have been reported to date. However, reports of mitochondrial-targeted DNA delivery are limited. To achieve this, the therapeutic agent must be taken up by the cell (1), after which, the multi-processes associated with intracellular trafficking must be sophisticatedly regulated so as to release the agent from the endosome and deliver it to the cytosol (2) and to pass through the mitochondrial membrane (3). We report herein on the mitochondrial delivery of oligo DNA as a model therapeutic using a Dual Function (DF)-MITO-Porter, an innovative nano carrier designed for mitochondrial delivery. The critical structural elements of the DF-MITO-Porter include mitochondria-fusogenic inner envelopes and endosome-fusogenic outer envelopes, modified with octaarginine which greatly assists in cellular uptake. Inside the cell, the carrier passes through the endosomal and mitochondrial membranes via step-wise membrane fusion. When the oligo DNA was packaged in the DF-MITO-Porter, cellular uptake efficiency was strongly enhanced. Intracellular observation using confocal laser scanning microscopy showed that the DF-MITO-Porter was effectively released from endosomes. Moreover, the findings confirmed that the mitochondrial targeting activity of the DF-MITO-Porter was significantly higher than that of a carrier without outer endosome-fusogenic envelopes. These results support the conclusion that mitochondrial-targeted DNA delivery using a DF-MITO-Porter can be achieved when intracellular trafficking is optimally regulated.

Defense Activated by 9-Lipoxygenase-Derived Oxylipins Requires Specific Mitochondrial Proteins1[W

PubMed Central

Vellosillo, Tamara; Aguilera, Verónica; Marcos, Ruth; Bartsch, Michael; Vicente, Jorge; Cascón, Tomas; Hamberg, Mats; Castresana, Carmen

2013-01-01

9-Lipoxygenases (9-LOXs) initiate fatty acid oxygenation, resulting in the formation of oxylipins activating plant defense against hemibiotrophic pathogenic bacteria. Previous studies using nonresponding to oxylipins (noxy), a series of Arabidopsis (Arabidopsis thaliana) mutants insensitive to the 9-LOX product 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT), have demonstrated the importance of cell wall modifications as a component of 9-LOX-induced defense. Here, we show that a majority (71%) of 41 studied noxy mutants have an added insensitivity to isoxaben, an herbicide inhibiting cellulose synthesis and altering the cell wall. The specific mutants noxy2, noxy15, and noxy38, insensitive to both 9-HOT and isoxaben, displayed enhanced susceptibility to Pseudomonas syringae DC3000 as well as reduced activation of salicylic acid-responding genes. Map-based cloning identified the mutation in noxy2 as At5g11630 encoding an uncharacterized mitochondrial protein, designated NOXY2. Moreover, noxy15 and noxy38 were mapped at the DYNAMIN RELATED PROTEIN3A and FRIENDLY MITOCHONDRIA loci, respectively. Fluorescence microscopy and molecular analyses revealed that the three noxy mutants characterized exhibit mitochondrial dysfunction and that 9-HOT added to wild-type Arabidopsis causes mitochondrial aggregation and loss of mitochondrial membrane potential. The results suggest that the defensive responses and cell wall modifications caused by 9-HOT are under mitochondrial retrograde control and that mitochondria play a fundamental role in innate immunity signaling. PMID:23370715

The Force Exerted by the Membrane Potential During Protein Import into the Mitochondrial Matrix

NASA Technical Reports Server (NTRS)

Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

2002-01-01

The electrostatic force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated and found to vary from 1.4 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 12 to 6.5 Angstroms, its measured range. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a nonaqueous pore gives a force of 3.1 pN per unit charge, which represents an upper limit. When applied to mitochondrial import experiments on the protein harness, these results imply that a force of 11 plus or minus 4 pN is sufficient to catalyze the unfolding of harness during import. Comparison of these results with unfolding forces measured using atomic force microscopy suggests that the two are not inconsistent.

Constriction of the mitochondrial inner compartment is a priming event for mitochondrial division

PubMed Central

Cho, Bongki; Cho, Hyo Min; Jo, Youhwa; Kim, Hee Dae; Song, Myungjae; Moon, Cheil; Kim, Hyongbum; Kim, Kyungjin; Sesaki, Hiromi; Rhyu, Im Joo; Kim, Hyun; Sun, Woong

2017-01-01

Mitochondrial division is critical for the maintenance and regulation of mitochondrial function, quality and distribution. This process is controlled by cytosolic actin-based constriction machinery and dynamin-related protein 1 (Drp1) on mitochondrial outer membrane (OMM). Although mitochondrial physiology, including oxidative phosphorylation, is also important for efficient mitochondrial division, morphological alterations of the mitochondrial inner-membrane (IMM) have not been clearly elucidated. Here we report spontaneous and repetitive constriction of mitochondrial inner compartment (CoMIC) associated with subsequent division in neurons. Although CoMIC is potentiated by inhibition of Drp1 and occurs at the potential division spots contacting the endoplasmic reticulum, it appears on IMM independently of OMM. Intra-mitochondrial influx of Ca2+ induces and potentiates CoMIC, and leads to K+-mediated mitochondrial bulging and depolarization. Synergistically, optic atrophy 1 (Opa1) also regulates CoMIC via controlling Mic60-mediated OMM–IMM tethering. Therefore, we propose that CoMIC is a priming event for efficient mitochondrial division. PMID:28598422

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

The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective Channel That Modulates Membrane Potential.

PubMed

Antonenkov, Vasily D; Isomursu, Antti; Mennerich, Daniela; Vapola, Miia H; Weiher, Hans; Kietzmann, Thomas; Hiltunen, J Kalervo

2015-05-29

The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel's selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17(-/-) mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective Channel That Modulates Membrane Potential*

PubMed Central

Antonenkov, Vasily D.; Isomursu, Antti; Mennerich, Daniela; Vapola, Miia H.; Weiher, Hans; Kietzmann, Thomas; Hiltunen, J. Kalervo

2015-01-01

The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel's selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17−/− mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions. PMID:25861990

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria.

PubMed

Chowdhury, Subir Roy; Djordjevic, Jelena; Albensi, Benedict C; Fernyhough, Paul

2015-12-08

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30-35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances. © 2016 Authors.

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.

Signal presequences increase mitochondrial permeability and open the multiple conductance channel.

PubMed

Kushnareva, Y E; Campo, M L; Kinnally, K W; Sokolove, P M

1999-06-01

We have reported that the signal presequence of cytochrome oxidase subunit IV from Neurospora crassa increases the permeability of isolated rat liver mitochondria [P. M. Sokolove and K. W. Kinnally (1996) Arch. Biochem. Biophys. 336, 69] and regulates the behavior of the mutiple conductance channel (MCC) of yeast inner mitochondrial membrane [T. A. Lohret and K. W. Kinnally (1995) J. Biol. Chem. 270, 15950]. Here we examine in greater detail the action of a number of mitochondrial presequences from various sources and of several control peptides on the permeability of isolated rat liver mitochondria and on MCC activity monitored via patch-clamp techniques in both mammalian mitoplasts and a reconstituted yeast system. The data indicate that the ability to alter mitochondrial permeability is a property of most, but not all, signal peptides. Furthermore, it is clear that, although signal peptides are characterized by positive charge and the ability to form amphiphilic alpha helices, these two characteristics are not sufficient to guarantee mitochondrial effects. Finally, the results reveal a strong correlation between peptide effects on the permeability of isolated mitochondria and on MCC activity: peptides that induced swelling of mouse and rat mitochondria also activated the quiescent MCC of mouse mitoplasts and induced flickering of active MCC reconstituted from yeast mitochondrial membranes. Moreover, relative peptide efficacies were very similar for mitochondrial swelling and both types of patch-clamp experiments. We propose that patch-clamp recordings of MCC activity and the high-amplitude swelling induced by signal peptides reflect the opening of a single channel. Based on the selective responsiveness of that channel to signal peptides and the dependence of its opening in isolated mitochondria on membrane potential, we further suggest that the channel is involved in the mitochondrial protein import process. Copyright 1999 Academic Press.

Involvement of mitochondrial Na+–Ca2+ exchange in intestinal pacemaking activity

PubMed Central

Kim, Byung Joo; Jun, Jae Yeoul; So, Insuk; Kim, Ki Whan

2006-01-01

AIM: Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. We have aimed to investigate the involvement of mitochondrial Na+-Ca2+ exchange in intestinal pacemaking activity in cultured interstitial cells of Cajal. METHODS: Enzymatic digestions were used to dissociate ICCs from the small intestine of a mouse. The whole-cell patch-clamp configuration was used to record membrane currents (voltage clamp) and potentials (current clamp) from cultured ICCs. RESULTS: Clonazepam and CGP37157 inhibited the pacemaking activity of ICCs in a dose-dependent manner. Clonazepam from 20 to 60 µmol/L and CGP37157 from 10 to 30 µmol/L effectively inhibited Ca2+ efflux from mitochondria in pacemaking activity of ICCs. The IC50s of clonazepam and CGP37157 were 37.1 and 18.2 µmol/L, respectively. The addition of 20 µmol/L NiCl2 to the internal solution caused a “wax and wane” phenomenon of pacemaking activity of ICCs. CONCLUSION: These results suggest that mitochondrial Na+-Ca2+ exchange has an important role in intestinal pacemaking activity. PMID:16521198

Crystallization of Mitochondrial Respiratory Complex II from Chicken Heart: a Membrane Protein Complex Diffracting to 2.0 Å.

PubMed Central

Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward

2006-01-01

Synopsis A multi-subunit mitochondrial membrane protein complex involved in the Krebs Cycle and respiratory chain has been crystallized in a form suitable for near-atomic resolution structure determination. A procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Å with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites. PMID:15805592

The oncolytic peptide LTX-315 kills cancer cells through Bax/Bak-regulated mitochondrial membrane permeabilization.

PubMed

Zhou, Heng; Forveille, Sabrina; Sauvat, Allan; Sica, Valentina; Izzo, Valentina; Durand, Sylvère; Müller, Kevin; Liu, Peng; Zitvogel, Laurence; Rekdal, Øystein; Kepp, Oliver; Kroemer, Guido

2015-09-29

LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells. Here, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins from the BCL-2 family, BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria (by transfection with Parkin combined with treatment with a protonophore causing mitophagy) were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes.

The oncolytic peptide LTX-315 kills cancer cells through Bax/Bak-regulated mitochondrial membrane permeabilization

PubMed Central

Zhou, Heng; Forveille, Sabrina; Sauvat, Allan; Sica, Valentina; Izzo, Valentina; Durand, Sylvère; Müller, Kevin; Liu, Peng; Zitvogel, Laurence; Rekdal, Øystein; Kepp, Oliver; Kroemer, Guido

2015-01-01

LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells. Here, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins from the BCL-2 family, BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria (by transfection with Parkin combined with treatment with a protonophore causing mitophagy) were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes. PMID:26378049

σ-1 Receptor at the Mitochondrial-Associated Endoplasmic Reticulum Membrane Is Responsible for Mitochondrial Metabolic Regulation

PubMed Central

Marriott, Karla-Sue C.; Prasad, Manoj; Thapliyal, Veena

2012-01-01

The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is a small section of the outer mitochondrial membrane tethered to the ER by lipid and protein filaments. One such MAM protein is the σ-1 receptor, which contributes to multiple signaling pathways. We found that short interfering RNA-mediated knockdown of σ-1 reduced pregnenolone synthesis by 95% without affecting expression of the inner mitochondrial membrane resident enzyme, 3-β-hydroxysteroid dehydrogenase 2. To explore the underlying mechanism of this effect, we generated a series of σ-receptor ligands: 5,6-dimethoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamide (KSCM-1), 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamide (KSCM-5), and 6-methoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl) propyl)benzofuran-2-carboxamide (KSCM-11) specifically bound to σ-1 in the nanomolar range, whereas KSCM-5 and KSCM-11 also bound to σ-2. Treatment of cells with the KSCM ligands led to decreased cell viability, with KSCM-5 having the most potent effect followed by KSCM-11. KSCM-1 increased σ-1 expression by 4-fold and progesterone synthesis, whereas the other compounds decreased progesterone synthesis. These differences probably are caused by ligand molecular structure. For example, KSCM-1 has two methoxy substituents at C-5 and C-6 of the benzofuran ring, whereas KSCM-11 has one at C-6. KSCM ligands or σ-1 knockdown did not alter the expression of ER resident enzymes that synthesize steroids. However, coimmunoprecipitation of the σ-1 receptor pulled down voltage-dependent anion channel 2 (VDAC2), whose expression was enhanced by KSCM-1. VDAC2 plays a key role in cholesterol transport into the mitochondria, suggesting that the σ-1 receptor at the MAM coordinates with steroidogenic acute regulatory protein for cholesterol trafficking into the mitochondria for metabolic regulation. PMID:22923735

Biogenesis of a Mitochondrial Outer Membrane Protein in Trypanosoma brucei: TARGETING SIGNAL AND DEPENDENCE ON A UNIQUE BIOGENESIS FACTOR.

PubMed

Bruggisser, Julia; Käser, Sandro; Mani, Jan; Schneider, André

2017-02-24

The mitochondrial outer membrane (OM) contains single and multiple membrane-spanning proteins that need to contain signals that ensure correct targeting and insertion into the OM. The biogenesis of such proteins has so far essentially only been studied in yeast and related organisms. Here we show that POMP10, an OM protein of the early diverging protozoan Trypanosoma brucei , is signal-anchored. Transgenic cells expressing variants of POMP10 fused to GFP demonstrate that the N-terminal membrane-spanning domain flanked by a few positively charged or neutral residues is both necessary and sufficient for mitochondrial targeting. Carbonate extraction experiments indicate that although the presence of neutral instead of positively charged residues did not interfere with POMP10 localization, it weakened its interaction with the OM. Expression of GFP-tagged POMP10 in inducible RNAi cell lines shows that its mitochondrial localization depends on pATOM36 but does not require Sam50 or ATOM40, the trypanosomal analogue of the Tom40 import pore. pATOM36 is a kinetoplastid-specific OM protein that has previously been implicated in the assembly of OM proteins and in mitochondrial DNA inheritance. In summary, our results show that although the features of the targeting signal in signal-anchored proteins are widely conserved, the protein machinery that mediates their biogenesis is not. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial bioenergetics decay in aging: beneficial effect of melatonin.

PubMed

Paradies, Giuseppe; Paradies, Valeria; Ruggiero, Francesca M; Petrosillo, Giuseppe

2017-11-01

Aging is a biological process characterized by progressive decline in physiological functions, increased oxidative stress, reduced capacity to respond to stresses, and increased risk of contracting age-associated disorders. Mitochondria are referred to as the powerhouse of the cell through their role in the oxidative phosphorylation to generate ATP. These organelles contribute to the aging process, mainly through impairment of electron transport chain activity, opening of the mitochondrial permeability transition pore and increased oxidative stress. These events lead to damage to proteins, lipids and mitochondrial DNA. Cardiolipin, a phospholipid of the inner mitochondrial membrane, plays a pivotal role in several mitochondrial bioenergetic processes as well as in mitochondrial-dependent steps of apoptosis and in mitochondrial membrane stability and dynamics. Cardiolipin alterations are associated with mitochondrial bienergetics decline in multiple tissues in a variety of physiopathological conditions, as well as in the aging process. Melatonin, the major product of the pineal gland, is considered an effective protector of mitochondrial bioenergetic function. Melatonin preserves mitochondrial function by preventing cardiolipin oxidation and this may explain, at least in part, the protective role of this compound in mitochondrial physiopathology and aging. Here, mechanisms through which melatonin exerts its protective role against mitochondrial dysfunction associated with aging and age-associated disorders are discussed.

Characterization of hydrophobic interaction and antioxidant properties of the phenothiazine nucleus in mitochondrial and model membranes.

PubMed

Borges, Marcelo B D; Dos Santos, Carolina G; Yokomizo, César H; Sood, Rohit; Vitovic, Pavol; Kinnunen, Paavo K J; Rodrigues, Tiago; Nantes, Iseli L

2010-09-01

The antioxidant properties of the phenothiazine nucleus (PHT) associated with mitochondrial membranes and liposomes were investigated. PHT exhibited hydrophobic interaction with lipid bilayers, as shown by the quenching of excited states of 1-palmitoyl-2[10-pyran-1-yl)]-decanoyl-sn-glycero-3-phophocholine (PPDPC) incorporated in phosphatidylcholine/phosphatidylethanolamine/cardiolipin liposomes, observed even in high ionic strength; and by the spectral changes of PHT following the addition of mitochondrial membranes. Inserted into bilayers, 5 microM PHT was able to protect lipids and cytochrome c against pro-oxidant agents and exhibited spectral changes suggestive of oxidative modifications promoted by the trapping of the reactive species. In this regard, PHT exhibited the ability to scavenge DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical. PHT was also able to protect rat liver mitochondria against peroxide- and iron-induced oxidative damage and consequent swelling. At the concentration range in which the antioxidant properties were observed, PHT did not cause alterations in the membrane structure and function. This study contributes to the comprehension of the correlation structure and function of phenothiazines and antioxidant properties.

Mitochondrial shaping cuts.

PubMed

Escobar-Henriques, Mafalda; Langer, Thomas

2006-01-01

A broad range of cellular processes are regulated by proteolytic events. Proteolysis has now also been established to control mitochondrial morphology which results from the balanced action of fusion and fission. Two out of three known core components of the mitochondrial fusion machinery are under proteolytic control. The GTPase Fzo1 in the outer membrane of mitochondria is degraded along two independent proteolytic pathways. One controls mitochondrial fusion in vegetatively growing cells, the other one acts upon mating factor-induced cell cycle arrest. Fusion also depends on proteolytic processing of the GTPase Mgm1 by the rhomboid protease Pcp1 in the inner membrane of mitochondria. Functional links of AAA proteases or other proteolytic components to mitochondrial dynamics are just emerging. This review summarises the current understanding of regulatory roles of proteolytic processes for mitochondrial plasticity.

Valproate Attenuates Nitroglycerin-Induced Trigeminovascular Activation by Preserving Mitochondrial Function in a Rat Model of Migraine

PubMed Central

Li, Ruxian; Liu, Yushuang; Chen, Nan; Zhang, Yitong; Song, Ge; Zhang, Zhongling

2016-01-01

Background Migraine is a chronic disease that interferes with life quality and work productivity. Valproate shows protective effects against migraine, yet the underlying mechanisms are unclear. This study aimed to evaluate the potential effect of valproate on migraine using a rat model of nitroglycerin-induced trigeminovascular activation, as well as to explore the underlying mechanism. Material/Methods Intraperitoneal injection of nitroglycerin was conducted to induce trigeminovascular activation in rats. To explore the protective effect of valproate, a low dose (100 mg/kg) or a high dose (200 mg/kg) of valproate was intraperitoneally injected into rats, and then the levels of 5-hydroxytryptamine and nitric oxide in the peripheral blood were examined. The mtDNA copy number and the protein levels of peroxisome proliferator-activated receptor-γ coactivator 1α, mitochondrial transcription factor A, and peroxisome proliferator-activated receptor-γ in the spinal trigeminal nucleus were detected to evaluate the biogenesis of mitochondria. The mitochondrial energy metabolism was determined by the mitochondrial membrane potential and the levels of adenosine triphosphate, cytochrome C oxidase, and reactive oxygen species. Results Valproate attenuated nitroglycerin-induced trigeminovascular activation in rats, with reduced scratching behavior and restored 5-hydroxytryptamine and nitric oxide levels. Moreover, the mitochondrial energy metabolism and the biogenesis of mitochondria were preserved by valproate in nitroglycerin-treated rats. Conclusions The protective effect of valproate against migraine may be achieved through the modulation of mitochondrial biogenesis and function. Our study provides evidence for the potential use of valproate in the treatment of migraine. PMID:27618395

Valproate Attenuates Nitroglycerin-Induced Trigeminovascular Activation by Preserving Mitochondrial Function in a Rat Model of Migraine.

PubMed

Li, Ruxian; Liu, Yushuang; Chen, Nan; Zhang, Yitong; Song, Ge; Zhang, Zhongling

2016-09-12

BACKGROUND Migraine is a chronic disease that interferes with life quality and work productivity. Valproate shows protective effects against migraine, yet the underlying mechanisms are unclear. This study aimed to evaluate the potential effect of valproate on migraine using a rat model of nitroglycerin-induced trigeminovascular activation, as well as to explore the underlying mechanism. MATERIAL AND METHODS Intraperitoneal injection of nitroglycerin was conducted to induce trigeminovascular activation in rats. To explore the protective effect of valproate, a low dose (100 mg/kg) or a high dose (200 mg/kg) of valproate was intraperitoneally injected into rats, and then the levels of 5-hydroxytryptamine and nitric oxide in the peripheral blood were examined. The mtDNA copy number and the protein levels of peroxisome proliferator-activated receptor-γ coactivator 1α, mitochondrial transcription factor A, and peroxisome proliferator-activated receptor-γ in the spinal trigeminal nucleus were detected to evaluate the biogenesis of mitochondria. The mitochondrial energy metabolism was determined by the mitochondrial membrane potential and the levels of adenosine triphosphate, cytochrome C oxidase, and reactive oxygen species. RESULTS Valproate attenuated nitroglycerin-induced trigeminovascular activation in rats, with reduced scratching behavior and restored 5-hydroxytryptamine and nitric oxide levels. Moreover, the mitochondrial energy metabolism and the biogenesis of mitochondria were preserved by valproate in nitroglycerin-treated rats. CONCLUSIONS The protective effect of valproate against migraine may be achieved through the modulation of mitochondrial biogenesis and function. Our study provides evidence for the potential use of valproate in the treatment of migraine.

PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells

PubMed Central

Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra

2016-01-01

Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2. PMID:27413259

PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells.

PubMed

Miranda, Dante; Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra; Montoya, Margarita

2016-01-01

Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.

Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase.

PubMed

Moreno, S N; Mason, R P; Docampo, R

1984-12-10

At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.

SLP-2 interacts with prohibitins in the mitochondrial inner membrane and contributes to their stability.

PubMed

Da Cruz, Sandrine; Parone, Philippe A; Gonzalo, Philippe; Bienvenut, Willy V; Tondera, Daniel; Jourdain, Alexis; Quadroni, Manfredo; Martinou, Jean-Claude

2008-05-01

Stomatin is a member of a large family of proteins including prohibitins, HflK/C, flotillins, mechanoreceptors and plant defense proteins, that are thought to play a role in protein turnover. Using different proteomic approaches, we and others have identified SLP-2, a member of the stomatin gene family, as a component of the mitochondria. In this study, we show that SLP-2 is strongly associated with the mitochondrial inner membrane and that it interacts with prohibitins. Depleting HeLa cells of SLP-2 lead to increased proteolysis of prohibitins and of subunits of the respiratory chain complexes I and IV. Further supporting the role of SLP-2 in regulating the stability of specific mitochondrial proteins, we found that SLP-2 is up-regulated under conditions of mitochondrial stress leading to increased protein turnover. These data indicate that SLP-2 plays a role in regulating the stability of mitochondrial proteins including prohibitins and subunits of respiratory chain complexes.

Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

PubMed

de Moura, Michelle Barbi; Uppala, Radha; Zhang, Yuxun; Van Houten, Bennett; Goetzman, Eric S

2014-01-01

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

Measurement of mitochondrial Ca2+ transport mediated by three transport proteins: VDAC1, the Na+/Ca2+ exchanger, and the Ca2+ uniporter.

PubMed

Ben-Hail, Danya; Palty, Raz; Shoshan-Barmatz, Varda

2014-02-01

Ca(2+) is a ubiquitous cellular signal, with changes in intracellular Ca(2+) concentration not only stimulating a number of intercellular events but also triggering cell death pathways, including apoptosis. Mitochondrial Ca(2+) uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca(2+) signaling, energy metabolism and cell death. Ca(2+) transport across the inner and outer mitochondrial membranes is mediated by several proteins, including channels, antiporters, and a uniporter. In this article, we present the background to several methods now established for assaying mitochondrial Ca(2+) transport activity across both mitochondrial membranes. The first of these is Ca(2+) transport mediated by the outer mitochondrial protein, the voltage-dependent anion-selective channel protein 1 (VDAC1, also known as porin 1), both as a purified protein reconstituted into a planar lipid bilayer (PLB) or into liposomes and as a mitochondrial membrane-embedded protein. The second method involves isolated mitochondria for assaying the activity of an inner mitochondrial membrane transport protein, the mitochondrial Ca(2+) uniporter (MCU) that transports Ca(2+) and is powered by the steep mitochondrial membrane potential. In the event of Ca(2+) overload, this leads to opening of the mitochondrial permeability transition pore (MPTP) and cell death. The third method describes how Na(+)-dependent mitochondrial Ca(2+) efflux mediated by mitochondrial NCLX, a member of the Na(+)/Ca(2+) exchanger superfamily, can be assayed in digitonin-permeabilized HEK-293 cells. The Ca(2+)-transport assays can be performed under various conditions and in combination with inhibitors, allowing detailed characterization of the transport activity of interest.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

PubMed Central

Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Houlden, Henry

2017-01-01

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. PMID:28360103

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

PubMed

Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

2017-05-26

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

SIRT1 activation inhibits hyperglycemia-induced apoptosis by reducing oxidative stress and mitochondrial dysfunction in human endothelial cells.

PubMed

Wang, Shengqiang; Wang, Jian; Zhao, Airong; Li, Jigang

2017-09-01

Sustained hyperglycemic stimulation of vascular cells is involved in the pathogenesis of diabetes mellitus‑induced cardiovascular complications. Silent information regulator T1 (SIRT1), a mammalian sirtuin, has been previously recognized to protect endothelial cells against hyperglycemia‑induced oxidative stress. In the present study, human umbilical vein endothelial cells (HUV‑EC‑C) were treated with D‑glucose, and the levels of oxidative stress, mitochondrial dysfunction, the rate of apoptosis and SIRT1 activity were measured. The effect of manipulated SIRT1 activity on hyperglycemia‑induced oxidative stress, mitochondrial dysfunction and apoptosis was then assessed using the SIRT1 activator, resveratrol (RSV), and the SIRT1 inhibitor, sirtinol. The present study confirmed that hyperglycemia promotes oxidative stress and mitochondrial dysfunction in HUV‑EC‑C cells. The accumulation of reactive oxygen species, the swelling of mitochondria, the ratio of adenosine 5'‑diphosphate to adenosine 5'‑triphosphate and localized mitochondrial superoxide levels were all increased following D‑glucose treatment, whereas the mitochondrial membrane potential was significantly reduced by >50 mg/ml D‑glucose treatment. In addition, hyperglycemia was confirmed to induce apoptosis in HUV‑EC‑C cells. Furthermore, the results confirmed the prevention and aggravation of hyperglycemia‑induced apoptosis by RSV treatment and sirtinol treatment, via the amelioration and enhancement of oxidative stress and mitochondrial dysfunction in HUV‑EC‑C cells, respectively. In conclusion, the present study revealed that hyperglycemia promotes oxidative stress, mitochondrial dysfunction and apoptosis in HUV‑EC‑C cells, and manipulation of SIRT1 activity regulated hyperglycemia‑induced mitochondrial dysfunction and apoptosis in HUV‑EC‑C cells. The data revealed the protective effect of SIRT1 against hyperglycemia‑induced apoptosis via the alleviation of

Lipid-Loving ANTs: Molecular Simulations of Cardiolipin Interactions and the Organization of the Adenine Nucleotide Translocase in Model Mitochondrial Membranes

PubMed Central

2016-01-01

The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large patches of a model mitochondrial membrane containing multiple copies of the translocase shows that CL interactions persist in the presence of protein–protein interactions and suggests CL may mediate interactions between translocases. This study provides a key example of how computational microscopy may be used to shed light on regulatory lipid–protein interactions. PMID:27786441

Lipid-Loving ANTs: Molecular Simulations of Cardiolipin Interactions and the Organization of the Adenine Nucleotide Translocase in Model Mitochondrial Membranes.

PubMed

Hedger, George; Rouse, Sarah L; Domański, Jan; Chavent, Matthieu; Koldsø, Heidi; Sansom, Mark S P

2016-11-15

The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large patches of a model mitochondrial membrane containing multiple copies of the translocase shows that CL interactions persist in the presence of protein-protein interactions and suggests CL may mediate interactions between translocases. This study provides a key example of how computational microscopy may be used to shed light on regulatory lipid-protein interactions.

Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

PubMed

Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

2015-06-15

High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Thermal acclimation in American alligators: Effects of temperature regime on growth rate, mitochondrial function, and membrane composition.

PubMed

Price, Edwin R; Sirsat, Tushar S; Sirsat, Sarah K G; Kang, Gurdeep; Keereetaweep, Jantana; Aziz, Mina; Chapman, Kent D; Dzialowski, Edward M

2017-08-01

We investigated the ability of juvenile American alligators (Alligator mississippiensis) to acclimate to temperature with respect to growth rate. We hypothesized that alligators would acclimate to cold temperature by increasing the metabolic capacity of skeletal muscles and the heart. Additionally, we hypothesized that lipid membranes in the thigh muscle and liver would respond to low temperature, either to maintain fluidity (via increased unsaturation) or to maintain enzyme reaction rates (via increased docosahexaenoic acid). Alligators were assigned to one of 3 temperature regimes beginning at 9 mo of age: constant warm (30°C), constant cold (20°C), and daily cycling for 12h at each temperature. Growth rate over the following 7 mo was highest in the cycling group, which we suggest occurred via high digestive function or feeding activity during warm periods and energy-saving during cold periods. The warm group also grew faster than the cold group. Heart and liver masses were proportional to body mass, while kidney was proportionately larger in the cold group compared to the warm animals. Whole-animal metabolic rate was higher in the warm and cycling groups compared to the cold group - even when controlling for body mass - when assayed at 30°C, but not at 20°C. Mitochondrial oxidative phosphorylation capacity in permeabilized fibers of thigh muscle and heart did not differ among treatments. Membrane fatty acid composition of the brain was largely unaffected by temperature treatment, but adjustments were made in the phospholipid headgroup composition that are consistent with homeoviscous adaptation. Thigh muscle cell membranes had elevated polyunsaturated fatty acids in the cold group relative to the cycling group, but this was not the case for thigh muscle mitochondrial membranes. Liver mitochondria from cold alligators had elevated docosahexaenoic acid, which might be important for maintenance of reaction rates of membrane-bound enzymes. Copyright © 2016

Ethanol Influences on Bax Associations with Mitochondrial Membrane Proteins in Neonatal Rat Cerebellum

PubMed Central

Heaton, Marieta Barrow; Siler-Marsiglio, Kendra; Paiva, Michael; Kotler, Alexandra; Rogozinski, Jonathan; Kubovec, Stacey; Coursen, Mary; Madorsky, Vladimir

2012-01-01

These studies investigated interactions taking place at the mitochondrial membrane in neonatal rat cerebellum following ethanol exposure, and focused on interactions between pro-apoptotic Bax and proteins of the permeability transition pore (PTP), voltage-dependent anion channel (VDAC), and adenine nucleotide translocator (ANT), of the outer and inner mitochondrial membranes, respectively. Cultured cerebellar granule cells were used to assess the role of these interactions in ethanol neurotoxicity. Analyses were made at the age of maximal cerebellar ethanol vulnerability (P4), compared to the later age of relative resistance (P7), to determine whether differential ethanol sensitivity was mirrored by differences in these molecular interactions. We found that following ethanol exposure, Bax pro-apoptotic associations with both VDAC and ANT were increased, particularly at the age of greater ethanol sensitivity, and these interactions were sustained at this age for at least two hours post-exposure. Since Bax:VDAC interactions disrupt protective VDAC interactions with mitochondrial hexokinase (HXK), we also assessed VDAC:HXK associations following ethanol treatment, and found such interactions were altered by ethanol treatment, but only at two-hours post-exposure, and only in the P4, ethanol-sensitive cerebellum. Ethanol neurotoxicity in cultured neuronal preparations was abolished by pharmacological inhibition of both VDAC and ANT interactions with Bax, but not by a Bax channel blocker. Therefore, we conclude that at this age, within the constraints of our experimental model, a primary mode of Bax-induced initiation of the apoptosis cascade following ethanol insult involves interactions with proteins of the PTP complex, and not channel formation independent of PTP constituents. PMID:22767450

The actions of mdivi-1, an inhibitor of mitochondrial fission, on rapidly activating delayed-rectifier K⁺ current and membrane potential in HL-1 murine atrial cardiomyocytes.

PubMed

So, Edmund Cheung; Hsing, Chung-Hsi; Liang, Chia-Hua; Wu, Sheng-Nan

2012-05-15

Mdivi-1 is an inhibitor of dynamin related protein 1- (drp1)-mediated mitochondrial fission. However, the mechanisms through which this compound interacts directly with ion currents in heart cells remain unknown. In this study, its effects on ion currents and membrane potential in murine HL-1 cardiomyocytes were investigated. In whole-cell recordings, the addition of mdivi-1 decreased the amplitude of tail current (I(tail)) for the rapidly activating delayed-rectifier K⁺ current (I(Kr)) in a concentration-dependent manner with an IC₅₀ value at 11.6 μM, a value that resembles the inhibition requirement for mitochondrial division. It shifted the activation curve of I(tail) to depolarized voltages with no change in the gating charge. However, mdivi-1 did not alter the rate of recovery from current inactivation. In cell-attached configuration, mdivi-1 inside the pipette suppressed the activity of acetylcholine-activated K⁺ channels without modifying the single-channel conductance. Mdivi-1 (30 μM) slightly depressed the peak amplitude of Na⁺ current with no change in the overall current-voltage relationship. Under current-clamp recordings, addition of mdivi-1 resulted in prolongation for the duration of action potentials (APs) and to increase the firing of spontaneous APs in HL-1 cells. Similarly, in pituitary GH₃ cells, mdivi-1 was effective in directly suppressing the amplitude of ether-à-go-go-related gene-mediated K⁺ current. Therefore, the lengthening of AP duration and increased firing of APs caused by mdivi-1 can be primarily explained by its inhibition of these K⁺ channels enriched in heart cells. The observed effects of mdivi-1 on ion currents were direct and not associated with its inhibition of mitochondrial division. Copyright © 2012 Elsevier B.V. All rights reserved.

The mitochondria targeted antioxidant MitoQ protects against fluoroquinolone-induced oxidative stress and mitochondrial membrane damage in human Achilles tendon cells.

PubMed

Lowes, Damon A; Wallace, Carol; Murphy, Michael P; Webster, Nigel R; Galley, Helen F

2009-04-01

Tendinitis and tendon rupture during treatment with fluoroquinolone antibiotics is thought to be mediated via oxidative stress. This study investigated whether ciprofloxacin and moxifloxacin cause oxidative stress and mitochondrial damage in cultured normal human Achilles' tendon cells and whether an antioxidant targeted to mitochondria (MitoQ) would protect against such damage better than a non-mitochondria targeted antioxidant. Human tendon cells from normal Achilles' tendons were exposed to 0-0.3 mM antibiotic for 24 h and 7 days in the presence of 1 microM MitoQ or an untargeted form, idebenone. Both moxifloxacin and ciprofloxacin resulted in up to a 3-fold increase in the rate of oxidation of dichlorodihydrofluorescein, a marker of general oxidative stress in tenocytes (p<0.0001) and loss of mitochondrial membrane permeability (p<0.001). In cells treated with MitoQ the oxidative stress was less and mitochondrial membrane potential was maintained. Mitochondrial damage to tenocytes during fluoroquinolone treatment may be involved in tendinitis and tendon rupture.

Phenyl-alpha-tert-butyl nitrone reverses mitochondrial decay in acute Chagas' disease.

PubMed

Wen, Jian-Jun; Bhatia, Vandanajay; Popov, Vsevolod L; Garg, Nisha Jain

2006-12-01

In this study, we investigated the mechanism(s) of mitochondrial functional decline in acute Chagas' disease. Our data show a substantial decline in respiratory complex activities (39 to 58%) and ATP (38%) content in Trypanosoma cruzi-infected murine hearts compared with normal controls. These metabolic alterations were associated with an approximately fivefold increase in mitochondrial reactive oxygen species production rate, substantial oxidative insult of mitochondrial membranes and respiratory complex subunits, and >60% inhibition of mtDNA-encoded transcripts for respiratory complex subunits in infected myocardium. The antioxidant phenyl-alpha-tert-butyl nitrone (PBN) arrested the oxidative damage-mediated loss in mitochondrial membrane integrity, preserved redox potential-coupled mitochondrial gene expression, and improved respiratory complex activities (47 to 95% increase) and cardiac ATP level (>or=40% increase) in infected myocardium. Importantly, PBN resulted twofold decline in mitochondrial reactive oxygen species production rate in infected myocardium. Taken together, our data demonstrate the pathological significance of oxidative stress in metabolic decay and energy homeostasis in acute chagasic myocarditis and further suggest that oxidative injuries affecting mitochondrial integrity-dependent expression and activity of the respiratory complexes initiate a feedback cycle of electron transport chain inefficiency, increased reactive oxygen species production, and energy homeostasis in acute chagasic hearts. PBN and other mitochondria-targeted antioxidants may be useful in altering mitochondrial decay and oxidative pathology in Chagas' disease.

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.

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.

A novel motif in the yeast mitochondrial dynamin Dnm1 is essential for adaptor binding and membrane recruitment

PubMed Central

Bui, Huyen T.; Karren, Mary A.; Bhar, Debjani

2012-01-01

To initiate mitochondrial fission, dynamin-related proteins (DRPs) must bind specific adaptors on the outer mitochondrial membrane. The structural features underlying this interaction are poorly understood. Using yeast as a model, we show that the Insert B domain of the Dnm1 guanosine triphosphatase (a DRP) contains a novel motif required for association with the mitochondrial adaptor Mdv1. Mutation of this conserved motif specifically disrupted Dnm1–Mdv1 interactions, blocking Dnm1 recruitment and mitochondrial fission. Suppressor mutations in Mdv1 that restored Dnm1–Mdv1 interactions and fission identified potential protein-binding interfaces on the Mdv1 β-propeller domain. These results define the first known function for Insert B in DRP–adaptor interactions. Based on the variability of Insert B sequences and adaptor proteins, we propose that Insert B domains and mitochondrial adaptors have coevolved to meet the unique requirements for mitochondrial fission of different organisms. PMID:23148233

Thioredoxin-2 Inhibits Mitochondrial ROS Generation and ASK1 Activity to Maintain Cardiac Function

PubMed Central

Huang, Qunhua; Zhou, Huanjiao Jenny; Zhang, Haifeng; Huang, Yan; Hinojosa-Kirschenbaum, Ford; Fan, Peidong; Yao, Lina; Belardinelli, Luiz; Tellides, George; Giordano, Frank J.; Budas, Grant R.; Min, Wang

2015-01-01

Background Thioredoxin 2 (Trx2) is a key mitochondrial protein which regulates cellular redox and survival by suppressing mitochondrial ROS generation and by inhibiting apoptosis stress kinase-1 (ASK1)-dependent apoptotic signaling. To date, the role of the mitochondrial Trx2 system in heart failure pathogenesis has not been investigated. Methods and Results Western blot and histological analysis revealed that Trx2 protein expression levels were reduced in hearts from patients with dilated cardiomyopathy (DCM), with a concomitant increase in increased ASK1 phosphorylation/activity. Cardiac-specific Trx2 knockout mice (Trx2-cKO). Trx2-cKO mice develop spontaneous DCM at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in left ventricular (LV) contractile function, resulting in mortality due to heart failure by ~4 months of age. The progressive decline in cardiac function observed in Trx2-cKO mice was accompanied by disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial ROS generation and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective ASK1 inhibitor improved cardiac phenotype and reduced maladaptive LV remodeling with significant reductions in oxidative stress, apoptosis, fibrosis and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial ROS generation. Conclusions Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial ROS production and ASK1-dependent apoptosis. Inhibition of ASK1 represents a promising therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure. PMID:25628390

A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c

PubMed Central

Mootha, Vamsi K.; Wei, Michael C.; Buttle, Karolyn F.; Scorrano, Luca; Panoutsakopoulou, Vily; Mannella, Carmen A.; Korsmeyer, Stanley J.

2001-01-01

Multiple apoptotic pathways release cytochrome c from the mitochondrial intermembrane space, resulting in the activation of downstream caspases. In vivo activation of Fas (CD95) resulted in increased permeability of the mitochondrial outer membrane and depletion of cytochrome c stores. Serial measurements of oxygen consumption, NADH redox state and membrane potential revealed a loss of respiratory state transitions. This tBID-induced respiratory failure did not require any caspase activity. At early time points, re-addition of exogenous cytochrome c markedly restored respiratory functions. Over time, however, mitochondria showed increasing irreversible respiratory dysfunction as well as diminished calcium buffering. Electron microscopy and tomographic reconstruction revealed asymmetric mitochondria with blebs of herniated matrix, distended inner membrane and partial loss of cristae structure. Thus, apoptogenic redistribution of cytochrome c is responsible for a distinct program of mitochondrial respiratory dysfunction, in addition to the activation of downstream caspases. PMID:11179211

SB203580 enhances the RV-induced loss of mitochondrial membrane potential and apoptosis in A549 cells

NASA Astrophysics Data System (ADS)

Li, Hai-yang; Zhuang, Cai-ping; Wang, Xiao-ping; Chen, Tong-sheng

2012-03-01

Resveratrol (RV), a naturally occurring phytoalexin, is known to possess a wide spectrum of chemopreventive and chemotherapeutic effects in various stages of human tumors. p38, a member of the mitogen-activated protein kinase (MAPK) superfamily, is always activated by some extracellular stimulus to regulate many cellular signal transduction pathways, such as apoptosis, proliferation, and inflammation and so on. In this report, we assessed the effect of SB203580, a specific inhibitor of p38 MAPK signaling pathway, on the RV-induced apoptosis in human lung adenocarcinoma (A549) cells. CCK-8 assay showed that pretreatment with SB203580 significantly enhanced the cytotoxicity of RV, which was further verified by analyzing the phosphatidylserine externalization using flow cytometry. In order to further confirm whether SB203580 accelerated apoptosis via the intrinsic apoptosis pathway, we analyzed the dysfunction of mitochondrial membrane potential (Δψm) of cells stained with rhodamine 123 by using flow cytometry after treatment with RV in the absence and presence of SB203580. Our data for the first time reported that p38 inhibitor SB203580 enhanced the RV-induced apoptosis via a mitochondrial pathway.

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

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins.

PubMed

Sanjuán Szklarz, Luiza K; Kozjak-Pavlovic, Vera; Vögtle, F-Nora; Chacinska, Agnieszka; Milenkovic, Dusanka; Vogel, Sandra; Dürr, Mark; Westermann, Benedikt; Guiard, Bernard; Martinou, Jean-Claude; Borner, Christoph; Pfanner, Nikolaus; Meisinger, Chris

2007-04-20

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.

Active Fragments from Pro- and Antiapoptotic BCL-2 Proteins Have Distinct Membrane Behavior Reflecting Their Functional Divergence

PubMed Central

Guillemin, Yannis; Lopez, Jonathan; Gimenez, Diana; Fuertes, Gustavo; Valero, Juan Garcia; Blum, Loïc; Gonzalo, Philippe; Salgado, Jesùs; Girard-Egrot, Agnès; Aouacheria, Abdel

2010-01-01

Background The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction. Methodology/Principal Findings Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death. Conclusion/Significance BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains. PMID:20140092

Millimeter wave treatment induces apoptosis via activation of the mitochondrial-dependent pathway in human osteosarcoma cells.

PubMed

Wu, Guangwen; Chen, Xuzheng; Peng, Jun; Cai, Qiaoyan; Ye, Jinxia; Xu, Huifeng; Zheng, Chunsong; Li, Xihai; Ye, Hongzhi; Liu, Xianxiang

2012-05-01

Millimeter wave (MW) is an electromagnetic wave with a wavelength between 1 and 10 mm and a frequency of 30-300 GHz that causes multiple biological effects and has been used as a major component in physiotherapies for the clinical treatment of various types of diseases including cancers. However, the precise molecular mechanism of the anticancer activity of millimeter wave remains to be elucidated. In the present study, we investigated the cellular effects of the MW in the U-2OS human osteosarcoma cell line. Our results showed that MW induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner suggesting that MW inhibited the growth of U-2OS cells as demonstrated. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, MW treatment caused loss of plasma membrane asymmetry, release of cytochrome c, collapse of mitochondrial membrane potential, activation of caspase-9 and -3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of MW was due to mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of millimeter wave treatment.

RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

PubMed Central

Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

2015-01-01

We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

Beneficial effect of the bioflavonoid quercetin on cholecystokinin-induced mitochondrial dysfunction in isolated rat pancreatic acinar cells.

PubMed

Weber, Heike; Jonas, Ludwig; Wakileh, Michael; Krüger, Burkhard

2014-03-01

The pathogenesis of acute pancreatitis (AP) is still poorly understood. Thus, a reliable pharmacological therapy is currently lacking. In recent years, an impairment of the energy metabolism of pancreatic acinar cells, caused by Ca(2+)-mediated depolarization of the inner mitochondrial membrane and a decreased ATP supply, has been implicated as an important pathological event. In this study, we investigated whether quercetin exerts protection against mitochondrial dysfunction. Following treatment with or without quercetin, rat pancreatic acinar cells were stimulated with supramaximal cholecystokinin-8 (CCK). CCK caused a decrease in the mitochondrial membrane potential (MMP) and ATP concentration, whereas the mitochondrial dehydrogenase activity was significantly increased. Quercetin treatment before CCK application exerted no protection on MMP but increased ATP to a normal level, leading to a continuous decrease in the dehydrogenase activity. The protective effect of quercetin on mitochondrial function was accompanied by a reduction in CCK-induced changes to the cell membrane. Concerning the molecular mechanism underlying the protective effect of quercetin, an increased AMP/ATP ratio suggests that the AMP-activated protein kinase system may be activated. In addition, quercetin strongly inhibited CCK-induced trypsin activity. The results indicate that the use of quercetin may be a therapeutic strategy for reducing the severity of AP.

tRNAs and proteins use the same import channel for translocation across the mitochondrial outer membrane of trypanosomes.

PubMed

Niemann, Moritz; Harsman, Anke; Mani, Jan; Peikert, Christian D; Oeljeklaus, Silke; Warscheid, Bettina; Wagner, Richard; Schneider, André

2017-09-12

Mitochondrial tRNA import is widespread, but the mechanism by which tRNAs are imported remains largely unknown. The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes, and thus imports all tRNAs from the cytosol. Here we show that in T. brucei in vivo import of tRNAs requires four subunits of the mitochondrial outer membrane protein translocase but not the two receptor subunits, one of which is essential for protein import. The latter shows that it is possible to uncouple mitochondrial tRNA import from protein import. Ablation of the intermembrane space domain of the translocase subunit, archaic translocase of the outer membrane (ATOM)14, on the other hand, while not affecting the architecture of the translocase, impedes both protein and tRNA import. A protein import intermediate arrested in the translocation channel prevents both protein and tRNA import. In the presence of tRNA, blocking events of single-channel currents through the pore formed by recombinant ATOM40 were detected in electrophysiological recordings. These results indicate that both types of macromolecules use the same import channel across the outer membrane. However, while tRNA import depends on the core subunits of the protein import translocase, it does not require the protein import receptors, indicating that the two processes are not mechanistically linked.

Overexpression of PGC-1α increases peroxisomal activity and mitochondrial fatty acid oxidation in human primary myotubes.

PubMed

Huang, Tai-Yu; Zheng, Donghai; Houmard, Joseph A; Brault, Jeffrey J; Hickner, Robert C; Cortright, Ronald N

2017-04-01

Peroxisomes are indispensable organelles for lipid metabolism in humans, and their biogenesis has been assumed to be under regulation by peroxisome proliferator-activated receptors (PPARs). However, recent studies in hepatocytes suggest that the mitochondrial proliferator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) also acts as an upstream transcriptional regulator for enhancing peroxisomal abundance and associated activity. It is unknown whether the regulatory mechanism(s) for enhancing peroxisomal function is through the same node as mitochondrial biogenesis in human skeletal muscle (HSkM) and whether fatty acid oxidation (FAO) is affected. Primary myotubes from vastus lateralis biopsies from lean donors (BMI = 24.0 ± 0.6 kg/m 2 ; n = 6) were exposed to adenovirus encoding human PGC-1α or GFP control. Peroxisomal biogenesis proteins (peroxins) and genes ( PEXs ) responsible for proliferation and functions were assessed by Western blotting and real-time qRT-PCR, respectively. [1- 14 C]palmitic acid and [1- 14 C]lignoceric acid (exclusive peroxisomal-specific substrate) were used to assess mitochondrial oxidation of peroxisomal-derived metabolites. After overexpression of PGC-1α, 1 ) peroxisomal membrane protein 70 kDa (PMP70), PEX19, and mitochondrial citrate synthetase protein content were significantly elevated ( P < 0.05), 2 ) PGC-1α , PMP70 , key PEXs , and peroxisomal β-oxidation mRNA expression levels were significantly upregulated ( P < 0.05), and 3 ) a concomitant increase in lignoceric acid oxidation by both peroxisomal and mitochondrial activity was observed ( P < 0.05). These novel findings demonstrate that, in addition to the proliferative effect on mitochondria, PGC-1α can induce peroxisomal activity and accompanying elevations in long-chain and very-long-chain fatty acid oxidation by a peroxisomal-mitochondrial functional cooperation, as observed in HSkM cells. Copyright © 2017 the American Physiological Society.

TAT-MTS-MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM-deficient cells.

PubMed

Erlich-Hadad, Tal; Hadad, Rita; Feldman, Anat; Greif, Hagar; Lictenstein, Michal; Lorberboum-Galski, Haya

2018-03-01

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

CED-9 and mitochondrial homeostasis in C. elegans muscle

PubMed Central

Tan, Frederick J.; Husain, Michelle; Manlandro, Cara Marie; Koppenol, Marijke; Fire, Andrew Z.; Hill, R. Blake

2009-01-01

Summary Mitochondrial homeostasis reflects a dynamic balance between membrane fission and fusion events thought essential for mitochondrial function. We report here that altered expression of the C. elegans BCL2 homolog CED-9 affects both mitochondrial fission and fusion. Although striated muscle cells lacking CED-9 have no alteration in mitochondrial size or ultrastructure, these cells appear more sensitive to mitochondrial fragmentation. By contrast, increased CED-9 expression in these cells produces highly interconnected mitochondria. This mitochondrial phenotype is partially suppressed by increased expression of the dynamin-related GTPase DRP-1, with suppression dependent on the BH3 binding pocket of CED-9. This suppression suggests that CED-9 directly regulates DRP-1, a model supported by our finding that CED-9 activates the GTPase activity of human DRP1. Thus, CED-9 is capable of regulating the mitochondrial fission-fusion cycle but is not essential for either fission or fusion. PMID:18827010

Inhibition of free radical scavenging enzymes affects mitochondrial membrane permeability transition during growth and aging of yeast cells.

PubMed

Deryabina, Yulia; Isakova, Elena; Sekova, Varvara; Antipov, Alexey; Saris, Nils-Erik L

2014-12-01

In this study, we investigated the change in the antioxidant enzymes activity, cell respiration, reactive oxygen species (ROS), and impairment of membrane mitochondria permeability in the Endomyces magnusii yeasts during culture growth and aging. We showed that the transition into stationary phase is the key tool to understanding interaction of these processes. This growth stage is distinguished by two-fold increase in ROS production and respiration rate as compared to those in the logarithmic phase. It results in induction of alternative oxidase (AO) in the stationary phase, decline of the main antioxidant enzymes activities, ROS-production, and mitochondria membrane permeability. Significant increase in the share of mitochondrial isoform of superoxide dismutase (SOD2) occurred in the stationary phase from 51.8% (24 h of cultivation) to 68.6% (48 h of cultivation). Upon blocking the essential ROS-scavenging enzymes, SODs and catalases (CATs) some heterogeneity of cell population was observed: 80-90% of cells displayed evident signs of early apoptosis (such as disorientation of mitochondria cristae, mitochondrial fragmentation and deformation of nuclear chromatine). However, 10-20% of the population were definitely healthy. It allowed to draw the conclusion that a complete system of cell antioxidant protection underlies normal mitochondria functioning while the E. magnusii yeasts grow and age. Moreover, this system provides unimpaired cell physiology under oxidative stress during culture aging in the stationary phase. Failures in mitochondria functions due to inhibition of ROS-scavenging enzymes of CATs and SODs could lead to damage of the cells and some signs of early apoptosis.

Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes

PubMed Central

Dimmer, Kai S

2018-01-01

Assembly and/or insertion of a subset of mitochondrial outer membrane (MOM) proteins, including subunits of the main MOM translocase, require the fungi-specific Mim1/Mim2 complex. So far it was unclear which proteins accomplish this task in other eukaryotes. Here, we show by reciprocal complementation that the MOM protein pATOM36 of trypanosomes is a functional analogue of yeast Mim1/Mim2 complex, even though these proteins show neither sequence nor topological similarity. Expression of pATOM36 rescues almost all growth, mitochondrial biogenesis, and morphology defects in yeast cells lacking Mim1 and/or Mim2. Conversely, co-expression of Mim1 and Mim2 restores the assembly and/or insertion defects of MOM proteins in trypanosomes ablated for pATOM36. Mim1/Mim2 and pATOM36 form native-like complexes when heterologously expressed, indicating that additional proteins are not part of these structures. Our findings indicate that Mim1/Mim2 and pATOM36 are the products of convergent evolution and arose only after the ancestors of fungi and trypanosomatids diverged. PMID:29923829

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.

Agmatine effects on mitochondrial membrane potential and NF-κB activation protect against rotenone-induced cell damage in human neuronal-like SH-SY5Y cells.

PubMed

Condello, Salvatore; Currò, Monica; Ferlazzo, Nadia; Caccamo, Daniela; Satriano, Joseph; Ientile, Riccardo

2011-01-01

Agmatine, an endogenous arginine metabolite, has been proposed as a novel neuromodulator that plays protective roles in the CNS in several models of cellular damage. However, the mechanisms involved in these protective effects in neurodegenerative diseases are poorly understood. The present study was undertaken to investigate the effects of agmatine on cell injury induced by rotenone, commonly used in establishing in vivo and in vitro models of Parkinson's disease, in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). We report that agmatine dose-dependently suppressed rotenone-induced cellular injury through a reduction of oxidative stress. Similar effects were obtained by spermine, suggesting a scavenging effect for these compounds. However, unlike spermine, agmatine also prevented rotenone-induced nuclear factor-κB nuclear translocation and mitochondrial membrane potential dissipation. Furthermore, rotenone-induced increase in apoptotic markers, such as caspase 3 activity, Bax expression and cytochrome c release, was significantly attenuated with agmatine treatment. These findings demonstrate mitochondrial preservation with agmatine in a rotenone model of apoptotic cell death, and that the neuroprotective action of agmatine appears because of suppressing apoptotic signalling mechanisms. Thus, agmatine may have therapeutic potential in the treatment of Parkinson's disease by protecting dopaminergic neurons.

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.

Lost region in amyloid precursor protein (APP) through TALEN-mediated genome editing alters mitochondrial morphology.

PubMed

Wang, Yajie; Wu, Fengyi; Pan, Haining; Zheng, Wenzhong; Feng, Chi; Wang, Yunfu; Deng, Zixin; Wang, Lianrong; Luo, Jie; Chen, Shi

2016-02-29

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) deposition in the brain. Aβ plaques are produced through sequential β/γ cleavage of amyloid precursor protein (APP), of which there are three main APP isoforms: APP695, APP751 and APP770. KPI-APPs (APP751 and APP770) are known to be elevated in AD, but the reason remains unclear. Transcription activator-like (TAL) effector nucleases (TALENs) induce mutations with high efficiency at specific genomic loci, and it is thus possible to knock out specific regions using TALENs. In this study, we designed and expressed TALENs specific for the C-terminus of APP in HeLa cells, in which KPI-APPs are predominantly expressed. The KPI-APP mutants lack a 12-aa region that encompasses a 5-aa trans-membrane (TM) region and 7-aa juxta-membrane (JM) region. The mutated KPI-APPs exhibited decreased mitochondrial localization. In addition, mitochondrial morphology was altered, resulting in an increase in spherical mitochondria in the mutant cells through the disruption of the balance between fission and fusion. Mitochondrial dysfunction, including decreased ATP levels, disrupted mitochondrial membrane potential, increased ROS generation and impaired mitochondrial dehydrogenase activity, was also found. These results suggest that specific regions of KPI-APPs are important for mitochondrial localization and function.

Mangiferin protects mitochondrial function by preserving mitochondrial hexokinase-II in vessel endothelial cells.

PubMed

Song, Junna; Li, Yi; Song, Junmei; Hou, Fangjie; Liu, Baolin; Li, Aiying

2017-07-01

Hexokinase-II (HK-II) confers protection against cell death and this study was designed to investigate the effect of mangiferin on the regulation of mitochondrial HK-II. In vessel endothelial cells, saturated fatty acid palmitate (PA) stimulation induced HK-II detachment from mitochondria due to cellular acidification. Mangiferin reduced lactate accumulation by improving pyruvate dehydrogenase activity, promoted Akt translocation to HK-II and prevented HK-II detachment from mitochondria. Knockdown of Akt2 diminished the protective effect of mangiferin on mitochondrial HK-II, confirming the role of Akt in the regulation of HK-II. Mangiferin prevented mitochondrial permeability transition pore opening, restored mitochondrial membrane potential and thereby protected cell from apoptosis. In high-fat diet fed mice, oral administration of mangiferin induced Akt phosphorylation, increased HK-II binding to mitochondria and resultantly protected vessel endothelial function, demonstrating its protective effect on endothelial integrity in vivo. This finding provided a novel strategy for the protection of mitochondrial function in the endothelium. Copyright © 2017 Elsevier B.V. All rights reserved.

Activation of G protein‐coupled oestrogen receptor 1 at the onset of reperfusion protects the myocardium against ischemia/reperfusion injury by reducing mitochondrial dysfunction and mitophagy

PubMed Central

Feng, Yansheng; Madungwe, Ngonidzashe B; da Cruz Junho, Carolina Victoria

2017-01-01

Background and Purpose Recent evidence indicates that GPER (G protein‐coupled oestrogen receptor 1) mediates acute pre‐ischaemic oestrogen‐induced protection of the myocardium from ischaemia/reperfusion injury via a signalling cascade that includes PKC translocation, ERK1/2/ GSK‐3β phosphorylation and inhibition of the mitochondrial permeability transition pore (mPTP) opening. Here, we investigated the impact and mechanism involved in post‐ischaemic GPER activation in ischaemia/reperfusion injury. We determined whether GPER activation at the onset of reperfusion confers cardioprotective effects by protecting against mitochondrial impairment and mitophagy. Experimental Approach In vivo rat hearts were subjected to ischaemia followed by reperfusion with oestrogen (17β‐oestradiol, E2), E2 + G15, a GPER antagonist, or vehicle. Myocardial infarct size, the threshold for the opening of mPTP, mitophagy, mitochondrial membrane potential, ROS production, proteins ubiquitinated including cyclophilin D, and phosphorylation levels of ERK and GSK‐3β were measured. Results We found that post‐ischaemic E2 administration to both male and female ovariectomized‐rats reduced myocardial infarct size. Post‐ischaemic E2 administration preserved mitochondrial structural integrity and this was associated with a decrease in ROS production and increased mitochondrial membrane potential, as well as an increase in the mitochondrial Ca2+ load required to induce mPTP opening via activation of the MEK/ERK/GSK‐3β axis. Moreover, E2 reduced mitophagy via the PINK1/Parkin pathway involving LC3I, LC3II and p62 proteins. All these post‐ischaemic effects of E2 were abolished by G15 suggesting a GPER‐dependent mechanism. Conclusion These results indicate that post‐ischaemic GPER activation induces cardioprotective effects against ischaemia/reperfusion injury in males and females by protecting mitochondrial structural integrity and function and reducing mitophagy. PMID

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.

Multitiered and Cooperative Surveillance of Mitochondrial Phosphatidylserine Decarboxylase 1.

PubMed

Ogunbona, Oluwaseun B; Onguka, Ouma; Calzada, Elizabeth; Claypool, Steven M

2017-09-01

Phosphatidylserine decarboxylase 1 (Psd1p), an ancient enzyme that converts phosphatidylserine to phosphatidylethanolamine in the inner mitochondrial membrane, must undergo an autocatalytic self-processing event to gain activity. Autocatalysis severs the protein into a large membrane-anchored β subunit that noncovalently associates with the small α subunit on the intermembrane space side of the inner membrane. Here, we determined that a temperature sensitive ( ts ) PSD1 allele is autocatalytically impaired and that its fidelity is closely monitored throughout its life cycle by multiple mitochondrial quality control proteases. Interestingly, the proteases involved in resolving misfolded Psd1 ts vary depending on its autocatalytic status. Specifically, the degradation of a Psd1 ts precursor unable to undergo autocatalysis requires the unprecedented cooperative and sequential actions of two inner membrane proteases, Oma1p and Yme1p. In contrast, upon heat exposure postautocatalysis, Psd1 ts β subunits accumulate in protein aggregates that are resolved by Yme1p acting alone, while the released α subunit is degraded in parallel by an unidentified protease. Importantly, the stability of endogenous Psd1p is also influenced by Yme1p. We conclude that Psd1p, the key enzyme required for the mitochondrial pathway of phosphatidylethanolamine production, is closely monitored at several levels and by multiple mitochondrial quality control mechanisms present in the intermembrane space. Copyright © 2017 American Society for Microbiology.

Caspase-8-mediated intracellular acidification precedes mitochondrial dysfunction in somatostatin-induced apoptosis.

PubMed

Liu, D; Martino, G; Thangaraju, M; Sharma, M; Halwani, F; Shen, S H; Patel, Y C; Srikant, C B

2000-03-31

Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH(i). (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH(i) is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.

Mitochondrial Protein Synthesis, Import, and Assembly

PubMed Central

Fox, Thomas D.

2012-01-01

The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

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.

Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

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

Marchissio, Maria Julia; Francés, Daniel Eleazar Antonio; Carnovale, Cristina Ester

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H{sub 2}O{sub 2} across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H{sub 2}O{sub 2} release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H{sub 2}O{sub 2} release, assessedmore » by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H{sub 2}O{sub 2} release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death. -- Highlights: ► Aquaporin-8 is expressed in mitochondria of human hepatoma HepG2 cells. ► Aquaporin-8 knockdown impairs mitochondrial H{sub 2}O{sub 2} release and increases ROS. �

Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology.

PubMed

Rubino, L; Di Franco, A; Russo, M

2000-01-01

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.

Identification of a glycoprotein from rat liver mitochondrial inner membrane and demonstration of its origin in the endoplasmic reticulum.

PubMed

Chandra, N C; Spiro, M J; Spiro, R G

1998-07-31

Employing antisera against various subfractions of rat liver mitochondria (mitoplast, inner membrane, intermembrane, and matrix) as well as metabolically radiolabeled BRL-3A rat liver cells, we undertook a search for the presence of glycoproteins in this major cellular compartment for which little information in regard to glycoconjugates was available. Subsequent to [35S]methionine labeling of BRL-3A cells, a peptide:N-glycosidase-sensitive protein (45 kDa) was observed by SDS-polyacrylamide gel electrophoresis of the inner membrane immunoprecipitate, which was reduced to a molecular mass of 42 kDa by this enzyme. The 45-kDa protein was readily labeled with [2-3H]mannose, and indeed the radioactivity of the inner membrane immunoprecipitate was almost exclusively present in this component. Moreover, antisera directed against mitochondrial NADH-ubiquinone oxidoreductase (complex I) or F1F0-ATPase (complex V) also precipitated a 45-kDa protein from BRL-3A cell lysates as the predominant mannose-radiolabeled constituent. Endo-beta-N-acetylglucosaminidase completely removed the radiolabel from this glycoprotein, and the released oligosaccharides were of the partially trimmed polymannose type (Glc1Man9GlcNAc to Man8GlcNAc). Cycloheximide as well as tunicamycin resulted in total inhibition of radiolabeling of the inner membrane glycoprotein, and moreover, pulse-chase studies employing metrizamide density gradient centrifugation demonstrated that the glycoprotein was initially present in the endoplasmic reticulum (ER) and subsequently appeared in a mitochondrial location. Early movement of the glycoprotein to the mitochondria after synthesis in the ER was also evident from the limited processing undergone by its N-linked oligosaccharides; this stood in contrast to lysosomal glycoproteins in which we noted extensive conversion to complex oligosaccharides. Our findings suggest that the 45-kDa glycoprotein migrates from ER to mitochondria by the previously observed contact

Zinc-dependent multi-conductance channel activity in mitochondria isolated from ischemic brain.

PubMed

Bonanni, Laura; Chachar, Mushtaque; Jover-Mengual, Teresa; Li, Hongmei; Jones, Adrienne; Yokota, Hidenori; Ofengeim, Dimitry; Flannery, Richard J; Miyawaki, Takahiro; Cho, Chang-Hoon; Polster, Brian M; Pypaert, Marc; Hardwick, J Marie; Sensi, Stefano L; Zukin, R Suzanne; Jonas, Elizabeth A

2006-06-21

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early post-ischemic period is enhanced permeability of mitochondrial membranes. The precise mechanisms by which mitochondrial function is disrupted are, as yet, unclear. Here we show that global ischemia promotes alterations in mitochondrial membrane contact points, a rise in intramitochondrial Zn2+, and activation of large, multi-conductance channels in mitochondrial outer membranes by 1 h after insult. Mitochondrial channel activity was associated with enhanced protease activity and proteolytic cleavage of BCL-xL to generate its pro-death counterpart, deltaN-BCL-xL. The findings implicate deltaN-BCL-xL in large, multi-conductance channel activity. Consistent with this, large channel activity was mimicked by introduction of recombinant deltaN-BCL-xL to control mitochondria and blocked by introduction of a functional BCL-xL antibody to post-ischemic mitochondria via the patch pipette. Channel activity was also inhibited by nicotinamide adenine dinucleotide, indicative of a role for the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. In vivo administration of the membrane-impermeant Zn2+ chelator CaEDTA before ischemia or in vitro application of the membrane-permeant Zn2+ chelator tetrakis-(2-pyridylmethyl) ethylenediamine attenuated channel activity, suggesting a requirement for Zn2+. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate deltaN-BCL-xL and VDAC in the large, Zn2+-dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria.

Zinc-Dependent Multi-Conductance Channel Activity in Mitochondria Isolated from Ischemic Brain

PubMed Central

Bonanni, Laura; Chachar, Mushtaque; Jover-Mengual, Teresa; Li, Hongmei; Jones, Adrienne; Yokota, Hidenori; Ofengeim, Dimitry; Flannery, Richard J.; Miyawaki, Takahiro; Cho, Chang-Hoon; Polster, Brian M.; Pypaert, Marc; Hardwick, J. Marie; Sensi, Stefano L.; Zukin, R. Suzanne; Jonas, Elizabeth A.

2015-01-01

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early post-ischemic period is enhanced permeability of mitochondrial membranes. The precise mechanisms by which mitochondrial function is disrupted are, as yet, unclear.Here we show that global ischemia promotes alterations in mitochondrial membrane contact points, a rise in intramitochondrial Zn2+, and activation of large, multi-conductance channels in mitochondrial outer membranes by 1 h after insult. Mitochondrial channel activity was associated with enhanced protease activity and proteolytic cleavage of BCL-xL to generate its pro-death counterpart, ΔN-BCL-xL. The findings implicate ΔN-BCL-xL in large, multi-conductance channel activity. Consistent with this, large channel activity was mimicked by introduction of recombinant ΔN-BCL-xL to control mitochondria and blocked by introduction of a functional BCL-xL antibody to post-ischemic mitochondria via the patch pipette. Channel activity was also inhibited by nicotinamide adenine dinucleotide, indicative of a role for the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. In vivo administration of the membrane-impermeant Zn2+ chelator CaEDTA before ischemia or in vitro application of the membrane-permeant Zn2+ chelator tetrakis-(2-pyridylmethyl) ethylenediamine attenuated channel activity, suggesting a requirement for Zn2+. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate ΔNBCL-xL and VDAC in the large, Zn2+-dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria. PMID:16793892

Citral exerts its antifungal activity against Penicillium digitatum by affecting the mitochondrial morphology and function.

PubMed

Zheng, Shiju; Jing, Guoxing; Wang, Xiao; Ouyang, Qiuli; Jia, Lei; Tao, Nengguo

2015-07-01

This work investigated the effect of citral on the mitochondrial morphology and function of Penicillium digitatum. Citral at concentrations of 2.0 or 4.0 μL/mL strongly damaged mitochondria of test pathogen by causing the loss of matrix and increase of irregular mitochondria. The deformation extent of the mitochondria of P. digitatum enhanced with increasing concentrations of citral, as evidenced by a decrease in intracellular ATP content and an increase in extracellular ATP content of P. digitatum cells. Oxygen consumption showed that citral resulted in an inhibition in the tricarboxylic acid cycle (TCA) pathway of P. digitatum cells, induced a decrease in activities of citrate synthetase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinodehydrogenase and the content of citric acid, while enhancing the activity of malic dehydrogenase in P. digitatum cells. Our present results indicated that citral could damage the mitochondrial membrane permeability and disrupt the TCA pathway of P. digitatum. Copyright © 2015 Elsevier Ltd. All rights reserved.

How Membrane-Active Peptides Get into Lipid Membranes.

PubMed

Sani, Marc-Antoine; Separovic, Frances

2016-06-21

The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

Oxidized Mitochondrial DNA Activates the NLRP3 Inflammasome During Apoptosis

PubMed Central

Shimada, Kenichi; Crother, Timothy R.; Karlin, Justin; Dagvadorj, Jargalsaikhan; Chiba, Norika; Chen, Shuang; Ramanujan, V. Krishnan; Wolf, Andrea J.; Vergnes, Laurent; Ojcius, David M.; Rentsendorj, Altan; Vargas, Mario; Guerrero, Candace; Wang, Yinsheng; Fitzgerald, Katherine A.; Underhill, David M.; Town, Terrence; Arditi, Moshe

2012-01-01

SUMMARY We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1β (IL-1β). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The anti-apoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1β production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1β secretion could be competitively inhibited by the oxidized nucleoside, 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome. PMID:22342844

Common effects of lithium and valproate on mitochondrial functions: protection against methamphetamine-induced mitochondrial damage.

PubMed

Bachmann, Rosilla F; Wang, Yun; Yuan, Peixiong; Zhou, Rulun; Li, Xiaoxia; Alesci, Salvatore; Du, Jing; Manji, Husseini K

2009-07-01

Accumulating evidence suggests that mitochondrial dysfunction plays a critical role in the progression of a variety of neurodegenerative and psychiatric disorders. Thus, enhancing mitochondrial function could potentially help ameliorate the impairments of neural plasticity and cellular resilience associated with a variety of neuropsychiatric disorders. A series of studies was undertaken to investigate the effects of mood stabilizers on mitochondrial function, and against mitochondrially mediated neurotoxicity. We found that long-term treatment with lithium and valproate (VPA) enhanced cell respiration rate. Furthermore, chronic treatment with lithium or VPA enhanced mitochondrial function as determined by mitochondrial membrane potential, and mitochondrial oxidation in SH-SY5Y cells. In-vivo studies showed that long-term treatment with lithium or VPA protected against methamphetamine (Meth)-induced toxicity at the mitochondrial level. Furthermore, these agents prevented the Meth-induced reduction of mitochondrial cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and mitochondrial cytochrome oxidase (COX) activity. Oligoarray analysis demonstrated that the gene expression of several proteins related to the apoptotic pathway and mitochondrial functions were altered by Meth, and these changes were attenuated by treatment with lithium or VPA. One of the genes, Bcl-2, is a common target for lithium and VPA. Knock-down of Bcl-2 with specific Bcl-2 siRNA reduced the lithium- and VPA-induced increases in mitochondrial oxidation. These findings illustrate that lithium and VPA enhance mitochondrial function and protect against mitochondrially mediated toxicity. These agents may have potential clinical utility in the treatment of other diseases associated with impaired mitochondrial function, such as neurodegenerative diseases and schizophrenia.

Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

PubMed Central

Ito, Hiroyuki; Uchida, Tokujiro; Makita, Koshi

2015-01-01

Purpose Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated. Methods Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD+ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy. Results Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P < 0.01), 14% increase in ROS production (P < 0.01), and 81% reduction in mitochondrial membrane potential (P < 0.01), compared with untreated cells. Lower concentration of ketamine (100 μM) decreased the ATP level (22%, P < 0.01) and increased the NADH/NAD+ ratio (46%, P < 0.05) without caspase activation. Transmission electron microscopy showed enhanced mitochondrial fission and autophagocytosis at the 100 μM ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation. Conclusions We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons. PMID:26020236

Ethanol Influences on Bax Translocation, Mitochondrial Membrane Potential, and Reactive Oxygen Species Generation are Modulated by Vitamin E and Brain-Derived Neurotrophic Factor

PubMed Central

Heaton, Marieta Barrow; Paiva, Michael; Siler-Marsiglio, Kendra

2011-01-01

Background This study investigated ethanol influences on intracellular events which predispose developing neurons toward apoptosis, and the capacity of the antioxidant α-tocopherol (vitamin E) and the neurotrophin brain-derived neurotrophic factor (BDNF) to modulate these effects. Assessments were made of the following: (1) ethanol-induced translocation of the pro-apoptotic Bax protein to the mitochondrial membrane, a key upstream event in the initiation of apoptotic cell death; (2) disruption of the mitochondrial membrane potential (MMP) as a result of ethanol exposure, an important process in triggering the apoptotic cascade; and (3) generation of damaging reactive oxygen species (ROS) as a function of ethanol exposure. Methods These interactions were investigated in cultured postnatal day 8 neonatal rat cerebellar granule cells, a population vulnerable to developmental ethanol exposure in vivo and in vitro. Bax mitochondrial translocation was analyzed via subcellular fractionation followed by Western blot, and mitochondrial membrane integrity was determined using the lipophilic dye, JC-1, which exhibits potential-dependent accumulation in the mitochondrial membrane as a function of the MMP. Results Brief ethanol exposure in these preparations precipitated Bax translocation, but both vitamin E and BDNF reduced this effect to control levels. Ethanol treatment also resulted in a disturbance of the MMP, and this effect was blunted by the antioxidant and the neurotrophin. ROS generation was enhanced by a short ethanol exposure in these cells, but the production of these harmful free radicals was diminished to control levels by co-treatment with either vitamin E or BDNF. Conclusions These results indicate that both antioxidants and neurotrophic factors have the potential to ameliorate ethanol neurotoxicity, and suggest possible interventions which could be implemented in preventing or lessening the severity of the damaging effects of ethanol in the developing central

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.

Soy lecithin interferes with mitochondrial function in frozen-thawed ram spermatozoa.

PubMed

Del Valle, I; Gómez-Durán, A; Holt, W V; Muiño-Blanco, T; Cebrián-Pérez, J A

2012-01-01

Egg yolk and milk are the 2 major membrane cryoprotectants commonly used in freezing media for the long-term preservation of semen (alone or in combination with others). However, in recent years, there have been increasing arguments against the use of egg yolk or milk because of the risk of introducing diseases through the use of cryopreserved semen. In this study, we analyzed the protective effect of lecithin as an alternative to egg yolk for the cryopreservation of ram semen, using a range of functional markers for sperm viability, motility, apoptosis, and mitochondrial functionality analyses (mitochondrial inner membrane surface [MIMS], mitochondrial inner membrane potential [MIMP], and cell membrane potential) as methods of assessment in samples diluted in 3 different media: Tris-citrate-glucose as control and 2 media supplemented with soy lecithin or egg yolk. The results showed that lecithin was able to effectively protect certain sperm quality characteristics against freezing-induced damage. However, lecithin induced loss of mitochondrial membrane potential or mitochondrial loss that was not reflected by modifications in sperm motility in fresh semen. MIMS and MIMP values decreased in thawed lecithin-treated samples, concomitant with a lower (P < .05) percentage of total and progressively motile cells, compared with those in egg yolk-containing samples. Further incubation of thawed samples revealed changes in motility and mitochondrial functionality that otherwise would not have been detected. These results indicated that lecithin may have affected the inner mitochondrial membrane in frozenthawed spermatozoa and confirmed that sublethal damages that seriously affect sperm functionality, not detected by classic sperm quality analyses, can be evidenced by changes in the inner mitochondrial membrane surface. These findings strengthen the relationship between mitochondrial membrane potential and motility and show that the mitochondrial alterations induced by the

Obligate intracellular bacterium Ehrlichia inhibiting mitochondrial activity

PubMed Central

Liu, Yan; Zhang, Zhikai; Jiang, Yongquan; Zhang, Lihong; Popov, Vsevolod L.; Zhang, Jianzhi; Walker, David H.; Yu, Xue-jie

2010-01-01

Ehrlichia are obligately intracellular bacteria that reside in a vacuole in the cytoplasm of phagocytes. We determined by confocal microscopy the interaction between Ehrlichia and mitochondria in DH82 cells to investigate the mechanism of Ehrlichia survival inside the phagocyte. The most remarkable finding of our study was that Ehrlichia morulae interacted with mitochondria and inhibited mitochondrial metabolism,. We showed that in E. chaffeensis-infected DH82 cells, mitochondria did not incorporate BrdU and transcriptional level of the mitochondrial gene NADPH2 was significantly reduced, indicating the inhibition of mitochondrial metabolism. This study demonstrates that Ehrlichia are able to inhibit mitochondrial activities, and it opens up a new avenue for the study of Ehrlichia pathogenesis. PMID:21070861

Neuronal Ca2+ sensor-1 contributes to stress tolerance in cardiomyocytes via activation of mitochondrial detoxification pathways.

PubMed

Nakamura, Tomoe Y; Nakao, Shu; Wakabayashi, Shigeo

2016-10-01

Identification of the molecules involved in cell death/survival pathways is important for understanding the mechanisms of cell loss in cardiac disease, and thus is clinically relevant. Ca 2+ -dependent signals are often involved in these pathways. Here, we found that neuronal Ca 2+ -sensor-1 (NCS-1), a Ca 2+ -binding protein, has an important role in cardiac survival during stress. Cardiomyocytes derived from NCS-1-deficient (Ncs1 -/- ) mice were more susceptible to oxidative and metabolic stress than wild-type (WT) myocytes. Cellular ATP levels and mitochondrial respiration rates, as well as the levels of mitochondrial marker proteins, were lower in Ncs1 -/- myocytes. Although oxidative stress elevated mitochondrial proton leak, which exerts a protective effect by inhibiting the production of reactive oxygen species in WT myocytes, this response was considerably diminished in Ncs1 -/- cardiomyocytes, and this would be a major reason for cell death. Consistently, H 2 O 2 -induced loss of mitochondrial membrane potential, a critical early event in cell death, was accelerated in Ncs1 -/- myocytes. Furthermore, NCS-1 was upregulated in hearts subjected to ischemia-reperfusion, and ischemia-reperfusion injury was more severe in Ncs1 -/- hearts. Activation of stress-induced Ca 2+ -dependent survival pathways, such as Akt and PGC-1α (which promotes mitochondrial biogenesis and function), was diminished in Ncs1 -/- hearts. Overall, these data demonstrate that NCS-1 contributes to stress tolerance in cardiomyocytes at least in part by activating certain Ca 2+ -dependent survival pathways that promote mitochondrial biosynthesis/function and detoxification pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mitochondrial morphology transitions and functions: implications for retrograde signaling?

PubMed Central

Picard, Martin; Shirihai, Orian S.; Gentil, Benoit J.

2013-01-01

In response to cellular and environmental stresses, mitochondria undergo morphology transitions regulated by dynamic processes of membrane fusion and fission. These events of mitochondrial dynamics are central regulators of cellular activity, but the mechanisms linking mitochondrial shape to cell function remain unclear. One possibility evaluated in this review is that mitochondrial morphological transitions (from elongated to fragmented, and vice-versa) directly modify canonical aspects of the organelle's function, including susceptibility to mitochondrial permeability transition, respiratory properties of the electron transport chain, and reactive oxygen species production. Because outputs derived from mitochondrial metabolism are linked to defined cellular signaling pathways, fusion/fission morphology transitions could regulate mitochondrial function and retrograde signaling. This is hypothesized to provide a dynamic interface between the cell, its genome, and the fluctuating metabolic environment. PMID:23364527

Pharmacologic Effects on Mitochondrial Function

ERIC Educational Resources Information Center

Cohen, Bruce H.

2010-01-01

The vast majority of energy necessary for cellular function is produced in mitochondria. Free-radical production and apoptosis are other critical mitochondrial functions. The complex structure, electrochemical properties of the inner mitochondrial membrane (IMM), and genetic control from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) are…

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.

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.

[Interaction of surface-active base with fraction of membrane-bound Williams's protons].

PubMed

Iaguzhinskiĭ, L S; Motovilov, K A; Volkov, E M; Eremeev, S A

2013-01-01

In the process of mitochondrial respiratory H(+)-pumps functioning, the fraction membrane-bound protons (R-protons), which have an excess of free energy is formed. According to R.J. Williams this fraction is included as energy source in the reaction of ATP synthesis. Previously, in our laboratory was found the formation of this fraction was found in the mitochondria and on the outer surface of mitoplast. On the mitoslast model we strictly shown that non-equilibrium R-proton fraction is localized on the surface of the inner mitochondrial membrane. In this paper a surface-active compound--anion of 2,4,6-trichloro-3-pentadecylphenol (TCP-C15) is described, which selectively interacts with the R-protons fraction in mitochondria. A detailed description of the specific interaction of the TCP-C15 with R-protons fraction in mitochondria is presented. Moreover, in this work it was found that phosphate transport system reacts with the R-protons fraction in mitochondria and plays the role of the endogenous volume regulation system of this fraction. The results of experiments are discussed in the terms of a local coupling model of the phosphorylation mechanism.

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.

6-HYDROXYDOPAMINE INDUCES MITOCHONDRIAL ERK ACTIVATION

PubMed Central

Kulich, Scott M.; Horbinski, Craig; Patel, Manisha; Chu, Charleen T.

2007-01-01

Reactive oxygen species (ROS) are implicated in 6-hydroxydopamine (6-OHDA) injury to catecholaminergic neurons; however, the mechanism(s) are unclear. In addition to ROS generated during autoxidation, 6-OHDA may initiate secondary cellular sources of ROS that contribute to toxicity. Using a neuronal cell line, we found that catalytic metalloporphyrin antioxidants conferred protection if added 1 hour after exposure to 6-OHDA, whereas the hydrogen peroxide scavenger catalase failed to protect if added more than 15 min after 6-OHDA. There was a temporal correspondence between loss of protection and loss of the ability of the antioxidant to inhibit 6-OHDA-induced ERK phosphorylation. Time course studies of aconitase inactivation, as an indicator of intracellular superoxide, and MitoSOX red, a mitochondria targeted ROS indicator, demonstrate early intracellular ROS followed by a delayed phase of mitochondrial ROS production, associated with phosphorylation of a mitochondrial pool of ERK. Furthermore, upon initiation of mitochondrial ROS and ERK activation, 6-OHDA-injured cells became refractory to rescue by metalloporphyrin antioxidants. Together with previous studies showing that inhibition of the ERK pathway confers protection from 6-OHDA toxicity, and that phosphorylated ERK accumulates in mitochondria of degenerating human Parkinson’s disease neurons, these studies implicate mitochondrial ERK activation in Parkinsonian oxidative neuronal injury. PMID:17602953

Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway.

PubMed

Wang, Wei-jia; Wang, Yuan; Chen, Hang-zi; Xing, Yong-zhen; Li, Feng-wei; Zhang, Qian; Zhou, Bo; Zhang, Hong-kui; Zhang, Jie; Bian, Xue-li; Li, Li; Liu, Yuan; Zhao, Bi-xing; Chen, Yan; Wu, Rong; Li, An-zhong; Yao, Lu-ming; Chen, Ping; Zhang, Yi; Tian, Xu-yang; Beermann, Friedrich; Wu, Mian; Han, Jiahuai; Huang, Pei-qiang; Lin, Tianwei; Wu, Qiao

2014-02-01

Autophagy is linked to cell death, yet the associated mechanisms are largely undercharacterized. We discovered that melanoma, which is generally resistant to drug-induced apoptosis, can undergo autophagic cell death with the participation of orphan nuclear receptor TR3. A sequence of molecular events leading to cellular demise is launched by a specific chemical compound, 1-(3,4,5-trihydroxyphenyl)nonan-1-one, newly acquired from screening a library of TR3-targeting compounds. The autophagic cascade comprises TR3 translocation to mitochondria through interaction with the mitochondrial outer membrane protein Nix, crossing into the mitochondrial inner membrane through Tom40 and Tom70 channel proteins, dissipation of mitochondrial membrane potential by the permeability transition pore complex ANT1-VDAC1 and induction of autophagy. This process leads to excessive mitochondria clearance and irreversible cell death. It implicates a new approach to melanoma therapy through activation of a mitochondrial signaling pathway that integrates a nuclear receptor with autophagy for cell death.

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

PubMed

Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

2015-12-29

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

Atypical mitochondrial fission upon bacterial infection

PubMed Central

Stavru, Fabrizia; Palmer, Amy E.; Wang, Chunxin; Youle, Richard J.; Cossart, Pascale

2013-01-01

We recently showed that infection by Listeria monocytogenes causes mitochondrial network fragmentation through the secreted pore-forming toxin listeriolysin O (LLO). Here, we examine factors involved in canonical fusion and fission. Strikingly, LLO-induced mitochondrial fragmentation does not require the traditional fission machinery, as Drp1 oligomers are absent from fragmented mitochondria following Listeria infection or LLO treatment, as the dynamin-like protein 1 (Drp1) receptor Mff is rapidly degraded, and as fragmentation proceeds efficiently in cells with impaired Drp1 function. LLO does not cause processing of the fusion protein optic atrophy protein 1 (Opa1), despite inducing a decrease in the mitochondrial membrane potential, suggesting a unique Drp1- and Opa1-independent fission mechanism distinct from that triggered by uncouplers or the apoptosis inducer staurosporine. We show that the ER marks LLO-induced mitochondrial fragmentation sites even in the absence of functional Drp1, demonstrating that the ER activity in regulating mitochondrial fission can be induced by exogenous agents and that the ER appears to regulate fission by a mechanism independent of the canonical mitochondrial fission machinery. PMID:24043775

Effect of tributyltin on trout blood cells: changes in mitochondrial morphology and functionality.

PubMed

Tiano, Luca; Fedeli, Donatella; Santoni, Giorgio; Davies, Ian; Falcioni, Giancarlo

2003-05-12

The aquatic environment is the largest sink for the highly toxic organotin compounds, particularly as one of the main sources is the direct release of organotins from marine antifouling paints. The aim of this study was to investigate the mitochondrial toxicity and proapoptotic activity of tributyltin chloride (TBTC) in teleost leukocytes and nucleated erythrocytes, by means of electron microscopy investigation and mitochondrial membrane potential evaluation, in order to provide an early indicator of aquatic environmental pollution. Erythrocytes and leukocytes were obtained from an inbred strain of rainbow trout (Oncorhynchus mykiss). Transmission electronic micrographs of trout red blood cells (RBC) incubated in the presence of TBTC at 1 and 5 microM for 60 min showed remarkable mitochondrial morphological changes. TBTC-mediated toxicity involved alteration of the cristae ultrastructure and mitochondrial swelling, in a dose-dependent manner. Both erythrocytes and leukocytes displayed a consistent drop in mitochondrial membrane potential following TBTC exposure at concentrations >1 microM. The proapoptotic effect of TBTC on fish blood cells, and involvement of mitochondrial pathways was also investigated by verifying the release of cytochrome c, activation of caspase-3 and the presence of "DNA laddering". Although mitochondrial activity was much more strongly affected in erythrocytes, leukocytes incubated in the presence of TBTC showed the characteristic features of apoptosis after only 1 h of incubation. Longer exposures, up to 12 h, were required to trigger an apoptotic response in erythrocytes.

[Effect of 3-bromopyruvate on mitochondrial membrane potential and apoptosis of human breast carcinoma SK-BR-3 cells].

PubMed

Zhang, Yuanyuan; Liu, Zhe; Zhang, Qianwen; Chao, Zhenhua; Zhang, Pei; Xia, Fei; Jiang, Chenchen; Liu, Hao; Jiang, Zhiwen

2013-09-01

To study the effect of glycolysis inhibitor 3-bromopyruvate (3-BrPA) in inducing apoptosis of human breast carcinoma cells SK-BR-3 and the possible mechanism. MTT assay was used to detect the growth inhibition induced by 3-BrPA in breast cancer cells SK-BR-3. The apoptotic cells were detected by flow cytometry with propidium iodide (PI). ATP levels in the cells were detected by ATP assay kit, and DHE fluorescent probe technique was used to determine superoxide anion levels; the mitochondrial membrane potential was assessed using JC-1 staining assay. MTT assay showed that the proliferation of SK-BR-3 cells was inhibited by 3-BrPA in a time- and concentration-dependent manner. Exposure to 80, 160, and 320 µmol·L(-1) 3-BrPA for 24 h resulted in cell apoptosis rates of 6.7%, 22.3%, and 79.6%, respectively, and the intracellular ATP levels of SK-BR-3 cells treated with 80, 160, 320 µmol·L(-1) 3-BrPA for 5 h were 87.7%, 60.6%, and 23.7% of the control levels. 3-BrPA at 160 µmol·L(-1) increased reactive oxygen levels and lowered mitochondrial membrane potential of SK-BR-3 cells. 3-BrPA can inhibit cell proliferation, reduce the mitochondrial membrane potential and induce apoptosis in SK-BR-3 cells, the mechanism of which may involve a reduced ATP level by inhibiting glycolysis and increasing the reactive oxygen level in the cells.

Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis.

PubMed

Shimada, Kenichi; Crother, Timothy R; Karlin, Justin; Dagvadorj, Jargalsaikhan; Chiba, Norika; Chen, Shuang; Ramanujan, V Krishnan; Wolf, Andrea J; Vergnes, Laurent; Ojcius, David M; Rentsendorj, Altan; Vargas, Mario; Guerrero, Candace; Wang, Yinsheng; Fitzgerald, Katherine A; Underhill, David M; Town, Terrence; Arditi, Moshe

2012-03-23

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1β (IL-1β). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1β production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1β secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome. Copyright © 2012 Elsevier Inc. All rights reserved.

Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant β-Glucocerebrosidase Activity and Mitochondrial Function in Neuronopathic Forms of Gaucher Disease

PubMed Central

de la Mata, Mario; Cotán, David; Oropesa-Ávila, Manuel; Garrido-Maraver, Juan; Cordero, Mario D.; Villanueva Paz, Marina; Delgado Pavón, Ana; Alcocer-Gómez, Elizabet; de Lavera, Isabel; Ybot-González, Patricia; Paula Zaderenko, Ana; Ortiz Mellet, Carmen; Fernández, José M. García; Sánchez-Alcázar, José A.

2015-01-01

Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes lysosomal β-glucocerebrosidase. Homozygosity for the L444P mutation in GBA1 is associated with high risk of neurological manifestations which are not improved by enzyme replacement therapy. Alternatively, pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the mutant enzyme represent promising alternative therapies.Here, we report on how the L444P mutation affects mitochondrial function in primary fibroblast derived from GD patients. Mitochondrial dysfunction was associated with reduced mitochondrial membrane potential, increased reactive oxygen species (ROS), mitophagy activation and impaired autophagic flux.Both abnormalities, mitochondrial dysfunction and deficient β-glucocerebrosidase activity, were partially restored by supplementation with coenzyme Q10 (CoQ) or a L-idonojirimycin derivative, N-[N’-(4-adamantan-1-ylcarboxamidobutyl)thiocarbamoyl]-1,6-anhydro-L-idonojirimycin (NAdBT-AIJ), and more markedly by the combination of both treatments. These data suggest that targeting both mitochondria function by CoQ and protein misfolding by PCs can be promising therapies in neurological forms of GD. PMID:26045184

Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy.

PubMed

Tigchelaar, Wardit; Yu, Hongjuan; de Jong, Anne Margreet; van Gilst, Wiek H; van der Harst, Pim; Westenbrink, B Daan; de Boer, Rudolf A; Silljé, Herman H W

2015-01-15

Recently, a locus at the mitochondrial exo/endonuclease EXOG gene, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and hypertrophy in cardiomyocytes. Depletion of EXOG in primary neonatal rat ventricular cardiomyocytes (NRVCs) induced a marked increase in cardiomyocyte hypertrophy. Depletion of EXOG, however, did not result in loss of mitochondrial DNA integrity. Although EXOG depletion did not induce fetal gene expression and common hypertrophy pathways were not activated, a clear increase in ribosomal S6 phosphorylation was observed, which readily explains increased protein synthesis. With the use of a Seahorse flux analyzer, it was shown that the mitochondrial oxidative consumption rate (OCR) was increased 2.4-fold in EXOG-depleted NRVCs. Moreover, ATP-linked OCR was 5.2-fold higher. This increase was not explained by mitochondrial biogenesis or alterations in mitochondrial membrane potential. Western blotting confirmed normal levels of the oxidative phosphorylation (OXPHOS) complexes. The increased OCR was accompanied by a 5.4-fold increase in mitochondrial ROS levels. These increased ROS levels could be normalized with specific mitochondrial ROS scavengers (MitoTEMPO, mnSOD). Remarkably, scavenging of excess ROS strongly attenuated the hypertrophic response. In conclusion, loss of EXOG affects normal mitochondrial function resulting in increased mitochondrial respiration, excess ROS production, and cardiomyocyte hypertrophy. Copyright © 2015 the American Physiological Society.

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

Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target.

PubMed

Hartuti, Endah Dwi; Inaoka, Daniel Ken; Komatsuya, Keisuke; Miyazaki, Yukiko; Miller, Russell J; Xinying, Wang; Sadikin, Mohamad; Prabandari, Erwahyuni Endang; Waluyo, Danang; Kuroda, Marie; Amalia, Eri; Matsuo, Yuichi; Nugroho, Nuki B; Saimoto, Hiroyuki; Pramisandi, Amila; Watanabe, Yoh-Ichi; Mori, Mihoko; Shiomi, Kazuro; Balogun, Emmanuel Oluwadare; Shiba, Tomoo; Harada, Shigeharu; Nozaki, Tomoyoshi; Kita, Kiyoshi

2018-03-01

Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc 1 complex inhibitor. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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

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 mitochondrial test to assess haemodialyser biocompatibility.

PubMed

Tabouy, L J; Chauvet-Monges, A M; Brunet, P J; Braguer, D L; García, P A; Berland, Y F; Crevat, A D

1997-08-01

This paper describes an in vitro mitochondrial test to assess the biocompatibility of haemodialysers. We tested on isolated liver mitochondria the effect of solutions obtained by an aqueous rinse of different haemodialysers (cuprophane, cellulose acetate, Hemophan, polyacrylonitrile, polymethylmethacrylate, polysulphone, polyamide). Moreover, to determine the penetration into the cell and the cytotoxicity of these solutions from haemodialysers, we examined the effect of rinse solutions on HT29-D4 cells. Our results showed that rinse solutions from haemodialysers decrease the mitochondrial ATP synthesis. Cuprophane has the most marked effect, and the synthetic membranes exhibited only mild effects. Rinse solutions penetrated the cell and were cytotoxic by acting on mitochondria in the cell. In this respect, cellulosic membranes were the most toxic. Taken together our findings lead to a classification of haemodialyser membranes which is identical to one based on criteria such as activation of complement (cuprophane > other cellulosics > synthetics). Moreover isolated mitochondria make it possible to differentiate among the synthetic membranes. Isolated mitochondria thus appear to be a good in vitro test to assess the biocompatibility of haemodialysers.

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

Acetyl-L-carnitine supplementation to old rats partially reverts the age-related mitochondrial decay of soleus muscle by activating peroxisome proliferator-activated receptor gamma coactivator-1alpha-dependent mitochondrial biogenesis.

PubMed

Pesce, Vito; Fracasso, Flavio; Cassano, Pierluigi; Lezza, Angela Maria Serena; Cantatore, Palmiro; Gadaleta, Maria Nicola

2010-01-01

The age-related decay of mitochondrial function is a major contributor to the aging process. We tested the effects of 2-month-daily acetyl-L-carnitine (ALCAR) supplementation on mitochondrial biogenesis in the soleus muscle of aged rats. This muscle is heavily dependent on oxidative metabolism. Mitochondrial (mt) DNA content, citrate synthase activity, transcript levels of some nuclear- and mitochondrial-coded genes (cytochrome c oxidase subunit IV [COX-IV], 16S rRNA, COX-I) and of some factors involved in the mitochondrial biogenesis signaling pathway (peroxisome proliferator-activated receptor gamma [PPARgamma] coactivator-1alpha [PGC-1alpha], mitochondrial transcription factor A mitochondrial [TFAM], mitochondrial transcription factor 2B [TFB2]), as well as the protein content of PGC-1alpha were determined. The results suggest that the ALCAR treatment in old rats activates PGC-1alpha-dependent mitochondrial biogenesis, thus partially reverting the age-related mitochondrial decay.

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein

PubMed Central

Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

2015-01-01

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins. DOI: http://dx.doi.org/10.7554/eLife.11897.001 PMID:26714107

Interaction of Mitochondria with the Endoplasmic Reticulum and Plasma Membrane in Calcium Homeostasis, Lipid Trafficking and Mitochondrial Structure.

PubMed

Szymański, Jędrzej; Janikiewicz, Justyna; Michalska, Bernadeta; Patalas-Krawczyk, Paulina; Perrone, Mariasole; Ziółkowski, Wiesław; Duszyński, Jerzy; Pinton, Paolo; Dobrzyń, Agnieszka; Więckowski, Mariusz R

2017-07-20

Studying organelles in isolation has been proven to be indispensable for deciphering the underlying mechanisms of molecular cell biology. However, observing organelles in intact cells with the use of microscopic techniques reveals a new set of different junctions and contact sites between them that contribute to the control and regulation of various cellular processes, such as calcium and lipid exchange or structural reorganization of the mitochondrial network. In recent years, many studies focused their attention on the structure and function of contacts between mitochondria and other organelles. From these studies, findings emerged showing that these contacts are involved in various processes, such as lipid synthesis and trafficking, modulation of mitochondrial morphology, endoplasmic reticulum (ER) stress, apoptosis, autophagy, inflammation and Ca 2 + handling. In this review, we focused on the physical interactions of mitochondria with the endoplasmic reticulum and plasma membrane and summarized present knowledge regarding the role of mitochondria-associated membranes in calcium homeostasis and lipid metabolism.

Critical role for the mitochondrial permeability transition pore and cyclophilin D in platelet activation and thrombosis

PubMed Central

Wilson, Katina M.; Leo, Lorie; Raimondi, Alejandro; Molkentin, Jeffery D.; Lentz, Steven R.; Di Paola, Jorge

2008-01-01

Many of the cellular responses that occur in activated platelets resemble events that take place following activation of cell-death pathways in nucleated cells. We tested the hypothesis that formation of the mitochondrial permeability transition pore (MPTP), a key signaling event during cell death, also plays a critical role in platelet activation. Stimulation of murine platelets with thrombin plus the glycoprotein VI agonist convulxin resulted in a rapid loss of mitochondrial transmembrane potential (Δψm) in a subpopulation of activated platelets. In the absence of cyclophilin D (CypD), an essential regulator of MPTP formation, murine platelet activation responses were altered. CypD-deficient platelets exhibited defects in phosphatidylserine externalization, high-level surface fibrinogen retention, membrane vesiculation, and procoagulant activity. Also, in CypD-deficient platelet-rich plasma, clot retraction was altered. Stimulation with thrombin plus H2O2, a known activator of MPTP formation, also increased high-level surface fibrinogen retention, phosphatidylserine externalization, and platelet procoagulant activity in a CypD-dependent manner. In a model of carotid artery photochemical injury, thrombosis was markedly accelerated in CypD-deficient mice. These results implicate CypD and the MPTP as critical regulators of platelet activation and suggest a novel CypD-dependent negative-feedback mechanism regulating arterial thrombosis. PMID:17989312

Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis.

PubMed

Parra, Valentina; Eisner, Veronica; Chiong, Mario; Criollo, Alfredo; Moraga, Francisco; Garcia, Alejandra; Härtel, Steffen; Jaimovich, Enrique; Zorzano, Antonio; Hidalgo, Cecilia; Lavandero, Sergio

2008-01-15

In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

Rebamipide suppresses diclofenac-induced intestinal permeability via mitochondrial protection in mice.

PubMed

Diao, Lei; Mei, Qiao; Xu, Jian-Ming; Liu, Xiao-Chang; Hu, Jing; Jin, Juan; Yao, Qiang; Chen, Mo-Li

2012-03-14

To investigate the protective effect and mechanism of rebamipide on small intestinal permeability induced by diclofenac in mice. Diclofenac (2.5 mg/kg) was administered once daily for 3 d orally. A control group received the vehicle by gavage. Rebamipide (100 mg/kg, 200 mg/kg, 400 mg/kg) was administered intragastrically once a day for 3 d 4 h after diclofenac administration. Intestinal permeability was evaluated by Evans blue and the FITC-dextran method. The ultrastructure of the mucosal barrier was evaluated by transmission electron microscopy (TEM). Mitochondrial function including mitochondrial swelling, mitochondrial membrane potential, mitochondrial nicotinamide adenine dinucleotide-reduced (NADH) levels, succinate dehydrogenase (SDH) and ATPase activities were measured. Small intestinal mucosa was collected for assessment of malondialdehyde (MDA) content and myeloperoxidase (MPO) activity. Compared with the control group, intestinal permeability was significantly increased in the diclofenac group, which was accompanied by broken tight junctions, and significant increases in MDA content and MPO activity. Rebamipide significantly reduced intestinal permeability, improved inter-cellular tight junctions, and was associated with decreases in intestinal MDA content and MPO activity. At the mitochondrial level, rebamipide increased SDH and ATPase activities, NADH level and decreased mitochondrial swelling. Increased intestinal permeability induced by diclofenac can be attenuated by rebamipide, which partially contributed to the protection of mitochondrial function.

Sulforaphane Inhibits Mitochondrial Permeability Transition and Oxidative Stress

PubMed Central

Greco, Tiffany; Shafer, Jonathan; Fiskum, Gary

2012-01-01

Exposure of mitochondria to oxidative stress and elevated Ca2+ promotes opening of the mitochondrial permeability transition pore (PTP), resulting in membrane depolarization, uncoupling of oxidative phosphorylation, and potentially cell death. This study tested the hypothesis that treatment of rats with sulforaphane (SFP), an activator of the Nrf2 pathway of antioxidant gene expression, increases the resistance of liver mitochondria to redox-regulated PTP opening and elevates mitochondrial levels of antioxidants. Rats were injected with SFP or drug vehicle and liver mitochondria were isolated 40 hr later. Respiring mitochondria actively accumulated added Ca2+, which was then released through PTP opening induced by agents that either cause an oxidized shift in the mitochondrial redox state or that directly oxidize protein thiol groups. SFP treatment of rats inhibited the rate of pro-oxidant-induced mitochondrial Ca2+ release and increased expression of the glutathione peroxidase/reductase system, thioredoxin, and malic enzyme. These results are the first to demonstrate that SFP treatment of animals increases liver mitochondrial antioxidant defenses and inhibits redox-sensitive PTP opening. This novel form of preconditioning could protect against a variety of pathologies that include oxidative stress and mitochondrial dysfunction in their etiologies. PMID:21986339

Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases

PubMed Central

Werner, Erica; Werb, Zena

2002-01-01

We show here the transient activation of the small GTPase Rac, followed by a rise in reactive oxygen species (ROS), as necessary early steps in a signal transduction cascade that lead to NFκB activation and collagenase-1 (CL-1)/matrix metalloproteinase-1 production after integrin-mediated cell shape changes. We show evidence indicating that this constitutes a new mechanism for ROS production mediated by small GTPases. Activated RhoA also induced ROS production and up-regulated CL-1 expression. A Rac mutant (L37) that prevents reorganization of the actin cytoskeleton prevented integrin-induced CL-1 expression, whereas mutations that abrogate Rac binding to the neutrophil NADPH membrane oxidase in vitro (H26 and N130) did not. Instead, ROS were produced by integrin-induced changes in mitochondrial function, which were inhibited by Bcl-2 and involved transient membrane potential loss. The cells showing this transient decrease in mitochondrial membrane potential were already committed to CL-1 expression. These results unveil a new molecular mechanism of signal transduction triggered by integrin engagement where a global mitochondrial metabolic response leads to gene expression rather than apoptosis. PMID:12119354

Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases.

PubMed

Werner, Erica; Werb, Zena

2002-07-22

We show here the transient activation of the small GTPase Rac, followed by a rise in reactive oxygen species (ROS), as necessary early steps in a signal transduction cascade that lead to NFkappaB activation and collagenase-1 (CL-1)/matrix metalloproteinase-1 production after integrin-mediated cell shape changes. We show evidence indicating that this constitutes a new mechanism for ROS production mediated by small GTPases. Activated RhoA also induced ROS production and up-regulated CL-1 expression. A Rac mutant (L37) that prevents reorganization of the actin cytoskeleton prevented integrin-induced CL-1 expression, whereas mutations that abrogate Rac binding to the neutrophil NADPH membrane oxidase in vitro (H26 and N130) did not. Instead, ROS were produced by integrin-induced changes in mitochondrial function, which were inhibited by Bcl-2 and involved transient membrane potential loss. The cells showing this transient decrease in mitochondrial membrane potential were already committed to CL-1 expression. These results unveil a new molecular mechanism of signal transduction triggered by integrin engagement where a global mitochondrial metabolic response leads to gene expression rather than apoptosis.

Multi-Parametric Analysis and Modeling of Relationships between Mitochondrial Morphology and Apoptosis

PubMed Central

Reis, Yara; Wolf, Thomas; Brors, Benedikt; Hamacher-Brady, Anne; Eils, Roland; Brady, Nathan R.

2012-01-01

Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis. PMID:22272225

Mitochondrial myopathies.

PubMed

DiMauro, Salvatore

2006-11-01

Our understanding of mitochondrial diseases (defined restrictively as defects of the mitochondrial respiratory chain) is expanding rapidly. In this review, I will give the latest information on disorders affecting predominantly or exclusively skeletal muscle. The most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency and mutations in genes controlling mitochondrial DNA abundance and structure, such as POLG, TK2, and MPV17. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with decreased amount and altered structure of cardiolipin, the main phospholipid of the inner mitochondrial membrane, but a secondary impairment of respiratory chain function is plausible. The role of mutations in protein-coding genes of mitochondrial DNA in causing isolated myopathies has been confirmed. Mutations in tRNA genes of mitochondrial DNA can also cause predominantly myopathic syndromes and--contrary to conventional wisdom--these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, cramps, recurrent myoglobinuria, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.

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

Mitochondrial Dysfunction in Schizophrenia: Determination of Mitochondrial Respiratory Activity in a Two-Hit Mouse Model.

PubMed

Monpays, Cécile; Deslauriers, Jessica; Sarret, Philippe; Grignon, Sylvain

2016-08-01

Schizophrenia is a chronic mental illness in which mitochondrial dysfunction has been suggested. Our laboratory recently developed a juvenile murine two-hit model (THM) of schizophrenia based on the combination of gestational inflammation, followed by juvenile restraint stress. We previously reported that relevant behaviors and neurochemical disturbances, including oxidative stress, were reversed by the antioxidant lipoic acid (LA), thereby pointing to the central role played by oxidative abnormalities and prompting us to investigate mitochondrial function. Mitochondrial activity was determined with the MitoXpress® commercial kit in two schizophrenia-relevant regions (prefrontal cortex (PFC) and striatum). Measurements were performed in state 3, with substrates for complex I- and complex II-induced respiratory activity (IRA). We observed an increase in complex I IRA in the PFC and striatum in both sexes but an increase in complex II activity only in males. LA treatment prevented this increase only in complex II IRA in males. Expression levels of the different respiratory chain complexes, as well as fission/fusion proteins and protein carbonylation, were unchanged. In conclusion, our juvenile schizophrenia THM shows an increase in mitochondrial activity reversed by LA, specifically in complex II IRA in males. Further investigations are required to determine the mechanisms of these modifications.

Mitochondrial Dysfunction in Lysosomal Storage Disorders

PubMed Central

de la Mata, Mario; Cotán, David; Villanueva-Paz, Marina; de Lavera, Isabel; Álvarez-Córdoba, Mónica; Luzón-Hidalgo, Raquel; Suárez-Rivero, Juan M.; Tiscornia, Gustavo; Oropesa-Ávila, Manuel

2016-01-01

Lysosomal storage diseases (LSDs) describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS), where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm), diminished ATP production and increased generation of reactive oxygen species (ROS). Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD), the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase). Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs. PMID:28933411

Mitochondrial targeting of HIF-1α inhibits hypoxia-induced apoptosis independently of its transcriptional activity.

PubMed

Li, Hong-Sheng; Zhou, Yan-Ni; Li, Lu; Li, Sheng-Fu; Long, Dan; Chen, Xue-Lu; Zhang, Jia-Bi; Li, You-Ping; Feng, Li

2018-04-25

The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates adaptive responses to hypoxia by nuclear translocation and regulation of gene expression. Mitochondrial changes are critical for the adaptive response to hypoxia. However, the transcriptional and non-transcriptional mechanisms by which HIF-1α regulates mitochondria under hypoxia are poorly understood. Here, we examined the subcellular localization of HIF-1α in human cells and identified a small fraction of HIF-1α that translocated to the mitochondria after exposure to hypoxia or hypoxia-mimicking pharmacological agents. To probe the function of this HIF-1α population, we ectopically expressed a mitochondrial-targeted form of HIF-1α (mito-HIF-1α). Expression of mito-HIF-1α was sufficient to attenuate apoptosis induced by exposure to hypoxia or H 2 O 2 -induced oxidative stress. Moreover, mito-HIF-1α expression reduced the production of reactive oxygen species, the collapse of mitochondrial membrane potential, and the expression of mitochondrial DNA-encoded mRNA in response to hypoxia. However, these functions of mito-HIF-1α were independent of its conventional transcriptional activity. Finally, the livers of mice with CCl 4 -induced fibrosis showed a progressive increase in HIF-1α association with the mitochondria, indicating the clinical relevance of this finding. These data suggested that mitochondrial HIF-1α protects against apoptosis independently of its well-known role as a transcription factor. Copyright © 2018. Published by Elsevier Inc.

The Roles of PINK1, Parkin and Mitochondrial Fidelity in Parkinson's Disease

PubMed Central

Pickrell, Alicia M.; Youle, Richard J.

2015-01-01

Understanding the function of genes mutated in hereditary forms of Parkinson's disease yields insight into disease etiology and reveals new pathways in cell biology. Although mutations or variants in many genes increase the susceptibility to Parkinson's disease, only a handful of monogenic causes of Parkinsonism have been identified. Biochemical and genetic studies reveal that the products of two genes that are mutated in autosomal recessive Parkinsonism, PINK1 and Parkin, normally work together in the same pathway to govern mitochondrial quality control, bolstering previous evidence that mitochondrial damage is involved in Parkinson's disease. PINK1 accumulates on the outer membrane of damaged mitochondria, activates Parkin's E3 ubiquitin ligase activity and recruits Parkin to the dysfunctional mitochondrion. Then, Parkin ubiquitinates outer mitochondrial membrane proteins to trigger selective autophagy. This review covers the normal functions that PINK1 and Parkin play within cells, their molecular mechanisms of action, and the pathophysiological consequences of their loss. PMID:25611507

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

Development of pharmacological strategies for mitochondrial disorders

PubMed Central

Kanabus, M; Heales, S J; Rahman, S

2014-01-01

Mitochondrial diseases are an unusually genetically and phenotypically heterogeneous group of disorders, which are extremely challenging to treat. Currently, apart from supportive therapy, there are no effective treatments for the vast majority of mitochondrial diseases. Huge scientific effort, however, is being put into understanding the mechanisms underlying mitochondrial disease pathology and developing potential treatments. To date, a variety of treatments have been evaluated by randomized clinical trials, but unfortunately, none of these has delivered breakthrough results. Increased understanding of mitochondrial pathways and the development of many animal models, some of which are accurate phenocopies of human diseases, are facilitating the discovery and evaluation of novel prospective treatments. Targeting reactive oxygen species has been a treatment of interest for many years; however, only in recent years has it been possible to direct antioxidant delivery specifically into the mitochondria. Increasing mitochondrial biogenesis, whether by pharmacological approaches, dietary manipulation or exercise therapy, is also currently an active area of research. Modulating mitochondrial dynamics and mitophagy and the mitochondrial membrane lipid milieu have also emerged as possible treatment strategies. Recent technological advances in gene therapy, including allotopic and transkingdom gene expression and mitochondrially targeted transcription activator-like nucleases, have led to promising results in cell and animal models of mitochondrial diseases, but most of these techniques are still far from clinical application. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8 PMID:24116962

Noninvasive probes of mitochondrial molecular motors

NASA Astrophysics Data System (ADS)

Nawarathna, Dharmakeerthna; Claycomb, James

2005-03-01

We report on a noninvasive method of probing mitochondrial molecular motors using nonlinear dielectric spectroscopy. It has been found previously that enzymes in the plasma membrane, particularly H+ ATPase, result in a strong low frequency (less than 100 Hz) nonlinear harmonic response. In this study, we find evidence that molecular motors located in the inner membranes of mitochondria cause the generation of harmonics at relatively high frequencies (1 - 30 kHz). In particular, we find that potassium cyanide (KCN), a respiratory inhibitor that binds to cytochrome c oxidase and thus prevents transport of protons across the mitochondrial inner membrane, suppresses the harmonic response. We observe this behavior in yeast (S. cerevisiae), a eucaryote that typically contains about 300 mitochondria, and B. indicas, a procaryote believed to be related to the ancient ancestor of mitochondria. Our current modeling efforts are focusing on a Brownian ratchet model of the F0 unit of ATP synthase, a remarkable molecular turbine driven by the proton gradient across the mitochondrial inner membrane.

Mitochondrial Dysfunction in Chemotherapy-Induced Peripheral Neuropathy (CIPN)

PubMed Central

Canta, Annalisa; Pozzi, Eleonora; Carozzi, Valentina Alda

2015-01-01

The mitochondrial dysfunction has a critical role in several disorders including chemotherapy-induced peripheral neuropathies (CIPN). This is due to a related dysregulation of pathways involving calcium signalling, reactive oxygen species and apoptosis. Vincristine is able to affect calcium movement through the Dorsal Root Ganglia (DRG) neuronal mitochondrial membrane, altering its homeostasis and leading to abnormal neuronal excitability. Paclitaxel induces the opening of the mitochondrial permeability transition pore in axons followed by mitochondrial membrane potential loss, increased reactive oxygen species generation, ATP level reduction, calcium release and mitochondrial swelling. Cisplatin and oxaliplatin form adducts with mitochondrial DNA producing inhibition of replication, disruption of transcription and morphological abnormalities within mitochondria in DRG neurons, leading to a gradual energy failure. Bortezomib is able to modify mitochondrial calcium homeostasis and mitochondrial respiratory chain. Moreover, the expression of a certain number of genes, including those controlling mitochondrial functions, was altered in patients with bortezomib-induced peripheral neuropathy. PMID:29056658

Molecular identity of cardiac mitochondrial chloride intracellular channel proteins.

PubMed

Ponnalagu, Devasena; Gururaja Rao, Shubha; Farber, Jason; Xin, Wenyu; Hussain, Ahmed Tafsirul; Shah, Kajol; Tanda, Soichi; Berryman, Mark; Edwards, John C; Singh, Harpreet

2016-03-01

Emerging evidences demonstrate significance of chloride channels in cardiac function and cardioprotection from ischemia-reperfusion (IR) injury. Unlike mitochondrial potassium channels sensitive to calcium (BKCa) and ATP (KATP), molecular identity of majority of cardiac mitochondrial chloride channels located at the inner membrane is not known. In this study, we report the presence of unique dimorphic chloride intracellular channel (CLIC) proteins namely CLIC1, CLIC4 and CLIC5 as abundant CLICs in the rodent heart. Further, CLIC4, CLIC5, and an ortholog present in Drosophila (DmCLIC) localize to adult cardiac mitochondria. We found that CLIC4 is enriched in the outer mitochondrial membrane, whereas CLIC5 is present in the inner mitochondrial membrane. Also, CLIC5 plays a direct role in regulating mitochondrial reactive oxygen species (ROS) generation. Our study highlights that CLIC5 is localized to the cardiac mitochondria and directly modulates mitochondrial function. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Identity and function of a cardiac mitochondrial small conductance Ca2+-activated K+ channel splice variant

PubMed Central

Yang, MeiYing; Camara, Amadou K.S.; Aldakkak, Mohammed; Kwok, Wai-Meng; Stowe, David F.

2017-01-01

We provide evidence for location and function of a small conductance, Ca2+-activated K+ (SKCa) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SKCa agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SKCa antagonists worsened IR injury. Intravenous infusion of a SKCa channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the Nterminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca2+ calmodulin binding domain is required for Ca2+ sensing to induce mK+ influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK+ influx was driven by adding CaCl2. Moreover, there was a greater fall in membrane potential (ΔΨm), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SKCa channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SKCa channel opening remains unclear. SKCa channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa2+ overload. PMID:28342809

miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor.

PubMed

Tak, Hyosun; Kim, Jihye; Jayabalan, Aravinth Kumar; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Ohn, Takbum; Nam, Suk Woo; Kim, Wook; Lee, Eun Kyung

2014-11-28

Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3'-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF.

Antibiotic tigecycline enhances cisplatin activity against human hepatocellular carcinoma through inducing mitochondrial dysfunction and oxidative damage

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

Tan, Jun; Song, Meijun; Zhou, Mi

Targeting mitochondrial metabolism has been recently demonstrated to be a promising therapeutic strategy for the treatment of various cancer. In this work, we demonstrate that antibiotic tigecycline is selectively against hepatocellular carcinoma (HCC) through inducing mitochondrial dysfunction and oxidative damage. Tigecycline is more effective in inhibiting proliferation and inducing apoptosis of HCC than normal liver cells. Importantly, tigecycline significantly enhances the inhibitory effects of chemotherapeutic drug cisplatin in HCC in vitro and in vivo. Mechanistically, tigecycline specifically inhibits mitochondrial translation as shown by the decreased protein levels of Cox-1 and -2 but not Cox-4 or Grp78, and increased mRNA levels of Cox-1more » and -2 but not Cox-4 in HCC cells exposed to tigecycline. In addition, tigecycline significantly induces mitochondrial dysfunction in HCC cells via decreasing mitochondrial membrane potential, complex I and IV activities, mitochondrial respiration and ATP levels. Tigecycline also increases levels of mitochondrial superoxide, hydrogen peroxide and ROS levels. Consistent with oxidative stress, oxidative damage on DNA, protein and lipid are also observed in tigecycline-treated cells. Importantly, antioxidant N-acetyl-L-cysteine (NAC) reverses the effects of tigecycline, suggesting that oxidative stress is required for the action of tigecycline in HCC cells. We further show that HCC cells have higher level of mitochondrial biogenesis than normal liver cells which might explain the different sensitivity to tigecycline between HCC and normal liver cells. Our work is the first to demonstrate that tigecycline is a promising candidate for HCC treatment and highlight the therapeutic value of targeting mitochondrial metabolism in HCC. - Highlights: • Tigecycline selectively targets HCC in vitro and in vivo. • Tigecycline enhances HCC cell response to chemotherapeutic drug. • Tigecycline inhibits mitochondrial

1,3-Butadiene-Induced Mitochondrial Dysfunction is Correlated with Mitochondrial CYP2E1 Activity in Collaborative Cross Mice

PubMed Central

Hartman, Jessica H.; Miller, Grover P.; Caro, Andres A.; Byrum, Stephanie D.; Orr, Lisa M.; Mackintosh, Samuel G.; Tackett, Alan J.; MacMillan-Crow, Lee Ann; Hallberg, Lance M.; Ameredes, Bill T.; Boysen, Gunnar

2017-01-01

Cytochrome P450 2E1 (CYP2E1) metabolizes low molecular weight hydrophobic compounds, including 1,3-butadiene, which is converted by CYP2E1 to electrophilic epoxide metabolites that covalently modify cellular proteins and DNA. Previous CYP2E1 studies have mainly focused on the enzyme localized in the endoplasmic reticulum (erCYP2E1); however, active CYP2E1 also localizes in mitochondria (mtCYP2E1) and the distribution of CYP2E1 between organelles can influence an individual's response to exposure. Relatively few studies have focused on the contribution of mtCYP2E1 to activation of chemical toxicants. We hypothesized that CYP2E1 bioactivation of butadiene within mitochondria adversely affects mitochondrial respiratory complexes I-IV. A population of Collaborative Cross mice were exposed to air (control) or 200 ppm butadiene. Subcellular fractions (mitochondria, DNA, and microsomes) were collected from frozen livers and CYP2E1 activity was measured in microsomes and mitochondria. Individual activities of mitochondrial respiratory complexes I-IV were measured using in vitro assays with purified mitochondrial fractions. In air- and butadiene-exposed mouse samples, mtDNA copy numbers were assessed by RT-PCR, and mtDNA integrity was assessed through a PCR-based assay. No significant change in mtDNA copy number or integrity were observed; however, there was a decrease in overall activity of mitochondrial respiratory complexes I, II, and IV after butadiene exposure. Additionally, higher mtCYP2E1 (but not erCYP2E1) activity was correlated with decreased mitochondrial respiratory complex activity (in complexes I-IV) in the butadiene-exposed (not control) animals. Together, these results represent the first in vivo link between mitochondrial CYP2E1 activity and mitochondrial toxicity. PMID:28082109

Nitrite activates protein kinase A in normoxia to mediate mitochondrial fusion and tolerance to ischaemia/reperfusion

PubMed Central

Pride, Christelle Kamga; Mo, Li; Quesnelle, Kelly; Dagda, Ruben K.; Murillo, Daniel; Geary, Lisa; Corey, Catherine; Portella, Rafael; Zharikov, Sergey; St Croix, Claudette; Maniar, Salony; Chu, Charleen T.; K. H. Khoo, Nicholas; Shiva, Sruti

2014-01-01

Aims Nitrite (NO2–), a dietary constituent and nitric oxide (NO) oxidation product, mediates cardioprotection after ischaemia/reperfusion (I/R) in a number of animal models when administered during ischaemia or as a pre-conditioning agent hours to days prior to the ischaemic episode. When present during ischaemia, the reduction of nitrite to bioactive NO by deoxygenated haem proteins accounts for its protective effects. However, the mechanism of nitrite-induced pre-conditioning, a normoxic response which does not appear to require reduction of nitrite to NO, remains unexplored. Methods and results Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocytes, we demonstrate that a transient (30 min) normoxic nitrite treatment significantly attenuates cell death after a hypoxic episode initiated 1 h later. Mechanistically, this protection depends on the activation of protein kinase A, which phosphorylates and inhibits dynamin-related protein 1, the predominant regulator of mitochondrial fission. This results morphologically, in the promotion of mitochondrial fusion and functionally in the augmentation of mitochondrial membrane potential and superoxide production. We identify AMP kinase (AMPK) as a downstream target of the mitochondrial reactive oxygen species (ROS) generated and show that its oxidation and subsequent phosphorylation are essential for cytoprotection, as scavenging of ROS prevents AMPK activation and inhibits nitrite-mediated protection after H/R. The protein kinase A-dependent protection mediated by nitrite is reproduced in an intact isolated rat heart model of I/R. Conclusions These data are the first to demonstrate nitrite-dependent normoxic modulation of both mitochondrial morphology and function and reveal a novel signalling pathway responsible for nitrite-mediated cardioprotection. PMID:24081164

Ebselen alters mitochondrial physiology and reduces viability of rat hippocampal astrocytes.

PubMed

Santofimia-Castaño, Patricia; Salido, Ginés M; González, Antonio

2013-04-01

The seleno-organic compound and radical scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) have been extensively employed as an anti-inflammatory and neuroprotective compound. However, its glutathione peroxidase activity at the expense of cellular thiols groups could underlie certain deleterious actions of the compound on cell physiology. In this study, we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular viability, the intracellular free-Ca(2+) concentration ([Ca(2+)]c), the mitochondrial free-Ca(2+) concentration ([Ca(2+)]m), and mitochondrial membrane potential (ψm) were analyzed. The caspase-3 activity was also assayed. Our results show that cell viability was reduced by treatment of cells with ebselen, depending on the concentration employed. In the presence of ebselen, we observed an initial transient increase in [Ca(2+)]c that was then followed by a progressive increase to an elevated plateau. We also observed a transient increase in [Ca(2+)]m in the presence of ebselen that returned toward a value over the prestimulation level. The compound induced depolarization of ψm and altered the permeability of the mitochondrial membrane. Additionally, a disruption of the mitochondrial network was observed. Finally, we did not detect changes in caspase-3 activation in response to ebselen treatment. Collectively, these data support the likelihood of ebselen, depending on the concentration employed, reduces viability of rat hippocampal astrocytes via its action on the mitochondrial activity. These may be early effects that do not involve caspase-3 activation. We conclude that, depending on the concentration used, ebselen might exert deleterious actions on astrocyte physiology that could compromise cell function.

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.

Elastocapillary Instability in Mitochondrial Fission

NASA Astrophysics Data System (ADS)

Gonzalez-Rodriguez, David; Sart, Sébastien; Babataheri, Avin; Tareste, David; Barakat, Abdul I.; Clanet, Christophe; Husson, Julien

2015-08-01

Mitochondria are dynamic cell organelles that constantly undergo fission and fusion events. These dynamical processes, which tightly regulate mitochondrial morphology, are essential for cell physiology. Here we propose an elastocapillary mechanical instability as a mechanism for mitochondrial fission. We experimentally induce mitochondrial fission by rupturing the cell's plasma membrane. We present a stability analysis that successfully explains the observed fission wavelength and the role of mitochondrial morphology in the occurrence of fission events. Our results show that the laws of fluid mechanics can describe mitochondrial morphology and dynamics.

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death

PubMed Central

Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A.; Quest, Andrew F.G.; Lavandero, Sergio

2014-01-01

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulatenumerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2+ overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2+ levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2+ influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2+ buffer capacity. These biochemical events increase cytosolic Ca2+ levels and trigger cardiomyocyte death via the activation of calpains. PMID:23602992

The matrix peptide exporter HAF-1 signals a mitochondrial unfolded protein response by activating the transcription factor ZC376.7 in C. elegans

PubMed Central

Haynes, Cole M.; Yang, Yun; Blais, Steven P.; Neubert, Thomas A.; Ron, David

2010-01-01

Summary Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear encoded mitochondrial chaperone genes in C. elegans (UPRmt). Here we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPRmt and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP-dependent and required HAF-1 and the protease ClpP. Defective UPRmt signaling in the haf-1 deleted worms was associated with failure of the bZIP protein, ZC376.7, to localize to nuclei in worms with perturbed mitochondrial protein folding, whereas zc376.7(RNAi) strongly inhibited the UPRmt. These observations suggest a simple model whereby perturbation of the protein-folding environment in the mitochondrial matrix promotes ClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPRmt signaling across the mitochondrial inner membrane. PMID:20188671

Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells.

PubMed

Szebeni, Gábor J; Balázs, Árpád; Madarász, Ildikó; Pócz, Gábor; Ayaydin, Ferhan; Kanizsai, Iván; Fajka-Boja, Roberta; Alföldi, Róbert; Hackler, László; Puskás, László G

2017-10-07

Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G₀/G₁ cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5 , ATF4, XBP1 , and DDIT3 . The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

Impaired cortical mitochondrial function following TBI precedes behavioral changes

PubMed Central

Watson, William D.; Buonora, John E.; Yarnell, Angela M.; Lucky, Jessica J.; D’Acchille, Michaela I.; McMullen, David C.; Boston, Andrew G.; Kuczmarski, Andrew V.; Kean, William S.; Verma, Ajay; Grunberg, Neil E.; Cole, Jeffrey T.

2014-01-01

Traumatic brain injury (TBI) pathophysiology can be attributed to either the immediate, primary physical injury, or the delayed, secondary injury which begins minutes to hours after the initial injury and can persist for several months or longer. Because these secondary cascades are delayed and last for a significant time period post-TBI, they are primary research targets for new therapeutics. To investigate changes in mitochondrial function after a brain injury, both the cortical impact site and ipsilateral hippocampus of adult male rats 7 and 17 days after a controlled cortical impact (CCI) injury were examined. State 3, state 4, and uncoupler-stimulated rates of oxygen consumption, respiratory control ratios (RCRs) were measured and membrane potential quantified, and all were significantly decreased in 7 day post-TBI cortical mitochondria. By contrast, hippocampal mitochondria at 7 days showed only non-significant decreases in rates of oxygen consumption and membrane potential. NADH oxidase activities measured in disrupted mitochondria were normal in both injured cortex and hippocampus at 7 days post-CCI. Respiratory and phosphorylation capacities at 17 days post-CCI were comparable to naïve animals for both cortical and hippocampus mitochondria. However, unlike oxidative phosphorylation, membrane potential of mitochondria in the cortical lining of the impact site did not recover at 17 days, suggesting that while diminished cortical membrane potential at 17 days does not adversely affect mitochondrial capacity to synthesize ATP, it may negatively impact other membrane potential-sensitive mitochondrial functions. Memory status, as assessed by a passive avoidance paradigm, was not significantly impaired until 17 days after injury. These results indicate pronounced disturbances in cortical mitochondrial function 7 days after CCI which precede the behavioral impairment observed at 17 days. PMID:24550822

Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia.

PubMed

Moshal, Karni S; Tipparaju, Srinivas M; Vacek, Thomas P; Kumar, Munish; Singh, Mahavir; Frank, Iluiana E; Patibandla, Phani K; Tyagi, Neetu; Rai, Jayesh; Metreveli, Naira; Rodriguez, Walter E; Tseng, Michael T; Tyagi, Suresh C

2008-08-01

Cardiomyocyte N-methyl-d-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (-dL/dt), and maximal rate of relaxation (+dL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, -dL/dt, and +dL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1.

Effect of the antitumoral alkylating agent 3-bromopyruvate on mitochondrial respiration: role of mitochondrially bound hexokinase.

PubMed

Rodrigues-Ferreira, Clara; da Silva, Ana Paula Pereira; Galina, Antonio

2012-02-01

The alkylating agent 3-Bromopyruvate (3-BrPA) has been used as an anti-tumoral drug due to its anti-proliferative property in hepatomas cells. This propriety is believed to disturb glycolysis and respiration, which leads to a decreased rate of ATP synthesis. In this study, we evaluated the effects of the alkylating agent 3-BrPA on the respiratory states and the metabolic steps of the mitochondria of mice liver, brain and in human hepatocarcinoma cell line HepG2. The mitochondrial membrane potential (ΔΨ(m)), O(2) consumption and dehydrogenase activities were rapidly dissipated/or inhibited by 3-BrPA in respiration medium containing ADP and succinate as respiratory substrate. 3-BrPA inhibition was reverted by reduced glutathione (GSH). Respiration induced by yeast soluble hexokinase (HK) was rapidly inhibited by 3-BrPA. Similar results were observed using mice brain mitochondria that present HK naturally bound to the outer mitochondrial membrane. When the adenine nucleotide transporter (ANT) was blocked by the carboxyatractiloside, the 3-BrPA effect was significantly delayed. In permeabilized human hepatoma HepG2 cells that present HK type II bound to mitochondria (mt-HK II), the inhibiting effect occurred faster when the endogenous HK activity was activated by 2-deoxyglucose (2-DOG). Inhibition of mt-HK II by glucose-6-phosphate retards the mitochondria to react with 3-BrPA. The HK activities recovered in HepG2 cells treated or not with 3-BrPA were practically the same. These results suggest that mitochondrially bound HK supporting the ADP/ATP exchange activity levels facilitates the 3-BrPA inhibition reaction in tumors mitochondria by a proton motive force-dependent dynamic equilibrium between sensitive and less sensitive SDH in the electron transport system.

Isoliquiritigenin reduces oxidative damage and alleviates mitochondrial impairment by SIRT1 activation in experimental diabetic neuropathy.

PubMed

Yerra, Veera Ganesh; Kalvala, Anil Kumar; Kumar, Ashutosh

2017-09-01

Sirtuin (SIRT1) inactivation underlies the pathogenesis of insulin resistance and hyperglycaemia-associated vascular complications, but its role in diabetic neuropathy (DN) has not been yet explored. We have evaluated hyperglycaemia-induced alteration of SIRT1 signalling and the effect of isoliquiritigenin (ILQ) on SIRT1-directed AMP kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) signalling in peripheral nerves of streptozotocin (STZ) (55 mg/kg, ip)-induced diabetic rats and in high glucose (30 mM)-exposed neuro2a (N2A) cells. Diabetic rats and high glucose-exposed N2A cells showed reduction in SIRT1 expression with consequent decline in mitochondrial biogenesis and autophagy. ILQ (10 & 20 mg/kg, po) administration to diabetic rats for 2 weeks and exposure to glucose-insulted N2A cells resulted in significant SIRT1 activation with concurrent increase in mitochondrial biogenesis and autophagy. ILQ administration also enhanced NAD + /NADH ratio in peripheral sciatic nerves which explains its possible SIRT1 modulatory effect. Functional and behavioural studies show beneficial effect of ILQ as it alleviated nerve conduction and nerve blood flow deficits in diabetic rats along with improvement in behavioural parameters (hyperalgesia and allodynia). ILQ treatment to N2A cells reduced high glucose-driven ROS production and mitochondrial membrane depolarization. Further, ILQ-mediated SIRT1 activation facilitated the Nrf2-directed antioxidant signalling. Overall, results from this study suggest that SIRT1 activation by ILQ mimic effects of calorie restriction, that is, PGC-1α-mediated mitochondrial biogenesis, FOXO3a mediated stress resistance and AMPK mediated autophagy effects to counteract the multiple manifestations in experimental DN. Copyright © 2017 Elsevier Inc. All rights reserved.

Mitochondrial loss, dysfunction and altered dynamics in Huntington's disease.

PubMed

Kim, Jinho; Moody, Jennifer P; Edgerly, Christina K; Bordiuk, Olivia L; Cormier, Kerry; Smith, Karen; Beal, M Flint; Ferrante, Robert J

2010-10-15

Although a direct causative pathway from the gene mutation to the selective neostriatal neurodegeneration remains unclear in Huntington's disease (HD), one putative pathological mechanism reported to play a prominent role in the pathogenesis of this neurological disorder is mitochondrial dysfunction. We examined mitochondria in preferentially vulnerable striatal calbindin-positive neurons in moderate-to-severe grade HD patients, using antisera against mitochondrial markers of COX2, SOD2 and cytochrome c. Combined calbindin and mitochondrial marker immunofluorescence showed a significant and progressive grade-dependent reduction in the number of mitochondria in spiny striatal neurons, with marked alteration in size. Consistent with mitochondrial loss, there was a reduction in COX2 protein levels using western analysis that corresponded with disease severity. In addition, both mitochondrial transcription factor A, a regulator of mtDNA, and peroxisome proliferator-activated receptor-co-activator gamma-1 alpha, a key transcriptional regulator of energy metabolism and mitochondrial biogenesis, were also significantly reduced with increasing disease severity. Abnormalities in mitochondrial dynamics were observed, showing a significant increase in the fission protein Drp1 and a reduction in the expression of the fusion protein mitofusin 1. Lastly, mitochondrial PCR array profiling in HD caudate nucleus specimens showed increased mRNA expression of proteins involved in mitochondrial localization, membrane translocation and polarization and transport that paralleled mitochondrial derangement. These findings reveal that there are both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion in HD. These findings provide further evidence that mitochondrial dysfunction plays a critical role in the pathogenesis of HD.

Ultrafine particulate matter impairs mitochondrial redox homeostasis and activates phosphatidylinositol 3-kinase mediated DNA damage responses in lymphocytes.

PubMed

Bhargava, Arpit; Tamrakar, Shivani; Aglawe, Aniket; Lad, Harsha; Srivastava, Rupesh Kumar; Mishra, Dinesh Kumar; Tiwari, Rajnarayan; Chaudhury, Koel; Goryacheva, Irina Yu; Mishra, Pradyumna Kumar

2018-03-01

Particulate matter (PM), broadly defined as coarse (2.5-10 μm), fine (0.1-2.5 μm) and ultrafine particles (≤0.1 μm), is a major constituent of ambient air pollution. Recent studies have linked PM exposure (coarse and fine particles) with several human diseases including cancer. However, the molecular mechanisms underlying ultrafine PM exposure induced cellular and sub-cellular repercussions are ill-defined. Since mitochondria are one of the major targets of different environmental pollutants, we herein aimed to understand the molecular repercussion of ultrafine PM exposure on mitochondrial machinery in peripheral blood lymphocytes. Upon comparative analysis, a significantly higher DCF fluorescence was observed in ultrafine PM exposed cells that confirmed the strong pro-oxidant nature of these particles. In addition, the depleted activity of antioxidant enzymes, glutathione reductase and superoxide dismutase suggested the strong association of ultrafine PM with oxidative stress. These results further coincided with mitochondrial membrane depolarization, altered mitochondrial respiratory chain enzyme activity and decline in mtDNA copy number. Moreover, the higher accumulation of DNA damage response proteins (γH2AX, pATM, p-p53), suggested that exposure to ultrafine PM induces DNA damage and triggers phosphatidylinositol 3 kinase mediated response pathway. Further, the alterations in mitochondrial machinery and redox balance among ultrafine PM exposed cells were accompanied by a considerably elevated pro-inflammatory cytokine response. Interestingly, the lower apoptosis levels observed in ultrafine particle treated cells suggest the possibility that the marked alterations may lead to the impairment of mitochondrial-nuclear cross talk. Together, our results showed that ultrafine PM, because of their smaller size possesses significant ability to disturb mitochondrial redox homeostasis and activates phosphatidylinositol 3 kinase mediated DNA damage response

Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death

PubMed Central

Rimmerman, N; Ben-Hail, D; Porat, Z; Juknat, A; Kozela, E; Daniels, M P; Connelly, P S; Leishman, E; Bradshaw, H B; Shoshan-Barmatz, V; Vogel, Z

2013-01-01

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD. PMID:24309936

Aspirin Increases Mitochondrial Fatty Acid Oxidation

PubMed Central

Uppala, Radha; Dudiak, Brianne; Beck, Megan E.; Bharathi, Sivakama S.; Zhang, Yuxun; Stolz, Donna B.; Goetzman, Eric S.

2016-01-01

The metabolic effects of salicylates are poorly understood. This study investigated the effects of aspirin on fatty acid oxidation. Aspirin increased mitochondrial long-chain fatty acid oxidation, but inhibited peroxisomal fatty acid oxidation, in two different cell lines. Aspirin increased mitochondrial protein acetylation and was found to be a stronger acetylating agent in vitro than acetyl-CoA. However, aspirin-induced acetylation did not alter the activity of fatty acid oxidation proteins, and knocking out the mitochondrial deacetylase SIRT3 did not affect the induction of long-chain fatty acid oxidation by aspirin. Aspirin did not change oxidation of medium-chain fatty acids, which can freely traverse the mitochondrial membrane. Together, these data indicate that aspirin does not directly alter mitochondrial matrix fatty acid oxidation enzymes, but most likely exerts its effects at the level of long-chain fatty acid transport into mitochondria. The drive on mitochondrial fatty acid oxidation may be a compensatory response to altered mitochondrial morphology and inhibited electron transport chain function, both of which were observed after 24 hr incubation of cells with aspirin. These studies provide insight into the pathophysiology of Reye Syndrome, which is known to be triggered by aspirin ingestion in patients with fatty acid oxidation disorders. PMID:27856258

Proapoptotic activity of Ukrain is based on Chelidonium majus L. alkaloids and mediated via a mitochondrial death pathway

PubMed Central

Habermehl, Daniel; Kammerer, Bernd; Handrick, René; Eldh, Therese; Gruber, Charlotte; Cordes, Nils; Daniel, Peter T; Plasswilm, Ludwig; Bamberg, Michael; Belka, Claus; Jendrossek, Verena

2006-01-01

Background The anticancer drug Ukrain (NSC-631570) which has been specified by the manufacturer as semisynthetic derivative of the Chelidonium majus L. alkaloid chelidonine and the alkylans thiotepa was reported to exert selective cytotoxic effects on human tumour cell lines in vitro. Few clinical trials suggest beneficial effects in the treatment of human cancer. Aim of the present study was to elucidate the importance of apoptosis induction for the antineoplastic activity of Ukrain, to define the molecular mechanism of its cytotoxic effects and to identify its active constituents by mass spectrometry. Methods Apoptosis induction was analysed in a Jurkat T-lymphoma cell model by fluorescence microscopy (chromatin condensation and nuclear fragmentation), flow cytometry (cellular shrinkage, depolarisation of the mitochondrial membrane potential, caspase-activation) and Western blot analysis (caspase-activation). Composition of Ukrain was analysed by mass spectrometry and LC-MS coupling. Results Ukrain turned out to be a potent inducer of apoptosis. Mechanistic analyses revealed that Ukrain induced depolarisation of the mitochondrial membrane potential and activation of caspases. Lack of caspase-8, expression of cFLIP-L and resistance to death receptor ligand-induced apoptosis failed to inhibit Ukrain-induced apoptosis while lack of FADD caused a delay but not abrogation of Ukrain-induced apoptosis pointing to a death receptor independent signalling pathway. In contrast, the broad spectrum caspase-inhibitor zVAD-fmk blocked Ukrain-induced cell death. Moreover, over-expression of Bcl-2 or Bcl-xL and expression of dominant negative caspase-9 partially reduced Ukrain-induced apoptosis pointing to Bcl-2 controlled mitochondrial signalling events. However, mass spectrometric analysis of Ukrain failed to detect the suggested trimeric chelidonine thiophosphortriamide or putative dimeric or monomeric chelidonine thiophosphortriamide intermediates from chemical synthesis

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.

Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

PubMed

Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer

2016-04-01

Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

The MEF2 gene is essential for yeast longevity, with a dual role in cell respiration and maintenance of mitochondrial membrane potential.

PubMed

Callegari, Sylvie; McKinnon, Ross A; Andrews, Stuart; de Barros Lopes, Miguel A

2011-04-20

The Saccharomyces cerevisiae MEF2 gene is a mitochondrial protein translation factor. Formerly believed to catalyze peptide elongation, evidence now suggests its involvement in ribosome recycling. This study confirms the role of the MEF2 gene for cell respiration and further uncovers a slow growth phenotype and reduced chronological lifespan. Furthermore, in comparison with cytoplasmic ρ(0) strains, mef2Δ strains have a marked reduction of the inner mitochondrial membrane potential and mitochondria show a tendency to aggregate, suggesting an additional role for the MEF2 gene in maintenance of mitochondrial health, a role that may also be shared by other mitochondrial protein synthesis factors. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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.

MFN1 deacetylation activates adaptive mitochondrial fusion and protects metabolically challenged mitochondria.

PubMed

Lee, Joo-Yong; Kapur, Meghan; Li, Ming; Choi, Moon-Chang; Choi, Sujin; Kim, Hak-June; Kim, Inhye; Lee, Eunji; Taylor, J Paul; Yao, Tso-Pang

2014-11-15

Fasting and glucose shortage activate a metabolic switch that shifts more energy production to mitochondria. This metabolic adaptation ensures energy supply, but also elevates the risk of mitochondrial oxidative damage. Here, we present evidence that metabolically challenged mitochondria undergo active fusion to suppress oxidative stress. In response to glucose starvation, mitofusin 1 (MFN1) becomes associated with the protein deacetylase HDAC6. This interaction leads to MFN1 deacetylation and activation, promoting mitochondrial fusion. Deficiency in HDAC6 or MFN1 prevents mitochondrial fusion induced by glucose deprivation. Unexpectedly, failure to undergo fusion does not acutely affect mitochondrial adaptive energy production; instead, it causes excessive production of mitochondrial reactive oxygen species and oxidative damage, a defect suppressed by an acetylation-resistant MFN1 mutant. In mice subjected to fasting, skeletal muscle mitochondria undergo dramatic fusion. Remarkably, fasting-induced mitochondrial fusion is abrogated in HDAC6-knockout mice, resulting in extensive mitochondrial degeneration. These findings show that adaptive mitochondrial fusion protects metabolically challenged mitochondria. © 2014. Published by The Company of Biologists Ltd.

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

miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor

PubMed Central

Tak, Hyosun; Kim, Jihye; Jayabalan, Aravinth Kumar; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Ohn, Takbum; Nam, Suk Woo; Kim, Wook; Lee, Eun Kyung

2014-01-01

Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3′-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF. PMID:25431021