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Sample records for endogenous murine parkin

  1. A Linkage Map of Endogenous Murine Leukemia Proviruses

    PubMed Central

    Frankel, W. N.; Stoye, J. P.; Taylor, B. A.; Coffin, J. M.

    1990-01-01

    Thirty endogenous proviruses belonging to the modified polytropic (Mpmv) class of murine leukemia virus (MLV) were identified by proviral-cellular DNA junction fragment segregation in several sets of recombinant inbred mice. Twenty-six Mpmv loci were mapped to chromosomal regions by matching proviral strain distribution patterns to those of previously assigned genes. Like other endogenous nonecotropic MLVs, Mpmv loci were present on several chromosomes in all strains examined. We pooled recombinant inbred strain linkage data from 110 MLV loci and selected marker genes in order to construct a chromosomal linkage map. Every mouse chromosome was found to harbor at least one proviral insertion, and several regions contained multiple integrations. However, the overall distribution of the 110 mapped proviruses did not deviate significantly from a random distribution. Because of their polymorphism in inbred strains of mice, and the ability to score as many as 57 proviruses per strain using only three hybridization probes, the nonecotropic MLVs mapped in common strains of mice offer a significant advantage over older methods (e.g., biochemical or individual restriction fragment polymorphisms) as genetic markers. These endogenous insertion elements should also be useful for assessing strain purity, and for studying the relatedness of common and not-so-common inbred strains. PMID:2155154

  2. Sequence heterogeneity of murine acquired immunodeficiency syndrome virus: the role of endogenous virus.

    PubMed

    Gayama, S; Vaupel, B A; Kanagawa, O

    1995-05-01

    A defective murine leukemia virus is the causative agent of murine acquired immunodeficiency syndrome (MAIDS). We have cloned cDNAs from both virus infected and non-infected cells using the PCR methods with primers corresponding to the franking sequence of the unique p12 gag gene. Sequence analysis of these cDNA clones revealed: (i) the presence of endogenous virus related to MAIDS virus in C57BL/6 mice, (ii) B cell lineage specific expression of endogenous virus and (iii) extensive heterogeneity of MAIDS virus recovered from virus infected cells due to the recombination of the related viruses (defective pathogenic virus, ecotropic virus and endogenous virus). These findings suggest that the creation of virus variants in infected cells may play an important role in virus pathogenesis and escape from immune attack during the development of MAIDS. PMID:7547712

  3. Structure, distribution, and expression of an ancient murine endogenous retroviruslike DNA family.

    PubMed Central

    Obata, M M; Khan, A S

    1988-01-01

    An endogenous retroviruslike DNA, B-26, was cloned from a BALB/c mouse embryo gene library by using a generalized murine leukemia virus DNA probe. Southern blot hybridization and nucleotide sequence analyses indicated that B-26 DNA might be a novel member of the GLN DNA family (A. Itin and E. Keshet, J. Virol. 59:301-307, 1986) which contains murine leukemia virus-related pol and env sequences. Northern analysis indicated that B-26-related RNAs of 8.4 and 3.0 kilobases were transcribed in thymus, spleen, brain, and liver tissues of 6-week-old BALB/c mice. Images PMID:3172346

  4. Parkin structure and function

    PubMed Central

    Seirafi, Marjan; Kozlov, Guennadi; Gehring, Kalle

    2015-01-01

    Mutations in the parkin or PINK1 genes are the leading cause of the autosomal recessive form of Parkinson’s disease. The gene products, the E3 ubiquitin ligase parkin and the serine/threonine kinase PINK1, are neuroprotective proteins, which act together in a mitochondrial quality control pathway. Here, we review the structure of parkin and mechanisms of its autoinhibition and function as a ubiquitin ligase. We present a model for the recruitment and activation of parkin as a key regulatory step in the clearance of depolarized or damaged mitochondria by autophagy (mitophagy). We conclude with a brief overview of other functions of parkin and considerations for drug discovery in the mitochondrial quality control pathway. PMID:25712550

  5. Parkin Modulates Endosomal Organization and Function of the Endo-Lysosomal Pathway.

    PubMed

    Song, Pingping; Trajkovic, Katarina; Tsunemi, Taiji; Krainc, Dimitri

    2016-02-24

    Mutations in PARK2 (parkin), which encodes Parkin protein, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson's disease (PD). While several studies implicated Parkin in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration upon Parkin loss of function remains incompletely understood. In this study, we found that Parkin modulates the endocytic pathway through the regulation of endosomal structure and function. We showed that loss of Parkin function led to decreased endosomal tubulation and membrane association of vesicle protein sorting 35 (VPS35) and sorting nexin 1 (SNX1), as well as decreased mannose 6 phosphate receptor (M6PR), suggesting the impairment of retromer pathway in Parkin-deficient cells. We also found increased formation of intraluminal vesicles coupled with enhanced release of exosomes in the presence of mutant Parkin. To elucidate the molecular mechanism of these alterations in the endocytic pathway in Parkin-deficient cells, we found that Parkin regulates the levels and activity of Rab7 by promoting its ubiquitination on lysine 38 residue. Both endogenous Rab7 in Parkin-deficient cells and overexpressed K38 R-Rab7 mutant displayed decreased effector binding and membrane association. Furthermore, overexpression of K38R-Rab7 in HEK293 cells phenocopied the increased secretion of exosomes observed in Parkin-deficient cells, suggesting that Rab7 deregulation may be at least partially responsible for the endocytic phenotype observed in Parkin-deficient cells. These findings establish a role for Parkin in regulating the endo-lysosomal pathway and retromer function and raise the possibility that alterations in these pathways contribute to the development of pathology in Parkin-linked Parkinson's disease. PMID:26911690

  6. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.

    PubMed

    Kane, Lesley A; Lazarou, Michael; Fogel, Adam I; Li, Yan; Yamano, Koji; Sarraf, Shireen A; Banerjee, Soojay; Youle, Richard J

    2014-04-28

    PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity. PMID:24751536

  7. Endogenous Murine BST-2/Tetherin Is Not a Major Restriction Factor of Influenza A Virus Infection

    PubMed Central

    Job, Emma R.; Moffat, Jessica M.; Wakim, Linda M.; Gonelli, Christopher A.; Purcell, Damien F. J.; Brooks, Andrew G.; Villadangos, Jose A.; Reading, Patrick C.; Mintern, Justine D.

    2015-01-01

    BST-2 (tetherin, CD317, HM1.24) restricts virus growth by tethering enveloped viruses to the cell surface. The role of BST-2 during influenza A virus infection (IAV) is controversial. Here, we assessed the capacity of endogenous BST-2 to restrict IAV in primary murine cells. IAV infection increased BST-2 surface expression by primary macrophages, but not alveolar epithelial cells (AEC). BST-2-deficient AEC and macrophages displayed no difference in susceptibility to IAV infection relative to wild type cells. Furthermore, BST-2 played little role in infectious IAV release from either AEC or macrophages. To examine BST-2 during IAV infection in vivo, we infected BST-2-deficient mice. No difference in weight loss or in viral loads in the lungs and/or nasal tissues were detected between BST-2-deficient and wild type animals. This study rules out a major role for endogenous BST-2 in modulating IAV in the mouse model of infection. PMID:26566124

  8. Noncanonical MicroRNAs and Endogenous siRNAs in Lytic Infection of Murine Gammaherpesvirus

    PubMed Central

    Xia, Jing; Zhang, Weixiong

    2012-01-01

    MicroRNA (miRNA) and endogenous small interfering RNA (endo-siRNA) are two essential classes of small noncoding RNAs (sncRNAs) in eukaryotes. The class of miRNA is diverse and there exist noncanonical miRNAs that bypass the canonical miRNA biogenesis pathway. In order to identify noncanonical miRNAs and endo-siRNAs responding to virus infection and study their potential function, we sequenced small-RNA species from cells lytically infected with murine gammaherpesvirus 68 (MHV68). In addition to three novel canonical miRNAs in mouse, two antisense miRNAs in virus and 25 novel noncanonical miRNAs, including miRNAs derived from transfer RNAs, small nucleolar RNAs and introns, in the host were identified. These noncanonical miRNAs exhibited features distinct from that of canonical miRNAs in lengths of hairpins, base pairings and first nucleotide preference. Many of the novel miRNAs are conserved in mammals. Besides several known murine endo-siRNAs detected by the sequencing profiling, a novel locus in the mouse genome was identified to produce endo-siRNAs. This novel endo-siRNA locus is comprised of two tandem inverted B4 short interspersed nuclear elements (SINEs). Unexpectedly, the SINE-derived endo-siRNAs were found in a variety of sequencing data and virus-infected cells. Moreover, a murine miRNA was up-regulated more than 35 fold in infected than in mock-treated cells. The putative targets of the viral and the up-regulated murine miRNAs were potentially involved in processes of gene transcription and protein phosphorylation, and localized to membranes, suggesting their potential role in manipulating the host basal immune system during lytic infection. Our results extended the number of noncanonical miRNAs in mammals and shed new light on their potential functions of lytic infection of MHV68. PMID:23110115

  9. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity

    PubMed Central

    Kane, Lesley A.; Lazarou, Michael; Fogel, Adam I.; Li, Yan; Yamano, Koji; Sarraf, Shireen A.; Banerjee, Soojay

    2014-01-01

    PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity. PMID:24751536

  10. Endogenous nitric oxide limits cytokine-induced damage of murine lung epithelial cells.

    PubMed

    Burke-Gaffney, A; Hellewell, P G

    1997-04-01

    This study investigated whether endogenous nitric oxide (NO) limits cytokine-induced damage to the murine lung epithelial cell line LA-4. NO production was assessed as nitrite using the Griess reaction, and cell damage was assessed using ethidium homodimer-1. Cytotoxicity was first detected after a 24-h incubation with a combination of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma (cytomix). Nitrite production increased to 78.0 +/- 0.5 nmol/10(6) cells at 24 h. Coincubation of LA-4 with cytomix and NO synthase inhibitors, aminoguanidine (3-1,000 microM) and N(G)-monomethyl-L-arginine (10-1,000 microM), but not N(G)-monomethyl-D-arginine, or a soluble guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one, reduced cytomix-induced nitrite production and increased cytotoxicity up to twofold (24 h). Removal of L-arginine from the medium increased damage; reintroduction of 1,000 microM L-arginine, but not D-arginine, reversed this. In aminoguanidine-treated cells, replacement of NO with an NO donor, S-nitrosoglutathione (30 microM), reversed, in part, the cell damage observed in aminoguanidine/cytomix-treated cells. These results suggest that endogenous NO limits cytokine-induced lung epithelial damage. PMID:9142945

  11. Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

    PubMed Central

    Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2011-01-01

    Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

  12. Intraventricular injections of mesenchymal stem cells activate endogenous functional remyelination in a chronic demyelinating murine model

    PubMed Central

    Cruz-Martinez, P; González-Granero, S; Molina-Navarro, M M; Pacheco-Torres, J; García-Verdugo, J M; Geijo-Barrientos, E; Jones, J; Martinez, S

    2016-01-01

    Current treatments for demyelinating diseases are generally only capable of ameliorating the symptoms, with little to no effect in decreasing myelin loss nor promoting functional recovery. Mesenchymal stem cells (MSCs) have been shown by many researchers to be a potential therapeutic tool in treating various neurodegenerative diseases, including demyelinating disorders. However, in the majority of the cases, the effect was only observed locally, in the area surrounding the graft. Thus, in order to achieve general remyelination in various brain structures simultaneously, bone marrow-derived MSCs were transplanted into the lateral ventricles (LVs) of the cuprizone murine model. In this manner, the cells may secrete soluble factors into the cerebrospinal fluid (CSF) and boost the endogenous oligodendrogenic potential of the subventricular zone (SVZ). As a result, oligodendrocyte progenitor cells (OPCs) were recruited within the corpus callosum (CC) over time, correlating with an increased myelin content. Electrophysiological studies, together with electron microscopy (EM) analysis, indicated that the newly formed myelin correctly enveloped the demyelinated axons and increased signal transduction through the CC. Moreover, increased neural stem progenitor cell (NSPC) proliferation was observed in the SVZ, possibly due to the tropic factors released by the MSCs. In conclusion, the findings of this study revealed that intraventricular injections of MSCs is a feasible method to elicit a paracrine effect in the oligodendrogenic niche of the SVZ, which is prone to respond to the factors secreted into the CSF and therefore promoting oligodendrogenesis and functional remyelination. PMID:27171265

  13. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    SciTech Connect

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D. )

    1991-05-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.

  14. Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation

    PubMed Central

    Xiong, Hui; Wang, Danling; Chen, Linan; Choo, Yeun Su; Ma, Hong; Tang, Chengyuan; Xia, Kun; Jiang, Wei; Ronai, Ze’ev; Zhuang, Xiaoxi; Zhang, Zhuohua

    2009-01-01

    Mutations in PARKIN, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of Parkinson disease (PD). Although mutations in these genes lead to the same disease state, the functional relationships between them and how their respective disease-associated mutations cause PD are largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed PPD complex) to promote ubiquitination and degradation of Parkin substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human brain lysates. Genetic ablation of either Pink1 or Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well as decreased degradation and increased accumulation of aberrantly expressed Parkin substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat shock–induced misfolded protein. In contrast, PD-pathogenic Parkin and PINK1 mutations showed reduced ability to promote degradation of Parkin substrates. This study identified a functional ubiquitin E3 ligase complex consisting of PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of the complex, which may constitute a mechanism underlying PD pathogenesis. PMID:19229105

  15. The Role of Parkin in the Differential Susceptibility of Tuberoinfundibular and Nigrostriatal Dopamine Neurons to Acute Toxicant Exposure

    PubMed Central

    Benskey, Matthew J.; Manfredsson, Fredric P.; Lookingland, Keith J.; Goudreau, John L.

    2014-01-01

    Parkinson Disease causes degeneration of nigrostriatal dopamine (DA) neurons, while tuberoinfundibular DA neurons remain unaffected. A similar pattern is observed following exposure to 1-methy-4-phenyl-1, 2, 3, 6-tetrahydropyradine (MPTP). The mechanism of tuberoinfundibular neuronal recovery from MPTP is associated with up-regulation of parkin protein. Here we tested if parkin mediates tuberoinfundibular neuronal recovery from MPTP by knocking-down parkin in tuberoinfundibular neurons using recombinant adeno-associated virus (rAAV), expressing a short hairpin RNA (shRNA) directed toward parkin. Following knockdown, axon terminal DA and tyrosine hydroxylase (TH) concentrations were analyzed 24 hours post-MPTP administration. rAAV-shRNA-mediated knockdown of endogenous parkin rendered tuberoinfundibular neurons susceptible to MPTP induced terminal DA loss, but not TH loss, within 24 hours post-MPTP. To determine if the neuroprotective benefits of parkin up-regulation could be translated to nigrostriatal neurons, rAAV expressing human parkin was injected into the substantia nigra of mice and axon terminal DA and TH concentrations were analyzed 24 hours post-MPTP. Nigral parkin over-expression prevented loss of TH in the axon terminals and soma of nigrostriatal neurons, but had no effect on terminal DA loss within 24h post-MPTP. These data show that parkin is necessary for the recovery of terminal DA concentrations within tuberoinfundibular neurons following acute MPTP administration, and parkin can rescue MPTP-induced decreases in TH within nigrostriatal neurons. PMID:25447324

  16. Parkin Regulation and Neurodegenerative Disorders

    PubMed Central

    Zhang, Cheng-Wu; Hang, Liting; Yao, Tso-Pang; Lim, Kah-Leong

    2016-01-01

    Parkin is a unique, multifunctional ubiquitin ligase whose various roles in the cell, particularly in neurons, are widely thought to be protective. The pivotal role that Parkin plays in maintaining neuronal survival is underscored by our current recognition that Parkin dysfunction represents not only a predominant cause of familial parkinsonism but also a formal risk factor for the more common, sporadic form of Parkinson’s disease (PD). Accordingly, keen research on Parkin over the past decade has led to an explosion of knowledge regarding its physiological roles and its relevance to PD. However, our understanding of Parkin is far from being complete. Indeed, surprises emerge from time to time that compel us to constantly update the paradigm of Parkin function. For example, we now know that Parkin’s function is not confined to mere housekeeping protein quality control (QC) roles but also includes mitochondrial homeostasis and stress-related signaling. Furthermore, emerging evidence also suggest a role for Parkin in several other major neurodegenerative diseases including Alzheimer’s disease (AD) and Amyotrophic Lateral Sclerosis (ALS). Yet, it remains truly amazing to note that a single enzyme could serve such multitude of functions and cellular roles. Clearly, its activity has to be tightly regulated. In this review, we shall discuss this and how dysregulated Parkin function may precipitate neuronal demise in various neurodegenerative disorders. PMID:26793099

  17. Central Parkin: The evolving role of Parkin in the heart.

    PubMed

    Dorn, Gerald W

    2016-08-01

    Parkin is familiar to many because of its link to Parkinson's disease, and to others because of its well-characterized role as a central factor mediating selective mitophagy of damaged mitochondria for mitochondrial quality control. The genetic connection between Parkin and Parkinson's disease derives from clinical gene-association studies, whereas our mechanistic understanding of Parkin functioning in mitophagy is based almost entirely on work performed in cultured cells. Surprisingly, experimental evidence linking the disease and the presumed mechanism derives almost entirely from fruit flies; germline Parkin deficient mice do not develop Parkinson's disease phenotypes. Moreover, genetic manipulation of Parkin signaling in mouse hearts does not support a central role for Parkin in homeostatic mitochondrial quality control in this mitochondria-rich and -dependent organ. Here, I provide an overview of data suggesting that (in mouse hearts at least) Parkin functions more as a stress-induced and developmentally-programmed facilitator of cardiomyocyte mitochondrial turnover. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016. PMID:26992930

  18. Generation of mink cell focus-forming viruses by Friend murine leukemia virus: recombination with specific endogenous proviral sequences.

    PubMed Central

    Evans, L H; Cloyd, M W

    1984-01-01

    A family of recombinant mink cell focus-forming viruses (MCF) was derived by inoculation of NFS mice with a Friend murine leukemia virus, and their genomes were analyzed by RNase T1-resistant oligonucleotide fingerprinting. The viruses were obtained from the thymuses and spleens of preleukemic and leukemic animals and were evaluated for dualtropism and oncogenicity. All these isolates induced cytopathic foci on mink cells but could be classified into two groups based on their relative infectivities for SC-1 (mouse) or mink (ATCC CCL64) cells. One group of Friend MCFs (F-MCFs) (group I) exhibited approximately equal infectivities for SC-1 and mink cells, whereas a second group (group II) infected mink cells 1,000- to 10,000-fold more efficiently than SC-1 cells. Structural analyses of the F-MCFs revealed that group I and group II viruses correlated with recombination of Friend murine leukemia virus with two distinct, but closely related, endogenous NFS proviral sequences. No correlation was found between the type of F-MCF and the tissue of origin or the disease state of the animal. Furthermore, none of the F-MCF isolates were found to be oncogenic in NFS/N or AKR/J mice. F-MCFs of both groups underwent extensive substitution of ecotropic sequences, involving much of the gag and env genes of group I F-MCFs and most of the gag, pol, and env genes of group II F-MCFs. All F-MCF isolates retained the 3' terminal U3 region of Friend murine leukemia virus. Comparison of the RNAs of the F-MCFs with RNAs of MCFs derived from NFS.Akv-1 or NFS.Akv-2 mice indicated that the F-MCFs were derived from NFS proviral sequences which are distinct from the sequences contained in NFS.Akv MCF isolates. This result suggested that recombination with particular endogenous proviral sequences to generate MCFs may be highly specific for a given murine leukemia virus. Images PMID:6422051

  19. Angiotensin II-induced hypertrophy of cultured murine proximal tubular cells is mediated by endogenous transforming growth factor-beta.

    PubMed Central

    Wolf, G; Mueller, E; Stahl, R A; Ziyadeh, F N

    1993-01-01

    Previous studies by our group have demonstrated that angiotensin II (ANG II), as a single factor in serum-free medium, induces cellular hypertrophy of a cultured murine proximal tubular cell line (MCT). The present study was performed to test the hypothesis that this growth effect was mediated by activation of endogenous transforming growth factor-beta (TGF-beta). Exogenous TGF-beta 1 (1 ng/ml) mimicked the growth effects observed with 10(-8) M ANG II (inhibition of DNA synthesis and induction of cellular hypertrophy). A neutralizing anti-TGF-beta antibody attenuated the ANG II-induced increase in de novo protein and total RNA synthesis as well as total protein content. This antibody also abolished the ANG II-mediated inhibition of [3H]thymidine incorporation into quiescent MCT cells. Control IgG or an unrelated antibody had no effect. A bioassay for TGF-beta using mink lung epithelial cells revealed that MCT cells treated with ANG II released active TGF-beta into the cell culture supernatant. Northern blot analysis and semi-quantitative cDNA amplification demonstrated increases in steady-state levels for TGF-beta 1 mRNA after ANG II stimulation of MCT cells, but not in a syngeneic murine mesangial cell line. Our data indicate that the ANG II-induced hypertrophy in MCT cells is mediated by synthesis and activation of endogenous TGF-beta. It is intriguing to speculate that TGF-beta may play a role in the early tubular cell hypertrophy and the subsequent interstitial scarring observed in several models of chronic renal injury that are characterized by increased activity of intrarenal ANG II. Images PMID:7690779

  20. Regulation of DNA repair by parkin

    SciTech Connect

    Kao, Shyan-Yuan

    2009-05-01

    Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant. Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.

  1. Identification of BC005512 as a DNA Damage Responsive Murine Endogenous Retrovirus of GLN Family Involved in Cell Growth Regulation

    PubMed Central

    Wu, Yuanfeng; Qi, Xinming; Gong, Likun; Xing, Guozhen; Chen, Min; Miao, Lingling; Yao, Jun; Suzuki, Takayoshi; Furihata, Chie; Luan, Yang; Ren, Jin

    2012-01-01

    Genotoxicity assessment is of great significance in drug safety evaluation, and microarray is a useful tool widely used to identify genotoxic stress responsive genes. In the present work, by using oligonucleotide microarray in an in vivo model, we identified an unknown gene BC005512 (abbreviated as BC, official full name: cDNA sequence BC005512), whose expression in mouse liver was specifically induced by seven well-known genotoxins (GTXs), but not by non-genotoxins (NGTXs). Bioinformatics revealed that BC was a member of the GLN family of murine endogenous retrovirus (ERV). However, the relationship to genotoxicity and the cellular function of GLN are largely unknown. Using NIH/3T3 cells as an in vitro model system and quantitative real-time PCR, BC expression was specifically induced by another seven GTXs, covering diverse genotoxicity mechanisms. Additionally, dose-response and linear regression analysis showed that expression level of BC in NIH/3T3 cells strongly correlated with DNA damage, measured using the alkaline comet assay,. While in p53 deficient L5178Y cells, GTXs could not induce BC expression. Further functional studies using RNA interference revealed that down-regulation of BC expression induced G1/S phase arrest, inhibited cell proliferation and thus suppressed cell growth in NIH/3T3 cells. Together, our results provide the first evidence that BC005512, a member from GLN family of murine ERV, was responsive to DNA damage and involved in cell growth regulation. These findings could be of great value in genotoxicity predictions and contribute to a deeper understanding of GLN biological functions. PMID:22514700

  2. Cure of murine thalassemia by bone marrow transplantation without eradication of endogenous stem cells

    SciTech Connect

    Wagemaker, G.; Visser, T.P.; van Bekkum, D.W.

    1986-09-01

    alpha-Thalassemic heterozygous (Hbath/+) mice were used to investigate the possible selective advantage of transplanted normal (+/+) hemopoietic cells. Without conditioning by total-body irradiation (TBI), infusion of large numbers of normal bone marrow cells failed to correct the thalassemic peripheral blood phenotype. Since the recipients' stem cells are normal with respect to number and differentiation capacity, it was thought that the transplanted stem cells were not able to lodge, or that they were not stimulated to proliferate. Therefore, a nonlethal dose of TBI was given to temporarily reduce endogenous stem cell numbers and hemopoiesis. TBI doses of 2 or 3 Gy followed by infusion of normal bone marrow cells proved to be effective in replacing the thalassemic red cells by normal red cells, whereas a dose of 1 Gy was ineffective. It is concluded that cure of thalassemia by bone marrow transplantation does not necessarily require eradication of thalassemic stem cells. Consequently, the objectives of conditioning regimens for bone marrow transplantation of thalassemic patients (and possibly other nonmalignant hemopoietic disorders) should be reconsidered.

  3. PINK1-Mediated Phosphorylation of Parkin Boosts Parkin Activity in Drosophila

    PubMed Central

    Shiba-Fukushima, Kahori; Inoshita, Tsuyoshi; Hattori, Nobutaka; Imai, Yuzuru

    2014-01-01

    Two genes linked to early onset Parkinson's disease, PINK1 and Parkin, encode a protein kinase and a ubiquitin-ligase, respectively. Both enzymes have been suggested to support mitochondrial quality control. We have reported that Parkin is phosphorylated at Ser65 within the ubiquitin-like domain by PINK1 in mammalian cultured cells. However, it remains unclear whether Parkin phosphorylation is involved in mitochondrial maintenance and activity of dopaminergic neurons in vivo. Here, we examined the effects of Parkin phosphorylation in Drosophila, in which the phosphorylation residue is conserved at Ser94. Morphological changes of mitochondria caused by the ectopic expression of wild-type Parkin in muscle tissue and brain dopaminergic neurons disappeared in the absence of PINK1. In contrast, phosphomimetic Parkin accelerated mitochondrial fragmentation or aggregation and the degradation of mitochondrial proteins regardless of PINK1 activity, suggesting that the phosphorylation of Parkin boosts its ubiquitin-ligase activity. A non-phosphorylated form of Parkin fully rescued the muscular mitochondrial degeneration due to the loss of PINK1 activity, whereas the introduction of the non-phosphorylated Parkin mutant in Parkin-null flies led to the emergence of abnormally fused mitochondria in the muscle tissue. Manipulating the Parkin phosphorylation status affected spontaneous dopamine release in the nerve terminals of dopaminergic neurons, the survivability of dopaminergic neurons and flight activity. Our data reveal that Parkin phosphorylation regulates not only mitochondrial function but also the neuronal activity of dopaminergic neurons in vivo, suggesting that the appropriate regulation of Parkin phosphorylation is important for muscular and dopaminergic functions. PMID:24901221

  4. BAC Transgenic Mice Expressing a Truncated Mutant Parkin Exhibit Age-dependent Hypokinetic Motor Deficits, Dopaminergic Neuron Degeneration, and Accumulation of Proteinase K-Resistant Alpha-Synuclein

    PubMed Central

    Lu, Xiao-Hong; Fleming, Sheila M.; Meurers, Bernhard; Ackerson, Larry C.; Mortazavi, Farzad; Lo, Victor; Hernandez, Daniela; Sulzer, David; Jackson, George R.; Maidment, Nigel T.; Chesselet, Marie-Francoise; Yang, X. William

    2009-01-01

    Summary Recessive mutations in parkin are the most common cause of familial early onset Parkinson's disease (PD). Recent studies suggest that certain parkin mutants may exert dominant toxic effects to cultured cells and such dominant toxicity can lead to progressive dopaminergic (DA) neuron degeneration in Drosophila. To explore whether mutant parkin could exert similar pathogenic effects to mammalian DA neurons in vivo, we developed a Bacterial Artificial Chromosome (BAC) transgenic mouse model expressing a C-terminal truncated human mutant parkin (Parkin-Q311X) in DA neurons driven by a dopamine transporter promoter. Parkin-Q311X mice exhibit multiple late-onset and progressive hypokinetic motor deficits. Stereological analyses reveal that the mutant mice develop age-dependent DA neuron degeneration in substantia nigra accompanied by a significant loss of DA neuron terminals in the striatum. Neurochemical analyses reveal a significant reduction of the striatal dopamine level in mutant mice, which is significantly correlated with their hypokinetic motor deficits. Finally, mutant Parkin-Q311X mice, but not wild-type controls, exhibit age-dependent accumulation of proteinase-K resistant endogenous α-synuclein in substantia nigra and co-localized with 3-nitrotyrosine, a marker for oxidative protein damage. Hence, our study provides the first mammalian genetic evidence that dominant toxicity of a parkin mutant is sufficient to elicit age-dependent hypokinetic motor deficits and DA neuron loss in vivo, and uncovers a causal relationship between dominant parkin toxicity and progressive α-synuclein accumulation in DA neurons. Our study underscores the need to further explore the putative link between parkin dominant toxicity and PD. PMID:19228951

  5. Parkin Somatic Mutations Link Melanoma and Parkinson's Disease.

    PubMed

    Levin, Lotan; Srour, Shani; Gartner, Jared; Kapitansky, Oxana; Qutob, Nouar; Dror, Shani; Golan, Tamar; Dayan, Roy; Brener, Ronen; Ziv, Tamar; Khaled, Mehdi; Schueler-Furman, Ora; Samuels, Yardena; Levy, Carmit

    2016-06-20

    Epidemiological studies suggest a direct link between melanoma and Parkinson's disease (PD); however, the underlying molecular basis is unknown. Since mutations in Parkin are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in 3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional protein association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson's disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases. PMID:27297116

  6. Parkin suppresses Drp1-independent mitochondrial division.

    PubMed

    Roy, Madhuparna; Itoh, Kie; Iijima, Miho; Sesaki, Hiromi

    2016-07-01

    The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson's disease-associated protein-parkin, which biochemically and genetically interacts with Drp1-in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division. PMID:27181353

  7. Parkin-dependent mitophagy in the heart.

    PubMed

    Dorn, Gerald W

    2016-06-01

    Mitochondria can undergo autophagic elimination for differing reasons, e.g. as part of a cell-wide macroautophagic response, as part of mitochondrial turnover during metabolic remodeling, or in the case of selective mitophagic destruction of dysfunctional mitochondria, during mitochondrial quality control. Multiple mechanistically distinct pathways converge upon, and activate, mitochondrial autophagy. Here, the evidence supporting a role for the prototypical mitochondrial quality control pathway, PINK1-Parkin mediated mitophagy, in cardiac homeostasis and heart disease is reviewed. Contrary to popular wisdom based on findings from non-cardiac systems, current data do not support a major role for Parkin-mediated mitophagy as a mechanism for constitutive mitochondrial housekeeping, and instead suggest that this pathway primarily functions in adult hearts as an inducible cardiac stress-response mechanism. Recent findings have also uncovered an unsuspected role for Parkin-mediated mitochondrial turnover in the normal perinatal transformation of myocardial metabolism. PMID:26611886

  8. Differential effects of endogenous and exogenous interferon-gamma on immunoglobulin E, cellular infiltration, and airway responsiveness in a murine model of allergic asthma.

    PubMed

    Hofstra, C L; Van Ark, I; Hofman, G; Nijkamp, F P; Jardieu, P M; Van Oosterhout, A J

    1998-11-01

    The inflammatory response as seen in human allergic asthma is thought to be regulated by Th2 cells. It has been shown that interferon-gamma (IFN-gamma) can downregulate the proliferation of Th2 cells and therefore might be of therapeutic use. In the present study we have investigated the in vivo role of endogenous and exogenous IFN-gamma in a murine model with features reminiscent of human allergic asthma. IFN-gamma gene knockout (GKO) and wild-type mice were sensitized with ovalbumin and exposed to repeated ovalbumin aerosol challenges. In addition, wild-type mice were treated with intraperitoneal or nebulized recombinant murine IFN-gamma during the challenge period. Sensitized wild-type mice exhibited upregulated ovalbumin-specific IgE in serum, and airway hyperresponsiveness and infiltration of eosinophils and mononuclear cells in the bronchoalveolar lavage fluid (BALF) after ovalbumin challenge. In contrast, in GKO mice only reduced eosinophilic infiltration in the BALF was observed after ovalbumin challenge. In wild-type mice, parenteral IFN-gamma treatment downregulated ovalbumin-specific IgE levels in serum, and airway hyperresponsiveness and cellular infiltration in the BALF, whereas aerosolized IFN-gamma treatment only suppressed airway hyperresponsiveness. In vitro experiments showed that these effects of IFN-gamma appear not to be mediated via a direct effect on the cytokine production of antigen-specific Th2 cells. These data indicate that airway hyperresponsiveness can be downregulated by IFN-gamma locally in the airways, whereas for downregulation of IgE and cellular infiltration systemic IFN-gamma is needed. The present study shows that exogenous IFN-gamma can downregulate the allergic response via an antigen-specific T-cell independent mechanism, but at the same time endogenous IFN-gamma plays a role in an optimal response. PMID:9806748

  9. Mechanism of phospho-ubiquitin-induced PARKIN activation.

    PubMed

    Wauer, Tobias; Simicek, Michal; Schubert, Alexander; Komander, David

    2015-08-20

    The E3 ubiquitin ligase PARKIN (encoded by PARK2) and the protein kinase PINK1 (encoded by PARK6) are mutated in autosomal-recessive juvenile Parkinsonism (AR-JP) and work together in the disposal of damaged mitochondria by mitophagy. PINK1 is stabilized on the outside of depolarized mitochondria and phosphorylates polyubiquitin as well as the PARKIN ubiquitin-like (Ubl) domain. These phosphorylation events lead to PARKIN recruitment to mitochondria, and activation by an unknown allosteric mechanism. Here we present the crystal structure of Pediculus humanus PARKIN in complex with Ser65-phosphorylated ubiquitin (phosphoUb), revealing the molecular basis for PARKIN recruitment and activation. The phosphoUb binding site on PARKIN comprises a conserved phosphate pocket and harbours residues mutated in patients with AR-JP. PhosphoUb binding leads to straightening of a helix in the RING1 domain, and the resulting conformational changes release the Ubl domain from the PARKIN core; this activates PARKIN. Moreover, phosphoUb-mediated Ubl release enhances Ubl phosphorylation by PINK1, leading to conformational changes within the Ubl domain and stabilization of an open, active conformation of PARKIN. We redefine the role of the Ubl domain not only as an inhibitory but also as an activating element that is restrained in inactive PARKIN and released by phosphoUb. Our work opens up new avenues to identify small-molecule PARKIN activators. PMID:26161729

  10. Mechanism of phospho-ubiquitin induced PARKIN activation

    PubMed Central

    Wauer, Tobias; Simicek, Michal; Schubert, Alexander; Komander, David

    2016-01-01

    Summary The E3 ubiquitin ligase PARKIN (encoded by PARK2) and the protein kinase PINK1 (encoded by PARK6) are mutated in autosomal recessive juvenile Parkinsonism (AR-JP) and work together in the disposal of damaged mitochondria by mitophagy1–3. PINK1 is stabilised on the outside of depolarised mitochondria, and phosphorylates poly-ubiquitin (polyUb)4–8 as well as the PARKIN Ub-like (Ubl) domain9,10. These phosphorylation events lead to PARKIN recruitment to mitochondria, and activation by an unknown allosteric mechanism4–12. Here we present the crystal structure of Pediculus humanus PARKIN in complex with Ser65-phosphorylated ubiquitin (phosphoUb), revealing the molecular basis for PARKIN recruitment and activation. The phosphoUb binding site on PARKIN comprises a conserved phosphate pocket and harbours residues mutated in AR-JP patients. PhosphoUb binding leads to straightening of a helix in the RING1 domain, and the resulting conformational changes release the Ubl domain from the PARKIN core; this activates PARKIN. Moreover, phosphoUb-mediated Ubl release enhances Ubl phosphorylation by PINK1, leading to conformational changes within the Ubl domain and stabilisation of an open, active conformation of PARKIN. We redefine the role of the Ubl domain not only as an inhibitory13 but also as an activating element that is restrained in inactive PARKIN and released by phosphoUb. Our work opens new avenues to identify small molecule PARKIN activators. PMID:26161729

  11. A Ubl/ubiquitin switch in the activation of Parkin

    PubMed Central

    Sauvé, Véronique; Lilov, Asparouh; Seirafi, Marjan; Vranas, Marta; Rasool, Shafqat; Kozlov, Guennadi; Sprules, Tara; Wang, Jimin; Trempe, Jean-François; Gehring, Kalle

    2015-01-01

    Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86–130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding. PMID:26254305

  12. A Ubl/ubiquitin switch in the activation of Parkin.

    PubMed

    Sauvé, Véronique; Lilov, Asparouh; Seirafi, Marjan; Vranas, Marta; Rasool, Shafqat; Kozlov, Guennadi; Sprules, Tara; Wang, Jimin; Trempe, Jean-François; Gehring, Kalle

    2015-10-14

    Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86-130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding. PMID:26254305

  13. Biochemical characterization of endogenous type C virus information in differentiated and undifferentiated murine teratocarcinoma-derived cell lines.

    PubMed Central

    Emanoil-Ravicovitch, R; Hojman-Montes-de-Oca, F; Robert, J; Garcette, M; Callahan, R; Peries, J; Boiron, M

    1980-01-01

    Undifferentiated teratocarcinoma cells express sixfold-higher levels of endogenous xenotropic type C virus-related RNA than differentiated cells. Three species of polyadenylated viral RNA (35S, 24S, and 14S) have been identified in the undifferentiated teratocarcinoma cells. Paradoxically, neither viral particles nor viral proteins have been detected in these cells. PMID:6246284

  14. Pathologic and Therapeutic Implications for the Cell Biology of Parkin

    PubMed Central

    Charan, Rakshita A.; LaVoie, Matthew J.

    2015-01-01

    Mutations in the E3 ligase parkin are the most common cause of autosomal recessive Parkinson's disease (PD), but it is believed that parkin dysfunction may also contribute to idiopathic PD. Since its discovery, parkin has been implicated in supporting multiple neuroprotective pathways, many revolving around the maintenance of mitochondrial health quality control and governance of cell survival. Recent advances across the structure, biochemistry, and cell biology of parkin have provided great insights into the etiology of parkin-linked and idiopathic PD and may ultimately generate novel therapeutic strategies to slow or halt disease progression. This review describes the various pathways in which parkin acts and the mechanisms by which parkin may be targeted for therapeutic intervention. PMID:25697646

  15. Parkin Regulates the Activity of Pyruvate Kinase M2.

    PubMed

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-05-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

  16. Parkin Regulates the Activity of Pyruvate Kinase M2*

    PubMed Central

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-01-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

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

    PubMed Central

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

    2012-01-01

    Summary 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 re-import of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation. PMID:22280891

  18. 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. PMID:22280891

  19. Dual Function of Phosphoubiquitin in E3 Activation of Parkin.

    PubMed

    Walinda, Erik; Morimoto, Daichi; Sugase, Kenji; Shirakawa, Masahiro

    2016-08-01

    Mutations in the gene encoding parkin, an auto-inhibited E3 ubiquitin ligase that functions in the clearance of damaged mitochondria, are the most common cause of autosomal recessive juvenile Parkinsonism. The mechanism regulating parkin activation remains poorly understood. Here we show, by using isothermal titration calorimetry, solution NMR, and fluorescence spectroscopy, that parkin can bind ubiquitin and phosphomimetic ubiquitin by recognizing the canonical hydrophobic patch and C terminus of ubiquitin. The affinity of parkin for both phosphomimetic and unmodified ubiquitin is markedly enhanced upon removal of the ubiquitin-like (UBL) domain of parkin. This suggests that the agonistic binding of ubiquitin to parkin in trans is counterbalanced by the antagonistic activity of the parkin UBL domain in cis Intriguingly, UBL binding is enthalpy-driven, whereas ubiquitin binding is driven by an increase in the total entropy of the system. These thermodynamic differences are explained by different chemistry in the ubiquitin- and UBL-binding pockets of parkin and, as shown by molecular dynamics simulations, are not a consequence of changes in protein conformational entropy. Indeed, comparison of conformational fluctuations reveals that the RING1-IBR element becomes considerably more rigid upon complex formation. A model of parkin activation is proposed in which E2∼Ub binding triggers large scale diffusional motion of the RING2 domain toward the ubiquitin-stabilized RING1-IBR assembly to complete formation of the active parkin-E2∼Ub transfer complex. Thus, ubiquitin plays a dual role in parkin activation by competing with the inhibitory UBL domain and stabilizing the active form of parkin. PMID:27284007

  20. Parkin deletion causes cerebral and systemic amyloidosis in human mutated tau over-expressing mice.

    PubMed

    Rodríguez-Navarro, Jose A; Gómez, Ana; Rodal, Izaskun; Perucho, Juan; Martinez, Armando; Furió, Vicente; Ampuero, Israel; Casarejos, María J; Solano, Rosa M; de Yébenes, Justo García; Mena, Maria A

    2008-10-15

    Deposition of proteins leading to amyloid takes place in some neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. Mutations of tau and parkin proteins produce neurofibrillary abnormalities without deposition of amyloid. Here we report that mature, parkin null, over-expressing human mutated tau (PK(-/-)/Tau(VLW)) mice have altered behaviour and dopamine neurotransmission, tau pathology in brain and amyloid deposition in brain and peripheral organs. PK(-/-)/Tau(VLW) mice have abnormal behaviour and severe drop out of dopamine neurons in the ventral midbrain, up to 70%, at 12 months and abundant phosphorylated tau positive neuritic plaques, neuro-fibrillary tangles, astrogliosis, microgliosis and plaques of murine beta-amyloid in the hippocampus. PK(-/-)/Tau(VLW) mice have organomegaly of the liver, spleen and kidneys. The electron microscopy of the liver confirmed the presence of a fibrillary protein deposits with amyloid characteristics. There is also accumulation of mouse tau in hepatocytes. These mice have lower levels of CHIP-HSP70, involved in the proteosomal degradation of tau, increased oxidative stress, measured as depletion of glutathione which, added to lack of parkin, could trigger tau accumulation and amyloidogenesis. This model is the first that demonstrates beta-amyloid deposits caused by over-expression of tau and without modification of the amyloid precursor protein, presenilins or secretases. PK(-/-)/Tau(VLW) mice provide a link between the two proteins more important for the pathogenesis of Alzheimer disease. PMID:18640988

  1. A model of secreting murine mammary epithelial HC11 cells comprising endogenous Bcrp/Abcg2 expression and function.

    PubMed

    Tallkvist, Jonas; Yagdiran, Yagmur; Danielsson, Louise; Oskarsson, Agneta

    2015-04-01

    Breast cancer resistance protein (Bcrp/Abcg2) and multidrug transporter 1 (Mdr1/Abcb1) are efflux proteins located in the apical membrane of mammary epithelial cells (MEC). Bcrp is induced in MEC during gestation and lactation, while Mdr1 is down-regulated during lactation. Numerous drugs and toxic compounds are known to be actively secreted into milk by Bcrp, but most chemicals have not been investigated in this respect, emphasizing the need for functional Bcrp studies in an established cell line with secreting mammary epithelial cells. The present study was undertaken to examine expressions of Bcrp and Mdr1 in mammary epithelial HC11 cells, derived from a mid-gestational murine mammary gland. In addition, Bcrp function was assessed by transport experiments with mitoxantrone (MX) in undifferentiated HC11 cells, in HC11 cells subjected to Bcrp RNA interference (RNAi), as well as in HC11 cells stimulated to differentiate by treatment with lactogenic hormones. Differentiated HC11 cells organized into alveolar-resembling structures and gene expression of the major milk protein β-casein was induced, whereas undifferentiated cells formed monolayers with lower β-casein expression. Bcrp and Mdr1 gene and protein were expressed in both undifferentiated and differentiated HC11 cells. Differentiation of HC11 cells resulted in increased Bcrp protein expression, while Mdr1 gene and protein expressions were reduced. The Bcrp inhibitor elacridar (GF120918) reduced secretion and increased accumulation of MX in both undifferentiated and differentiated HC11 cells. Silencing of the Bcrp gene caused an increased accumulation of MX. The results indicate that the HC11 cell model provides a promising tool to investigate transport of potential Bcrp substrates in mammary epithelial cells. PMID:25791223

  2. Restriction by APOBEC3 proteins of endogenous retroviruses with an extracellular life cycle: ex vivo effects and in vivo "traces" on the murine IAPE and human HERV-K elements

    PubMed Central

    Esnault, Cécile; Priet, Stéphane; Ribet, David; Heidmann, Odile; Heidmann, Thierry

    2008-01-01

    Background APOBEC3 cytosine deaminases have been demonstrated to restrict infectivity of a series of retroviruses, with different efficiencies depending on the retrovirus. In addition, APOBEC3 proteins can severely restrict the intracellular transposition of a series of retroelements with a strictly intracellular life cycle, including the murine IAP and MusD LTR-retrotransposons. Results Here we show that the IAPE element, which is the infectious progenitor of the strictly intracellular IAP elements, and the infectious human endogenous retrovirus HERV-K are restricted by both murine and human APOBEC3 proteins in an ex vivo assay for infectivity, with evidence in most cases of strand-specific G-to-A editing of the proviruses, with the expected signatures. In silico analysis of the naturally occurring genomic copies of the corresponding endogenous elements performed on the mouse and human genomes discloses "traces" of APOBEC3-editing, with the specific signature of the murine APOBEC3 and human APOBEC3G enzymes, respectively, and to a variable extent depending on the family member. Conclusion These results indicate that the IAPE and HERV-K elements, which can only replicate via an extracellular infection cycle, have been restricted at the time of their entry, amplification and integration into their target host genomes by definite APOBEC3 proteins, most probably acting in evolution to limit the mutagenic effect of these endogenized extracellular parasites. PMID:18702815

  3. Identification and Molecular Characterization of Parkin in Clonorchis sinensis

    PubMed Central

    Bai, Xuelian; Kim, Tae Im; Lee, Ji-Yun; Dai, Fuhong; Hong, Sung-Jong

    2015-01-01

    Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis. PMID:25748711

  4. Identification and molecular characterization of Parkin in Clonorchis sinensis.

    PubMed

    Bai, Xuelian; Kim, Tae Im; Lee, Ji-Yun; Dai, Fuhong; Hong, Sung-Jong

    2015-02-01

    Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn(2+) were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis. PMID:25748711

  5. Parkin protein deficiency exacerbates cardiac injury and reduces survival following myocardial infarction.

    PubMed

    Kubli, Dieter A; Zhang, Xiaoxue; Lee, Youngil; Hanna, Rita A; Quinsay, Melissa N; Nguyen, Christine K; Jimenez, Rebecca; Petrosyan, Susanna; Murphy, Anne N; Gustafsson, Asa B

    2013-01-11

    It is known that loss-of-function mutations in the gene encoding Parkin lead to development of Parkinson disease. Recently, Parkin was found to play an important role in the removal of dysfunctional mitochondria via autophagy in neurons. Although Parkin is expressed in the heart, its functional role in this tissue is largely unexplored. In this study, we have investigated the role of Parkin in the myocardium under normal physiological conditions and in response to myocardial infarction. We found that Parkin-deficient (Parkin(-/-)) mice had normal cardiac function for up to 12 months of age as determined by echocardiographic analysis. Although ultrastructural analysis revealed that Parkin-deficient hearts had disorganized mitochondrial networks and significantly smaller mitochondria, mitochondrial function was unaffected. However, Parkin(-/-) mice were much more sensitive to myocardial infarction when compared with wild type mice. Parkin(-/-) mice had reduced survival and developed larger infarcts when compared with wild type mice after the infarction. Interestingly, Parkin protein levels and mitochondrial autophagy (mitophagy) were rapidly increased in the border zone of the infarct in wild type mice. In contrast, Parkin(-/-) myocytes had reduced mitophagy and accumulated swollen, dysfunctional mitochondria after the infarction. Overexpression of Parkin in isolated cardiac myocytes also protected against hypoxia-mediated cell death, whereas nonfunctional Parkinson disease-associated mutants ParkinR42P and ParkinG430D had no effect. Our results suggest that Parkin plays a critical role in adapting to stress in the myocardium by promoting removal of damaged mitochondria. PMID:23152496

  6. Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

    PubMed Central

    Kazlauskaite, Agne; Kelly, Van; Johnson, Clare; Baillie, Carla; Hastie, C. James; Peggie, Mark; Macartney, Thomas; Woodroof, Helen I.; Alessi, Dario R.; Pedrioli, Patrick G. A.; Muqit, Miratul M. K.

    2014-01-01

    Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser65 by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser65 phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin's ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser65 is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson's disease. PMID:24647965

  7. Short Mitochondrial ARF Triggers Parkin/PINK1-dependent Mitophagy*

    PubMed Central

    Grenier, Karl; Kontogiannea, Maria; Fon, Edward A.

    2014-01-01

    Parkinson disease (PD) is a complex neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra. Multiple genes have been associated with PD, including Parkin and PINK1. Recent studies have established that the Parkin and PINK1 proteins function in a common mitochondrial quality control pathway, whereby disruption of the mitochondrial membrane potential leads to PINK1 stabilization at the mitochondrial outer surface. PINK1 accumulation leads to Parkin recruitment from the cytosol, which in turn promotes the degradation of the damaged mitochondria by autophagy (mitophagy). Most studies characterizing PINK1/Parkin mitophagy have relied on high concentrations of chemical uncouplers to trigger mitochondrial depolarization, a stimulus that has been difficult to adapt to neuronal systems and one unlikely to faithfully model the mitochondrial damage that occurs in PD. Here, we report that the short mitochondrial isoform of ARF (smARF), previously identified as an alternate translation product of the tumor suppressor p19ARF, depolarizes mitochondria and promotes mitophagy in a Parkin/PINK1-dependent manner, both in cell lines and in neurons. The work positions smARF upstream of PINK1 and Parkin and demonstrates that mitophagy can be triggered by intrinsic signaling cascades. PMID:25217637

  8. Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation*

    PubMed Central

    Iguchi, Masahiro; Kujuro, Yuki; Okatsu, Kei; Koyano, Fumika; Kosako, Hidetaka; Kimura, Mayumi; Suzuki, Norihiro; Uchiyama, Shinichiro; Tanaka, Keiji; Matsuda, Noriyuki

    2013-01-01

    PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation. PMID:23754282

  9. Non-motor behavioural impairments in parkin-deficient mice.

    PubMed

    Zhu, Xin-Ran; Maskri, Lyutha; Herold, Christina; Bader, Verian; Stichel, Christine C; Güntürkün, Onur; Lübbert, Hermann

    2007-10-01

    Mutations in the parkin gene are the major cause of early-onset familial Parkinson's disease (PD). We previously reported the generation and analysis of a knockout mouse carrying a deletion of exon 3 in the parkin gene. F1 hybrid pa+/- mice were backcrossed to wild-type C57Bl/6 for three more generations to establish a pa-/-(F4) mouse line. The appearance of tyrosine hydroxylase-positive neurons was normal in young and aged pa-/- (F4) animals. Loss of parkin function in mice did not enhance vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. However, the pa-/- (F4) mice displayed impaired exploration and habituation to a new environment and exhibited thigmotaxis behaviour in the open field and Morris water maze. Abnormal anxiety-related behaviour of pa-/- (F4) mice was also observed in the light/dark exploration test paradigm. Dopamine metabolism was enhanced in the striatum of pa-/- (F4) mice, as revealed by increased homovanillic acid (HVA) content and a reduced ratio of dihydroxyphenylacetic acid (DOPAC)/HVA. The alterations found in the dopaminergic system could be responsible for the behavioural impairments of pa-/- (F4) mice. Consistent with a recent observation of cognitive dysfunction in parkin-linked patients with PD, our findings provide evidence of a physiological role of parkin in non-motor behaviour, possibly representing a disease stage that precedes dopaminergic neuron loss. PMID:17883413

  10. Parkin and PINK1: much more than mitophagy.

    PubMed

    Scarffe, Leslie A; Stevens, Daniel A; Dawson, Valina L; Dawson, Ted M

    2014-06-01

    Parkinson's disease (PD) is a progressive neurodegenerative disease that causes a debilitating movement disorder. Although most cases of PD appear to be sporadic, rare Mendelian forms have provided tremendous insight into disease pathogenesis. Accumulating evidence suggests that impaired mitochondria underpin PD pathology. In support of this theory, data from multiple PD models have linked Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and parkin, two recessive PD genes, in a common pathway impacting mitochondrial health, prompting a flurry of research to identify their mitochondrial targets. Recent work has focused on the role of PINK1 and parkin in mediating mitochondrial autophagy (mitophagy); however, emerging evidence casts parkin and PINK1 as key players in multiple domains of mitochondrial health and quality control. PMID:24735649

  11. Parkin and PINK1: Much More than Mitophagy

    PubMed Central

    Scarffe, Leslie A.; Stevens, Daniel A.; Dawson, Valina L.; Dawson, Ted M.

    2014-01-01

    Parkinson’s disease (PD) is a progressive neurodegenerative disease that causes a debilitating movement disorder. While most cases of PD appear to be sporadic, rare Mendelian forms have provided tremendous insight into disease pathogenesis. Accumulating evidence suggests that impaired mitochondria underpin PD pathology. In support of this theory, data from multiple PD models has linked PINK1 and parkin, two recessive PD genes, in a common pathway impacting mitochondrial health, prompting a flurry of research to identify their mitochondrial targets. Recent work has focused on the role of PINK1 and parkin in mediating mitochondrial autophagy (mitophagy), however, emerging evidence casts parkin and PINK1 as key players in multiple domains of mitochondrial health and quality control. PMID:24735649

  12. Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain

    PubMed Central

    Kageyama, Yusuke; Hoshijima, Masahiko; Seo, Kinya; Bedja, Djahida; Sysa-Shah, Polina; Andrabi, Shaida A; Chen, Weiran; Höke, Ahmet; Dawson, Valina L; Dawson, Ted M; Gabrielson, Kathleen; Kass, David A; Iijima, Miho; Sesaki, Hiromi

    2014-01-01

    Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease-associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals. PMID:25349190

  13. Parkin degrades estrogen-related receptors to limit the expression of monoamine oxidases.

    PubMed

    Ren, Yong; Jiang, Houbo; Ma, Dingyuan; Nakaso, Kazuhiro; Feng, Jian

    2011-03-15

    Parkin, whose mutations cause Parkinson disease (PD), controls oxidative stress by limiting the expression of monoamine oxidases (MAO)--mitochondrial enzymes responsible for the oxidative de-amination of dopamine. Here, we show that parkin performed this function by increasing the ubiquitination and degradation of estrogen-related receptors (ERR), orphan nuclear receptors that play critical roles in the transcription regulation of many nuclear-encoded mitochondrial proteins. All three ERRs (α, β and γ) increased the transcription of MAOs A and B; the effects were abolished by parkin, but not by its PD-linked mutants. Parkin bound to ERRs and increased their ubiquitination and degradation. In fibroblasts from PD patients with parkin mutations or brain slices from parkin knockout mice, degradation of ERRs was significantly attenuated. The results reveal the molecular mechanism by which parkin suppresses the transcription of MAOs to control oxidative stress induced by dopamine oxidation. PMID:21177257

  14. Parkin degrades estrogen-related receptors to limit the expression of monoamine oxidases

    PubMed Central

    Ren, Yong; Jiang, Houbo; Ma, Dingyuan; Nakaso, Kazuhiro; Feng, Jian

    2011-01-01

    Parkin, whose mutations cause Parkinson disease (PD), controls oxidative stress by limiting the expression of monoamine oxidases (MAO)—mitochondrial enzymes responsible for the oxidative de-amination of dopamine. Here, we show that parkin performed this function by increasing the ubiquitination and degradation of estrogen-related receptors (ERR), orphan nuclear receptors that play critical roles in the transcription regulation of many nuclear-encoded mitochondrial proteins. All three ERRs (α, β and γ) increased the transcription of MAOs A and B; the effects were abolished by parkin, but not by its PD-linked mutants. Parkin bound to ERRs and increased their ubiquitination and degradation. In fibroblasts from PD patients with parkin mutations or brain slices from parkin knockout mice, degradation of ERRs was significantly attenuated. The results reveal the molecular mechanism by which parkin suppresses the transcription of MAOs to control oxidative stress induced by dopamine oxidation. PMID:21177257

  15. Expression profile of parkin isoforms in human gliomas.

    PubMed

    Maugeri, Grazia; D'Amico, Agata Grazia; Magro, Gaetano; Salvatorelli, Lucia; Barbagallo, Giuseppe M V; Saccone, Salvatore; Drago, Filippo; Cavallaro, Sebastiano; D'Agata, Velia

    2015-10-01

    Mutations of parkin gene are not restricted to familial forms of Parkinsonism but they also occur in a wide variety of malignancies including gliomas. Parkin over-expression reduces glioma cells proliferation and analysis of its expression is predictive for the survival outcome of patients with glioma. To date have been identified 21 parkin alternative splice variants. However, most of the studies have focused their attention exclusively on full-length protein. In the present study, the expression profile of parkin isoforms in different grades of astrocytomas was analyzed for the first time, in order to evaluate their involvement in this malignancy. Furthermore, to investigate their role in cellular processes, their expression in three glioblastoma cell lines was analyzed following treatment with the proteasome inhibitor MG132, or induction of mitophagy with CCCP, or after serum deprivation. Results suggested that H20, H1 and H5 isoforms are always expressed in tumors both in vivo and in vitro models. Therefore, these isoforms might be used as specific biomarkers to develop a prognostic tool for brain tumors. PMID:26238155

  16. Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis

    PubMed Central

    Kumar, Atul; Aguirre, Jacob D; Condos, Tara EC; Martinez-Torres, R Julio; Chaugule, Viduth K; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Mercier, Pascal; Knebel, Axel; Spratt, Donald E; Barber, Kathryn R; Shaw, Gary S; Walden, Helen

    2015-01-01

    The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2∼Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2∼Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin. PMID:26254304

  17. Parkin cooperates with GDNF/RET signaling to prevent dopaminergic neuron degeneration

    PubMed Central

    Meka, Durga Praveen; Müller-Rischart, Anne Kathrin; Nidadavolu, Prakash; Mohammadi, Behnam; Motori, Elisa; Ponna, Srinivas Kumar; Aboutalebi, Helia; Bassal, Mahmoud; Annamneedi, Anil; Finckh, Barbara; Miesbauer, Margit; Rotermund, Natalie; Lohr, Christian; Tatzelt, Jörg; Winklhofer, Konstanze F.; Kramer, Edgar R.

    2015-01-01

    Parkin and the glial cell line–derived neurotrophic factor (GDNF) receptor RET have both been independently linked to the dopaminergic neuron degeneration that underlies Parkinson’s disease (PD). In the present study, we demonstrate that there is genetic crosstalk between parkin and the receptor tyrosine kinase RET in two different mouse models of PD. Mice lacking both parkin and RET exhibited accelerated dopaminergic cell and axonal loss compared with parkin-deficient animals, which showed none, and RET-deficient mice, in which we found moderate degeneration. Transgenic expression of parkin protected the dopaminergic systems of aged RET-deficient mice. Downregulation of either parkin or RET in neuronal cells impaired mitochondrial function and morphology. Parkin expression restored mitochondrial function in GDNF/RET-deficient cells, while GDNF stimulation rescued mitochondrial defects in parkin-deficient cells. In both cases, improved mitochondrial function was the result of activation of the prosurvival NF-κB pathway, which was mediated by RET through the phosphoinositide-3-kinase (PI3K) pathway. Taken together, these observations indicate that parkin and the RET signaling cascade converge to control mitochondrial integrity and thereby properly maintain substantia nigra pars compacta dopaminergic neurons and their innervation in the striatum. The demonstration of crosstalk between parkin and RET highlights the interplay in the protein network that is altered in PD and suggests potential therapeutic targets and strategies to treat PD. PMID:25822020

  18. Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

    PubMed Central

    Ko, Han Seok; Lee, Yunjong; Shin, Joo-Ho; Karuppagounder, Senthilkumar S.; Gadad, Bharathi Shrikanth; Koleske, Anthony J.; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.

    2010-01-01

    Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571’s protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD. PMID:20823226

  19. Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras.

    PubMed

    Papapetrou, Eirini P; Kovalovsky, Damian; Beloeil, Laurent; Sant'angelo, Derek; Sadelain, Michel

    2009-01-01

    MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy. PMID:19033646

  20. Mitochondrial Quality Control Mediated by PINK1 and Parkin: Links to Parkinsonism

    PubMed Central

    Narendra, Derek; Walker, John E.; Youle, Richard

    2012-01-01

    Mutations in Parkin or PINK1 are the most common cause of recessive familial parkinsonism. Recent studies suggest that PINK1 and Parkin form a mitochondria quality control pathway that identifies dysfunctional mitochondria, isolates them from the mitochondrial network, and promotes their degradation by autophagy. In this pathway the mitochondrial kinase PINK1 senses mitochondrial fidelity and recruits Parkin selectively to mitochondria that lose membrane potential. Parkin, an E3 ligase, subsequently ubiquitinates outer mitochondrial membrane proteins, notably the mitofusins and Miro, and induces autophagic elimination of the impaired organelles. Here we review the recent rapid progress in understanding the molecular mechanisms of PINK1- and Parkin-mediated mitophagy and the identification of Parkin substrates suggesting how mitochondrial fission and trafficking are involved. We also discuss how defects in mitophagy may be linked to Parkinson's disease. PMID:23125018

  1. Parkin loss leads to PARIS-dependent declines in mitochondrial mass and respiration.

    PubMed

    Stevens, Daniel A; Lee, Yunjong; Kang, Ho Chul; Lee, Byoung Dae; Lee, Yun-Il; Bower, Aaron; Jiang, Haisong; Kang, Sung-Ung; Andrabi, Shaida A; Dawson, Valina L; Shin, Joo-Ho; Dawson, Ted M

    2015-09-15

    Mutations in parkin lead to early-onset autosomal recessive Parkinson's disease (PD) and inactivation of parkin is thought to contribute to sporadic PD. Adult knockout of parkin in the ventral midbrain of mice leads to an age-dependent loss of dopamine neurons that is dependent on the accumulation of parkin interacting substrate (PARIS), zinc finger protein 746 (ZNF746), and its transcriptional repression of PGC-1α. Here we show that adult knockout of parkin in mouse ventral midbrain leads to decreases in mitochondrial size, number, and protein markers consistent with a defect in mitochondrial biogenesis. This decrease in mitochondrial mass is prevented by short hairpin RNA knockdown of PARIS. PARIS overexpression in mouse ventral midbrain leads to decreases in mitochondrial number and protein markers and PGC-1α-dependent deficits in mitochondrial respiration. Taken together, these results suggest that parkin loss impairs mitochondrial biogenesis, leading to declining function of the mitochondrial pool and cell death. PMID:26324925

  2. Parkin Controls Dopamine Utilization in Human Midbrain Dopaminergic Neurons Derived from Induced Pluripotent Stem Cells

    PubMed Central

    Jiang, Houbo; Ren, Yong; Yuen, Eunice Y; Zhong, Ping; Ghaedi, Mahboobe; Hu, Zhixing; Azabdaftari, Gissou; Nakaso, Kazuhiro; Yan, Zhen; Feng, Jian

    2012-01-01

    Parkinson’s disease (PD) is defined by the degeneration of nigral dopaminergic (DA) neurons and can be caused by monogenic mutations of genes such as parkin. The lack of phenotype in parkin knockout mice suggests that human nigral DA neurons have unique vulnerabilities. Through the generation and analyses of induced pluripotent stem cells (iPSCs) from normal subjects and PD patients with parkin mutations, we show here that loss of parkin in human midbrain DA neurons greatly increased the transcription of monoamine oxidases and oxidative stress, significantly reduced DA uptake and increased spontaneous DA release. Lentiviral expression of parkin, but not its PD-linked mutant, rescued all the phenotypes. The results suggest that parkin controls dopamine utilization in human midbrain DA neurons by enhancing the precision of dopaminergic neurotransmission and suppressing dopamine oxidation. Thus, the study provides novel targets and a physiologically relevant screening platform for disease-modifying therapies of PD. PMID:22314364

  3. Parkin controls dopamine utilization in human midbrain dopaminergic neurons derived from induced pluripotent stem cells.

    PubMed

    Jiang, Houbo; Ren, Yong; Yuen, Eunice Y; Zhong, Ping; Ghaedi, Mahboobe; Hu, Zhixing; Azabdaftari, Gissou; Nakaso, Kazuhiro; Yan, Zhen; Feng, Jian

    2012-01-01

    Parkinson's disease (PD) is defined by the degeneration of nigral dopaminergic (DA) neurons and can be caused by monogenic mutations of genes such as parkin. The lack of phenotype in parkin knockout mice suggests that human nigral DA neurons have unique vulnerabilities. Here we generate induced pluripotent stem cells from normal subjects and PD patients with parkin mutations. We demonstrate that loss of parkin in human midbrain DA neurons greatly increases the transcription of monoamine oxidases and oxidative stress, significantly reduces DA uptake and increases spontaneous DA release. Lentiviral expression of parkin, but not its PD-linked mutant, rescues these phenotypes. The results suggest that parkin controls dopamine utilization in human midbrain DA neurons by enhancing the precision of DA neurotransmission and suppressing dopamine oxidation. Thus, the study provides novel targets and a physiologically relevant screening platform for disease-modifying therapies of PD. PMID:22314364

  4. Homozygous partial genomic triplication of the parkin gene in early-onset parkinsonism.

    PubMed

    Mata, Ignacio F; Alvarez, Victoria; Coto, Eliecer; Blazquez, Marta; Guisasola, Luis M; Salvador, Carlos; Kachergus, Jennifer M; Lincoln, Sarah J; Farrer, Matthew

    2005-06-01

    Autosomal recessive mutations in the parkin gene are the predominant cause of familial, early-onset parkinsonism; missense mutations involving one or a few nucleotides, exonic deletions and duplications have been described. Here we report a family with two affected brothers. Direct sequencing of parkin did not detect mutations, but semi-quantitative analysis identified a novel exonic rearrangement of exons 2-4. Both patients were homozygous for unique genomic triplications of the parkin gene. PMID:15862897

  5. Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization

    PubMed Central

    Sarraf, Shireen A.; Raman, Malavika; Guarani-Pereira, Virginia; Sowa, Mathew E.; Huttlin, Edward L.; Gygi, Steven P.; Harper, J. Wade

    2013-01-01

    The PARKIN (PARK2) ubiquitin ligase and its regulatory kinase PINK1 (PARK6), often mutated in familial early onset Parkinson’s Disease (PD), play central roles in mitochondrial homeostasis and mitophagy.1–3 While PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM,1,4–11 the full repertoire of PARKIN substrates – the PARKIN-dependent ubiquitylome - remains poorly defined. Here we employ quantitative diGLY capture proteomics12,13 to elucidate the ubiquitylation site-specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization. Hundreds of dynamically regulated ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, we found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of PARKIN’s active site residue C431, which has been found mutated in PD patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and D. melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis. PMID:23503661

  6. PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria.

    PubMed

    Chen, Yun; Dorn, Gerald W

    2013-04-26

    Senescent and damaged mitochondria undergo selective mitophagic elimination through mechanisms requiring two Parkinson's disease factors, the mitochondrial kinase PINK1 (PTEN-induced putative kinase protein 1; PTEN is phosphatase and tensin homolog) and the cytosolic ubiquitin ligase Parkin. The nature of the PINK-Parkin interaction and the identity of key factors directing Parkin to damaged mitochondria are unknown. We show that the mitochondrial outer membrane guanosine triphosphatase mitofusin (Mfn) 2 mediates Parkin recruitment to damaged mitochondria. Parkin bound to Mfn2 in a PINK1-dependent manner; PINK1 phosphorylated Mfn2 and promoted its Parkin-mediated ubiqitination. Ablation of Mfn2 in mouse cardiac myocytes prevented depolarization-induced translocation of Parkin to the mitochondria and suppressed mitophagy. Accumulation of morphologically and functionally abnormal mitochondria induced respiratory dysfunction in Mfn2-deficient mouse embryonic fibroblasts and cardiomyocytes and in Parkin-deficient Drosophila heart tubes, causing dilated cardiomyopathy. Thus, Mfn2 functions as a mitochondrial receptor for Parkin and is required for quality control of cardiac mitochondria. PMID:23620051

  7. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

    PubMed Central

    Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

    2015-01-01

    Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of

  8. Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation.

    PubMed

    Yamano, Koji; Queliconi, Bruno B; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

    2015-10-16

    Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser(65) by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

  9. NH2-truncated human tau induces deregulated mitophagy in neurons by aberrant recruitment of Parkin and UCHL-1: implications in Alzheimer's disease.

    PubMed

    Corsetti, V; Florenzano, F; Atlante, A; Bobba, A; Ciotti, M T; Natale, F; Della Valle, F; Borreca, A; Manca, A; Meli, G; Ferraina, C; Feligioni, M; D'Aguanno, S; Bussani, R; Ammassari-Teule, M; Nicolin, V; Calissano, P; Amadoro, G

    2015-06-01

    Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance. PMID:25687137

  10. Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

    PubMed Central

    2010-01-01

    Background Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated

  11. Parkin gene causing benign autosomal recessive juvenile parkinsonism.

    PubMed

    Nisipeanu, P; Inzelberg, R; Abo Mouch, S; Carasso, R L; Blumen, S C; Zhang, J; Matsumine, H; Hattori, N; Mizuno, Y

    2001-06-12

    Autosomal recessive juvenile parkinsonism (AR-JP) is an early-onset parkinsonism caused by exonic deletions or point mutations in the parkingene. The relationship between the type of the genetic defect and the clinical presentation, the response to therapy, and the evolution have not been yet determined. The authors describe a single-basepair deletion at nucleotide 202 in exon 2 of the parkin gene in a kindred with a benign clinical course. PMID:11402119

  12. Mitochondrial Dynamics and Parkinson's Disease: Focus on Parkin

    PubMed Central

    Ng, Xiao-Hui; Grace, Lim Gui-Yin; Yao, Tso-Pang

    2012-01-01

    Abstract Significance: Parkinson’s disease (PD) is a prevalent neurodegenerative disease affecting millions of individuals worldwide. Despite intensive efforts devoted to drug discovery, the disease remains incurable. To provide more effective medical therapy for PD, better understanding of the underlying causes of the disease is clearly necessary. Recent Advances: A broad range of studies conducted over the past few decades have collectively implicated aberrant mitochondrial homeostasis as a key contributor to the development of PD. Supporting this, mutations in several PD-linked genes are directly or indirectly linked to mitochondrial dysfunction. In particular, recent discoveries have identified parkin, whose mutations are causative of recessive parkinsonism, as a key regulator of mitochondrial homeostasis. Critical Issues: Parkin appears to be involved in the entire spectrum of mitochondrial dynamics, including organelle biogenesis, fusion/fission, and clearance via mitophagy. How a single protein can regulate such diverse mitochondrial events is as intriguing as it is amazing; the mechanism underlying this is currently under intense research. Here, we provide an overview of mitochondrial dynamics and its relationship with neurodegenerative diseases and discuss current evidence and controversies surrounding the role of parkin in mitochondrial quality control and its relevance to PD pathogenesis. Future Directions: Although the emerging field of parkin-mediated mitochondrial quality control has proven to be exciting, it is important to recognize that PD pathogenesis is likely to involve an intricate network of interacting pathways. Elucidating the reciprocity of pathways, particularly how other PD-related pathways potentially influence mitochondrial homeostasis, may hold the key to therapeutic development. Antioxid. Redox Signal. 16, 935–949. PMID:21668405

  13. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

    PubMed Central

    Kazlauskaite, Agne; Kondapalli, Chandana; Gourlay, Robert; Campbell, David G.; Ritorto, Maria Stella; Hofmann, Kay; Alessi, Dario R.; Knebel, Axel; Trost, Matthias; Muqit, Miratul M. K.

    2014-01-01

    We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65

  14. Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress

    PubMed Central

    Bouman, L; Schlierf, A; Lutz, A K; Shan, J; Deinlein, A; Kast, J; Galehdar, Z; Palmisano, V; Patenge, N; Berg, D; Gasser, T; Augustin, R; Trümbach, D; Irrcher, I; Park, D S; Wurst, W; Kilberg, M S; Tatzelt, J; Winklhofer, K F

    2011-01-01

    Loss of parkin function is responsible for the majority of autosomal recessive parkinsonism. Here, we show that parkin is not only a stress-protective, but also a stress-inducible protein. Both mitochondrial and endoplasmic reticulum (ER) stress induce an increase in parkin-specific mRNA and protein levels. The stress-induced upregulation of parkin is mediated by ATF4, a transcription factor of the unfolded protein response (UPR) that binds to a specific CREB/ATF site within the parkin promoter. Interestingly, c-Jun can bind to the same site, but acts as a transcriptional repressor of parkin gene expression. We also present evidence that mitochondrial damage can induce ER stress, leading to the activation of the UPR, and thereby to an upregulation of parkin expression. Vice versa, ER stress results in mitochondrial damage, which can be prevented by parkin. Notably, the activity of parkin to protect cells from stress-induced cell death is independent of the proteasome, indicating that proteasomal degradation of parkin substrates cannot explain the cytoprotective activity of parkin. Our study supports the notion that parkin has a role in the interorganellar crosstalk between the ER and mitochondria to promote cell survival under stress, suggesting that both ER and mitochondrial stress can contribute to the pathogenesis of Parkinson's disease. PMID:21113145

  15. Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

    PubMed Central

    Ch'ang, L Y; Yang, W K; Myer, F E; Yang, D M

    1989-01-01

    Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs. Images PMID:2542587

  16. Quantitative proteomics reveal a feed-forward model for mitochondrial PARKIN translocation and UB chain synthesis

    PubMed Central

    Ordureau, Alban; Sarraf, Shireen A.; Duda, David M.; Heo, Jin-Mi; Jedrykowski, Mark P.; Sviderskiy, Vladislav; Olszewski, Jennifer L.; Koerber, James T.; Xie, Tiao; Beausoleil, Sean A.; Wells, James A.; Gygi, Steven P.; Schulman, Brenda A.; Harper, J. Wade

    2014-01-01

    Phosphorylation is often used to promote protein ubiquitylation, yet we rarely understand quantitatively how ligase activation and ubiquitin (UB) chain assembly are integrated with phospho-regulation. Here we employ quantitative proteomics and live-cell imaging to dissect individual steps in the PINK1 kinase-PARKIN UB ligase mitochondrial control pathway disrupted in Parkinson’s Disease. PINK1 plays a dual role by phosphorylating PARKIN on its UB-like domain and poly-UB chains on mitochondria. PARKIN activation by PINK1 produces canonical and non-canonical UB chains on mitochondria, and PARKIN-dependent chain assembly is required for accumulation of poly-phospho-UB (poly-p-UB) on mitochondria. In vitro, PINK1 directly activates PARKIN’s ability to assemble canonical and non-canonical UB chains, and promotes association of PARKIN with both p-UB and poly-p-UB. Our data reveal a feed-forward mechanism that explains how PINK1 phosphorylation of both PARKIN and poly-UB chains synthesized by PARKIN drives a program of PARKIN recruitment and mitochondrial ubiquitylation in response to mitochondrial damage. PMID:25284222

  17. PINK1 and Parkin Target Miro for Phosphorylation and Degradation to Arrest Mitochondrial Motility

    PubMed Central

    Wang, Xinnan; Winter, Dominic; Ashrafi, Ghazaleh; Schlehe, Julia; Wong, Yao Liang; Selkoe, Dennis; Rice, Sarah; Steen, Judith; LaVoie, Matthew J.; Schwarz, Thomas L.

    2011-01-01

    SUMMARY Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson’s disease proteins, the Ser/Thr-kinase PINK1 and ubiquitin-ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome. PMID:22078885

  18. PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility.

    PubMed

    Wang, Xinnan; Winter, Dominic; Ashrafi, Ghazaleh; Schlehe, Julia; Wong, Yao Liang; Selkoe, Dennis; Rice, Sarah; Steen, Judith; LaVoie, Matthew J; Schwarz, Thomas L

    2011-11-11

    Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson's disease proteins, the Ser/Thr kinase PINK1 and ubiquitin ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin, but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome. PMID:22078885

  19. Mitochondrial Contagion Induced by Parkin Deficiency in Drosophila Hearts and Its Containment by Suppressing Mitofusin

    PubMed Central

    Bhandari, Poonam; Song, Moshi; Chen, Yun; Burelle, Yan; Dorn, Gerald W.

    2015-01-01

    Rationale Dysfunctional Parkin-mediated mitophagic culling of senescent or damaged mitochondria is a major pathological process underlying Parkinson disease and a potential genetic mechanism of cardiomyopathy. Despite epidemiological associations between Parkinson disease and heart failure, the role of Parkin and mitophagic quality control in maintaining normal cardiac homeostasis is poorly understood. Objective We used germline mutants and cardiac-specific RNA interference to interrogate Parkin regulation of cardiomyocyte mitochondria and examine functional crosstalk between mitophagy and mitochondrial dynamics in Drosophila heart tubes. Methods and Results Transcriptional profiling of Parkin knockout mouse hearts revealed compensatory upregulation of multiple related E3 ubiquitin ligases. Because Drosophila lack most of these redundant genes, we examined heart tubes of parkin knockout flies and observed accumulation of enlarged hollow donut mitochondria with dilated cardiomyopathy, which could be rescued by cardiomyocyte-specific Parkin expression. Identical abnormalities were induced by cardiomyocyte-specific Parkin suppression using 2 different inhibitory RNAs. Parkin-deficient cardiomyocyte mitochondria exhibited dysmorphology, depolarization, and reactive oxygen species generation without calcium cycling abnormalities, pointing to a primary mitochondrial defect. Suppressing cardiomyocyte mitochondrial fusion in Parkin-deficient fly heart tubes completely prevented the cardiomyopathy and corrected mitochondrial dysfunction without normalizing mitochondrial dysmorphology, demonstrating a central role for mitochondrial fusion in the cardiomyopathy provoked by impaired mitophagy. Conclusions Parkin deficiency and resulting mitophagic disruption produces cardiomyopathy in part by contamination of the cardiomyocyte mitochondrial pool through fusion between improperly retained dysfunctional/senescent and normal mitochondria. Limiting mitochondrial contagion by

  20. Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 iPS cells

    PubMed Central

    Seibler, Philip; Graziotto, John; Jeong, Hyun; Simunovic, Filip; Klein, Christine; Krainc, Dimitri

    2011-01-01

    Genetic Parkinson disease (PD) has been associated with mutations in PINK1, a gene encoding a mitochondrial kinase implicated in the regulation of mitochondrial degradation. While the studies so far examined PINK1 function in non-neuronal systems or through PINK1 knockdown approaches, there is an imperative to examine the role of endogenous PINK1 in appropriate human-derived and biologically relevant cell models. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblasts taken from three PD patients with nonsense (c.1366C>T; p.Q456X) or missense mutations (c.509T>G; p.V170G) in the PINK1 gene. These cells were differentiated into dopaminergic neurons that upon mitochondrial depolarization showed impaired recruitment of lentivirally expressed Parkin to mitochondria, increased mitochondrial copy number and upregulation of PGC-1α, an important regulator of mitochondrial biogenesis. Importantly, these alterations were corrected by lentiviral expression of wild-type PINK1 in mutant iPS cell-derived PINK1 neurons. In conclusion, our studies suggest that fibroblasts from genetic PD can be reprogrammed and differentiated into neurons. These neurons exhibit distinct phenotypes that should be amenable to further mechanistic studies in this relevant biological context. PMID:21508222

  1. PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding.

    PubMed

    Lazarou, Michael; Narendra, Derek P; Jin, Seok Min; Tekle, Ephrem; Banerjee, Soojay; Youle, Richard J

    2013-01-21

    Genetic studies indicate that the mitochondrial kinase PINK1 and the RING-between-RING E3 ubiquitin ligase Parkin function in the same pathway. In concurrence, mechanistic studies show that PINK1 can recruit Parkin from the cytosol to the mitochondria, increase the ubiquitination activity of Parkin, and induce Parkin-mediated mitophagy. Here, we used a cell-free assay to recapitulate PINK1-dependent activation of Parkin ubiquitination of a validated mitochondrial substrate, mitofusin 1. We show that PINK1 activated the formation of a Parkin-ubiquitin thioester intermediate, a hallmark of HECT E3 ligases, both in vitro and in vivo. Parkin HECT-like ubiquitin ligase activity was essential for PINK1-mediated Parkin translocation to mitochondria and mitophagy. Using an inactive Parkin mutant, we found that PINK1 stimulated Parkin self-association and complex formation upstream of mitochondrial translocation. Self-association occurred independent of ubiquitination activity through the RING-between-RING domain, providing mechanistic insight into how PINK1 activates Parkin. PMID:23319602

  2. CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity.

    PubMed

    Imai, Yuzuru; Soda, Mariko; Hatakeyama, Shigetsugu; Akagi, Takumi; Hashikawa, Tsutomu; Nakayama, Kei Ichi; Takahashi, Ryosuke

    2002-07-01

    Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity. PMID:12150907

  3. USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin

    PubMed Central

    Durcan, Thomas M; Tang, Matthew Y; Pérusse, Joëlle R; Dashti, Eman A; Aguileta, Miguel A; McLelland, Gian-Luca; Gros, Priti; Shaler, Thomas A; Faubert, Denis; Coulombe, Benoit; Fon, Edward A

    2014-01-01

    Mutations in the Park2 gene, encoding the E3 ubiquitin-ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin-mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin-mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin-mediated mitophagy. USP8 preferentially removes non-canonical K6-linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8-mediated deubiquitination of K6-linked ubiquitin conjugates from parkin in mitochondrial quality control. PMID:25216678

  4. Parkin Protects against Oxygen-Glucose Deprivation/Reperfusion Insult by Promoting Drp1 Degradation.

    PubMed

    Tang, Jiayu; Hu, Zhiping; Tan, Jieqiong; Yang, Sonlin; Zeng, Liuwang

    2016-01-01

    Ischemic stroke results in severe brain damage and remains one of the leading causes of death and disability worldwide. Effective neuroprotective therapies are needed to reduce brain damage resulting from ischemic stroke. Mitochondria are crucial for cellular energy production and homeostasis. Modulation of mitochondrial function mediates neuroprotection against ischemic brain damage. Dynamin-related protein 1 (Drp1) and parkin play a key role in regulating mitochondrial dynamics. They are potential therapeutic targets for neuroprotection in ischemic stroke. Protective effects of parkin-Drp1 pathway on mitochondria were assessed in a cellular ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a) cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. OGDR induces mitochondrial fragmentation. The expression of Drp1 protein is increased after OGDR insult, while the parkin protein level is decreased. The altered protein level of Drp1 after OGDR injury is mediated by parkin through ubiquitin proteasome system (UPS). Drp1 depletion protects against OGDR induced mitochondrial damage and apoptosis. Meanwhile, parkin overexpression protects against OGDR induced apoptosis and mitochondrial dysfunction, which is attenuated by increased expression of Drp1. Our data demonstrate that parkin protects against OGDR insult through promoting degradation of Drp1. This neuroprotective potential of parkin-Drp1 pathway against OGDR insult will pave the way for developing novel neuroprotective agents for cerebral ischemia-reperfusion related disorders. PMID:27597885

  5. Structural insights into Parkin substrate lysine targeting from minimal Miro substrates.

    PubMed

    Klosowiak, Julian L; Park, Sungjin; Smith, Kyle P; French, Michael E; Focia, Pamela J; Freymann, Douglas M; Rice, Sarah E

    2016-01-01

    Hereditary Parkinson's disease is commonly caused by mutations in the protein kinase PINK1 or the E3 ubiquitin ligase Parkin, which function together to eliminate damaged mitochondria. PINK1 phosphorylates both Parkin and ubiquitin to stimulate ubiquitination of dozens of proteins on the surface of the outer mitochondrial membrane. However, the mechanisms by which Parkin recognizes specific proteins for modification remain largely unexplored. Here, we show that the C-terminal GTPase (cGTPase) of the Parkin primary substrate human Miro is necessary and sufficient for efficient ubiquitination. We present several new X-ray crystal structures of both human Miro1 and Miro2 that reveal substrate recognition and ubiquitin transfer to be specific to particular protein domains and lysine residues. We also provide evidence that Parkin substrate recognition is functionally separate from substrate modification. Finally, we show that prioritization for modification of a specific lysine sidechain of the cGTPase (K572) within human Miro1 is dependent on both its location and chemical microenvironment. Activation of Parkin by phosphorylation or by binding of pUb is required for prioritization of K572 for modification, suggesting that Parkin activation and acquisition of substrate specificity are coupled. PMID:27605430

  6. Parkin Protects against Oxygen-Glucose Deprivation/Reperfusion Insult by Promoting Drp1 Degradation

    PubMed Central

    Tang, Jiayu; Hu, Zhiping; Tan, Jieqiong; Yang, Sonlin

    2016-01-01

    Ischemic stroke results in severe brain damage and remains one of the leading causes of death and disability worldwide. Effective neuroprotective therapies are needed to reduce brain damage resulting from ischemic stroke. Mitochondria are crucial for cellular energy production and homeostasis. Modulation of mitochondrial function mediates neuroprotection against ischemic brain damage. Dynamin-related protein 1 (Drp1) and parkin play a key role in regulating mitochondrial dynamics. They are potential therapeutic targets for neuroprotection in ischemic stroke. Protective effects of parkin-Drp1 pathway on mitochondria were assessed in a cellular ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a) cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. OGDR induces mitochondrial fragmentation. The expression of Drp1 protein is increased after OGDR insult, while the parkin protein level is decreased. The altered protein level of Drp1 after OGDR injury is mediated by parkin through ubiquitin proteasome system (UPS). Drp1 depletion protects against OGDR induced mitochondrial damage and apoptosis. Meanwhile, parkin overexpression protects against OGDR induced apoptosis and mitochondrial dysfunction, which is attenuated by increased expression of Drp1. Our data demonstrate that parkin protects against OGDR insult through promoting degradation of Drp1. This neuroprotective potential of parkin-Drp1 pathway against OGDR insult will pave the way for developing novel neuroprotective agents for cerebral ischemia-reperfusion related disorders. PMID:27597885

  7. Structural insights into Parkin substrate lysine targeting from minimal Miro substrates

    PubMed Central

    Klosowiak, Julian L.; Park, Sungjin; Smith, Kyle P.; French, Michael E.; Focia, Pamela J.; Freymann, Douglas M.; Rice, Sarah E.

    2016-01-01

    Hereditary Parkinson’s disease is commonly caused by mutations in the protein kinase PINK1 or the E3 ubiquitin ligase Parkin, which function together to eliminate damaged mitochondria. PINK1 phosphorylates both Parkin and ubiquitin to stimulate ubiquitination of dozens of proteins on the surface of the outer mitochondrial membrane. However, the mechanisms by which Parkin recognizes specific proteins for modification remain largely unexplored. Here, we show that the C-terminal GTPase (cGTPase) of the Parkin primary substrate human Miro is necessary and sufficient for efficient ubiquitination. We present several new X-ray crystal structures of both human Miro1 and Miro2 that reveal substrate recognition and ubiquitin transfer to be specific to particular protein domains and lysine residues. We also provide evidence that Parkin substrate recognition is functionally separate from substrate modification. Finally, we show that prioritization for modification of a specific lysine sidechain of the cGTPase (K572) within human Miro1 is dependent on both its location and chemical microenvironment. Activation of Parkin by phosphorylation or by binding of pUb is required for prioritization of K572 for modification, suggesting that Parkin activation and acquisition of substrate specificity are coupled. PMID:27605430

  8. Behavioral phenotyping of Parkin-deficient mice: looking for early preclinical features of Parkinson's disease.

    PubMed

    Rial, Daniel; Castro, Adalberto A; Machado, Nuno; Garção, Pedro; Gonçalves, Francisco Q; Silva, Henrique B; Tomé, Angelo R; Köfalvi, Attila; Corti, Olga; Raisman-Vozari, Rita; Cunha, Rodrigo A; Prediger, Rui D

    2014-01-01

    There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson's disease (PD) begin many years before the appearance of the characteristic motor symptoms. Neuropsychiatric, sensorial and cognitive deficits are recognized as early non-motor manifestations of PD, and are not attenuated by the current anti-parkinsonian therapy. Although loss-of-function mutations in the parkin gene cause early-onset familial PD, Parkin-deficient mice do not display spontaneous degeneration of the nigrostriatal pathway or enhanced vulnerability to dopaminergic neurotoxins such as 6-OHDA and MPTP. Here, we employed adult homozygous C57BL/6 mice with parkin gene deletion on exon 3 (parkin-/-) to further investigate the relevance of Parkin in the regulation of non-motor features, namely olfactory, emotional, cognitive and hippocampal synaptic plasticity. Parkin-/- mice displayed normal performance on behavioral tests evaluating olfaction (olfactory discrimination), anxiety (elevated plus-maze), depressive-like behavior (forced swimming and tail suspension) and motor function (rotarod, grasping strength and pole). However, parkin-/- mice displayed a poor performance in the open field habituation, object location and modified Y-maze tasks suggestive of procedural and short-term spatial memory deficits. These behavioral impairments were accompanied by impaired hippocampal long-term potentiation (LTP). These findings indicate that the genetic deletion of parkin causes deficiencies in hippocampal synaptic plasticity, resulting in memory deficits with no major olfactory, emotional or motor impairments. Therefore, parkin-/- mice may represent a promising animal model to study the early stages of PD and for testing new therapeutic strategies to restore learning and memory and synaptic plasticity impairments in PD. PMID:25486126

  9. Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson's Disease Patients

    PubMed Central

    Swart, Chrisna; van der Westhuizen, Francois; van Dyk, Hayley; van der Merwe, Lize; van der Merwe, Celia; Loos, Ben; Carr, Jonathan; Kinnear, Craig; Bardien, Soraya

    2016-01-01

    Mutations in the parkin gene are the most common cause of early-onset Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (p = 0.0304). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p = 0.0001). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation. PMID:27034887

  10. PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding

    PubMed Central

    Lazarou, Michael; Narendra, Derek P.; Jin, Seok Min; Tekle, Ephrem; Banerjee, Soojay

    2013-01-01

    Genetic studies indicate that the mitochondrial kinase PINK1 and the RING-between-RING E3 ubiquitin ligase Parkin function in the same pathway. In concurrence, mechanistic studies show that PINK1 can recruit Parkin from the cytosol to the mitochondria, increase the ubiquitination activity of Parkin, and induce Parkin-mediated mitophagy. Here, we used a cell-free assay to recapitulate PINK1-dependent activation of Parkin ubiquitination of a validated mitochondrial substrate, mitofusin 1. We show that PINK1 activated the formation of a Parkin–ubiquitin thioester intermediate, a hallmark of HECT E3 ligases, both in vitro and in vivo. Parkin HECT-like ubiquitin ligase activity was essential for PINK1-mediated Parkin translocation to mitochondria and mitophagy. Using an inactive Parkin mutant, we found that PINK1 stimulated Parkin self-association and complex formation upstream of mitochondrial translocation. Self-association occurred independent of ubiquitination activity through the RING-between-RING domain, providing mechanistic insight into how PINK1 activates Parkin. PMID:23319602

  11. Parkin Enhances the Expression of Cyclin-dependent Kinase 6 and Negatively Regulates the Proliferation of Breast Cancer Cells*

    PubMed Central

    Tay, Shiam-Peng; Yeo, Calvin W. S.; Chai, Chou; Chua, Pei-Jou; Tan, Hui-Mei; Ang, Alex X. Y.; Yip, Daniel L. H.; Sung, Jian-Xiong; Tan, Puay Hoon; Bay, Boon-Huat; Wong, Siew-Heng; Tang, Carol; Tan, Jeanne M. M.; Lim, Kah-Leong

    2010-01-01

    Although mutations in the parkin gene are frequently associated with familial Parkinsonism, emerging evidence suggests that parkin also plays a role in cancers as a putative tumor suppressor. Supporting this, we show here that parkin expression is dramatically reduced in several breast cancer-derived cell lines as well as in primary breast cancer tissues. Importantly, we found that ectopic parkin expression in parkin-deficient breast cancer cells mitigates their proliferation rate both in vitro and in vivo, as well as reduces the capacity of these cells to migrate. Cell cycle analysis revealed the arrestment of a significant percentage of parkin-expressing breast cancer cells at the G1-phase. However, we did not observe significant changes in the levels of the G1-associated cyclin D1 and E. On the other hand, the level of cyclin-dependent kinase 6 (CDK6) is dramatically and selectively elevated in parkin-expressing breast cancer cells, the extent of which correlates well with the expression of parkin. Interestingly, a recent study demonstrated that CDK6 restrains the proliferation of breast cancer cells. Taken together, our results support a negative role for parkin in tumorigenesis and provide a potential mechanism by which parkin exerts its suppressing effects on breast cancer cell proliferation. PMID:20630868

  12. DMXAA causes tumor site-specific vascular disruption in murine non-small cell lung cancer, and like the endogenous non-canonical cyclic dinucleotide STING agonist, 2'3'-cGAMP, induces M2 macrophage repolarization.

    PubMed

    Downey, Charlene M; Aghaei, Mehrnoosh; Schwendener, Reto A; Jirik, Frank R

    2014-01-01

    The vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a murine agonist of the stimulator of interferon genes (STING), appears to target the tumor vasculature primarily as a result of stimulating pro-inflammatory cytokine production from tumor-associated macrophages (TAMs). Since there were relatively few reports of DMXAA effects in genetically-engineered mutant mice (GEMM), and models of non-small cell lung cancer (NSCLC) in particular, we examined both the effectiveness and macrophage dependence of DMXAA in various NSCLC models. The DMXAA responses of primary adenocarcinomas in K-rasLA1/+ transgenic mice, as well as syngeneic subcutaneous and metastatic tumors, generated by a p53R172HΔg/+; K-rasLA1/+ NSCLC line (344SQ-ELuc), were assessed both by in vivo bioluminescence imaging as well as by histopathology. Macrophage-dependence of DMXAA effects was explored by clodronate liposome-mediated TAM depletion. Furthermore, a comparison of the vascular structure between subcutaneous tumors and metastases was carried out using micro-computed tomography (micro-CT). Interestingly, in contrast to the characteristic hemorrhagic necrosis produced by DMXAA in 344SQ-ELuc subcutaneous tumors, this agent failed to cause hemorrhagic necrosis of either 344SQ-ELuc-derived metastases or autochthonous K-rasLA1/+ NSCLCs. In addition, we found that clodronate liposome-mediated depletion of TAMs in 344SQ-ELuc subcutaneous tumors led to non-hemorrhagic necrosis due to tumor feeding-vessel occlusion. Since NSCLC were comprised exclusively of TAMs with anti-inflammatory M2-like phenotype, the ability of DMXAA to re-educate M2-polarized macrophages was examined. Using various macrophage phenotypic markers, we found that the STING agonists, DMXAA and the non-canonical endogenous cyclic dinucleotide, 2'3'-cGAMP, were both capable of re-educating M2 cells towards an M1 phenotype. Our findings demonstrate that the choice of preclinical model and the anatomical site of a

  13. DMXAA Causes Tumor Site-Specific Vascular Disruption in Murine Non-Small Cell Lung Cancer, and like the Endogenous Non-Canonical Cyclic Dinucleotide STING Agonist, 2′3′-cGAMP, Induces M2 Macrophage Repolarization

    PubMed Central

    Downey, Charlene M.; Aghaei, Mehrnoosh; Schwendener, Reto A.; Jirik, Frank R.

    2014-01-01

    The vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a murine agonist of the stimulator of interferon genes (STING), appears to target the tumor vasculature primarily as a result of stimulating pro-inflammatory cytokine production from tumor-associated macrophages (TAMs). Since there were relatively few reports of DMXAA effects in genetically-engineered mutant mice (GEMM), and models of non-small cell lung cancer (NSCLC) in particular, we examined both the effectiveness and macrophage dependence of DMXAA in various NSCLC models. The DMXAA responses of primary adenocarcinomas in K-rasLA1/+ transgenic mice, as well as syngeneic subcutaneous and metastatic tumors, generated by a p53R172HΔg/+; K-rasLA1/+ NSCLC line (344SQ-ELuc), were assessed both by in vivo bioluminescence imaging as well as by histopathology. Macrophage-dependence of DMXAA effects was explored by clodronate liposome-mediated TAM depletion. Furthermore, a comparison of the vascular structure between subcutaneous tumors and metastases was carried out using micro-computed tomography (micro-CT). Interestingly, in contrast to the characteristic hemorrhagic necrosis produced by DMXAA in 344SQ-ELuc subcutaneous tumors, this agent failed to cause hemorrhagic necrosis of either 344SQ-ELuc-derived metastases or autochthonous K-rasLA1/+ NSCLCs. In addition, we found that clodronate liposome-mediated depletion of TAMs in 344SQ-ELuc subcutaneous tumors led to non-hemorrhagic necrosis due to tumor feeding-vessel occlusion. Since NSCLC were comprised exclusively of TAMs with anti-inflammatory M2-like phenotype, the ability of DMXAA to re-educate M2-polarized macrophages was examined. Using various macrophage phenotypic markers, we found that the STING agonists, DMXAA and the non-canonical endogenous cyclic dinucleotide, 2′3′-cGAMP, were both capable of re-educating M2 cells towards an M1 phenotype. Our findings demonstrate that the choice of preclinical model and the anatomical site of a

  14. Parkinson's Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation.

    PubMed

    Matheoud, Diana; Sugiura, Ayumu; Bellemare-Pelletier, Angélique; Laplante, Annie; Rondeau, Christiane; Chemali, Magali; Fazel, Ali; Bergeron, John J; Trudeau, Louis-Eric; Burelle, Yan; Gagnon, Etienne; McBride, Heidi M; Desjardins, Michel

    2016-07-14

    Antigen presentation is essential for establishing immune tolerance and for immune responses against infectious disease and cancer. Although antigen presentation can be mediated by autophagy, here we demonstrate a pathway for mitochondrial antigen presentation (MitAP) that relies on the generation and trafficking of mitochondrial-derived vesicles (MDVs) rather than on autophagy/mitophagy. We find that PINK1 and Parkin, two mitochondrial proteins linked to Parkinson's disease (PD), actively inhibit MDV formation and MitAP. In absence of PINK1 or Parkin, inflammatory conditions trigger MitAP in immune cells, both in vitro and in vivo. MitAP and the formation of MDVs require Rab9 and Sorting nexin 9, whose recruitment to mitochondria is inhibited by Parkin. The identification of PINK1 and Parkin as suppressors of an immune-response-eliciting pathway provoked by inflammation suggests new insights into PD pathology. PMID:27345367

  15. Parkin protects dopaminergic neurons from excessive Wnt/{beta}-catenin signaling

    SciTech Connect

    Rawal, Nina; Corti, Olga; Sacchetti, Paola; Ardilla-Osorio, Hector; Sehat, Bita; Brice, Alexis; Arenas, Ernest

    2009-10-23

    Parkinson's disease (PD) is caused by degeneration of the dopaminergic (DA) neurons of the substantia nigra but the molecular mechanisms underlying the degenerative process remain elusive. Several reports suggest that cell cycle deregulation in post-mitotic neurons could lead to neuronal cell death. We now show that Parkin, an E3 ubiquitin ligase linked to familial PD, regulates {beta}-catenin protein levels in vivo. Stabilization of {beta}-catenin in differentiated primary ventral midbrain neurons results in increased levels of cyclin E and proliferation, followed by increased levels of cleaved PARP and loss of DA neurons. Wnt3a signaling also causes death of post-mitotic DA neurons in parkin null animals, suggesting that both increased stabilization and decreased degradation of {beta}-catenin results in DA cell death. These findings demonstrate a novel regulation of Wnt signaling by Parkin and suggest that Parkin protects DA neurons against excessive Wnt signaling and {beta}-catenin-induced cell death.

  16. Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

    PubMed Central

    McLelland, Gian-Luca; Soubannier, Vincent; Chen, Carol X; McBride, Heidi M; Fon, Edward A

    2014-01-01

    Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondria-derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin- and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD. PMID:24446486

  17. Loss of locus coeruleus neurons and reduced startle in parkin null mice

    PubMed Central

    von Coelln, Rainer; Thomas, Bobby; Savitt, Joseph M.; Lim, Kah Leong; Sasaki, Masayuki; Hess, Ellen J.; Dawson, Valina L.; Dawson, Ted M.

    2004-01-01

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized pathologically by degeneration of catecholaminergic neurons of the substantia nigra pars compacta and locus coeruleus, among other regions. Autosomal-recessive juvenile Parkinsonism (ARJP) is caused by mutations in the PARK2 gene coding for parkin and constitutes the most common familial form of PD. The majority of ARJP-associated parkin mutations are thought to be loss of function-mutations; however, the pathogenesis of ARJP remains poorly understood. Here, we report the generation of parkin null mice by targeted deletion of parkin exon 7. These mice show a loss of catecholaminergic neurons in the locus coeruleus and an accompanying loss of norepinephrine in discrete regions of the central nervous system. Moreover, there is a dramatic reduction of the norepinephrine-dependent startle response. The nigrostriatal dopaminergic system does not show any impairment. This mouse model will help gain a better understanding of parkin function and the mechanisms underlying parkin-associated PD. PMID:15249681

  18. Direct binding with HDAC6 mediates the reversible recruitment of parkin to the centrosome

    PubMed Central

    Jiang, Qian; Ren, Yong; Feng, Jian

    2009-01-01

    Histone deacetylase 6 (HDAC6), a microtubule-associated tubulin deacetylase, plays a significant role in the formation of protein aggregates in many neurodegenerative disorders. Parkin, a protein-ubiquitin E3 ligase linked to Parkinson’s disease, accumulates at the centrosome in a microtubule-dependent manner in response to proteasome inhibition. Here, we show that the centrosome recruitment of parkin was mediated by its direct binding to HDAC6 through multiple interaction domains. The tubulin deacetylase activity of HDAC6 was required for the accumulation of parkin as well as its dispersion upon the reversal of proteasome inhibition. The bidirectional movements of parkin required intact microtubule network and were dependent on dynein and kinesin 1, respectively. Tubulin deacetylation increases microtubule dynamicity and may thus facilitate microtubule-based trafficking of the parkin-HDAC6 complex. The results suggest that HDAC6 acts as a sensor of proteasome inhibition and directs the trafficking of parkin by utilizing different motor proteins. PMID:19036992

  19. Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases

    PubMed Central

    Riley, B.E.; Lougheed, J.C.; Callaway, K.; Velasquez, M.; Brecht, E.; Nguyen, L.; Shaler, T.; Walker, D.; Yang, Y.; Regnstrom, K.; Diep, L.; Zhang, Z.; Chiou, S.; Bova, M.; Artis, D.R.; Yao, N.; Baker, J.; Yednock, T.; Johnston, J.A.

    2013-01-01

    Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson’s disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed ‘RING/HECT hybrid’ enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin’s cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein. PMID:23770887

  20. Structure of the human Parkin ligase domain in an autoinhibited state

    PubMed Central

    Wauer, Tobias; Komander, David

    2013-01-01

    Mutations in the protein Parkin are associated with Parkinson's disease (PD), the second most common neurodegenerative disease in men. Parkin is an E3 ubiquitin (Ub) ligase of the structurally uncharacterized RING-in-between-RING(IBR)-RING (RBR) family, which, in an HECT-like fashion, forms a catalytic thioester intermediate with Ub. We here report the crystal structure of human Parkin spanning the Unique Parkin domain (UPD, also annotated as RING0) and RBR domains, revealing a tightly packed structure with unanticipated domain interfaces. The UPD adopts a novel elongated Zn-binding fold, while RING2 resembles an IBR domain. Two key interactions keep Parkin in an autoinhibited conformation. A linker that connects the IBR with the RING2 over a 50-Å distance blocks the conserved E2∼Ub binding site of RING1. RING2 forms a hydrophobic interface with the UPD, burying the catalytic Cys431, which is part of a conserved catalytic triad. Opening of intra-domain interfaces activates Parkin, and enables Ub-based suicide probes to modify Cys431. The structure further reveals a putative phospho-peptide docking site in the UPD, and explains many PD-causing mutations. PMID:23727886

  1. PKA Regulates PINK1 Stability and Parkin Recruitment to Damaged Mitochondria through Phosphorylation of MIC60.

    PubMed

    Akabane, Shiori; Uno, Midori; Tani, Naoki; Shimazaki, Shunta; Ebara, Natsumi; Kato, Hiroki; Kosako, Hidetaka; Oka, Toshihiko

    2016-05-01

    A mitochondrial kinase, PTEN-induced putative kinase 1 (PINK1), selectively recruits the ubiquitin ligase Parkin to damaged mitochondria, which modifies mitochondria by polyubiquitination, leading to mitochondrial autophagy. Here, we report that treatment with an adenylate cyclase agonist or expression of protein kinase A (PKA) impairs Parkin recruitment to damaged mitochondria and decreases PINK1 protein levels. We identified a mitochondrial membrane protein, MIC60 (also known as mitofilin), as a PKA substrate. Mutational and mass spectrometric analyses revealed that the Ser528 residue of MIC60 undergoes PKA-dependent phosphorylation. MIC60 transiently interacts with PINK1, and MIC60 downregulation leads to a reduction in PINK1 and mislocalization of Parkin. Phosphorylation-mimic mutants of MIC60 fail to restore the defect in Parkin recruitment in MIC60-knocked down cells, whereas a phosphorylation-deficient MIC60 mutant facilitates the mitochondrial localization of Parkin. Our findings indicate that PKA-mediated phosphorylation of MIC60 negatively regulates mitochondrial clearance that is initiated by PINK1 and Parkin. PMID:27153535

  2. Exon dosage analysis of parkin gene in Chinese sporadic Parkinson's disease.

    PubMed

    Guo, Ji-Feng; Dong, Xiao-Li; Xu, Qian; Li, Nan; Yan, Xin-Xiang; Xia, Kun; Tang, Bei-Sha

    2015-09-14

    Parkin gene mutations are by far the most common mutations in both familial Parkinson's disease (PD) and sporadic PD. Approximately, 50% of parkin mutations is exon dosage mutations (i.e., deletions and duplications of entire exons). Here, we first established a MLPA assay for quick detection of parkin exon rearrangements. Then, we studied parkin exon dosage mutations in 755 Chinese sporadic PDdisease patients using the established MLPA assay. We found that there were 25 (3.3%) patients with exon dosage alterations including deletions and duplications, 20 (11.4%) patients with exon rearrangements in 178 early-onset patients, and 5 (0.86%) patients with exon rearrangement mutations in 579 later-onset patients. The percentage of individuals with parkin dosage mutations is more than 33% when the age at onset is less than 30 years old, but less than 7% when the age at onset is more than 30. In these mutations, deletion is the main mutational style, especially in exon 2-5. Our results indicated that exon dosage mutations in parkin gene might be the main cause for sporadic PD, especially in EOP. PMID:26240990

  3. Mitochondrial impairment observed in fibroblasts from South African Parkinson’s disease patients with parkin mutations

    SciTech Connect

    Merwe, Celia van der; Loos, Ben; Swart, Chrisna; Kinnear, Craig; Merwe, Lize van der; Pillay, Komala; Muller, Nolan; Zaharie, Dan; Engelbrecht, Lize; Carr, Jonathan; and others

    2014-05-02

    Highlights: • Mitochondrial dysfunction observed in patients with parkin-null mutations. • Mitochondrial ATP levels were decreased. • Electron-dense vacuoles were observed in the patients. • Mitochondria from muscle biopsies appeared within normal limits. • One patient did not show these defects possibly due to compensatory mechanisms. - Abstract: Parkinson’s disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients’ fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD

  4. Nitric Oxide Induction of Parkin Translocation in PTEN-induced Putative Kinase 1 (PINK1) Deficiency

    PubMed Central

    Han, Ji-Young; Kang, Min-Ji; Kim, Kyung-Hee; Han, Pyung-Lim; Kim, Hyun-Seok; Ha, Ji-Young; Son, Jin H.

    2015-01-01

    The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser1412) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease. PMID:25716315

  5. Oxidation of the cysteine-rich regions of parkin perturbs its E3 ligase activity and contributes to protein aggregation

    PubMed Central

    2011-01-01

    Background Accumulation of aberrant proteins to form Lewy bodies (LBs) is a hallmark of Parkinson's disease (PD). Ubiquitination-mediated degradation of aberrant, misfolded proteins is critical for maintaining normal cell function. Emerging evidence suggests that oxidative/nitrosative stress compromises the precisely-regulated network of ubiquitination in PD, particularly affecting parkin E3 ligase activity, and contributes to the accumulation of toxic proteins and neuronal cell death. Results To gain insight into the mechanism whereby cell stress alters parkin-mediated ubiquitination and LB formation, we investigated the effect of oxidative stress. We found significant increases in oxidation (sulfonation) and subsequent aggregation of parkin in SH-SY5Y cells exposed to the mitochondrial complex I inhibitor 1-methyl-4-phenlypyridinium (MPP+), representing an in vitro cell-based PD model. Exposure of these cells to direct oxidation via pathological doses of H2O2 induced a vicious cycle of increased followed by decreased parkin E3 ligase activity, similar to that previously reported following S-nitrosylation of parkin. Pre-incubation with catalase attenuated H2O2 accumulation, parkin sulfonation, and parkin aggregation. Mass spectrometry (MS) analysis revealed that H2O2 reacted with specific cysteine residues of parkin, resulting in sulfination/sulfonation in regions of the protein similar to those affected by parkin mutations in hereditary forms of PD. Immunohistochemistry or gel electrophoresis revealed an increase in aggregated parkin in rats and primates exposed to mitochondrial complex I inhibitors, as well as in postmortem human brain from patients with PD with LBs. Conclusion These findings show that oxidative stress alters parkin E3 ligase activity, leading to dysfunction of the ubiquitin-proteasome system and potentially contributing to LB formation. PMID:21595948

  6. Parkin regulates mitophagy and mitochondrial function to protect against alcohol-induced liver injury and steatosis in mice.

    PubMed

    Williams, Jessica A; Ni, Hong-Min; Ding, Yifeng; Ding, Wen-Xing

    2015-09-01

    Alcoholic liver disease claims two million lives per year. We previously reported that autophagy protected against alcohol-induced liver injury and steatosis by removing damaged mitochondria. However, the mechanisms for removal of these mitochondria are unknown. Parkin is an evolutionarily conserved E3 ligase that is recruited to damaged mitochondria to initiate ubiquitination of mitochondrial outer membrane proteins and subsequent mitochondrial degradation by mitophagy. In addition to its role in mitophagy, Parkin has been shown to have other roles in maintaining mitochondrial function. We investigated whether Parkin protected against alcohol-induced liver injury and steatosis using wild-type (WT) and Parkin knockout (KO) mice treated with alcohol by the acute-binge and Gao-binge (chronic plus acute-binge) models. We found that Parkin protected against liver injury in both alcohol models, likely because of Parkin's role in maintaining a population of healthy mitochondria. Alcohol caused greater mitochondrial damage and oxidative stress in Parkin KO livers compared with WT livers. After alcohol treatment, Parkin KO mice had severely swollen and damaged mitochondria that lacked cristae, which were not seen in WT mice. Furthermore, Parkin KO mice had decreased mitophagy, β-oxidation, mitochondrial respiration, and cytochrome c oxidase activity after acute alcohol treatment compared with WT mice. Interestingly, liver mitochondria seemed able to adapt to alcohol treatment, but Parkin KO mouse liver mitochondria had less capacity to adapt to Gao-binge treatment compared with WT mouse liver mitochondria. Overall, our findings indicate that Parkin is an important mediator of protection against alcohol-induced mitochondrial damage, steatosis, and liver injury. PMID:26159696

  7. Parkin regulates mitophagy and mitochondrial function to protect against alcohol-induced liver injury and steatosis in mice

    PubMed Central

    Williams, Jessica A.; Ni, Hong-Min; Ding, Yifeng

    2015-01-01

    Alcoholic liver disease claims two million lives per year. We previously reported that autophagy protected against alcohol-induced liver injury and steatosis by removing damaged mitochondria. However, the mechanisms for removal of these mitochondria are unknown. Parkin is an evolutionarily conserved E3 ligase that is recruited to damaged mitochondria to initiate ubiquitination of mitochondrial outer membrane proteins and subsequent mitochondrial degradation by mitophagy. In addition to its role in mitophagy, Parkin has been shown to have other roles in maintaining mitochondrial function. We investigated whether Parkin protected against alcohol-induced liver injury and steatosis using wild-type (WT) and Parkin knockout (KO) mice treated with alcohol by the acute-binge and Gao-binge (chronic plus acute-binge) models. We found that Parkin protected against liver injury in both alcohol models, likely because of Parkin's role in maintaining a population of healthy mitochondria. Alcohol caused greater mitochondrial damage and oxidative stress in Parkin KO livers compared with WT livers. After alcohol treatment, Parkin KO mice had severely swollen and damaged mitochondria that lacked cristae, which were not seen in WT mice. Furthermore, Parkin KO mice had decreased mitophagy, β-oxidation, mitochondrial respiration, and cytochrome c oxidase activity after acute alcohol treatment compared with WT mice. Interestingly, liver mitochondria seemed able to adapt to alcohol treatment, but Parkin KO mouse liver mitochondria had less capacity to adapt to Gao-binge treatment compared with WT mouse liver mitochondria. Overall, our findings indicate that Parkin is an important mediator of protection against alcohol-induced mitochondrial damage, steatosis, and liver injury. PMID:26159696

  8. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy

    PubMed Central

    Ordureau, Alban; Heo, Jin-Mi; Duda, David M.; Paulo, Joao A.; Olszewski, Jennifer L.; Yanishevski, David; Rinehart, Jesse; Schulman, Brenda A.; Harper, J. Wade

    2015-01-01

    The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using “UBS65A-replacement,” we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UBS65A-replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains. PMID:25969509

  9. The principal PINK1 and Parkin cellular events triggered in response to dissipation of mitochondrial membrane potential occur in primary neurons

    PubMed Central

    Koyano, Fumika; Okatsu, Kei; Ishigaki, Shinsuke; Fujioka, Yusuke; Kimura, Mayumi; Sobue, Gen; Tanaka, Keiji; Matsuda, Noriyuki

    2013-01-01

    PINK1 and PARKIN are causal genes for hereditary Parkinsonism. Recent studies have shown that PINK1 and Parkin play a pivotal role in the quality control of mitochondria, and dysfunction of either protein likely results in the accumulation of low-quality mitochondria that triggers early-onset familial Parkinsonism. As neurons are destined to degenerate in PINK1/Parkin-associated Parkinsonism, it is imperative to investigate the function of PINK1 and Parkin in neurons. However, most studies investigating PINK1/Parkin have used non-neuronal cell lines. Here we show that the principal PINK1 and Parkin cellular events that have been documented in non-neuronal lines in response to mitochondrial damage also occur in primary neurons. We found that dissipation of the mitochondrial membrane potential triggers phosphorylation of both PINK1 and Parkin and that, in response, Parkin translocates to depolarized mitochondria. Furthermore, Parkin's E3 activity is re-established concomitant with ubiquitin–ester formation at Cys431 of Parkin. As a result, mitochondrial substrates in neurons become ubiquitylated. These results underscore the relevance of the PINK1/Parkin-mediated mitochondrial quality control pathway in primary neurons and shed further light on the underlying mechanisms of the PINK1 and Parkin pathogenic mutations that predispose Parkinsonism in vivo. PMID:23751051

  10. Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin

    PubMed Central

    Fiesel, Fabienne C.; Caulfield, Thomas R.; Moussaud-Lamodière, Elisabeth L.; Ogaki, Kotaro; Dourado, Daniel F.A.R.; Flores, Samuel C.; Ross, Owen A.; Springer, Wolfdieter

    2015-01-01

    Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility and protein aggregation. However pathogenic effects can also result from misregulation of Parkin auto-inhibition and of its enzymatic functions. In addition, interference of binding to co-enzymes, substrates and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin. PMID:25939424

  11. ATM mediates spermidine-induced mitophagy via PINK1 and Parkin regulation in human fibroblasts

    PubMed Central

    Qi, Yongmei; Qiu, Qian; Gu, Xueyan; Tian, Yihong; Zhang, Yingmei

    2016-01-01

    The ATM (ataxia telangiectasia mutated) protein has recently been proposed to play critical roles in the response to mitochondrial dysfunction by initiating mitophagy. Here, we have used ATM-proficient GM00637 cells and ATM-deficient GM05849 cells to investigate the mitophagic effect of spermidine and to elucidate the role of ATM in spermdine-induced mitophagy. Our results indicate that spermidine induces mitophagy by eliciting mitochondrial depolarization, which triggers the formation of mitophagosomes and mitolysosomes, thereby promoting the accumulation of PINK1 and translocation of Parkin to damaged mitochondria, finally leading to the decreased mitochondrial mass in GM00637 cells. However, in GM05849 cells or GM00637 cells pretreated with the ATM kinase inhibitor KU55933, the expression of full-length PINK1 and the translocation of Parkin are blocked, and the colocalization of Parkin with either LC3 or PINK1 is disrupted. These results suggest that ATM drives the initiation of the mitophagic cascade. Our study demonstrates that spermidine induces mitophagy through ATM-dependent activation of the PINK1/Parkin pathway. These findings underscore the importance of a mitophagy regulatory network of ATM and PINK1/Parkin and elucidate a novel mechanism by which ATM influences spermidine-induced mitophagy. PMID:27089984

  12. The relationship between Obsessive-Compulsive symptoms and PARKIN genotype: The CORE-PD study

    PubMed Central

    Sharp, ME; Caccappolo, E; Mejia-Santana, H; Tang, M–X; Rosado, L; Orbe Reilly, M; Ruiz, D; Louis, ED; Comella, C; Nance, M; Bressman, S; Scott, WK; Tanner, C; Waters, C; Fahn, S; Cote, L; Ford, B; Rezak, M; Novak, K; Friedman, JH; Pfeiffer, R; Payami, H; Molho, E; Factor, SA; Nutt, J; Serrano, C; Arroyo, M; Pauciulo, MW; Nichols, WC; Clark, LN; Alcalay, RN; Marder, KS

    2014-01-01

    Background Few studies have systematically investigated the association between PARKIN genotype and psychiatric co-morbidities of PD. PARKIN-associated PD is characterized by severe nigral dopaminergic neuronal loss, a finding that may have implications for behaviors rooted in dopaminergic circuits such as obsessive-compulsive symptoms (OCS). Methods The Schedule of Compulsions and Obsessions Patient Inventory (SCOPI) was administered to 104 patients with early-onset PD and 257 asymptomatic first-degree relatives. Carriers of one and two PARKIN mutations were compared to non-carriers. Results Among patients, carriers scored lower than non-carriers in adjusted models (one-mutation: 13.9 point difference, p=0.03; two-mutation: 24.1, p=0.001), where lower scores indicate less OCS. Among asymptomatic relatives, there was a trend towards the opposite: mutation carriers scored higher than non-carriers (one mutation p = 0.05; two mutations p = 0.13). Conclusions First, there was a significant association between PARKIN mutation status and obsessive-compulsive symptom level in both PD and asymptomatics, suggesting that OCS might represent an early non-motor dopamine-dependent feature. Second, irrespective of disease status, heterozygotes were significantly different that non-carriers suggesting that PARKIN heterozygosity may contribute to phenotype. PMID:25393808

  13. Mitofusin-mediated ER stress triggers neurodegeneration in pink1/parkin models of Parkinson's disease.

    PubMed

    Celardo, I; Costa, A C; Lehmann, S; Jones, C; Wood, N; Mencacci, N E; Mallucci, G R; Loh, S H Y; Martins, L M

    2016-01-01

    Mutations in PINK1 and PARKIN cause early-onset Parkinson's disease (PD), thought to be due to mitochondrial toxicity. Here, we show that in Drosophila pink1 and parkin mutants, defective mitochondria also give rise to endoplasmic reticulum (ER) stress signalling, specifically to the activation of the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response (UPR). We show that enhanced ER stress signalling in pink1 and parkin mutants is mediated by mitofusin bridges, which occur between defective mitochondria and the ER. Reducing mitofusin contacts with the ER is neuroprotective, through suppression of PERK signalling, while mitochondrial dysfunction remains unchanged. Further, both genetic inhibition of dPerk-dependent ER stress signalling and pharmacological inhibition using the PERK inhibitor GSK2606414 were neuroprotective in both pink1 and parkin mutants. We conclude that activation of ER stress by defective mitochondria is neurotoxic in pink1 and parkin flies and that the reduction of this signalling is neuroprotective, independently of defective mitochondria. A video abstract for this article is available online in the supplementary information. PMID:27336715

  14. Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice.

    PubMed

    Gong, Guohua; Song, Moshi; Csordas, Gyorgy; Kelly, Daniel P; Matkovich, Scot J; Dorn, Gerald W

    2015-12-01

    In developing hearts, changes in the cardiac metabolic milieu during the perinatal period redirect mitochondrial substrate preference from carbohydrates to fatty acids. Mechanisms responsible for this mitochondrial plasticity are unknown. Here, we found that PINK1-Mfn2-Parkin-mediated mitophagy directs this metabolic transformation in mouse hearts. A mitofusin (Mfn) 2 mutant lacking PINK1 phosphorylation sites necessary for Parkin binding (Mfn2 AA) inhibited mitochondrial Parkin translocation, suppressing mitophagy without impairing mitochondrial fusion. Cardiac Parkin deletion or expression of Mfn2 AA from birth, but not after weaning, prevented postnatal mitochondrial maturation essential to survival. Five-week-old Mfn2 AA hearts retained a fetal mitochondrial transcriptional signature without normal increases in fatty acid metabolism and mitochondrial biogenesis genes. Myocardial fatty acylcarnitine levels and cardiomyocyte respiration induced by palmitoylcarnitine were concordantly depressed. Thus, instead of transcriptional reprogramming, fetal cardiomyocyte mitochondria undergo perinatal Parkin-mediated mitophagy and replacement by mature adult mitochondria. Mitophagic mitochondrial removal underlies developmental cardiomyocyte mitochondrial plasticity and metabolic transitioning of perinatal hearts. PMID:26785495

  15. The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process

    SciTech Connect

    Park, Jeehye; Lee, Gina; Chung, Jongkyeong

    2009-01-16

    The two Parkinson's disease (PD) genes, PTEN-induced kinase 1 (PINK1) and parkin, are linked in a common pathway which affects mitochondrial integrity and function. However, it is still not known what this pathway does in the mitochondria. Therefore, we investigated its physiological function in Drosophila. Because Drosophila PINK1 and parkin mutants show changes in mitochondrial morphology in both indirect flight muscles and dopaminergic neurons, we here investigated whether the PINK1-Parkin pathway genetically interacts with the regulators of mitochondrial fusion and fission such as Drp1, which promotes mitochondrial fission, and Opa1 or Marf, which induces mitochondrial fusion. Surprisingly, DrosophilaPINK1 and parkin mutant phenotypes were markedly suppressed by overexpression of Drp1 or downregulation of Opa1 or Marf, indicating that the PINK1-Parkin pathway regulates mitochondrial remodeling process in the direction of promoting mitochondrial fission. Therefore, we strongly suggest that mitochondrial fusion and fission process could be a prominent therapeutic target for the treatment of PD.

  16. Triggering of Parkin Mitochondrial Translocation in Mitophagy: Implications for Liver Diseases

    PubMed Central

    Eid, Nabil; Ito, Yuko; Otsuki, Yoshinori

    2016-01-01

    A growing body of evidence based on in vitro studies indicates that mitophagy (selective autophagic clearance of damaged mitochondria) is a prosurvival mechanism associated with cellular exposure to various mitochondrial stressors. Very recently, a limited number of publications on animal-based models of alcoholic fatty liver diseases have reported that Parkin-mediated mitophagy may mitigate hepatocyte apoptosis, improve mitochondrial quality and suppress steatosis (lipid accumulation). From this perspective, the authors focus on the mechanisms of Parkin mitochondrial translocation (a key consideration in mitophagy activation) and therapeutic implications of mitophagy in liver disease. DNA repair and other functions of Parkin beyond mitophagy are also briefly discussed. The paper additionally shows original data from the authors’ current research indicating enhanced hepatic mitophagy in ethanol-treated rats, which is associated with Parkin mitochondrial translocation triggered by oxidative mitochondrial DNA damage. Natural or pharmaceutical products that may trigger Parkin mitochondrial translocation in hepatocytes and/or suppress repressors of such translocation could be a potential therapeutic target in alcoholic and non-alcoholic fatty liver disease. PMID:27199746

  17. Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin

    PubMed Central

    Moussaud-Lamodière, Elisabeth L.; Dourado, Daniel F. A. R.; Flores, Samuel C.; Springer, Wolfdieter

    2014-01-01

    Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through

  18. Parp mutations protect against mitochondrial dysfunction and neurodegeneration in a PARKIN model of Parkinson's disease

    PubMed Central

    Lehmann, S; Costa, A C; Celardo, I; Loh, S H Y; Martins, L M

    2016-01-01

    The co-enzyme nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular energy generation in mitochondria as well as for DNA repair mechanisms in the cell nucleus involving NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Mitochondrial function is compromised in animal models of Parkinson's disease (PD) associated with PARKIN mutations. Here, we uncovered alterations in NAD+ salvage metabolism in Drosophila parkin mutants. We show that a dietary supplementation with the NAD+ precursor nicotinamide rescues mitochondrial function and is neuroprotective. Further, by mutating Parp in parkin mutants, we show that this increases levels of NAD+ and its salvage metabolites. This also rescues mitochondrial function and suppresses dopaminergic neurodegeneration. We conclude that strategies to enhance NAD+ levels by administration of dietary precursors or the inhibition of NAD+-dependent enzymes, such as PARP, that compete with mitochondria for NAD+ could be used to delay neuronal death associated with mitochondrial dysfunction. PMID:27031963

  19. Mutational analysis of parkin and PINK1 in multiple system atrophy

    PubMed Central

    Brooks, Janet A.; Houlden, Henry; Melchers, Anna; Islam, Ansha J.; Ding, Jinhui; Li, Abi; Paudel, Reema; Revesz, Tamas; Holton, Janice L.; Wood, Nick; Lees, Andrew; Singleton, Andrew B.; Scholz, Sonja W.

    2009-01-01

    Multiple system atrophy (MSA) and Parkinson’s disease (PD) are progressive neurodegenerative disorders with overlapping clinical, biochemical and genetic features. To test the hypothesis that the Parkinson’s disease genes parkin and PINK1 also play a role in the pathogenesis of MSA, we performed a mutational screening study involving 87 pathology-proven MSA cases. In parkin we identified eight sequence variants and four heterozygous deletions, and in PINK1 we identified nine variants of which two silent mutations have not been previously reported (p.Gly189Gly and p.Arg337Arg). The frequencies of the observed variants were not significantly different from previously published control data and none of the possibly pathogenic variants were found in a homozygous state. Our results indicate that genetic variants at the parkin and PINK1 loci do not play a critical role in the pathogenesis of MSA. PMID:20034704

  20. Structure and Function of Parkin, PINK1, and DJ-1, the Three Musketeers of Neuroprotection

    PubMed Central

    Trempe, Jean-François; Fon, Edward A.

    2013-01-01

    Autosomal recessive forms of Parkinson’s disease are caused by mutations in three genes: Parkin, PINK1, and DJ-1. These genes encode for proteins with distinct enzymatic activities that may work together to confer neuroprotection. Parkin is an E3 ubiquitin ligase that has been shown to ubiquitinate substrates and to trigger proteasome-dependent degradation or autophagy, two crucial homeostatic processes in neurons. PINK1 is a mitochondrial protein kinase whose activity is required for Parkin-dependent mitophagy, a process that has been linked to neurodegeneration. Finally, DJ-1 is a protein homologous to a broad class of bacterial enzymes that may function as a sensor and modulator of reactive oxygen species, which have been implicated in neurodegenerative diseases. Here, we review the literature on the structure and biochemical functions of these three proteins. PMID:23626584

  1. Parkin Binds to α/β Tubulin and Increases their Ubiquitination and Degradation

    PubMed Central

    Ren, Yong; Zhao, Jinghui; Feng, Jian

    2007-01-01

    In addition to inhibiting the mitochondrial respiratory chain, toxins known to cause Parkinson’s disease (PD), such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone, also strongly depolymerize microtubules and increase tubulin degradation. Microtubules are polymers of tubulin α/β heterodimers, whose correct folding requires coordinated actions of cellular chaperonins and co-factors. Misfolded tubulin monomers are highly toxic and quickly degraded through a hitherto unknown mechanism. Here we report that parkin, a protein-ubiquitin E3 ligase linked to PD, was tightly bound to microtubules in taxol-mediated microtubule co-assembly assays. In lysates from the rat brain or transfected HEK293 cells, α-tubulin and β-tubulin were strongly co-immunoprecipitated with parkin at 4°C in the presence of colchicine, a condition where tubulin exits as α/β heterodimers. At the subcellular level, parkin exhibited punctate immunostaining along microtubules in rat brain sections, cultured primary neurons, glial cells and cell lines. This pattern of subcellular localization was abolished in cells treated with the microtubule-depolymerizing drug colchicine. The binding between parkin and tubulin apparently led to increased ubiquitination and accelerated degradation of α- and β-tubulins in HEK293 cells. Similarly ubiquitinated tubulins were also observed in rat brain lysates. Furthermore, parkin mutants found in PD patients did not ubiquitinate or degrade either tubulin. Taken together, our results show that parkin is a novel tubulin-binding protein, as well as a microtubule-associated protein (MAP). Its ability to enhance the ubiquitination and degradation of misfolded tubulins may play a significant role in protecting neurons from toxins that cause PD. PMID:12716939

  2. Endogenous ochronosis.

    PubMed

    Turgay, E; Canat, D; Gurel, M S; Yuksel, T; Baran, M F; Demirkesen, C

    2009-12-01

    Endogenous ochronosis or alkaptonuria is a rare, autosomal recessive disease of tyrosine metabolism that is caused by a deficiency of the enzyme homogentisic acid oxidase. The disease results in the accumulation and deposition of homogentisic acid in the cartilage, eyelids, forehead, cheeks, axillae, genital region, buccal mucosa, larynx, tympanic membranes, and tendons. The disease generally presents in adults with arthritis and skin abnormalities; occasionally, involvement of other organs may be seen. A 49-year-old man was referred to our clinic with verrucous lesions on his hands. On physical examination, caviar-like ochronotic papules were found around his eyes and the helix cartilage of his ears, and on the dorsa of both hands. There were brown macules on the sclera (Osler's sign). The patient had arthritis and nephrolithiasis, and a sample of his urine darkened upon standing. Histopathological examination showed deposition of ochronotic pigment. High-dose ascorbic acid was given, and the patient showed improvement on follow-up examination 6 months later. PMID:20055850

  3. parkin-induced defects in neurophysiology and locomotion are generated by metabolic dysfunction and not oxidative stress

    PubMed Central

    Vincent, Amanda; Briggs, Laura; Chatwin, Griff F.J.; Emery, Elizabeth; Tomlins, Rose; Oswald, Matt; Middleton, C. Adam; Evans, Gareth J.O.; Sweeney, Sean T.; Elliott, Christopher J.H.

    2012-01-01

    Parkinson's disease (PD) is characterized by movement disorders, including bradykinesia. Analysis of inherited, juvenile PD, identified several genes linked via a common pathway to mitochondrial dysfunction. In this study, we demonstrate that the larva of the Drosophila parkin mutant faithfully models the locomotory and metabolic defects of PD and is an excellent system for investigating their inter-relationship. parkin larvae displayed a marked bradykinesia that was caused by a reduction in both the frequency of peristalsis and speed of muscle contractions. Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle. Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit. This was supported by our observations in parkin larvae that the resting potential was depolarized, oxygen consumption and ATP concentration were drastically reduced while lactate was increased. These findings suggest that neuronal mitochondrial respiration is severely compromised and there is a compensatory switch to glycolysis for energy production. parkin mutants also possessed overgrown neuromuscular synapses, indicative of oxidative stress, which could be rescued by overexpression of parkin or scavengers of reactive oxygen species (ROS). Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes. We therefore propose that mitochondrial dysfunction in parkin mutants induces Parkinsonian bradykinesia via a neuronal energy deficit and resulting synaptic failure, rather than as a consequence of downstream oxidative stress. PMID:22215442

  4. MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

    PubMed Central

    Nezich, Catherine L.; Wang, Chunxin; Fogel, Adam I.

    2015-01-01

    The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats–generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy. PMID:26240184

  5. Lithium prevents parkinsonian behavioral and striatal phenotypes in an aged parkin mutant transgenic mouse model.

    PubMed

    Lieu, Christopher A; Dewey, Colleen M; Chinta, Shankar J; Rane, Anand; Rajagopalan, Subramanian; Batir, Sean; Kim, Yong-Hwan; Andersen, Julie K

    2014-12-01

    Lithium has long been used as a treatment for the psychiatric disease bipolar disorder. However, previous studies suggest that lithium provides neuroprotective effects in neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease. The exact mechanism by which lithium exerts these effects still remains unclear. In the present study, we evaluated the effects of low-dose lithium treatment in an aged mouse model expressing a parkin mutation within dopaminergic neurons. We found that low-dose lithium treatment prevented motor impairment as demonstrated by the open field test, pole test, and rearing behavior. Furthermore, lithium prevented dopaminergic striatal degeneration in parkin animals. We also found that parkin-induced striatal astrogliosis and microglial activation were prevented by lithium treatment. Our results further corroborate the use of this parkin mutant transgenic mouse line as a model for PD for testing novel therapeutics. The findings of the present study also provide further validation that lithium could be re-purposed as a therapy for PD and suggest that anti-inflammatory effects may contribute to its neuroprotective mechanisms. PMID:25452026

  6. Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin

    PubMed Central

    Ashrafi, Ghazaleh; Schlehe, Julia S.; LaVoie, Matthew J.

    2014-01-01

    To minimize oxidative damage to the cell, malfunctioning mitochondria need to be removed by mitophagy. In neuronal axons, mitochondrial damage may occur in distal regions, far from the soma where most lysosomal degradation is thought to occur. In this paper, we report that PINK1 and Parkin, two Parkinson’s disease–associated proteins, mediate local mitophagy of dysfunctional mitochondria in neuronal axons. To reduce cytotoxicity and mimic physiological levels of mitochondrial damage, we selectively damaged a subset of mitochondria in hippocampal axons. Parkin was rapidly recruited to damaged mitochondria in axons followed by formation of LC3-positive autophagosomes and LAMP1-positive lysosomes. In PINK1−/− axons, damaged mitochondria did not accumulate Parkin and failed to be engulfed in autophagosomes. Similarly, initiation of mitophagy was blocked in Parkin−/− axons. Our findings demonstrate that the PINK1–Parkin-mediated pathway is required for local mitophagy in distal axons in response to focal damage. Local mitophagy likely provides rapid neuroprotection against oxidative stress without a requirement for retrograde transport to the soma. PMID:25154397

  7. Metformin Restores Parkin-Mediated Mitophagy, Suppressed by Cytosolic p53.

    PubMed

    Song, Young Mi; Lee, Woo Kyung; Lee, Yong-Ho; Kang, Eun Seok; Cha, Bong-Soo; Lee, Byung-Wan

    2016-01-01

    Metformin is known to alleviate hepatosteatosis by inducing 5' adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1) ad libitum feeding of a standard chow diet; (2) intraperitoneal injections of metformin 300 mg/kg; and (3) 3 g/day caloric restriction (CR). HepG2 cells were treated with palmitate (PA) plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER) stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins. PMID:26784190

  8. Metformin Restores Parkin-Mediated Mitophagy, Suppressed by Cytosolic p53

    PubMed Central

    Song, Young Mi; Lee, Woo Kyung; Lee, Yong-ho; Kang, Eun Seok; Cha, Bong-Soo; Lee, Byung-Wan

    2016-01-01

    Metformin is known to alleviate hepatosteatosis by inducing 5’ adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1) ad libitum feeding of a standard chow diet; (2) intraperitoneal injections of metformin 300 mg/kg; and (3) 3 g/day caloric restriction (CR). HepG2 cells were treated with palmitate (PA) plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER) stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins. PMID:26784190

  9. Naturally- and experimentally-designed restorations of the Parkin gene deficit in autosomal recessive juvenile parkinsonism

    SciTech Connect

    Asai, Hirohide; Hirano, Makito; Kiriyama, Takao; Ikeda, Masanori; Ueno, Satoshi

    2010-01-01

    Intranuclear events due to mutations in the Parkin gene remain elusive in autosomal recessive juvenile parkinsonism (ARJP). We identified a mutant PARKIN protein in fibroblast cultures from a pair of siblings with ARJP who were homozygous for the exon 4-deleted Parkin gene. Disease was mild in one patient and debilitating in the other. The detected mutant, encoded by a transcript lacking exon 3 as well as exon 4, is an in-frame deletion that removes 121 aa, resulting in a 344-aa protein (PaDel3,4). Cell culture and transfection studies revealed negative correlations between expression levels of PaDel3,4 and those of cell cycle proteins, including cyclin E, CDK2, ppRb, and E2F-1, and demonstrated that GFP-PaDel3,4 entered nucleus and ubiquitinated cyclin E as a part of SCF{sup hSel-10} ligase complex in the patient cells. In addition, nuclear localization signal-tagged PaDel3,4 expressed in the transfected patient cells most effectively ubiquitinated cyclin E and reduced DNA damage, protecting cells from oxidative stress. Antisense-oligonucleotide treatment promoted skipping of exon 3 and thus generated PaDel3,4, increasing cell survival. Collectively, we propose that naturally- and experimentally-induced exon skipping at least partly restores the mutant Parkin gene deficit, providing a molecular basis for the development of therapeutic exon skipping.

  10. A Dimeric PINK1-containing Complex on Depolarized Mitochondria Stimulates Parkin Recruitment*

    PubMed Central

    Okatsu, Kei; Uno, Midori; Koyano, Fumika; Go, Etsu; Kimura, Mayumi; Oka, Toshihiko; Tanaka, Keiji; Matsuda, Noriyuki

    2013-01-01

    Parkinsonism typified by sporadic Parkinson disease is a prevalent neurodegenerative disease. Mutations in PINK1 (PTEN-induced putative kinase 1), a mitochondrial Ser/Thr protein kinase, or PARKIN, a ubiquitin-protein ligase, cause familial parkinsonism. The accumulation and autophosphorylation of PINK1 on damaged mitochondria results in the recruitment of Parkin, which ultimately triggers quarantine and/or degradation of the damaged mitochondria by the proteasome and autophagy. However, the molecular mechanism of PINK1 in dissipation of the mitochondrial membrane potential (ΔΨm) has not been fully elucidated. Here we show by fluorescence-based techniques that the PINK1 complex formed following a decrease in ΔΨm is composed of two PINK1 molecules and is correlated with intermolecular phosphorylation of PINK1. Disruption of complex formation by the PINK1 S402A mutation weakened Parkin recruitment onto depolarized mitochondria. The most disease-relevant mutations of PINK1 inhibit the complex formation. Taken together, these results suggest that formation of the complex containing dyadic PINK1 is an important step for Parkin recruitment onto damaged mitochondria. PMID:24189060

  11. Glutamate excitotoxicity in neurons triggers mitochondrial and endoplasmic reticulum accumulation of Parkin, and, in the presence of N-acetyl cysteine, mitophagy

    PubMed Central

    Van Laar, Victor S.; Roy, Nikita; Liu, Annie; Rajprohat, Swati; Arnold, Beth; Dukes, April A.; Holbein, Cory D.; Berman, Sarah B.

    2014-01-01

    Disruption of the dynamic properties of mitochondria (fission, fusion, transport, degradation, and biogenesis) has been implicated in the pathogenesis of neurodegenerative disorders, including Parkinson’s disease (PD). Parkin, the product of gene PARK2 whose mutation causes familial PD, has been linked to mitochondrial quality control via its role in regulating mitochondrial dynamics, including mitochondrial degradation via mitophagy. Models using mitochondrial stressors in numerous cell types have elucidated a PINK1-dependent pathway whereby Parkin accumulates on damaged mitochondria and targets them for mitophagy. However, the role Parkin plays in regulating mitochondrial homeostasis specifically in neurons has been less clear. We examined whether a stressor linked to neurodegeneration, glutamate excitotoxicity, elicits Parkin-mitochondrial translocation and mitophagy in neurons. We found that brief, acute exposure to glutamate causes Parkin translocation to mitochondria in neurons, in a calcium- and N-methyl-D-aspartate (NMDA) receptor-dependent manner. In addition, we found that Parkin accumulates on endoplasmic reticulum (ER) and mitochondrial/ER junctions following excitotoxicity, supporting a role for Parkin in mitochondrial-ER crosstalk in mitochondrial homeostasis. Despite significant Parkin-mitochondria translocation, however, we did not observe mitophagy under these conditions. To further investigate, we examined the role of glutamate-induced oxidative stress in Parkin-mitochondria accumulation. Unexpectedly, we found that glutamate-induced accumulation of Parkin on mitochondria was promoted by the antioxidant N-acetyl cysteine (NAC), and that co-treatment with NAC facilitated Parkin-associated mitophagy. These results suggest the possibility that mitochondrial depolarization and oxidative damage may have distinct pathways associated with Parkin function in neurons, which may be critical in understanding the role of Parkin in neurodegeneration. PMID

  12. Dysregulation of Parkin-mediated mitophagy in thyroid Hürthle cell tumors.

    PubMed

    Lee, Junguee; Ham, Sujin; Lee, Min Hee; Kim, Soung Jung; Park, Ji Hoon; Lee, Seong Eun; Chang, Joon Young; Joung, Kyong Hye; Kim, Tae Yong; Kim, Jin Man; Sul, Hae Joung; Kweon, Gi Ryang; Jo, Young Suk; Kim, Koon Soon; Shong, Young Kee; Gasparre, Giuseppe; Chung, Jong Kyeong; Porcelli, Anna Maria; Shong, Minho

    2015-11-01

    Abnormal accumulation of defective mitochondria is the hallmark of oncocytes, which are frequently observed in thyroid Hürthle cell lesions. Autophagy is an essential cellular catabolic mechanism for the degradation of dysfunctional organelles and has been implicated in several human diseases. It is yet unknown how autophagic turnover of defective mitochondria in Hürthle cell tumors is regulated. We characterized the expression patterns of molecular markers including Beclin1, LC3, PINK1 and Parkin, which are required for autophagy or mitophagy, in human oncocytic lesions of the thyroid. To undertake mechanistic studies, we investigated autophagy and mitophagy using XTC.UC1 cells, the only in vitro model of Hürthle cell tumors. Beclin1 and LC3 were highly expressed in oncocytes of Hürthle cell tumors. XTC.UC1 showed autophagic responses to starvation and rapamycin treatment, whereas they displayed ineffective activation of mitophagy, which is triggered by the coordinated action of PINK1 and Parkin in response to CCCP. This resulted in a decreased turnover of abnormal mitochondria. The mechanisms underlying defective mitophagy and mitochondrial turnover were investigated by genetic analysis of the PARK2 gene in XTC.UC1 and Hürthle cell tumor tissues. XTC.UC1 and several tumors harbored the V380L mutation, resulting in dysfunctional autoubiquitination and decreased E3 ligase activity. Consistently, oncocytes in Hürthle cell tumors displayed comparable expression of PINK1 but decreased Parkin expression in comparison to normal thyrocytes. The introduction of wild-type Parkin sensitized XTC.UC1 to death induced by CCCP. This study provides a possible etiological basis for oncocytic formation in heterogeneous Hürthle cell tumors through insufficient mitophagy leading to ineffective turnover of aberrant mitochondria caused by dysfunctional Parkin-mediated pathways of mitochondria quality control. PMID:26354775

  13. Phosphatase and tensin homolog-induced putative kinase 1 and Parkin in diabetic heart: Role of mitophagy.

    PubMed

    Tang, Ying; Liu, Jiankang; Long, Jiangang

    2015-05-01

    Diabetes is an independent risk factor for cardiovascular morbidity and mortality. Diabetes-associated cardiac pathophysiology is recognized to be due to reasons including metabolic consequences on the myocardium. The heart is a highly energy-demanding tissue, with mitochondria supplying over 90% of adenosine triphosphate. The involvement of mitochondrial dysfunction in diabetes-related cardiac pathogenesis has been studied. Phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and Parkin, initially identified to be associated with the pathogenesis of a familiar form of Parkinson's disease, have recently been recognized to play a critical role in mediating cardiomyocytes' adaption to stresses. Extensive studies have suggested PINK1 and Parkin as key regulators of mitophagy. In the present review article, we will first summarize the new findings on PINK1/Parkin acting in cardioprotection, and then discuss the potential role of PINK1/Parkin in diabetic heart by mediating mitophagy. PMID:25969707

  14. AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1

    PubMed Central

    Strappazzon, F; Nazio, F; Corrado, M; Cianfanelli, V; Romagnoli, A; Fimia, G M; Campello, S; Nardacci, R; Piacentini, M; Campanella, M; Cecconi, F

    2015-01-01

    Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in ‘general' autophagy. In basal conditions, a pool of AMBRA1 – an upstream autophagy regulator and a PARKIN interactor – is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways. PMID:25215947

  15. Parkin depletion delays motor decline dose-dependently without overtly affecting neuropathology in α-synuclein transgenic mice

    PubMed Central

    2013-01-01

    Background Mutations of the gene encoding the major component of Lewy bodies (LB), α-synuclein (α-syn), cause autosomal dominant forms of Parkinson’s disease (PD), whereas loss-of-function mutations of the gene encoding the multifunctional E3 ubiquitin-protein ligase Parkin account for autosomal recessive forms of the disease. Parkin overproduction protects against α-syn-dependent neurodegeneration in various in vitro and in vivo models, but it remains unclear whether this process is affected by Parkin deficiency. We addressed this issue, by carrying out more detailed analyses of transgenic mice overproducing the A30P variant of human α-syn (hA30Pα-syn) and with two, one or no parkin knockout alleles. Results Longitudinal behavioral follow-up of these mice indicated that Parkin depletion delayed disease-predictive sensorimotor impairment due to α-syn accumulation, in a dose-dependent fashion. At the end stage of the disease, neuronal deposits containing fibrillar α-syn species phosphorylated at S129 (PS129α-syn) were the predominant neuropathological feature in hA30Pα-syn mice, regardless of their parkin expression. Some of these deposits colocalized with the LB markers ubiquitin and α-syn truncated at D135 (α-synD135), indicating that PS129α-syn is subjected to secondary posttranslational modification (PTM); these features were not significantly affected by parkin dysfunction. Conclusions These findings suggest that Parkin deficiency acts as a protective modifier in α-syn-dependent neurodegeneration, without overtly affecting the composition and characteristics of α-syn deposits in end-stage disease. PMID:24192137

  16. PINK1-Parkin-Mediated Mitophagy Protects Mitochondrial Integrity and Prevents Metabolic Stress-Induced Endothelial Injury.

    PubMed

    Wu, Weiwei; Xu, Hao; Wang, Zemin; Mao, Yun; Yuan, Liangshuai; Luo, Wei; Cui, Zhaoqiang; Cui, Taixing; Wang, Xing Li; Shen, Ying H

    2015-01-01

    Mitochondrial injury and dysfunction, a significant feature in metabolic syndrome, triggers endothelial cell dysfunction and cell death. Increasing evidence suggests that mitophagy, a process of autophagic turnover of damaged mitochondria, maintains mitochondrial integrity. PINK1 (phosphatase and tensin homolog (PTEN)-induced putative kinase 1) and Parkin signaling is a key pathway in mitophagy control. In this study, we examined whether this pathway could protect mitochondria under metabolic stress. We found that palmitic acid (PA) induced significant mitophagy and activated PINK1 and Parkin in endothelial cells. Knocking down PINK1 or Parkin reduced mitophagy, leading to impaired clearance of damaged mitochondria and intracellular accumulation of mitochondrial fragments. Furthermore, PINK1 and Parkin prevented PA-induced mitochondrial dysfunction, ROS production and apoptosis. Finally, we show that PINK1 and Parkin were up-regulated in vascular wall of obese mice and diabetic mice. Our study demonstrates that PINK1-Parkin pathway is activated in response to metabolic stress. Through induction of mitophagy, this pathway protects mitochondrial integrity and prevents metabolic stress-induced endothelial injury. PMID:26161534

  17. S-Nitrosylation of parkin as a novel regulator of p53-mediated neuronal cell death in sporadic Parkinson’s disease

    PubMed Central

    2013-01-01

    Background Mutations in the gene encoding parkin, a neuroprotective protein with dual functions as an E3 ubiquitin ligase and transcriptional repressor of p53, are linked to familial forms of Parkinson’s disease (PD). We hypothesized that oxidative posttranslational modification of parkin by environmental toxins may contribute to sporadic PD. Results We first demonstrated that S-nitrosylation of parkin decreased its activity as a repressor of p53 gene expression, leading to upregulation of p53. Chromatin immunoprecipitation as well as gel-shift assays showed that parkin bound to the p53 promoter, and this binding was inhibited by S-nitrosylation of parkin. Additionally, nitrosative stress induced apoptosis in cells expressing parkin, and this death was, at least in part, dependent upon p53. In primary mesencephalic cultures, pesticide-induced apoptosis was prevented by inhibition of nitric oxide synthase (NOS). In a mouse model of pesticide-induced PD, both S-nitrosylated (SNO-)parkin and p53 protein levels were increased, while administration of a NOS inhibitor mitigated neuronal death in these mice. Moreover, the levels of SNO-parkin and p53 were simultaneously elevated in postmortem human PD brain compared to controls. Conclusions Taken together, our data indicate that S-nitrosylation of parkin, leading to p53-mediated neuronal cell death, contributes to the pathophysiology of sporadic PD. PMID:23985028

  18. USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death.

    PubMed

    Liang, Jin-Rui; Martinez, Aitor; Lane, Jon D; Mayor, Ugo; Clague, Michael J; Urbé, Sylvie

    2015-05-01

    Mitochondria play a pivotal role in the orchestration of cell death pathways. Here, we show that the control of ubiquitin dynamics at mitochondria contributes to the regulation of apoptotic cell death. The unique mitochondrial deubiquitylase, USP30, opposes Parkin-dependent ubiquitylation of TOM20, and its depletion enhances depolarization-induced cell death in Parkin-overexpressing cells. Importantly, USP30 also regulates BAX/BAK-dependent apoptosis, and its depletion sensitizes cancer cells to BH3-mimetics. These results provide the first evidence for a fundamental role of USP30 in determining the threshold for mitochondrial cell death and suggest USP30 as a potential target for combinatorial anti-cancer therapy. PMID:25739811

  19. USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death

    PubMed Central

    Liang, Jin-Rui; Martinez, Aitor; Lane, Jon D; Mayor, Ugo; Clague, Michael J; Urbé, Sylvie

    2015-01-01

    Mitochondria play a pivotal role in the orchestration of cell death pathways. Here, we show that the control of ubiquitin dynamics at mitochondria contributes to the regulation of apoptotic cell death. The unique mitochondrial deubiquitylase, USP30, opposes Parkin-dependent ubiquitylation of TOM20, and its depletion enhances depolarization-induced cell death in Parkin-overexpressing cells. Importantly, USP30 also regulates BAX/BAK-dependent apoptosis, and its depletion sensitizes cancer cells to BH3-mimetics. These results provide the first evidence for a fundamental role of USP30 in determining the threshold for mitochondrial cell death and suggest USP30 as a potential target for combinatorial anti-cancer therapy. PMID:25739811

  20. Effects of partial suppression of parkin on huntingtin mutant R6/1 mice.

    PubMed

    Rubio, Isabel; Rodríguez-Navarro, José Antonio; Tomás-Zapico, Cristina; Ruíz, Carolina; Casarejos, María José; Perucho, Juan; Gómez, Ana; Rodal, Izaskun; Lucas, José J; Mena, María Angeles; de Yébenes, Justo García

    2009-07-24

    Huntington's disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines which makes huntingtin more resistant to degradation. Parkin is an ubiquitin ligase which promotes proteosomal degradation of abnormal proteins. We investigated whether partial suppression of parkin increases HD phenotype. We studied the behavior and brain histology and biochemistry of the mice produced by interbreeding of R6/1 (model of HD in mice) with Park-2(-/-) (parkin null mice): R6/1, WT (wild-type), PK(+/-) (hemizygotic deletion of Park-2) and R6/1/PK(+/-). R6/1 and R6/1/PK(+/-) mice had abnormal motor and exploratory behavior. R6/1/PK(+/-) mice were more akinetic. These two groups of mice had severe but similar loss of nigrostriatal dopamine neurons and monoamine levels in striatum. R6/1/PK(+/-) mice had fewer huntingtin inclusions and a greater number of TUNEL(+) cells than R6/1 in striatum but there were no differences in the hippocampus. DARPP-32 protein was equally reduced in striatum of R6/1 and R6/1/PK(+/-) mice. Striatal levels of GSH were increased, of HSP-70 reduced and of CHIP unchanged in both R6/1 and R6/1/PK(+/-) mice. LC-3 II/I ratios were significantly increased in striatum of R6/1/PK(+/-) mice. Partial suppression of parkin slightly aggravates the phenotype in R6/1 mice, confirming a pathogenic role of the UPS in the processing of mutant huntingtin. The absence of massive additional cellular lesions in R6/1/PK(+/-) mice suggests the existence of compensatory mechanisms, such as autophagy, for the processing of huntingtin. PMID:19464273

  1. Tau accumulation impairs mitophagy via increasing mitochondrial membrane potential and reducing mitochondrial Parkin

    PubMed Central

    Wang, Zhi-hao; Luo, Yu; Zhang, Xiangnan; Liu, Xiu-Ping; Feng, Qiong; Wang, Qun; Yue, Zhenyu; Chen, Zhong; Ye, Keqiang; Wang, Jian-Zhi; Liu, Gong-Ping

    2016-01-01

    Intracellular accumulation of wild type tau is a hallmark of sporadic Alzheimer's disease (AD). However, the molecular mechanisms underlying tau toxicity is not fully understood. Here, we detected mitophagy deficits evidenced by the increased levels of mitophagy markers, including COX IV, TOMM20, and the ratio of mtDNA to genomic DNA indexed as mt-Atp6/Rpl13, in the AD brains and in the human wild type full-length tau (htau) transgenic mice. More interestingly, the mitophagy deficit was only shown in the AD patients who had an increased total tau level. Further studies demonstrated that overexpression of htau induced mitophagy deficits in HEK293 cells, the primary hippocampal neurons and in the brains of C57 mice. Upon overexpression of htau, the mitochondrial membrane potential was increased and the levels of PTEN-induced kinase 1 (PINK1) and Parkin decreased in the mitochondrial fraction, while upregulation of Parkin attenuated the htau-induced mitophagy deficits. Finally, we detected a dose-dependent allocation of tau proteins into the mitochondrial outer membrane fraction along with its cytoplasmic accumulation. These data suggest that intracellular accumulation of htau induces mitophagy deficits by direct inserting into the mitochondrial membrane and thus increasing the membrane potential, which impairs the mitochondrial residence of PINK1/Parkin. Our findings reveal a novel mechanism underlying the htau-induced neuronal toxicities in AD and other tauopathies. PMID:26943044

  2. Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1

    PubMed Central

    Burbulla, L F; Fitzgerald, J C; Stegen, K; Westermeier, J; Thost, A-K; Kato, H; Mokranjac, D; Sauerwald, J; Martins, L M; Woitalla, D; Rapaport, D; Riess, O; Proikas-Cezanne, T; Rasse, T M; Krüger, R

    2014-01-01

    The mitochondrial chaperone mortalin was implicated in Parkinson's disease (PD) because of its reduced levels in the brains of PD patients and disease-associated rare genetic variants that failed to rescue impaired mitochondrial integrity in cellular knockdown models. To uncover the molecular mechanisms underlying mortalin-related neurodegeneration, we dissected the cellular surveillance mechanisms related to mitochondrial quality control, defined the effects of reduced mortalin function at the molecular and cellular levels and investigated the functional interaction of mortalin with Parkin and PINK1, two PD-related proteins involved in mitochondrial homeostasis. We found that reduced mortalin function leads to: (1) activation of the mitochondrial unfolded protein response (UPR(mt)), (2) increased susceptibility towards intramitochondrial proteolytic stress, (3) increased autophagic degradation of fragmented mitochondria and (4) reduced mitochondrial mass in human cells in vitro and ex vivo. These alterations caused increased vulnerability toward apoptotic cell death. Proteotoxic perturbations induced by either partial loss of mortalin or chemical induction were rescued by complementation with native mortalin, but not disease-associated mortalin variants, and were independent of the integrity of autophagic pathways. However, Parkin and PINK1 rescued loss of mortalin phenotypes via increased lysosomal-mediated mitochondrial clearance and required intact autophagic machinery. Our results on loss of mortalin function reveal a direct link between impaired mitochondrial proteostasis, UPR(mt) and PD and show that effective removal of dysfunctional mitochondria via either genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes. PMID:24743735

  3. Murine Typhus

    PubMed Central

    Dzul-Rosado, Karla R; Zavala Velázquez, Jorge Ernesto; Zavala-Castro, Jorge

    2012-01-01

    Rickettsia typhi: is an intracellular bacteria who causes murine typhus. His importance is reflected in the high frequency founding specific antibodies against Rickettsia typhi in several worldwide seroepidemiological studies, the seroprevalence ranging between 3-36%. Natural reservoirs of R. typhi are rats (some species belonging the Rattus Genus) and fleas (Xenopsylla cheopis) are his vector. This infection is associated with overcrowding, pollution and poor hygiene. Typically presents fever, headache, rash on trunk and extremities, in some cases may occur organ-specific complications, affecting liver, kidney, lung or brain. Initially the disease is very similar to other diseases, is very common to confuse the murine typhus with Dengue fever, therefore, ignorance of the disease is a factor related to complications or non-specific treatments for the resolution of this infection. This paper presents the most relevant information to consider about the rickettsiosis caused by Rickettsia typhi. PMID:24893060

  4. Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity.

    PubMed

    Ham, Su Jin; Lee, Soo Young; Song, Saera; Chung, Ju-Ryung; Choi, Sekyu; Chung, Jongkyeong

    2016-01-22

    Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis. PMID:26631732

  5. Altered α-synuclein, parkin, and synphilin isoform levels in multiple system atrophy brains.

    PubMed

    Brudek, Tomasz; Winge, Kristian; Rasmussen, Nadja Bredo; Bahl, Justyna Maria Czarna; Tanassi, Julia; Agander, Tina Klitmøller; Hyde, Thomas M; Pakkenberg, Bente

    2016-01-01

    Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease-specific transcription patterns in frontal cortex in PD, dementia with Lewy bodies, and MSA, and thus may mediate the development of α-synucleinopathies. In this study, the differential expression of α-synuclein isoforms on transcriptional and translational levels was ascertained in MSA patients in comparison with PD cases and normal controls using isoform-specific primers and exon-specific antibodies in substantia nigra, striatum, cerebellar cortex, and nucleus dentatus. These regions are severely affected by α-synuclein pathology and neurodegeneration. Furthermore, we have also investigated transcript levels for parkin and synphilin-1 isoforms. In MSA brains, α-synuclein140 and α-synuclein 112 isoform levels were significantly increased, whereas levels of the α-synuclein 126 isoform were decreased in the substantia nigra, striatum, cerebellar cortex, and nucleus dentatus versus controls. Moreover, in MSA cases, we showed increased levels of parkin isoforms lacking the N-terminal ubiquitin-like domain and an aggregation-prone synphilin-1A isoform that causes neuronal toxicity in MSA. In PD brains, parkin transcript variant 3, 7, and 11 were significantly and specifically over-expressed in the striatum and cerebellar cortex, together with synphilin-1A and 1C. The changes of isoform expression profiles in neurodegenerative diseases suggest alterations in the regulation of transcription and/or splicing events, leading to regional/cellular events that may be important for the highly increased aggregation of α-synuclein in the brain. We report differential expression of α-synuclein, parkin, and synphilin-1 isoforms in multiple system atrophy (MSA) versus Parkinson's disease and normal

  6. Cross-functional E3 ligases Parkin and C-terminus Hsp70-interacting protein in neurodegenerative disorders.

    PubMed

    Kumar, Pravir; Pradhan, Kaveri; Karunya, R; Ambasta, Rashmi K; Querfurth, Henry W

    2012-02-01

    The study of neurodegenerative disorders has had a major impact on our understanding of more fundamental mechanisms underlying neurobiology. Breakthroughs in the genetics of Alzheimer's (AD) and Parkinson's diseases (PD) has resulted in new knowledge in the areas of axonal transport, energy metabolism, protein trafficking/clearance and synaptic physiology. The major neurodegenerative diseases have in common a regional or network pathology associated with abnormal protein accumulation(s) and various degrees of motor or cognitive decline. In AD, β-amyloids are deposited in extracellular diffuse and compacted plaques as well as intracellularly. There is a major contribution to the disease by the co-existence of an intraneuronal tauopathy. Additionally, PD-like Lewy Bodies (LBs) bearing aggregated α-synuclein is present in 40-60% of all AD cases, especially involving amygdala. Amyloid deposits can be degraded or cleared by several mechanisms, including immune-mediated and transcytosis across the blood-brain barrier. Another avenue for disposal involves the lysosome pathway via autophagy. Enzymatic pathways include insulin degradative enzyme and neprilysin. Finally, the co-operative actions of C-terminus Hsp70 interacting protein (CHIP) and Parkin, components of a multiprotein E3 ubiquitin ligase complex, may be a portal to proteasome-mediated degradation. Mutations in the Parkin gene are the most common genetic link to autosomal recessive Parkinson's disease. Parkin catalyzes the post-translational modification of proteins with polyubiquitin, targeting them to the 26S proteasome. Parkin reduces intracellular Aβ(1-42) peptide levels, counteracts its effects on cell death, and reverses its effect to inhibit the proteasome. Additionally, Parkin has intrinsic cytoprotective activity to promote proteasome function and defend against oxidative stress to mitochondria. Parkin and CHIP are also active in amyloid clearance and cytoprotection in vivo. Parkin has cross

  7. Parkin induces upregulation of 40S ribosomal protein SA and posttranslational modification of cytokeratins 8 and 18 in human cervical cancer cells.

    PubMed

    Song, Dae-Geun; Kim, Yoon Suk; Jung, Byung Chul; Rhee, Ki-Jong; Pan, Cheol-Ho

    2013-12-01

    Parkin was originally identified as a protein associated with Parkinson's disease. Recently, numerous research studies have suggested that parkin acts as a tumor suppressor. In accordance with these studies, we previously reported that overexpression of parkin in HeLa cells induced growth inhibition. To elucidate possible mechanisms by which parkin may inhibit cell growth, HeLa cells were infected with adenoviruses expressing either the parkin gene or adenovirus alone for 72 h and a total proteomic analysis was performed using 2-D gel electrophoresis followed by LC-MS/MS. We identified three proteins whose expression changed between the two groups: the 40S ribosomal protein SA (RPSA) was downregulated in parkin virus-infected cells, and cytokeratins 8 and 18 exhibited an acid shift in pI value without a change in molecular weight, suggesting that these proteins became phosphorylated in parkin virus-infected cells. The changes in these three proteins were first observed at 60 h postinfection and were most dramatic at 72 h postinfection. Because upregulation of RPSA and dephosphorylation of cytokeratins 8/18 have been linked with tumor progression, these data suggest that parkin may inhibit cell growth, at least in part, by decreasing RPSA expression and inducing phosphorylation of cytokeratin 8/18. PMID:23990477

  8. PINK1/Parkin-mediated mitophagy alleviates chlorpyrifos-induced apoptosis in SH-SY5Y cells.

    PubMed

    Dai, Hongmei; Deng, Yuanying; Zhang, Jie; Han, Hailong; Zhao, Mingyi; Li, Ying; Zhang, Chen; Tian, Jing; Bing, Guoying; Zhao, Lingling

    2015-08-01

    Chlorpyrifos (CPF) is one of the most widely used organophosphorous insecticides. There are links between CPF exposure and neurological disorders. Mitochondrial damage has been implicated to play a key role in CPF-induced neurotoxicity. Mitophagy, the selective autophagic elimination of mitochondria, is an important mitochondrial quality control mechanism. However, the role of mitophagy in CPF-induced neurotoxicity remains unclear. In this study, CPF-caused mitochondrial damage, role and mechanism of mitophagy on CPF-induced neuroapoptosis were extensively studied by using SH-SY5Y cells. We showed that CPF treatment caused mitochondrial fragmentation, excessive ROS generation and mitochondrial depolarization, thus led to cell apoptosis. Moreover, CPF treatment also resulted in increased colocalizaton of mitochondria with LC3, decreased levels of mitochondrial proteins, PINK1 stabilization and mitochondrial accumulation of Parkin. These data suggested that CPF treatment induced PINK1/Parkin-mediated mitophagy in SH-SY5Y cells. Furthermore, knockdown of Parkin dramatically increased CPF-induced neuroapoptosis. On the other hand, overexpression of Parkin markedly alleviated CPF-induced SH-SY5Y cell apoptosis. Together, these findings implicate a protective role of PINK1/Parkin-mediated mitophagy against neuroapoptosis and that enhancing mitophagy provides a potential therapeutic strategy for CPF-induced neurological disorders. PMID:26070385

  9. TAT-ODD-p53 enhances the radiosensitivity of hypoxic breast cancer cells by inhibiting Parkin-mediated mitophagy

    PubMed Central

    Du, Shasha; Ren, Chen; Wang, Yuxia; Yuan, Yawei

    2015-01-01

    Radiation therapy has an important role in the treatment of breast cancer. Dysfunction p53 and hypoxia are typical biological characteristics of breast cancer that constitute barriers to the efficacy of radiotherapy. Mitophagy plays a protective role in cellular homeostasis under hypoxic conditions, while mitophagy is inhibited by p53 in normal cells. We explored the effects of a p53 fusion protein, TAT-ODD-p53, on the radiosensitivity of hypoxic breast cancer cells both in vitro and in vivo, as well as investigating the related molecular mechanisms. We found that selective accumulation of TAT-ODD-p53 occurred under hypoxic conditions and significantly increased tumor cell radiosensitivity both in vitro and in vivo. Mitophagy had an important role in maintaining hypoxia-induced radioresistance. Mitophagy was inhibited by TAT-ODD-p53 and this inhibition was suppressed by over-expression of Parkin in hypoxic irradiated breast cancer cells. In addition, mitophagy was induced by deletion of p53, with this effect being weakened by Parkin knockdown at a low oxygen tension. By interacting with Parkin, p53 inhibited the translocation of Parkin to the mitochondria, disrupting the protective mitophagy process. These results suggest that TAT-ODD-p53 has a significant and preferential radiosensitizing effect on hypoxic breast cancer cells by inhibition of Parkin-mediated mitophagy. PMID:26025927

  10. Changes in the expression of neurotransmitter receptors in Parkin and DJ-1 knockout mice--A quantitative multireceptor study.

    PubMed

    Cremer, J N; Amunts, K; Schleicher, A; Palomero-Gallagher, N; Piel, M; Rösch, F; Zilles, K

    2015-12-17

    Parkinson's disease (PD) is a well-characterized neurological disorder with regard to its neuropathological and symptomatic appearance. At the genetic level, mutations of particular genes, e.g. Parkin and DJ-1, were found in human hereditary PD with early onset. Neurotransmitter receptors constitute decisive elements in neural signal transduction. Furthermore, since they are often altered in neurological and psychiatric diseases, receptors have been successful targets for pharmacological agents. However, the consequences of PD-associated gene mutations on the expression of transmitter receptors are largely unknown. Therefore, we studied the expression of 16 different receptor binding sites of the neurotransmitters glutamate, GABA, acetylcholine, adrenaline, serotonin, dopamine and adenosine by means of quantitative receptor autoradiography in Parkin and DJ-1 knockout mice. These knockout mice exhibit electrophysiological and behavioral deficits, but do not show the typical dopaminergic cell loss. We demonstrated differential changes of binding site densities in eleven brain regions. Most prominently, we found an up-regulation of GABA(B) and kainate receptor densities in numerous cortical areas of Parkin and DJ-1 knockout mice, as well as increased NMDA but decreased AMPA receptor densities in different brain regions of the Parkin knockout mice. The alterations of three different glutamate receptor types may indicate the potential relevance of the glutamatergic system in the pathogenesis of PD. Furthermore, the cholinergic M1, M2 and nicotinic receptors as well as the adrenergic α2 and the adenosine A(2A) receptors showed differentially increased densities in Parkin and DJ-1 knockout mice. Taken together, knockout of the PD-associated genes Parkin or DJ-1 results in differential changes of neurotransmitter receptor densities, highlighting a possible role of altered non-dopaminergic, and in particular of glutamatergic neurotransmission in PD pathogenesis. PMID

  11. Suppression of Parkin enhances nigrostriatal and motor neuron lesion in mice over-expressing human-mutated tau protein.

    PubMed

    Menéndez, J; Rodríguez-Navarro, J A; Solano, R M; Casarejos, M J; Rodal, I; Guerrero, R; Sánchez, M P; Avila, J; Mena, M A; de Yébenes, J G

    2006-07-01

    Abnormal deposition of protein tau takes place in the brain of patients with several neurodegenerative diseases. Few of these patients present frontotemporal dementia with parkinsonism and amyotrophy (FTDPA-17), an autosomal dominant tauopathy related to mutations of the gene that codes for protein tau, localized in chromosome 17. The great majority of patients with tauopathies such as Alzheimer's disease, sporadic frontotemporal dementia or progressive supranuclear palsy do not show a Mendelian pattern of inheritance. We have occasionally seen tauopathies in patients with parkin mutations and, therefore, hypothesized that the protein tau interacts with parkin. We have tested that hypothesis in mice with combined genetic modifications of tau (over-expression of human tau with three mutations known to produce FTDPA-17) and parkin (deleted) proteins. Homozygote parkin null or over-expressing mutated-human tau mice have subtle behavioral and molecular abnormalities but do not express a clinical phenotype of neurodegenerative disease. Mice with combined homozygous mutations of these two genes show progressively abnormal walking already noticeable at 3 months of age, loss of dopamine and dopamine markers in striatum, nuclear tau immunoreactive deposits in motor neurons of the spinal cord, abnormal expression of glial markers and enhanced levels of pro-apoptotic proteins; findings that were absent or less pronounced in homozygote animals with deletions of parkin or over-expression of tau. The double transgenic mice do not express normal mechanisms of adaptation to stress such as increased levels of GSH and Hsp-70. In addition, they have reduced levels of CHIP-Hsc70, a complex known to attenuate aggregation of tau and to enhance ubiquitination of phosphorylated tau. We have found high levels of phosphorylated tau in parkin-/-+tau(VLW) mice and a relative decrease of the inactivated pSer9 to total GSK-3 levels. Our data reveal that there are interactions between tau and

  12. PINK1/Parkin-mediated mitophagy play a protective role in manganese induced apoptosis in SH-SY5Y cells.

    PubMed

    Zhang, Hong-Tao; Mi, Lan; Wang, Ting; Yuan, Lan; Li, Xue-Hui; Dong, Li-Sha; Zhao, Peng; Fu, Juan-Ling; Yao, Bi-Yun; Zhou, Zong-Can

    2016-08-01

    Manganese (Mn) as an environmental risk factor of Parkinson's disease (PD) is considered to cause manganism. Mitophagy is thought to play a key role in elimination the injured mitochondria. The goal of this paper was to explore whether the PINK1/Parkin-mediated mitophagy is activated and its role in Mn-induced mitochondrial dysfunction and cell death in SH-SY5Y cells. Here, we investigated effects of MnCl2 on ROS generation, mitochondrial membrane potential (MMP/ΔΨm) and apoptosis by FACS and examined PINK1/Parkin-mediated mitophagy by western-blotting and the co-localization of mitochondria and acidic lysosomes. Further, we explore the role of mitophagy in Mn-induced apoptosis by inhibition the mitophagy by knockdown Parkin level. Results show that MnCl2 dose-dependently caused ΔΨm decrease, ROS generation and apoptosis of dopaminergic SH-SY5Y cells. Moreover, Mn could induce mitophagy and PINK1/Parkin-mediated pathway was activated in SH-SY5Y cells. Transient transfection of Parkin siRNA knockdown the expressing level of parkin inhibited Mn-induced mitophagy and aggravated apoptosis of SH-SY5Y cells. In conclusion, our study demonstrated that Mn may induce PINK1/Parkin-mediated mitophagy, which may exert significant neuro-protective effect against Mn-induced dopaminergic neuronal cells apoptosis. PMID:27091500

  13. Polychlorinated biphenyls alter expression of alpha-synuclein, synaptophysin and parkin in the rat brain.

    PubMed

    Malkiewicz, Katarzyna; Mohammed, Roma; Folkesson, Ronnie; Winblad, Bengt; Szutowski, Miroslaw; Benedikz, Eirikur

    2006-02-20

    Polychlorinated Biphenyls (PCBs)-induced changes in synaptic transmission are one of the effects of their neurotoxicity but the mechanism remains unknown. We assessed the in vivo effects of the PCBs mixture, Aroclor 1254 on the expression of neuronal proteins that are involved in the synaptic function and/or are associated with neurodegeneration. Wistar rats were treated orally with repeated doses of Aroclor 1254 and the levels of soluble alpha-synuclein, parkin, synaptophysin and amyloid precursor protein (APP) in the brain were determined by Western blotting. The results showed that Aroclor did not cause changes in the expression and processing of APP but at a dose 100 microg/g/day repeated for 6 days caused a decrease in the expression of alpha-synuclein in the cerebellum, cortex, hippocampus and hypothalamus of the animals sacrificed 2 days after treatment. The decrease in alpha-synuclein was accompanied by a transient increase in parkin and synaptophysin levels. Interestingly, in the hypothalamus the levels of alpha-synuclein remained decreased after 21 days post treatment perhaps due to regional differences in the PCBs elimination or perhaps a more specific interaction with the dopaminergic cells that are present in the hypothalamus that needs to be investigated further. PMID:16174552

  14. [CK2beta promotes Pink1/Parkin-mediated MIRO1 degradation].

    PubMed

    Zhang, Chenliang; Qin, Siyue; Jiang, Chang'an

    2014-12-01

    PTEN-induced putative kinase 1 (PINK1), a Parkinson's disease (PD)-related protein, has two isoforms, the mitochondria-localized full-length isoform PINK1FL and the cytoplasm-localized short isoform PINK1-cyto. Studies have suggested that PINK1FL can selectively accumulate at the surface of damaged mitochondria and cooperate with another Parkinson's Disease-related protein PARKIN to trigger the degradation of MIRO1, a mitochondria trafficking regulator. The functions of PINK1-cyto are, however, not yet clear. To investigate the functions of PINK1-cyto, we expressed different proteins in cultured HEK293 cells by transfecting it with different plasmids, and detected the protein levels by Western blot after expressing for 24 h. We found that in cultured HEK293 cells, PINK1-cyto could also cooperate with PARKIN degrade MIRO1 in the presence of CK23, and the regulatory subunit of Casein Kinase II. Interestingly, this function of CK2P was not dependent on CK2alpha, the catalytic subunit of Casein Kinase II. We also found that CK2P could promote the direct interaction between PINK1-cyto and MIRO1 by immunocoprecipitation analysis. This result suggested that in addition to CK2alpha, CK2beta could also form a kinase complex. PMID:25868250

  15. Syntaxin-17 delivers PINK1/parkin-dependent mitochondrial vesicles to the endolysosomal system.

    PubMed

    McLelland, Gian-Luca; Lee, Sydney A; McBride, Heidi M; Fon, Edward A

    2016-08-01

    Mitochondria are considered autonomous organelles, physically separated from endocytic and biosynthetic pathways. However, recent work uncovered a PINK1/parkin-dependent vesicle transport pathway wherein oxidized or damaged mitochondrial content are selectively delivered to the late endosome/lysosome for degradation, providing evidence that mitochondria are indeed integrated within the endomembrane system. Given that mitochondria have not been shown to use canonical soluble NSF attachment protein receptor (SNARE) machinery for fusion, the mechanism by which mitochondrial-derived vesicles (MDVs) are targeted to the endosomal compartment has remained unclear. In this study, we identify syntaxin-17 as a core mitochondrial SNARE required for the delivery of stress-induced PINK1/parkin-dependent MDVs to the late endosome/lysosome. Syntaxin-17 remains associated with mature MDVs and forms a ternary SNARE complex with SNAP29 and VAMP7 to mediate MDV-endolysosome fusion in a manner dependent on the homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Syntaxin-17 can be traced to the last eukaryotic common ancestor, hinting that the removal of damaged mitochondrial content may represent one of the earliest vesicle transport routes in the cell. PMID:27458136

  16. Dopamine-dependent neurodegeneration in rats induced by viral vector-mediated overexpression of the parkin target protein, CDCrel-1

    PubMed Central

    Dong, Zhizhong; Ferger, Boris; Paterna, Jean-Charles; Vogel, Denise; Furler, Sven; Osinde, Maribel; Feldon, Joram; Büeler, Hansruedi

    2003-01-01

    Mutations in the parkin gene are linked to autosomal-recessive juvenile parkinsonism (AR-JP). Parkin functions as a ubiquitin protein ligase in the degradation of several proteins, including the neuron-specific septin CDCrel-1. AR-JP-associated parkin mutations inhibit ubiquitination and degradation of CDCrel-1 and other parkin target proteins. Here we show that recombinant adeno-associated virus-mediated CDCrel-1 gene transfer to the substantia nigra of rats results in a rapid onset (6-10 days) of nigral and striatal CDCrel-1 expression that is followed by a progressive loss of nigral dopaminergic neurons and a decline of the striatal dopamine levels. In contrast, neurons of the globus pallidus are spared from CDCrel-1 toxicity. Furthermore, CDCrel-1 inhibits the release of dopamine from stably-transfected PC12 cells, and pharmacological inhibition of tyrosine hydroxylase and dopamine synthesis in rats prevents CDCrel-1-induced nigral neurodegeneration. These results show that CDCrel-1 overexpression exerts dopamine-dependent neurotoxicity and suggest that inhibition of dopamine secretion by CDCrel-1 may contribute to the development of AR-JP. PMID:14530399

  17. Endogenous Pyrogen Physiology.

    ERIC Educational Resources Information Center

    Beisel, William R.

    1980-01-01

    Discusses the physiology of endogenous pyrogen (EP), the fever-producing factor of cellular origin. Included are: its hormone-like role, its molecular nature, bioassay procedures, cellular production and mechanisms of EP action. (SA)

  18. Bacillus cereus endogenous panophthalmitis.

    PubMed

    Bouza, E; Grant, S; Jordan, C; Yook, R H; Sulit, H L

    1979-03-01

    A case of severe suppurative endogenous panophthalmitis caused by Bacillus cereus resulted from intravenously administered medications. This is the first, to our knowledge, well-documented case of endogenous endophthalmitis associated with this organism. It is recommended that if on Gram's stain of the anterior chamber fluid, Gram-positive rods are seen, chloramphenicol should be administered in addition to penicillin because of the possibility of B cereus infection. PMID:105693

  19. Clinical, 18F-dopa PET, and genetic analysis of an ethnic Chinese kindred with early-onset parkinsonism and parkin gene mutations.

    PubMed

    Wu, Ruey-Meei; Shan, Din-E; Sun, Chen-Ming; Liu, Ren-Shyan; Hwu, Wuh-Liang; Tai, Chun-Hwei; Hussey, Jennifer; West, Andrew; Gwinn-Hardy, Katrina; Hardy, John; Chen, Judy; Farrer, Matt; Lincoln, Sarah

    2002-07-01

    We report on clinical (18)F-labeled 6-fluorodopa ((18)F-dopa) positron emission tomography (PET) and molecular genetic analyses of an ethnic Chinese family in which three siblings presented with early-onset Parkinson's disease. As described in some parkin patients, neither sleep benefit nor diurnal fluctuation was noted. Interestingly, depression, anxiety, and obsessive-compulsive disorders were manifest. The (18)F-dopa PET scans showed bilateral presynaptic dopaminergic dysfunction without marked lateralization. Molecular genetic analysis showed identical chromosome 6 haplotypes inherited by affected subjects, with alternate allelic deletions of parkin exons 3 and 4. Furthermore, mRNA analyses identified aberrantly spliced parkin transcripts, suggesting that unusual parkin protein isoforms may be expressed in the brain and retain some function. PMID:12210855

  20. Cinnamon Treatment Upregulates Neuroprotective Proteins Parkin and DJ-1 and Protects Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease

    PubMed Central

    Khasnavis, Saurabh

    2014-01-01

    Upregulation and/or maintenance of Parkinson’s disease (PD)-related beneficial proteins such as Parkin and DJ-1 in astrocytes during neurodegenerative insults may have therapeutic efficacy in PD. Cinnamon is a commonly used natural spice and flavoring material throughout the world. Here we have explored a novel use of cinnamon in upregulating Parkin and DJ-1 and protecting dopaminergic neurons in MPTP mouse model of PD. Recently we have delineated that oral feeding of cinnamon (Cinnamonum verum) powder produces sodium benzoate (NaB) in blood and brain of mice. Proinflammatory cytokine IL-1β decreased the level of Parkin/DJ-1 in mouse astrocytes. However, cinnamon metabolite NaB abrogated IL-1β-induced loss of these proteins. Inability of TNF-α to produce nitric oxide (NO) and decrease the level of Parkin/DJ-1 in wild type (WT) astrocytes, failure of IL-1β to reduce Parkin/DJ-1 in astrocytes isolated from iNOS (−/−) mice, and decrease in Parkin/DJ-1 in WT astrocytes by NO donor DETA-NONOate suggest that NO is a negative regulator of Parkin/DJ-1. Furthermore, suppression of IL-1β-induced expression of iNOS in astrocytes by NaB and reversal of NaB-mediated protection of Parkin/DJ-1 by DETA-NONOate in astrocytes indicate that NaB protects Parkin/DJ-1 in activated astrocytes via suppressing iNOS. Similarly MPTP intoxication also increased the level of iNOS and decreased the level of Parkin/DJ-1 in vivo in the nigra. However, oral treatment of MPTP-intoxicated mice with cinnamon powder and NaB reduced the expression of iNOS and protected Parkin/DJ-1 in the nigra. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions by cinnamon in MPTP-intoxicated mice. These results suggest that cinnamon may be beneficial for PD patients. PMID:24946862

  1. Parkin-Dependent Degradation of the F-Box Protein Fbw7β Promotes Neuronal Survival in Response to Oxidative Stress by Stabilizing Mcl-1

    PubMed Central

    Ekholm-Reed, Susanna; Goldberg, Matthew S.; Schlossmacher, Michael G.

    2013-01-01

    Parkinson's disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons resulting in motor dysfunction. While most PD is sporadic in nature, a significant subset can be linked to either dominant or recessive germ line mutations. PARK2, encoding the ubiquitin ligase parkin, is the most frequently mutated gene in hereditary Parkinson's disease. Here, we present evidence for a neuronal ubiquitin ligase cascade involving parkin and the multisubunit ubiquitin ligase SCFFbw7β. Specifically, parkin targets the SCF substrate adapter Fbw7β for proteasomal degradation. Furthermore, we show that the physiological role of parkin-mediated regulation of Fbw7β levels is the stabilization of the mitochondrial prosurvival factor Mcl-1, an SCFFbw7β target in neurons. We show that neurons depleted of parkin become acutely sensitive to oxidative stress due to an inability to maintain adequate levels of Mcl-1. Therefore, loss of parkin function through biallelic mutation of PARK2 may lead to death of dopaminergic neurons through unregulated SCFFbw7β-mediated ubiquitylation-dependent proteolysis of Mcl-1. PMID:23858059

  2. Parkin modulates expression of HIF-1α and HIF-3α during hypoxia in gliobastoma-derived cell lines in vitro.

    PubMed

    Maugeri, Grazia; D'Amico, Agata Grazia; Reitano, Rita; Saccone, Salvatore; Federico, Concetta; Cavallaro, Sebastiano; D'Agata, Velia

    2016-06-01

    Mutation of the Parkin gene causes an autosomal recessive juvenile-onset form of Parkinson's disease. However, recently, it has been also linked to a wide variety of malignancies, including glioblastoma multiforme (GBM). In this pathology, Parkin exhibits a tumor suppressor role by mitigating the proliferation rate in both in vitro and in vivo models. However, Parkin involvement in the hypoxic process has not as yet been investigated. GBM is the most common and aggressive primary brain tumor in adults and is characterized by hypoxic areas. The low oxygen supply causes the expression of hypoxia-inducible factors (HIFs) leading to an accumulation of pro-angiogenic factors and tumoral invasiveness. We assess the relationship between Parkin and two HIFs expressed during hypoxic conditions, namely HIF-1α and HIF-3α. Our data show that Parkin is downregulated under hypoxia and that it interferes with HIF expression based on cellular oxygen tension. These results suggest a role for the involvement of Parkin in GBM, although further studies will be needed to understand the mechanism by which it modulates HIF-1α and HIF-3α expression. PMID:26742768

  3. The Endogenous Exposome

    PubMed Central

    Nakamura, Jun; Mutlu, Esra; Sharma, Vyom; Collins, Leonard; Bodnar, Wanda; Yu, Rui; Lai, Yongquan; Moeller, Benjamin; Lu, Kun; Swenberg, James

    2014-01-01

    The concept of the Exposome, is a compilation of diseases and one’s lifetime exposure to chemicals, whether the exposure comes from environmental, dietary, or occupational exposures; or endogenous chemicals that are formed from normal metabolism, inflammation, oxidative stress, lipid peroxidation, infections, and other natural metabolic processes such as alteration of the gut microbiome. In this review, we have focused on the Endogenous Exposome, the DNA damage that arises from the production of endogenous electrophilic molecules in our cells. It provides quantitative data on endogenous DNA damage and its relationship to mutagenesis, with emphasis on when exogenous chemical exposures that produce identical DNA adducts to those arising from normal metabolism cause significant increases in total identical DNA adducts. We have utilized stable isotope labeled chemical exposures of animals and cells, so that accurate relationships between endogenous and exogenous exposures can be determined. Advances in mass spectrometry have vastly increased both the sensitivity and accuracy of such studies. Furthermore, we have clear evidence of which sources of exposure drive low dose biology that results in mutations and disease. These data provide much needed information to impact quantitative risk assessments, in the hope of moving towards the use of science, rather than default assumptions. PMID:24767943

  4. Tissue- and Cell-Specific Mitochondrial Defect in Parkin-Deficient Mice

    PubMed Central

    Bulteau, Anne-Laure; Ferrando-Miguel, Rosa; Gouarne, Caroline; Paoli, Marc Giraudon; Pruss, Rebecca; Auchère, Françoise; L'Hermitte-Stead, Caroline; Bouillaud, Frédéric; Brice, Alexis; Corti, Olga; Lombès, Anne

    2014-01-01

    Loss of Parkin, encoded by PARK2 gene, is a major cause of autosomal recessive Parkinson's disease. In Drosophila and mammalian cell models Parkin has been shown in to play a role in various processes essential to maintenance of mitochondrial quality, including mitochondrial dynamics, biogenesis and degradation. However, the relevance of altered mitochondrial quality control mechanisms to neuronal survival in vivo is still under debate. We addressed this issue in the brain of PARK2−/− mice using an integrated mitochondrial evaluation, including analysis of respiration by polarography or by fluorescence, respiratory complexes activity by spectrophotometric assays, mitochondrial membrane potential by rhodamine 123 fluorescence, mitochondrial DNA content by real time PCR, and oxidative stress by total glutathione measurement, proteasome activity, SOD2 expression and proteins oxidative damage. Respiration rates were lowered in PARK2−/− brain with high resolution but not standard respirometry. This defect was specific to the striatum, where it was prominent in neurons but less severe in astrocytes. It was present in primary embryonic cells and did not worsen in vivo from 9 to 24 months of age. It was not associated with any respiratory complex defect, including complex I. Mitochondrial inner membrane potential in PARK2−/− mice was similar to that of wild-type mice but showed increased sensitivity to uncoupling with ageing in striatum. The presence of oxidative stress was suggested in the striatum by increased mitochondrial glutathione content and oxidative adducts but normal proteasome activity showed efficient compensation. SOD2 expression was increased only in the striatum of PARK2−/− mice at 24 months of age. Altogether our results show a tissue-specific mitochondrial defect, present early in life of PARK2−/− mice, mildly affecting respiration, without prominent impact on mitochondrial membrane potential, whose underlying mechanisms remain to be

  5. Endogenous opioids and reward.

    PubMed

    Van Ree, J M; Niesink, R J; Van Wolfswinkel, L; Ramsey, N F; Kornet, M M; Van Furth, W R; Vanderschuren, L J; Gerrits, M A; Van den Berg, C L

    2000-09-29

    The discovery of endogenous opioids has markedly influenced the research on the biology of addiction and reward brain processes. Evidence has been presented that these brain substances modulate brain stimulation reward, self-administration of different drugs of abuse, sexual behaviour and social behaviour. There appears to be two different domains in which endogenous opioids, present in separate and distinct brain regions, are involved. One is related to the modulation of incentive motivational processes and the other to the performance of certain behaviours. It is concluded that endogenous opioids may play a role in the vulnerability to certain diseases, such as addiction and autism, but also when the disease is present, such as alcoholism. PMID:11033317

  6. Melatonin rescues zebrafish embryos from the parkinsonian phenotype restoring the parkin/PINK1/DJ-1/MUL1 network.

    PubMed

    Díaz-Casado, María E; Lima, Elena; García, José A; Doerrier, Carolina; Aranda, Paula; Sayed, Ramy Ka; Guerra-Librero, Ana; Escames, Germaine; López, Luis C; Acuña-Castroviejo, Darío

    2016-08-01

    Multiple studies reporting mitochondrial impairment in Parkinson's disease (PD) involve knockout or knockdown models to inhibit the expression of mitochondrial-related genes, including parkin, PINK1, and DJ-1 ones. Melatonin has significant neuroprotective properties, which have been related to its ability to boost mitochondrial bioenergetics. The meaning and molecular targets of melatonin in PD are yet unclear. Zebrafish are an outstanding model of PD because they are vertebrates, their dopaminergic system is comparable to the nigrostriatal system of humans, and their brains express the same genes as mammals. The exposure of 24 hpf zebrafish embryos to MPTP leads to a significant inhibition of the mitochondrial complex I and the induction of sncga gene, responsible for enhancing γ-synuclein accumulation, which is related to mitochondrial dysfunction. Moreover, MPTP inhibited the parkin/PINK1/DJ-1 expression, impeding the normal function of the parkin/PINK1/DJ-1/MUL1 network to remove the damaged mitochondria. This situation remains over time, and removing MPTP from the treatment did not stop the neurodegenerative process. On the contrary, mitochondria become worse during the next 2 days without MPTP, and the embryos developed a severe motor impairment that cannot be rescued because the mitochondrial-related gene expression remained inhibited. Melatonin, added together with MPTP or added once MPTP was removed, prevented and recovered, respectively, the parkinsonian phenotype once it was established, restoring gene expression and normal function of the parkin/PINK1/DJ-1/MUL1 loop and also the normal motor activity of the embryos. The results show, for the first time, that melatonin restores brain function in zebrafish suffering with Parkinson-like disease. PMID:27064726

  7. A homozygous parkin p.G284R mutation in a Chinese family with autosomal recessive juvenile parkinsonism.

    PubMed

    Chen, Han; Huang, Xiangjun; Yuan, Lamei; Xia, Hong; Xu, Hongbo; Yang, Yan; Zheng, Wen; Deng, Hao

    2016-06-15

    Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and neuropathologic entity characterized by early onset parkinsonism and localized neuronal degeneration in the substantia nigra without Lewy bodies. The purpose of this study is to identify the genetic defect in a Chinese pedigree with familial AR-JP and to explore genotype-phenotype correlation. A three-generation Chinese Han pedigree with familial AR-JP was recruited in this study, and the patients in the pedigree presented with typical but heterogeneous clinical features of AR-JP and with different ages of disease onset. Exome sequencing and Sanger sequencing were conducted in the index case diagnosed as juvenile parkinsonism and a homozygous variant, c.850G>C (p.G284R), in the parkin gene was identified. The homozygous variant co-segregated with the disease in the family and was absent in 800 controls. The homozygous variant, c.850G>C (p.G284R), in the parkin gene is possibly responsible for AR-JP in this pedigree. Heterozygous c.850G>C mutation carriers were free of any neurological symptoms, consistent with a loss-of-function mechanism of the parkin mutations. These findings may provide new insights into the cause and diagnosis of AR-JP and have implications for genetic counseling. PMID:27177722

  8. PINK1 and Parkin cooperatively protect neurons against constitutively active TRP channel-induced retinal degeneration in Drosophila

    PubMed Central

    Huang, Z; Ren, S; Jiang, Y; Wang, T

    2016-01-01

    Calcium has an important role in regulating numerous cellular activities. However, extremely high levels of intracellular calcium can lead to neurotoxicity, a process commonly associated with degenerative diseases. Despite the clear role of calcium cytotoxicity in mediating neuronal cell death in this context, the pathological mechanisms remain controversial. We used a well-established Drosophila model of retinal degeneration, which involves the constitutively active TRPP365 channels, to study calcium-induced neurotoxicity. We found that the disruption of mitochondrial function was associated with the degenerative process. Further, increasing autophagy flux prevented cell death in TrpP365 mutant flies, and this depended on the PINK1/Parkin pathway. In addition, the retinal degeneration process was also suppressed by the coexpression of PINK1 and Parkin. Our results provide genetic evidence that mitochondrial dysfunction has a key role in the pathology of cellular calcium neurotoxicity. In addition, the results demonstrated that maintaining mitochondrial homeostasis via PINK1/Parkin-dependent mitochondrial quality control can potentially alleviate cell death in a wide range of neurodegenerative diseases. PMID:27054334

  9. Rescue of mitochondrial function in parkin-mutant fibroblasts using drug loaded PMPC-PDPA polymersomes and tubular polymersomes.

    PubMed

    Yealland, G; Battaglia, G; Bandmann, O; Mortiboys, H

    2016-09-01

    Mutations in parkin cause autosomal recessive Parkinsonism and mitochondrial defects. A recent drug screen identified a class of steroid-like hydrophobic compounds able to rescue mitochondrial function in parkin-mutant fibroblasts. Whilst these possess therapeutic potential, the size and high hydrophobicity of some may limit their ability to penetrate the blood-brain barrier from systemic circulation, something that could be improved by novel drug formulations. In the present study, the steroid-like compounds Ursolic Acid (UA) and Ursocholanic Acid (UCA) were successfully encapsulated within nanoscopic polymersomes formed by poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-di-isopropylamino)ethyl methacrylate) (PMPC-PDPA) and separated into spherical and tubular morphologies to assess the effects of nanoparticle mediated delivery on drug efficacy. Following incubation with either morphology, parkin-mutant fibroblasts demonstrated time and concentration dependent increases in intracellular ATP levels, resembling those resulting from treatment with nascent UA and UCA formulated in 0.1% DMSO, as used in the original drug screen. Empty PMPC-PDPA polymersomes did not alter physiological measures related to mitochondrial function or induce cytotoxicity. In combination with other techniques such as ligand functionalisation, PMPC-PDPA nanoparticles of well-defined morphology may prove a promising platform for tailoring the pharmacokinetic profile and organ specific bio-distribution of highly hydrophobic compounds. PMID:27412236

  10. Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

    PubMed

    Sun, Lei; Zhao, Mei; Yang, Yang; Xue, Run-Qing; Yu, Xiao-Jiang; Liu, Jian-Kang; Zang, Wei-Jin

    2016-05-01

    Acetylcholine (ACh) protected against cardiac injury via promoting autophagy and mitochondrial biogenesis, however, the involvement of mitophagy in ACh-elicited cardioprotection remains unknown. In the present study, H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) and ACh treatment during reoxygenation. Mitophagy markers PTEN-induced kinase 1 (PINK1) and Parkin translocation were examined using western blot and confocal fluorescence microscopy. Mitochondrial membrane potential and reactive oxygen species (ROS) were detected with fluorescence staining. We found that H/R-treated cells exhibited reduced levels of PINK1 and Parkin in mitochondria, accompanied with decreased autophagy flux (reduced LC3-II/LC3-I and increased p62). Conversely, ACh increased PINK1 and Parkin translocation to mitochondria and enhanced autophagy proteins. Confocal imaging of Parkin and MitoTracker Green-labeled mitochondria further confirmed ACh-induced mitochondrial translocation of Parkin, which was reversed by M2 receptor antagonist methoctramine and M2 receptor siRNA, suggesting ACh could induce mitophagy by M2 receptor after H/R. Mitophagy inhibitor 3-methaladenine abolished ACh-induced mitoprotection, manifesting as aggravated mitochondrial morphology disruption, ATP and membrane potential depletion, increased ROS overproduction, and apoptosis. Furthermore, PINK1/Parkin siRNA attenuated the protective effects of ACh against ATP loss and oxidative stress due to mitochondrial-dependent injury. Taken together, ACh promoted mitochondrial translocation of PINK1/Parkin to stimulate cytoprotective mitophagy via M2 receptor, which may provide beneficial targets in the preservation of cardiac homeostasis against H/R injury. PMID:26465230

  11. Stimulating endogenous cardiac repair

    PubMed Central

    Finan, Amanda; Richard, Sylvain

    2015-01-01

    The healthy adult heart has a low turnover of cardiac myocytes. The renewal capacity, however, is augmented after cardiac injury. Participants in cardiac regeneration include cardiac myocytes themselves, cardiac progenitor cells, and peripheral stem cells, particularly from the bone marrow compartment. Cardiac progenitor cells and bone marrow stem cells are augmented after cardiac injury, migrate to the myocardium, and support regeneration. Depletion studies of these populations have demonstrated their necessary role in cardiac repair. However, the potential of these cells to completely regenerate the heart is limited. Efforts are now being focused on ways to augment these natural pathways to improve cardiac healing, primarily after ischemic injury but in other cardiac pathologies as well. Cell and gene therapy or pharmacological interventions are proposed mechanisms. Cell therapy has demonstrated modest results and has passed into clinical trials. However, the beneficial effects of cell therapy have primarily been their ability to produce paracrine effects on the cardiac tissue and recruit endogenous stem cell populations as opposed to direct cardiac regeneration. Gene therapy efforts have focused on prolonging or reactivating natural signaling pathways. Positive results have been demonstrated to activate the endogenous stem cell populations and are currently being tested in clinical trials. A potential new avenue may be to refine pharmacological treatments that are currently in place in the clinic. Evidence is mounting that drugs such as statins or beta blockers may alter endogenous stem cell activity. Understanding the effects of these drugs on stem cell repair while keeping in mind their primary function may strike a balance in myocardial healing. To maximize endogenous cardiac regeneration, a combination of these approaches could ameliorate the overall repair process to incorporate the participation of multiple cellular players. PMID:26484341

  12. Mitochondrial and lysosomal biogenesis are activated following PINK1/parkin-mediated mitophagy.

    PubMed

    Ivankovic, Davor; Chau, Kai-Yin; Schapira, Anthony H V; Gegg, Matthew E

    2016-01-01

    Impairment of the autophagy-lysosome pathway is implicated with the changes in α-synuclein and mitochondrial dysfunction observed in Parkinson's disease (PD). Damaged mitochondria accumulate PINK1, which then recruits parkin, resulting in ubiquitination of mitochondrial proteins. These can then be bound by the autophagic proteins p62/SQSTM1 and LC3, resulting in degradation of mitochondria by mitophagy. Mutations in PINK1 and parkin genes are a cause of familial PD. We found a significant increase in the expression of p62/SQSTM1 mRNA and protein following mitophagy induction in human neuroblastoma SH-SY5Y cells. p62 protein not only accumulated on mitochondria, but was also greatly increased in the cytosol. Increased p62/SQSMT1 expression was prevented in PINK1 knock-down cells, suggesting increased p62 expression was a consequence of mitophagy induction. The transcription factors Nrf2 and TFEB, which play roles in mitochondrial and lysosomal biogenesis, respectively, can regulate p62/SQSMT1. We report that both Nrf2 and TFEB translocate to the nucleus following mitophagy induction and that the increase in p62 mRNA levels was significantly impaired in cells with Nrf2 or TFEB knockdown. TFEB translocation also increased expression of itself and lysosomal proteins such as glucocerebrosidase and cathepsin D following mitophagy induction. We also report that cells with increased TFEB protein have significantly higher PGC-1α mRNA levels, a regulator of mitochondrial biogenesis, resulting in increased mitochondrial content. Our data suggests that TFEB is activated following mitophagy to maintain autophagy-lysosome pathway and mitochondrial biogenesis. Therefore, strategies to increase TFEB may improve both the clearance of α-synuclein and mitochondrial dysfunction in PD. Damaged mitochondria are degraded by the autophagy-lysosome pathway and is termed mitophagy. Following mitophagy induction, the transcription factors Nrf2 and TFEB translocate to the nucleus, inducing

  13. Endogenous retroviruses and human cancer: is there anything to the rumors?

    PubMed

    Bhardwaj, Neeru; Coffin, John M

    2014-03-12

    Xenotropic murine leukemia virus-related virus (XMRV) infection was incorrectly associated with prostate cancer and chronic fatigue syndrome (CFS) in recent years. In this forum, we discuss the story of XMRV and how we can apply lessons learned here to inform the debate surrounding cancers associated with human endogenous retroviruses (HERVs). PMID:24629332

  14. Endogenous Erythropoietin Protects Neuroretinal Function in Ischemic Retinopathy

    PubMed Central

    Mowat, Freya M.; Gonzalez, Francisco; Luhmann, Ulrich F.O.; Lange, Clemens A.; Duran, Yanai; Smith, Alexander J.; Maxwell, Patrick H.; Ali, Robin R.; Bainbridge, James W.B.

    2012-01-01

    Because retinal ischemia is a common cause of vision loss, we sought to determine the effects of ischemia on neuroretinal function and survival in murine oxygen-induced retinopathy (OIR) and to define the role of endogenous erythropoietin (EPO) in this model. OIR is a reproducible model of ischemia-induced retinal neovascularization; it is used commonly to develop antiangiogenic strategies. We investigated the effects of ischemia in murine OIR on retinal function and neurodegeneration by electroretinography and detailed morphology. OIR was associated with significant neuroretinal dysfunction, with reduced photopic and scotopic ERG responses and reduced b-wave/a-wave ratios consistent with specific inner-retinal dysfunction. OIR resulted in significantly increased apoptosis and atrophy of the inner retina in areas of ischemia. EPO deficiency in heterozygous Epo-Tag transgenic mice was associated with more profound retinal dysfunction after OIR, indicated by a significantly greater suppression of ERG amplitudes, but had no measurable effect on the extent of retinal ischemia, preretinal neovascularization, or neuroretinal degeneration in OIR. Systemic administration of recombinant EPO protected EPO-deficient mice against this additional suppression, but EPO supplementation in wild-type animals with OIR did not rescue neuroretinal dysfunction or degeneration. Murine OIR offers a valuable model of ischemic neuroretinal dysfunction and degeneration in which to investigate adaptive tissue responses and evaluate novel therapeutic approaches. Endogenous EPO can protect neuroretinal function in ischemic retinopathy. PMID:22342523

  15. PDR-1/hParkin negatively regulates the phagocytosis of apoptotic cell corpses in Caenorhabditis elegans

    PubMed Central

    Cabello, J; Sämann, J; Gómez-Orte, E; Erazo, T; Coppa, A; Pujol, A; Büssing, I; Schulze, B; Lizcano, J M; Ferrer, I; Baumeister, R; Dalfo, E

    2014-01-01

    Apoptotic cell death is an integral part of cell turnover in many tissues, and proper corpse clearance is vital to maintaining tissue homeostasis in all multicellular organisms. Even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. In Caenorhabditis elegans, two parallel and partly redundant conserved pathways act in cell corpse engulfment. The pathway for cytoskeletal rearrangement requires the small GTPase CED-10 Rac1 acting for an efficient surround of the dead cell. The CED-10 Rac pathway is also required for the proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. Parkin, the mammalian homolog of the C. elegans PDR-1, interacts with Rac1 in aged human brain and it is also implicated with actin dynamics and cytoskeletal rearrangements in Parkinsons's disease, suggesting that it might act on engulfment. Our genetic and biochemical studies indicate that PDR-1 inhibits apoptotic cell engulfment and DTC migration by ubiquitylating CED-10 for degradation. PMID:24625979

  16. Park2-null/tau transgenic mice reveal a functional relationship between parkin and tau.

    PubMed

    Guerrero, Rosa; Navarro, Paloma; Gallego, Eva; Avila, Jesus; de Yebenes, Justo G; Sanchez, Marina P

    2008-03-01

    Mutations, haplotypes, and polymorphisms of tau and Park-2 genes constitute risk factors for developing tauopathies. In order to analyze the possible relationship between parkin and tau we generated a double-mutant mouse deficient for Park-2 expression and overexpressing a mutant tau protein (hTauVLW). Mice develop normally, although the median survival rate is considerably reduced with respect to wild type (45%). Aggregates of phosphorylated tau in neurons and reactive gliosis are quite abundant in cortex and hippocampus of these mice. Moreover, while in young transgenic mice the hTauVLW immunostained transgene product is observed in both cell bodies and dendrites, the hTauVLW mutant protein is only detected in the neuronal cell bodies when Park-2 gene is additionally deleted. Moreover, DNA fragmentation was detected by the TUNEL method, and cerebral atrophy is also present in these regions. The levels of phosphorylated tau and Hsp70 are increased in the double-mutant mice, while CHIP expression in hippocampus is lower when the Park-2 gene is deleted. Thus, the combination of Park-2 gene deletion with hTauVLW transgene overexpression in mice produces serious neuropathological effects, which reflect the existence of some relationship between both proteins. PMID:18376058

  17. Unconventional PINK1 localization to the outer membrane of depolarized mitochondria drives Parkin recruitment

    PubMed Central

    Okatsu, Kei; Kimura, Mayumi; Oka, Toshihiko; Tanaka, Keiji; Matsuda, Noriyuki

    2015-01-01

    ABSTRACT Dysfunction of PTEN-induced putative kinase 1 (PINK1), a Ser/Thr kinase with an N-terminal mitochondrial-targeting sequence (MTS), causes familial recessive parkinsonism. Reduction of the mitochondrial membrane potential limits MTS-mediated matrix import and promotes PINK1 accumulation on the outer mitochondrial membrane (OMM) of depolarized mitochondria. PINK1 then undergoes autophosphorylation and phosphorylates ubiquitin and Parkin, a cytosolic ubiquitin ligase, for clearance of damaged mitochondria. The molecular basis for PINK1 localization on the OMM of depolarized mitochondria rather than release to the cytosol is poorly understood. Here, we disentangle the PINK1 localization mechanism using deletion mutants and a newly established constitutively active PINK1 mutant. Disruption of the MTS through N-terminal insertion of aspartic acid residues results in OMM localization of PINK1 in energized mitochondria. Unexpectedly, the MTS and putative transmembrane domain (TMD) are dispensable for OMM localization, whereas mitochondrial translocase Tom40 (also known as TOMM40) and an alternative mitochondrial localization signal that resides between the MTS and TMD are required. PINK1 utilizes a mitochondrial localization mechanism that is distinct from that of conventional MTS proteins and that presumably functions in conjunction with the Tom complex in OMM localization when the conventional N-terminal MTS is inhibited. PMID:25609704

  18. Hepatitis B Virus-Induced Parkin-Dependent Recruitment of Linear Ubiquitin Assembly Complex (LUBAC) to Mitochondria and Attenuation of Innate Immunity

    PubMed Central

    Khan, Mohsin; Syed, Gulam Hussain; Kim, Seong-Jun; Siddiqui, Aleem

    2016-01-01

    Hepatitis B virus (HBV) suppresses innate immune signaling to establish persistent infection. Although HBV is a DNA virus, its pre-genomic RNA (pgRNA) can be sensed by RIG-I and activates MAVS to mediate interferon (IFN) λ synthesis. Despite of the activation of RIG-I-MAVS axis by pgRNA, the underlying mechanism explaining how HBV infection fails to induce interferon-αβ (IFN) synthesis remained uncharacterized. We demonstrate that HBV induced parkin is able to recruit the linear ubiquitin assembly complex (LUBAC) to mitochondria and abrogates IFN β synthesis. Parkin interacts with MAVS, accumulates unanchored linear polyubiquitin chains on MAVS via LUBAC, to disrupt MAVS signalosome and attenuate IRF3 activation. This study highlights the novel role of parkin in antiviral signaling which involves LUBAC being recruited to the mitochondria. These results provide avenues of investigations on the role of mitochondrial dynamics in innate immunity. PMID:27348524

  19. p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both

    PubMed Central

    Narendra, Derek P; Kane, Lesley A; Hauser, David N; Fearnley, Ian M

    2010-01-01

    Mitochondria sustain damage with aging, and the resulting mitochondrial dysfunction has been implicated in a number of diseases including parkinson disease. We recently demonstrated that the E3 ubiquitin ligase Parkin, which is linked to recessive forms of parkinsonism, causes a dramatic increase in mitophagy and a change in mitochondrial distribution, following its translocation from the cytosol to mitochondria. Investigating how Parkin induces these changes may offer insight into the mechanisms that lead to the sequestration and elimination of damaged mitochondria. We report that following Parkin's translocation from the cytosol to mitochondria, Parkin (but not a pathogenic mutant) promotes the K63-linked polyubiquitination of mitochondrial substrate(s) and recruits the ubiquitin- and LC3-binding protein, p62/SQSTM1, to mitochondria. After its recruitment, p62/SQSTM1 mediates the aggregation of dysfunctional mitochondria through polymerization via its PB1 domain, in a manner analogous to its aggregation of polyubiquitinated proteins. Surprisingly and in contrast to what has been recently reported for ubiquitin-induced pexophagy and xenophagy, p62 appears to be dispensable for mitophagy. Similarly, mitochondrial-anchored ubiquitin is sufficient to recruit p62 and promote mitochondrial clustering, but does not promote mitophagy. Although VDAC1 (but not VDAC2) is ubiquitinated following mitochondrial depolarization, we find VDAC1 cannot fully account for the mitochondrial K63-linked ubiquitin immunoreactivity observed following depolarization, as it is also observed in VDAC1/3−/− mouse embryonic fibroblasts. Additionally, we find VDAC1 and VDAC3 are dispensable for the recruitment of p62, mitochondrial clustering and mitophagy. These results demonstrate that mitochondria are aggregated by p62, following its recruitment by Parkin in a VDAC1-independent manner. They also suggest that proteins other than p62 are likely required for mitophagy downstream of Parkin

  20. Mammalian Endogenous Retroviruses.

    PubMed

    Mager, Dixie L; Stoye, Jonathan P

    2015-02-01

    Over 40% of mammalian genomes comprise the products of reverse transcription. Among such retrotransposed sequences are those characterized by the presence of long terminal repeats (LTRs), including the endogenous retroviruses (ERVs), which are inherited genetic elements closely resembling the proviruses formed following exogenous retrovirus infection. Sequences derived from ERVs make up at least 8 to 10% of the human and mouse genomes and range from ancient sequences that predate mammalian divergence to elements that are currently still active. In this chapter we describe the discovery, classification and origins of ERVs in mammals and consider cellular mechanisms that have evolved to control their expression. We also discuss the negative effects of ERVs as agents of genetic disease and cancer and review examples of ERV protein domestication to serve host functions, as in placental development. Finally, we address growing evidence that the gene regulatory potential of ERV LTRs has been exploited multiple times during evolution to regulate genes and gene networks. Thus, although recently endogenized retroviral elements are often pathogenic, those that survive the forces of negative selection become neutral components of the host genome or can be harnessed to serve beneficial roles. PMID:26104559

  1. Cloning of murine ferrochelatase.

    PubMed Central

    Brenner, D A; Frasier, F

    1991-01-01

    Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron and protoporphyrin to form heme. The activity of ferrochelatase is deficient in the inherited disease protoporphyria. In this study, murine ferrochelatase cDNAs were obtained by screening cDNA libraries with an oligonucleotide probe. The derived amino acid sequence of murine ferrochelatase has 47% identity with the recently cloned Saccharomyces cerevisiae ferrochelatase, but it is not significantly similar to other published sequences. Results of Southern blotting are consistent with a single murine ferrochelatase gene, while Northern blotting demonstrates two ferrochelatase transcripts in all tissues examined. The ferrochelatase protein and mRNAs have different relative concentrations in different tissues. The cloning of murine ferrochelatase cDNAs provides the basis for future studies on ferrochelatase gene expression and on the identification of the molecular defect in protoporphyria. Images PMID:1704134

  2. Endogenous pulmonary antibiotics.

    PubMed

    Gibbons, M A; Bowdish, D M; Davidson, D J; Sallenave, J M; Simpson, A J

    2006-05-01

    The human lung produces a variety of peptides and proteins which have intrinsic antimicrobial activity. In general these molecules have broad spectra of antimicrobial activity, kill micro-organisms rapidly, and evade resistance generated by pathogens. In recent years it has become increasingly apparent that the antimicrobial peptides (AMPs) simultaneously possess immunomodulatory functions, suggesting complex roles for these molecules in regulating the clearance of, and immune response to, invading pathogens. These collective properties have stimulated considerable interest in the potential clinical application of endogenous AMPs. This article outlines the biology of AMPs, their pattern of expression in the lung, and their functions, with reference to both antimicrobial and immunomodulatory activity. We then consider the biological importance of AMPs, before concentrating on the potential to use AMPs to therapeutic effect. The principles discussed in the article apply to innate immune defence throughout the body, but particular emphasis is placed on AMPs in the lung and the potential application to pulmonary infection. PMID:16722137

  3. Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila

    PubMed Central

    Sen, Aditya; Kalvakuri, Sreehari; Bodmer, Rolf; Cox, Rachel T.

    2015-01-01

    ABSTRACT Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD). However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control. PMID:26035866

  4. Angiostatin and endostatin: endogenous inhibitors of tumor growth.

    PubMed

    Sim, B K; MacDonald, N J; Gubish, E R

    2000-01-01

    Considerable progress has been made in the understanding of the molecular structure and mechanistic aspects of Angiostatin and Endostatin, endogenous angiogenesis inhibitors that have been shown to regress tumors in murine models. The growing body of literature surrounding these molecules and on the efficacy of these proteins is in part due to the ability to generate these proteins in recombinant systems as well characterized molecules. Recombinant human Angiostatin and Endostatin are in Phase I trials, following the manufacture of clinical grade material at large scale. This review highlights the recent advances made on understanding the structure and function of Angiostatin and Endostatin. PMID:11191058

  5. Exon Dosage Variations in Brazilian Patients with Parkinson’s Disease: Analysis of SNCA, PARKIN, PINK1 and DJ-1 Genes

    PubMed Central

    Moura, Karla Cristina Vasconcelos; Junior, Mário Campos; de Rosso, Ana Lúcia Zuma; Nicaretta, Denise Hack; Pereira, João Santos; Silva, Delson José; Santos-Rebouças, Cíntia Barros; Pimentel, Márcia Mattos Gonçalves

    2012-01-01

    Parkinson’s disease is one of the most common neurodegenerative disorders associated with aging, reaching ∼ 2% of individuals over 65 years. Knowledge achieved in the last decade about the genetic basis of Parkinson’s disease clearly shows that genetic factors play an important role in the etiology of this disorder. Exon dosage variations account for a high proportion of Parkinson’s disease mutations, mainly for PARKIN gene. In the present study, we screened genomic rearrangements in SNCA, PARKIN, PINK1 and DJ-1 genes in 102 Brazilian Parkinson’s disease patients with early onset (age of onset ≤ 50 years), using the multiplex ligation-dependent probe amplification method. Family history was reported by 24 patients, while 78 were sporadic cases. Screening of exon dosage revealed PARKIN and PINK1 copy number variations, but no dosage alteration was found in SNCA and DJ-1 genes. Most of the carriers harbor heterozygous deletions or duplications in the PARKIN gene and only one patient was found to have a deletion in PINK1 exon 1. Data about dosage changes are scarce in the Brazilian population, which stresses the importance of including exon dosage analysis in Parkinson’s disease genetic studies. PMID:22377733

  6. Detrimental effects of oxidative losses in parkin activity in a model of sporadic Parkinson's disease are attenuated by restoration of PGC1alpha.

    PubMed

    Siddiqui, Almas; Rane, Anand; Rajagopalan, Subramanian; Chinta, Shankar J; Andersen, Julie K

    2016-09-01

    Loss of parkin E3 ligase activity as a result of parkin gene mutation in rare familial forms of Parkinson's disease (PD) has been shown to be detrimental to mitochondrial function and to contribute to ensuing neurodegeneration. This has been shown by ourselves and others to be in part due to reductions in parkin-mediated ubiquitination of the transcriptional repressor PARIS, limiting the protein's subsequent degradation by the proteasome. Subsequent elevations in PARIS protein levels result in reduced expression of the master mitochondrial regulator PGC-1α, impacting in turn on mitochondrial function. Here, we report that oxidatively-mediated reductions in parkin solubility and function in a mouse model of age-related sporadic PD coincides with increased PARIS levels and reduced PGC-1α signaling. Furthermore, restoration of PGC-1α expression was found to abrogate losses in mitochondrial function and degeneration of dopaminergic (DAergic) neurons within the substantia nigra pars compacta (SNpc) associated with this particular model. These findings suggest that the PGC-1α signaling pathway constitutes a viable therapeutic target for the treatment of not only familial PD, but also more common sporadic forms of the disorder. PMID:27185595

  7. [Endogenous bacterial endophthalmitis].

    PubMed

    Cornut, P-L; Chiquet, C

    2011-01-01

    Endogenous bacterial endophthalmitis, also called metastatic bacterial endophthalmitis, remains a diagnostic and therapeutic challenge. It is a rare and potentially sight-threatening ocular infection that occurs when bacteria reach the eye via the bloodstream, cross the blood-ocular barrier, and multiply within the eye. It usually affects immunocompromised patients and those suffering from diabetes mellitus, malignancy, or cardiac disease, but has also been reported after invasive procedures or in previously healthy people. In most cases, the ocular symptoms occur after the diagnosis of septicemia or systemic infection. Ocular symptoms include decreased vision, redness, discharge, pain, and floaters. The ocular inflammatory signs may be anterior and/or posterior. Bilateral involvement occurs in nearly 25% of cases. A wide range of microorganisms are involved, with differences in their frequency according to geography as well as the patient's age and past medical history, because of variations in the predisposing conditions and the source of the sepsis. The majority of patients are initially misdiagnosed, and ophthalmologists should be aware of this because prompt local and general management is required to save the eye and/or the patient's life. PMID:21145128

  8. Endogenous strategy in exploration.

    PubMed

    Solman, Grayden J F; Kingstone, Alan

    2015-12-01

    We examined the characteristics of endogenous exploratory behaviors in a generalized search task in which guidance signals (e.g., landmarks, semantics, visual saliency, layout) were limited or precluded. Individuals looked for the highest valued cell in an array and were scored on the quality of the best value they could find. Exploration was guided only by the cells that had been previously examined, and the value of this guidance was manipulated by adjusting spatial autocorrelation to produce relatively smooth and rough landscapes-that is, arrays in which nearby cells had unrelated values (low correlation = rough) or similar values (high correlation = smooth). For search in increasingly rough as compared with smooth arrays, we found reduced performance despite increased sampling and increased time spent searching after revelation of a searcher's best cell. Spatially, sampling strategies tended toward more excursive, branching, and space-filling patterns as correlation decreased. Using a novel generalized-recurrence analysis, we report that these patterns reflect an increase in systematic search paths, characterized by regularized sweeps with localized infilling. These tendencies were likewise enhanced for high-performance as compared with low-performance participants. The results suggest a trade-off between guidance (in smooth arrays) and systematicity (in rough arrays), and they provide insight into the particular strategic approaches adopted by searchers when exogenous guiding information is minimized. PMID:26214501

  9. Ethanol-induced mitophagy in liver is associated with activation of the PINK1-Parkin pathway triggered by oxidative DNA damage.

    PubMed

    Eid, Nabil; Ito, Yuko; Horibe, Akio; Otsuki, Yoshinori

    2016-10-01

    Mitophagy is a cytoprotective mechanism against mitochondrial damaging agents. Studies demonstrating morphological evidence for the involvement of the PINK1-Parkin pathway in the hepatocyte mitophagic response to ethanol toxicity, and potential links to apoptosis and mitochondrial alterations such as spheroid formation are still lacking. We addressed these unresolved issues using a rat model of binge alcohol exposure. Adult rats were injected with ethanol (5g/kg) and liver samples were taken at 0, 3, 6, and 24 hours after ethanol administration and processed for light and electron microscopic studies. Ethanol induced a low level of hepatocyte apoptosis, peaking at 3 h and decreasing significantly by 24 h. In contrast, there was enhanced formation of mitophagic vacuoles in the majority of normal hepatocytes of ethanol-treated rats (ETRs), which peaked at 6 h and was maintained up to 24 h based on electron microscopy and TUNEL/LC3 double labelling. Moreover, enhanced mitophagy in ETR hepatocytes was confirmed by increased LC3 puncta formation, and co-localization of Parkin and LC3 with mitochondrial and lysosomal markers. Immunoelectron microscopy demonstrated the localization of PINK1 and Parkin to damaged mitochondria of ETR hepatocytes, which was consistent with co-localization of Parkin with 8-OHdG, a marker of oxidative mitochondrial DNA damage. Furthermore, electron microscopy showed enhanced formation of mitochondrial spheroids in ETR hepatocytes. These data are the first direct morphological evidence linking PINK1-Parkin pathway activation to the enhanced mitophagic response of hepatocytes to ethanol toxicity. Ethanol-induced hepatic mitophagy may be a prosurvival mechanism, which may have therapeutic implications. PMID:26935412

  10. Epigenetic upregulation of endogenous VEGF-A reduces myocardial infarct size in mice.

    PubMed

    Turunen, Mikko P; Husso, Tiia; Musthafa, Haja; Laidinen, Svetlana; Dragneva, Galina; Laham-Karam, Nihay; Honkanen, Sanna; Paakinaho, Anne; Laakkonen, Johanna P; Gao, Erhe; Vihinen-Ranta, Maija; Liimatainen, Timo; Ylä-Herttuala, Seppo

    2014-01-01

    "Epigenetherapy" alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral in vivo delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed in vivo that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes. PMID:24587164

  11. Identification of receptors for pig endogenous retrovirus

    PubMed Central

    Ericsson, Thomas A.; Takeuchi, Yasuhiro; Templin, Christian; Quinn, Gary; Farhadian, Shelli F.; Wood, James C.; Oldmixon, Beth A.; Suling, Kristen M.; Ishii, Jennifer K.; Kitagawa, Yoshinori; Miyazawa, Takayuki; Salomon, Daniel R.; Weiss, Robin A.; Patience, Clive

    2003-01-01

    Xenotransplantation of porcine tissues has the potential to treat a wide variety of major health problems including organ failure and diabetes. Balanced against the potential benefits of xenotransplantation, however, is the risk of human infection with a porcine microorganism. In particular, the transmission of porcine endogenous retrovirus (PERV) is a major concern [Chapman, L. E. & Bloom, E. T. (2001) J. Am. Med. Assoc. 285, 2304–2306]. Here we report the identification of two, sequence-related, human proteins that act as receptors for PERV-A, encoded by genes located on chromosomes 8 and 17. We also describe homologs from baboon and porcine cells that also are active as receptors. Conversely, activity could not be demonstrated with a syntenic murine receptor homolog. Sequence analysis indicates that PERV-A receptors [human PERV-A receptor (HuPAR)-1, HuPAR-2, baboon PERV-A receptor 2, and porcine PERV-A receptor] are multiple membrane-spanning proteins similar to receptors for other gammaretroviruses. Expression is widespread in human tissues including peripheral blood mononuclear cells, but their biological functions are unknown. The identification of the PERV-A receptors opens avenues of research necessary for a more complete assessment of the retroviral risks of pig to human xenotransplantation. PMID:12740431

  12. Human and murine erythropoiesis

    PubMed Central

    An, Xiuli; Schulz, Vincent P.; Mohandas, Narla; Gallagher, Patrick G.

    2015-01-01

    Purpose of review Research into the fundamental mechanisms of erythropoiesis has provided critical insights into inherited and acquired disorders of the erythrocyte. Studies of human erythropoiesis have primarily utilized in-vitro systems, whereas murine models have provided insights from in-vivo studies. This report reviews recent insights into human and murine erythropoiesis gained from transcriptome-based analyses. Recent findings The availability of high-throughput genomic methodologies has allowed attainment of detailed gene expression data from cells at varying developmental and differentiation stages of erythropoiesis. Transcriptome analyses of human and murine reveal both stage and species-specific similarities and differences across terminal erythroid differentiation. Erythroid-specific long noncoding RNAs exhibit poor sequence conservation between human and mouse. Genome-wide analyses of alternative splicing reveal that complex, dynamic, stage-specific programs of alternative splicing program are utilized during terminal erythroid differentiation. Transcriptome data provide a significant resource for understanding mechanisms of normal and perturbed erythropoiesis. Understanding these processes will provide innovative strategies to detect, diagnose, prevent, and treat hematologic disease. Summary Understanding the shared and different mechanisms controlling human and murine erythropoiesis will allow investigators to leverage the best model system to provide insights in normal and perturbed erythropoiesis. PMID:25719574

  13. Endogenous rhythms influence interpersonal synchrony.

    PubMed

    Zamm, Anna; Wellman, Chelsea; Palmer, Caroline

    2016-05-01

    Interpersonal synchrony, the temporal coordination of actions between individuals, is fundamental to social behaviors from conversational speech to dance and music-making. Animal models indicate constraints on synchrony that arise from endogenous rhythms: Intrinsic periodic behaviors or processes that continue in the absence of change in external stimulus conditions. We report evidence for a direct causal link between endogenous rhythms and interpersonal synchrony in a music performance task, which places high demands on temporal coordination. We first establish that endogenous rhythms, measured by spontaneous rates of individual performance, are stable within individuals across stimulus materials, limb movements, and time points. We then test a causal link between endogenous rhythms and interpersonal synchrony by pairing each musician with a partner who is either matched or mismatched in spontaneous rate and by measuring their joint behavior up to 1 year later. Partners performed melodies together, using either the same or different hands. Partners who were matched for spontaneous rate showed greater interpersonal synchrony in joint performance than mismatched partners, regardless of hand used. Endogenous rhythms offer potential to predict optimal group membership in joint behaviors that require temporal coordination. (PsycINFO Database Record PMID:26820249

  14. Nematode endogenous small RNA pathways

    PubMed Central

    Hoogstrate, Suzanne W; Volkers, Rita JM; Sterken, Mark G; Kammenga, Jan E; Snoek, L Basten

    2014-01-01

    The discovery of small RNA silencing pathways has greatly extended our knowledge of gene regulation. Small RNAs have been presumed to play a role in every field of biology because they affect many biological processes via regulation of gene expression and chromatin remodeling. Most well-known examples of affected processes are development, fertility, and maintenance of genome stability. Here we review the role of the three main endogenous small RNA silencing pathways in Caenorhabditis elegans: microRNAs, endogenous small interfering RNAs, and PIWI-interacting RNAs. After providing an entry-level overview on how these pathways function, we discuss research on other nematode species providing insight into the evolution of these small RNA pathways. In understanding the differences between the endogenous small RNA pathways and their evolution, a more comprehensive picture is formed of the functions and effects of small RNAs. PMID:25340013

  15. Quantitative analysis of endogenous compounds.

    PubMed

    Thakare, Rhishikesh; Chhonker, Yashpal S; Gautam, Nagsen; Alamoudi, Jawaher Abdullah; Alnouti, Yazen

    2016-09-01

    Accurate quantitative analysis of endogenous analytes is essential for several clinical and non-clinical applications. LC-MS/MS is the technique of choice for quantitative analyses. Absolute quantification by LC/MS requires preparing standard curves in the same matrix as the study samples so that the matrix effect and the extraction efficiency for analytes are the same in both the standard and study samples. However, by definition, analyte-free biological matrices do not exist for endogenous compounds. To address the lack of blank matrices for the quantification of endogenous compounds by LC-MS/MS, four approaches are used including the standard addition, the background subtraction, the surrogate matrix, and the surrogate analyte methods. This review article presents an overview these approaches, cite and summarize their applications, and compare their advantages and disadvantages. In addition, we discuss in details, validation requirements and compatibility with FDA guidelines to ensure method reliability in quantifying endogenous compounds. The standard addition, background subtraction, and the surrogate analyte approaches allow the use of the same matrix for the calibration curve as the one to be analyzed in the test samples. However, in the surrogate matrix approach, various matrices such as artificial, stripped, and neat matrices are used as surrogate matrices for the actual matrix of study samples. For the surrogate analyte approach, it is required to demonstrate similarity in matrix effect and recovery between surrogate and authentic endogenous analytes. Similarly, for the surrogate matrix approach, it is required to demonstrate similar matrix effect and extraction recovery in both the surrogate and original matrices. All these methods represent indirect approaches to quantify endogenous compounds and regardless of what approach is followed, it has to be shown that none of the validation criteria have been compromised due to the indirect analyses. PMID

  16. Endogenized viral sequences in mammals.

    PubMed

    Parrish, Nicholas F; Tomonaga, Keizo

    2016-06-01

    Reverse-transcribed RNA molecules compose a significant portion of the human genome. Many of these RNA molecules were retrovirus genomes either infecting germline cells or having done so in a previous generation but retaining transcriptional activity. This mechanism itself accounts for a quarter of the genomic sequence information of mammals for which there is data. We understand relatively little about the causes and consequences of retroviral endogenization. This review highlights functions ascribed to sequences of viral origin endogenized into mammalian genomes and suggests some of the most pressing questions raised by these observations. PMID:27128186

  17. [Endogenous hyperlactatemia and insulin secretion].

    PubMed

    Ribes, G; Valette, G; Lignon, F; Loubatières-Mariani, M M

    1978-01-01

    In the normal anesthetized dog, the endogenous hyperlactatemia induced either by intense muscular work or by a high dose of phenformin (20 mg/kg subtucaneously) is followed by an increase in the pancreaticoduodenal insulin output. A previous perfusion of sodium dichloroacetate (50 mg/kg. h) opposes the hyperlactatemia, and reduces or suppresses the increase in insulin output. PMID:150887

  18. Upregulating endogenous genes by an RNA-programmable artificial transactivator

    PubMed Central

    Fimiani, Cristina; Goina, Elisa; Mallamaci, Antonello

    2015-01-01

    To promote expression of endogenous genes ad libitum, we developed a novel, programmable transcription factor prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action. PMID:26152305

  19. Endogenous opiates and behavior: 2014.

    PubMed

    Bodnar, Richard J

    2016-01-01

    This paper is the thirty-seventh consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2014 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (endogenous opioids and receptors), and the roles of these opioid peptides and receptors in pain and analgesia (pain and analgesia); stress and social status (human studies); tolerance and dependence (opioid mediation of other analgesic responses); learning and memory (stress and social status); eating and drinking (stress-induced analgesia); alcohol and drugs of abuse (emotional responses in opioid-mediated behaviors); sexual activity and hormones, pregnancy, development and endocrinology (opioid involvement in stress response regulation); mental illness and mood (tolerance and dependence); seizures and neurologic disorders (learning and memory); electrical-related activity and neurophysiology (opiates and conditioned place preferences (CPP)); general activity and locomotion (eating and drinking); gastrointestinal, renal and hepatic functions (alcohol and drugs of abuse); cardiovascular responses (opiates and ethanol); respiration and thermoregulation (opiates and THC); and immunological responses (opiates and stimulants). This paper is the thirty-seventh consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2014 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular

  20. Endogenous respiration of Polyporus sulphureus

    SciTech Connect

    Li, S.M.W.; Siehr, D.J.

    1980-01-01

    Thirty percent of the dry weight of the basidiomycete Polyporus sulphureus is triterpenoid acid. The endogenous respiratory quotient of this organism is 0.8 indicating that the triterpenoid is being used as an endogenous storage material. Monosaccharides did not seem to be utilized as exogenous substrates but Krebs-cycle intermediates stimulated oxygen uptake. Pyruvic acid inhibited oxygen uptake. Studies with /sup 14/C-labeled glucose indicated that 27% of the glucose was metabolized by way of glycolysis. The hexose-monophosphate pathway was the major metabolic path for the utilization of glucose. Despite the fact that P. sulphureus is associated with brown rot, its carbon metabolism suggests that it utilizes substances associated with the degradation of lignin more readily than it does glucose.

  1. Endogenous Mechanisms of Cardiac Regeneration.

    PubMed

    Xiang, M S W; Kikuchi, K

    2016-01-01

    Zebrafish possess a remarkable capacity for cardiac regeneration throughout their lifetime, providing a model for investigating endogenous cellular and molecular mechanisms regulating myocardial regeneration. By contrast, adult mammals have an extremely limited capacity for cardiac regeneration, contributing to mortality and morbidity from cardiac diseases such as myocardial infarction and heart failure. However, the viewpoint of the mammalian heart as a postmitotic organ was recently revised based on findings that the mammalian heart contains multiple undifferentiated cell types with cardiogenic potential as well as a robust regenerative capacity during a short period early in life. Although it occurs at an extremely low level, continuous cardiomyocyte turnover has been detected in adult mouse and human hearts, which could potentially be enhanced to restore lost myocardium in damaged human hearts. This review summarizes and discusses recent advances in the understanding of endogenous mechanisms of cardiac regeneration. PMID:27572127

  2. Endogenous opiates and behavior: 2004.

    PubMed

    Bodnar, Richard J; Klein, Gad E

    2005-12-01

    This paper is the 27th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over 30 years of research. It summarizes papers published during 2004 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses. PMID:16039752

  3. Endogenous opiates and behavior: 2013.

    PubMed

    Bodnar, Richard J

    2014-12-01

    This paper is the thirty-sixth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2013 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses. PMID:25263178

  4. Endogenous opiates and behavior: 2007.

    PubMed

    Bodnar, Richard J

    2008-12-01

    This paper is the thirtieth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2007 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses. PMID:18851999

  5. Intramembrane protease PARL defines a negative regulator of PINK1- and PARK2/Parkin-dependent mitophagy.

    PubMed

    Meissner, Cathrin; Lorenz, Holger; Hehn, Beate; Lemberg, Marius K

    2015-01-01

    Mutations in PINK1 and PARK2/Parkin are a main risk factor for familial Parkinson disease. While the physiological mechanism of their activation is unclear, these proteins have been shown in tissue culture cells to serve as a key trigger for autophagy of depolarized mitochondria. Here we show that ablation of the mitochondrial rhomboid protease PARL leads to retrograde translocation of an intermembrane space-bridging PINK1 import intermediate. Subsequently, it is rerouted to the outer membrane in order to recruit PARK2, which phenocopies mitophagy induction by uncoupling agents. Consistent with a role of this retrograde translocation mechanism in neurodegenerative disease, we show that pathogenic PINK1 mutants which are not cleaved by PARL affect PINK1 kinase activity and the ability to induce PARK2-mediated mitophagy. Altogether we suggest that PARL is an important intrinsic player in mitochondrial quality control, a system substantially impaired in Parkinson disease as indicated by reduced removal of damaged mitochondria in affected patients. PMID:26101826

  6. Endogenous endostatin inhibits choroidal neovascularization.

    PubMed

    Marneros, Alexander G; She, Haicheng; Zambarakji, Hadi; Hashizume, Hiroya; Connolly, Edward J; Kim, Ivana; Gragoudas, Evangelos S; Miller, Joan W; Olsen, Bjorn R

    2007-12-01

    Endostatin, a fragment of the basement membrane component collagen XVIII, exhibits antiangiogenic properties in vitro and in vivo when high doses are administered. It is not known whether endogenous endostatin at physiological levels has a protective role as an inhibitor of pathological angiogenesis, such as choroidal neovascularization (CNV) in age-related macular degeneration. Using a laser injury model, we induced CNV in mice lacking collagen XVIII/endostatin and in control mice. CNV lesions in mutant mice were approximately 3-fold larger than in control mice and showed increased vascular leakage. These differences were independent of age-related changes at the choroid-retina interface. Ultrastructural analysis of the choroidal vasculature in mutant mice excluded morphological vascular abnormalities as a cause for the larger CNV lesions. When recombinant endostatin was administered to collagen XVIII/endostatin-deficient mice, CNV lesions were similar to those seen in control mice. In control mice treated with recombinant endostatin, CNV lesions were almost undetectable. These findings demonstrate that endogenous endostatin is an inhibitor of induced angiogenesis and that administration of endostatin potently inhibits CNV growth and vascular leakage. Endostatin may have a regulatory role in the pathogenesis of CNV and could be used therapeutically to inhibit growth and leakage of CNV lesions. PMID:17526870

  7. Endogenous Opiates and Behavior: 2006

    PubMed Central

    Bodnar, Richard J.

    2009-01-01

    This paper is the twenty-ninth consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning thirty years of research. It summarizes papers published during 2006 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurological disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17). PMID:17949854

  8. Endogenous opiates and behavior: 2012.

    PubMed

    Bodnar, Richard J

    2013-12-01

    This paper is the thirty-fifth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2012 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17). PMID:24126281

  9. Endogenous opiates and behavior: 2009.

    PubMed

    Bodnar, Richard J

    2010-12-01

    This paper is the 32nd consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2009 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17). PMID:20875476

  10. Endogenous opiates and behavior: 2005.

    PubMed

    Bodnar, Richard J; Klein, Gad E

    2006-12-01

    This paper is the 28th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over a quarter-century of research. It summarizes papers published during 2005 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity, neurophysiology and transmitter release (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); immunological responses (Section 17). PMID:16973239

  11. Endogenous opiates and behavior: 2008.

    PubMed

    Bodnar, Richard J

    2009-12-01

    This paper is the 31st consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2008 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17). PMID:19793543

  12. Endogenous opiates and behavior: 2010.

    PubMed

    Bodnar, Richard J

    2011-12-01

    This paper is the thirty-third consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2010 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration (Section 16); and immunological responses (Section 17). PMID:21983105

  13. Endogenous opiates and behavior: 2006.

    PubMed

    Bodnar, Richard J

    2007-12-01

    This paper is the 29th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning 30 years of research. It summarizes papers published during 2006 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurological disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17). PMID:17949854

  14. Endogenous opiates and behavior: 2011.

    PubMed

    Bodnar, Richard J

    2012-12-01

    This paper is the thirty-fourth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2011 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration (Section 16); and immunological responses (Section 17). PMID:23041439

  15. Endogenous opiates and behavior: 2002.

    PubMed

    Bodnar, Richard J; Hadjimarkou, Maria M

    2003-08-01

    This paper is the twenty-fifth consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over a quarter-century of research. It summarizes papers published during 2002 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17). PMID:14612197

  16. Endogenous opiates and behavior: 2003.

    PubMed

    Bodnar, Richard J; Klein, Gad E

    2004-12-01

    This paper is the 26th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over a quarter-century of research. It summarizes papers published during 2003 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17). PMID:15572211

  17. Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy.

    PubMed

    Di Sante, Gabriele; Casimiro, Mathew C; Pestell, Timothy G; Pestell, Richard G

    2016-01-01

    Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach. PMID:27168174

  18. Functional Analysis of the env Open Reading Frame in Human Endogenous Retrovirus IDDMK1,222 Encoding Superantigen Activity

    PubMed Central

    Lapatschek, Matthias; Dürr, Susanne; Löwer, Roswitha; Magin, Christine; Wagner, Hermann; Miethke, Thomas

    2000-01-01

    Mice harbor a family of endogenous retroviruses, the mouse mammary tumor viruses (MMTV), which encode superantigens. These superantigens are responsible for the deletion of T cells expressing certain Vβ chains of the T-cell receptor in the thymus. Human T cells are able to recognize MMTV-encoded superantigens presented by human major histocompatibility complex class II-positive cells. Owing to this and to the similarity of the human and murine immune systems, it was speculated that human endogenous retroviruses might also code for superantigens. Recently, it was reported that a proviral clone (IDDMK1,222) of the human endogenous retrovirus family HTDV/HERV-K encodes a superantigen. The putative superantigen gene was located within the env region of the virus. Stimulated by these findings, we amplified by PCR and cloned into eucaryotic expression vectors open reading frames (ORFs) which were identical or very similar to IDDMK1,222. When we transfected these vectors into A20 cells, a murine B-cell lymphoma, we were able to demonstrate mRNA expression and protein production. However, we did not find any evidence that the ORF stimulated human or murine T cells in a Vβ-specific fashion, the most prominent feature of superantigens. PMID:10864649

  19. Endogenous Retroviruses and Human Evolution

    PubMed Central

    Lebedev, Yuri; Sverdlov, Eugene

    2002-01-01

    Humans share about 99% of their genomic DNA with chimpanzees and bonobos; thus, the differences between these species are unlikely to be in gene content but could be caused by inherited changes in regulatory systems. Endogenous retroviruses (ERVs) comprise ∼ 5% of the human genome. The LTRs of ERVs contain many regulatory sequences, such as promoters, enhancers, polyadenylation signals and factor-binding sites. Thus, they can influence the expression of nearby human genes. All known human-specific LTRs belong to the HERV-K (human ERV) family, the most active family in the human genome. It is likely that some of these ERVs could have integrated into regulatory regions of the human genome, and therefore could have had an impact on the expression of adjacent genes, which have consequently contributed to human evolution. This review discusses possible functional consequences of ERV integration in active coding regions. PMID:18629260

  20. Endogenous opiates and behavior: 2001.

    PubMed

    Bodnar, Richard J; Hadjimarkou, Maria M

    2002-12-01

    This paper is the twenty-fourth installment of the annual review of research concerning the opiate system. It summarizes papers published during 2001 that studied the behavioral effects of the opiate peptides and antagonists. The particular topics covered this year include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology(Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17). PMID:12535711

  1. Murine leukemia virus envelope gp70 is a shared biomarker for the high-sensitivity quantification of murine tumor burden

    PubMed Central

    Scrimieri, Francesca; Askew, David; Corn, David J; Eid, Saada; Bobanga, Iuliana D; Bjelac, Jaclyn A; Tsao, Matthew L; Allen, Frederick; Othman, Youmna S; Wang, Shih-Chung G; Huang, Alex Y

    2013-01-01

    The preclinical development of anticancer drugs including immunotherapeutics and targeted agents relies on the ability to detect minimal residual tumor burden as a measure of therapeutic efficacy. Real-time quantitative (qPCR) represents an exquisitely sensitive method to perform such an assessment. However, qPCR-based applications are limited by the availability of a genetic defect associated with each tumor model under investigation. Here, we describe an off-the-shelf qPCR-based approach to detect a broad array of commonly used preclinical murine tumor models. In particular, we report that the mRNA coding for the envelope glycoprotein 70 (gp70) encoded by the endogenous murine leukemia virus (MuLV) is universally expressed in 22 murine cancer cell lines of disparate histological origin but is silent in 20 out of 22 normal mouse tissues. Further, we detected the presence of as few as 100 tumor cells in whole lung extracts using qPCR specific for gp70, supporting the notion that this detection approach has a higher sensitivity as compared with traditional tissue histology methods. Although gp70 is expressed in a wide variety of tumor cell lines, it was absent in inflamed tissues, non-transformed cell lines, or pre-cancerous lesions. Having a high-sensitivity biomarker for the detection of a wide range of murine tumor cells that does not require additional genetic manipulations or the knowledge of specific genetic alterations present in a given neoplasm represents a unique experimental tool for investigating metastasis, assessing antitumor therapeutic interventions, and further determining tumor recurrence or minimal residual disease. PMID:24482753

  2. Angiostatin and endostatin: endothelial cell-specific endogenous inhibitors of angiogenesis and tumor growth.

    PubMed

    Sim, B K

    1998-01-01

    Angiostatin and Endostatin are potent inhibitors of angiogenesis. These proteins are endogenously produced and specifically target endothelial cells resulting in angiogenesis inhibition. Recombinant preparations of these proteins inhibit the growth of metastases and regress primary tumors to dormant microscopic lesions. A variety of murine tumors as well as human breast, prostate and colon tumors in human xenograft models regress when treated with Angiostatin or Endostatin. Regression of tumors upon systemic treatment with these proteins is in part due to increased tumor cell apoptosis. Repeated cycles of Endostatin therapy lead to prolonged tumor dormancy without further treatment and are not associated with any apparent toxicity or acquired drug resistance. PMID:14517374

  3. ARHGAP18: an endogenous inhibitor of angiogenesis, limiting tip formation and stabilizing junctions

    PubMed Central

    Chang, Garry HK; Lay, Angelina J; Ting, Ka Ka; Zhao, Yang; Coleman, Paul R; Powter, Elizabeth E; Formaz-Preston, Ann; Jolly, Christopher J; Bower, Neil I; Hogan, Benjamin M; Rinkwitz, Silke; Becker, Thomas S; Vadas, Mathew A; Gamble, Jennifer R

    2014-01-01

    The formation of the vascular network requires a tightly controlled balance of pro-angiogenic and stabilizing signals. Perturbation of this balance can result in dysregulated blood vessel morphogenesis and drive pathologies including cancer. Here, we have identified a novel gene, ARHGAP18, as an endogenous negative regulator of angiogenesis, limiting pro-angiogenic signaling and promoting vascular stability. Loss of ARHGAP18 promotes EC hypersprouting during zebrafish and murine retinal vessel development and enhances tumor vascularization and growth. Endogenous ARHGAP18 acts specifically on RhoC and relocalizes to the angiogenic and destabilized EC junctions in a ROCK dependent manner, where it is important in reaffirming stable EC junctions and suppressing tip cell behavior, at least partially through regulation of tip cell genes, Dll4, Flk-1 and Flt-4. These findings highlight ARHGAP18 as a specific RhoGAP to fine tune vascular morphogenesis, limiting tip cell formation and promoting junctional integrity to stabilize the angiogenic architecture. PMID:25425145

  4. Murine typhus in travelers returning from Indonesia.

    PubMed Central

    Parola, P.; Vogelaers, D.; Roure, C.; Janbon, F.; Raoult, D.

    1998-01-01

    We report the first three documented cases of murine typhus imported into Europe from Indonesia, discuss clues for the diagnosis of the disease, and urge that murine fever be considered in the diagnosis of febrile disease in travelers. PMID:9866749

  5. Antibacterial activity of recombinant murine beta interferon.

    PubMed Central

    Fujiki, T; Tanaka, A

    1988-01-01

    Recombinant murine beta interferon was protective and therapeutic for mice against Listeria monocytogenes infection in vivo. The recombinant murine beta interferon caused enhanced H2O2 release by macrophages in vivo, but not in vitro. PMID:3343048

  6. Immunosuppressive effect of murine cytomegalovirus.

    PubMed Central

    Loh, L; Hudson, J B

    1980-01-01

    Murine cytomegalovirus suppressed the ability of spleen cells to respond to mitogens in vitro. The degree of suppression was proportional to the multiplicity of infection. This effect could not be explained by cytolysis of lymphocytes, an alteration in the kinetics of the response to mitogen, or a direct competition between virions and mitogen molecules for cell-surface receptors. Nor was it due to simple contact between cell and virus, since ultraviolet-inactivated murine cytomegalovirus failed to suppress the response to mitogens. Reconstitution experiments were performed which involved mixing various combinations of infected and uninfected macrophages and lymphocytes. Under these conditions, it was found that the infected macrophages and lymphocytes. Under these conditions, it was found that the infected macrophages had an impaired capacity to mediate the response ot T lymphocytes to concanavalin A. This suggests that murine cytomegalovirus may cause immunosuppression indirectly by interfering with macrophage function. PMID:6244228

  7. Endogenous Peer Effects: Fact or Fiction?

    ERIC Educational Resources Information Center

    Yeung, Ryan; Nguyen-Hoang, Phuong

    2016-01-01

    The authors examine endogenous peer effects, which occur when a student's behavior or outcome is a function of the behavior or outcome of his or her peer group. Endogenous peer effects have important implications for educational policies such as busing, school choice and tracking. In this study, the authors quantitatively review the literature on…

  8. Endogenous timing factors in bird migration

    NASA Technical Reports Server (NTRS)

    Gwinner, E. G.

    1972-01-01

    Several species of warbler birds were observed in an effort to determine what initiates and terminates migration. Environmental and endogenous timing mechanisms were analyzed. The results indicate that endogenous stimuli are dominant factors for bird migration especially for long distances. It was concluded that environmental factors act as an assist mechanism.

  9. Epigenetic Upregulation of Endogenous VEGF-A Reduces Myocardial Infarct Size in Mice

    PubMed Central

    Musthafa, Haja; Laidinen, Svetlana; Dragneva, Galina; Laham-Karam, Nihay; Honkanen, Sanna; Paakinaho, Anne; Laakkonen, Johanna P.; Gao, Erhe; Vihinen-Ranta, Maija; Liimatainen, Timo; Ylä-Herttuala, Seppo

    2014-01-01

    “Epigenetherapy” alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral in vivo delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed in vivo that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes. PMID:24587164

  10. Antimicrobial proteins of murine macrophages.

    PubMed Central

    Hiemstra, P S; Eisenhauer, P B; Harwig, S S; van den Barselaar, M T; van Furth, R; Lehrer, R I

    1993-01-01

    Three murine microbicidal proteins (MUMPs) were purified from cells of the murine macrophage cell line RAW264.7 that had been activated by gamma interferon. Similar proteins were also present in nonactivated RAW264.7 cells, in cells of the murine macrophage cell line J774A.1, and in resident and activated murine peritoneal macrophages. MUMP-1, MUMP-2, and MUMP-3 killed Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Mycobacterium fortuitum, and Cryptococcus neoformans in vitro. MUMP-1 resembled an H1 histone but was unusual because its N-terminal residue (serine) was not N acetylated. Although MUMP-2 was N terminally blocked, its high lysine/arginine ratio and its reactivity with an antibody to H1 histones suggested that it also belonged to the H1 histone family. MUMP-3 was identical to histone H2B in 30 of 30 amino-terminal residues. Although the antimicrobial properties of histones have been recognized for decades, this is the first evidence that such proteins may endow the lysosomal apparatus of macrophages with nonoxidative antimicrobial potential. Other MUMPs, including some with a more restricted antimicrobial spectrum and one that appeared to be induced in RAW264.7 cells after gamma interferon stimulation, were noted but remain to be characterized. Images PMID:8514411

  11. Gravity effects on endogenous movements

    NASA Astrophysics Data System (ADS)

    Johnsson, Anders; Antonsen, Frank

    Gravity effects on endogenous movements A. Johnsson * and F. Antonsen *+ * Department of Physics, Norwegian University of Science and Technology,NO-7491, Trond-heim, Norway, E-mail: anders.johnsson@ntnu.no + Present address: Statoil Research Center Trondheim, NO-7005, Trondheim, Norway Circumnutations in stems/shoots exist in many plants and often consists of more or less regular helical movements around the plumb line under Earth conditions. Recent results on circumnu-tations of Arabidopsis in space (Johnsson et al. 2009) showed that minute amplitude oscilla-tions exist in weightlessness, but that centripetal acceleration (mimicking the gravity) amplified and/or created large amplitude oscillations. Fundamental mechanisms underlying these results will be discussed by modeling the plant tissue as a cylinder of cells coupled together. As a starting point we have modeled (Antonsen 1998) standing waves on a ring of biological cells, as first discussed in a classical paper (Turing 1952). If the coupled cells can change their water content, an `extension' wave could move around the ring. We have studied several, stacked rings of cells coupled into a cylinder that together represent a cylindrical plant tissue. Waves of extensions travelling around the cylinder could then represent the observable circumnutations. The coupling between cells can be due to cell-to-cell diffusion, or to transport via channels, and the coupling can be modeled to vary in both longitudinal and transversal direction of the cylinder. The results from ISS experiments indicate that this cylindrical model of coupled cells should be able to 1) show self-sustained oscillations without the impact of gravity (being en-dogenous) and 2) show how an environmental factor like gravity can amplify or generate the oscillatory movements. Gravity has been introduced in the model by a negative, time-delayed feed-back transport across the cylinder. This represents the physiological reactions to acceler

  12. Infectivity and insertional mutagenesis of endogenous retrovirus in autoimmune NZB and B/W mice.

    PubMed

    Beck-Engeser, Gabriele B; Ahrends, Tomasz; Knittel, Gero; Wabl, Rafael; Metzner, Mirjam; Eilat, Dan; Wabl, Matthias

    2015-11-01

    Murine leukaemia virus has been suggested to contribute to both autoimmune disease and leukaemia in the NZB mouse and in the (NZB × NZW) F1 (abbreviated B/W) mouse. However, with apparently only xenotropic but no ecotropic virus constitutively expressed in these mice, few mechanisms could explain the aetiology of either disease in either mouse strain. Because pseudotyped and/or inducible ecotropic virus may play a role, we surveyed the ability of murine leukaemia virus in NZB, NZW and B/W mice to infect and form a provirus. From the spleen of NZB mice, we isolated circular cDNA of xenotropic and polytropic virus, which indicates ongoing infection by these viruses. From a B/W lymphoma, we isolated and determined the complete sequence of a putative ecotropic NZW virus. From B/W mice, we recovered de novo endogenous retroviral integration sites (tags) from the hyperproliferating cells of the spleen and the peritoneum. The tagged genes seemed to be selected to aid cellular proliferation, as several of them are known cancer genes. The insertions are consistent with the idea that endogenous retrovirus contributes to B-cell hyperproliferation and progression to lymphoma in B/W mice. PMID:26315139

  13. Selenium suppresses leukemia through the action of endogenous eicosanoids.

    PubMed

    Gandhi, Ujjawal H; Kaushal, Naveen; Hegde, Shailaja; Finch, Emily R; Kudva, Avinash K; Kennett, Mary J; Jordan, Craig T; Paulson, Robert F; Prabhu, K Sandeep

    2014-07-15

    Eradicating cancer stem-like cells (CSC) may be essential to fully eradicate cancer. Metabolic changes in CSC could hold a key to their targeting. Here, we report that the dietary micronutrient selenium can trigger apoptosis of CSC derived from chronic or acute myelogenous leukemias when administered at supraphysiologic but nontoxic doses. In leukemia CSC, selenium treatment activated ATM-p53-dependent apoptosis accompanied by increased intracellular levels of reactive oxygen species. Importantly, the same treatment did not trigger apoptosis in hematopoietic stem cells. Serial transplantation studies with BCR-ABL-expressing CSC revealed that the selenium status in mice was a key determinant of CSC survival. Selenium action relied upon the endogenous production of the cyclooxygenase-derived prostaglandins Δ(12)-PGJ2 and 15d-PGJ2. Accordingly, nonsteroidal anti-inflammatory drugs and NADPH oxidase inhibitors abrogated the ability of selenium to trigger apoptosis in leukemia CSC. Our results reveal how selenium-dependent modulation of arachidonic acid metabolism can be directed to trigger apoptosis of primary human and murine CSC in leukemia. PMID:24872387

  14. Accelerated Human Mutant Tau Aggregation by Knocking Out Murine Tau in a Transgenic Mouse Model

    PubMed Central

    Ando, Kunie; Leroy, Karelle; Héraud, Céline; Yilmaz, Zehra; Authelet, Michèle; Suain, Valèrie; De Decker, Robert; Brion, Jean-Pierre

    2011-01-01

    Many models of human tauopathies have been generated in mice by expression of a human mutant tau with maintained expression of mouse endogenous tau. Because murine tau might interfere with the toxic effects of human mutant tau, we generated a model in which a pathogenic human tau protein is expressed in the absence of wild-type tau protein, with the aim of facilitating the study of the pathogenic role of the mutant tau and to reproduce more faithfully a human tauopathy. The Tg30 line is a tau transgenic mouse model overexpressing human 1N4R double-mutant tau (P301S and G272V) that develops Alzheimer's disease-like neurofibrillary tangles in an age-dependent manner. By crossing Tg30 mice with mice invalidated for their endogenous tau gene, we obtained Tg30xtau−/− mice that express only exogenous human double-mutant 1N4R tau. Although Tg30xtau−/− mice express less tau protein compared with Tg30, they exhibit signs of decreased survival, increased proportion of sarkosyl-insoluble tau in the brain and in the spinal cord, increased number of Gallyas-positive neurofibrillary tangles in the hippocampus, increased number of inclusions in the spinal cord, and a more severe motor phenotype. Deletion of murine tau accelerated tau aggregation during aging of this mutant tau transgenic model, suggesting that murine tau could interfere with the development of tau pathology in transgenic models of human tauopathies. PMID:21281813

  15. Induction of murine p30 by superinfecting herpesviruses.

    PubMed Central

    Reed, C L; Rapp, F

    1976-01-01

    The interaction of endogenous type C viruses with superinfecting herpes simplex virus type 2 (HSV-2) was investigated in two murine cell lines. Replication of HSV-2 was suboptimal in random-bred Swiss/3T3A cells and, in initial experiments, infection with a low virus-to-cell ratio resulted in carrier cultures with enhanced murine leukemia virus (MuLV) p30 expression. Immunofluorescence tests with Swiss/3T3A cells productively infected with HSV-2 also showed HSV-associated cytoplasmic antigens and enhanced MuLV p30 expression when compared with uninfected controls. Inactivation of HSV-2 with UV light did not abolish this reaction, although the number of cells expressing p30 was reduced. HSV-2 replicated more efficiently in a line of NIH Swiss cells (N c1 A c1 10). These cells are not readily inducible for type C expression by conventional methods; however, untreated and UV-inactivated HSV-2 induced both HSV-2-associated antigens and MuLV p30 in these cells. Although the Birch strain of human cytomegalovirus induced MuLV p30, neither mouse cytomegalovirus nor vesicular stomatitis virus induced MuLV p30 in either cell line. Images PMID:184296

  16. Mechanism of endogenous myc gene down-regulation in E mu-N-myc tumors.

    PubMed

    Ma, A; Smith, R K; Tesfaye, A; Achacoso, P; Dildrop, R; Rosenberg, N; Alt, F W

    1991-01-01

    Transgenic mouse lines carrying the N-myc oncogene deregulated by the immunoglobulin heavy-chain enhancer spontaneously develop B-lymphoid tumors (R. Dildrop, A. Ma, K. Zimmerman, E. Hsu, A. Tesfaye, R. DePinho, and F. W. Alt, EMBO J. 8:1121-1128, 1989; H. Rosenbaum, E. Webb, J. M. Adams, S. Cory, and A. W. Harris, EMBO J. 8:749-755). Permanent cell lines derived from these tumors (E mu-N-myc cell lines) express extremely high levels of the N-myc transgene but little or no detectable endogenous N-myc or c-myc. We have employed nuclear run-on assays to show that down-regulation of endogenous N- and c-myc expression occurs at the transcriptional level. To determine whether the lack of endogenous myc gene transcription is a direct effect of high-level N-myc transgene expression, we have generated Abelson murine leukemia virus (A-MuLV)-transformed cell lines from prelymphomatous E mu-N-myc mice (A-MuLV/E mu-N-myc cell lines). Although these A-MuLV/E mu-N-myc lines express very high levels of the N-myc transgene, they continue to transcribe the endogenous c-myc gene. These findings demonstrate that high-level N-myc gene expression alone does not necessarily lead to down-regulation of endogenous myc gene expression and suggest that events associated with transformation by N-myc may be critical to this process. PMID:1986238

  17. Commensal bacteria drive endogenous transformation and tumour stem cell marker expression through a bystander effect

    PubMed Central

    Wang, Xingmin; Yang, Yonghong; Huycke, Mark M

    2015-01-01

    Objective Commensal bacteria and innate immunity play a major role in the development of colorectal cancer (CRC). We propose that selected commensals polarise colon macrophages to produce endogenous mutagens that initiate chromosomal instability (CIN), lead to expression of progenitor and tumour stem cell markers, and drive CRC through a bystander effect. Design Primary murine colon epithelial cells were repetitively exposed to Enterococcus faecalis-infected macrophages, or purified trans-4-hydroxy-2-nonenal (4-HNE)—an endogenous mutagen and spindle poison produced by macrophages. CIN, gene expression, growth as allografts in immunodeficient mice were examined for clones and expression of markers confirmed using interleukin (IL) 10 knockout mice colonised by E. faecalis. Results Primary colon epithelial cells exposed to polarised macrophages or 4-hydroxy-2-nonenal developed CIN and were transformed after 10 weekly treatments. In immunodeficient mice, 8 of 25 transformed clones grew as poorly differentiated carcinomas with 3 tumours invading skin and/or muscle. All tumours stained for cytokeratins confirming their epithelial cell origin. Gene expression profiling of clones showed alterations in 3 to 7 cancer driver genes per clone. Clones also strongly expressed stem/progenitor cell markers Ly6A and Ly6E. Although not differentially expressed in clones, murine allografts positively stained for the tumour stem cell marker doublecortin-like kinase 1. Doublecortin-like kinase 1 and Ly6A/E were expressed by epithelial cells in colon biopsies for areas of inflamed and dysplastic tissue from E. faecalis-colonised IL-10 knockout mice. Conclusions These results validate a novel mechanism for CRC that involves endogenous CIN and cellular transformation arising through a microbiome-driven bystander effect. PMID:24906974

  18. Endogenous Optical Signals Reveal Changes of Elastin and Collagen Organization During Differentiation of Mouse Embryonic Stem Cells.

    PubMed

    Thimm, Terra N; Squirrell, Jayne M; Liu, Yuming; Eliceiri, Kevin W; Ogle, Brenda M

    2015-10-01

    Components of the extracellular matrix (ECM) have recently been shown to influence stem cell specification. However, it has been challenging to assess the spatial and temporal dynamics of stem cell-ECM interactions because most methodologies utilized to date require sample destruction or fixation. We examined the efficacy of utilizing the endogenous optical signals of two important ECM proteins, elastin (Eln), through autofluorescence, and type I collagen (ColI), through second harmonic generation (SHG), during mouse embryonic stem cell differentiation. After finding favorable overlap between antibody labeling and the endogenous fluorescent signal of Eln, we used this endogenous signal to map temporal changes in Eln and ColI during murine embryoid body differentiation and found that Eln increases until day 9 and then decreases slightly by day 12, while Col1 steadily increases over the 12-day period. Furthermore, we combined endogenous fluorescence imaging and SHG with antibody labeling of cardiomyocytes to examine the spatial relationship between Eln and ColI accumulation and cardiomyocyte differentiation. Eln was ubiquitously present, with enrichment in regions with cardiomyocyte differentiation, while there was an inverse correlation between ColI and cardiomyocyte differentiation. This work provides an important first step for utilizing endogenous optical signals, which can be visualized in living cells, to understand the relationship between the ECM and cardiomyocyte development and sets the stage for future studies of stem cell-ECM interactions and dynamics relevant to stem cells as well as other cell and tissue types. PMID:25923353

  19. Rat sequences of the Kirsten and Harvey murine sarcoma virus genomes: nature, origin, and expression in rat tumor RNA.

    PubMed Central

    Anderson, G R; Robbins, K C

    1976-01-01

    Two murine sarcoma viruses, the Kirsten and the Harvey, were isolated by passage of mouse type C leukemia viruses through rats. These sarcoma viruses have genomes containing portions of their parental type C mouse leukemia virus genomes, in stable association with specific rat cellular sequences that we find to be quite likely not those of a rat type C leukemia virus. To determine if these murine sarcoma viruses provide a model relevant to the events occurring in spontaneous tumors, we have hybridized DNA and RNA prepared from rat tumors and normal rat tissues to [3H]DNA prepared from the Kirsten murine sarcoma virus. We have also hybridized these rat tissue nucleic acids to [3H]DNA prepared from a respresentative endogenous rat type C leukemia virus, the WFU (Wistar-Furth). Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected in the DNA of both tumor and normal tissues, with no evidence of either gene amplification or additional sequences being present in tumor DNA. Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected at elevated concentrations in the RNA of many rat tumors and in specific groups of normal tissues. PMID:176419

  20. Effect of caffeine on induction of endogenous type C virus in mouse cells in vitro

    SciTech Connect

    Niwa, O.; Sugahara, T.

    1981-08-01

    The effect of caffeine on the expression of murine endogenous virus in mouse cells induced by radiation and chemicals was studied. Postirradiation treatment of K-BALB cells with caffeine enhanced cell killing as well as the induction of xenotropic virus after ultraviolet light irradiation. The degree of enhancement for the virus induction was comparable to that for cell killing. On the other hand, colony-forming ability and the expression of xenotropic virus of K-BALB cells after X-irradiation were unaffected by caffeine. These data suggest a linear relationship between the degree of endogenous virus expression and the amount of lethal damages after irradiation. For induction by halogenated pyrimidines, a 24-hr incubation of AKR2B cells with caffeine after 5-iodo-2'-deoxyuridine treatment resulted in marked suppression of the expression of ecotropic virus. On the contrary, in K-BALB cells, caffeine exerted only a small effect on 5-iodo-2'-deoxyuridine-induced expression of ecotropic and xenotropic viruses. These results indicate that, although using the same inducing agent, the pathway of endogenous virus induction may be different for AKR2B cells and for K-BALB cells.

  1. SMADs and FOXL2 synergistically regulate murine FSHbeta transcription via a conserved proximal promoter element.

    PubMed

    Tran, Stella; Lamba, Pankaj; Wang, Ying; Bernard, Daniel J

    2011-07-01

    Pituitary FSH regulates ovarian and testicular function. Activins stimulate FSHβ subunit (Fshb) gene transcription in gonadotrope cells, the rate-limiting step in mature FSH synthesis. Activin A-induced murine Fshb gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor FOXL2 (FOXL2). Here, we demonstrate that FOXL2 synergizes with SMAD2, SMAD3, and SMAD4 to stimulate murine Fshb promoter-reporter activity in heterologous cells. Moreover, SMAD3-induction of Fshb promoter activity or endogenous mRNA expression is dependent upon endogenous FOXL2 in homologous cells. FOXL2/SMAD synergy requires binding of both FOXL2 and SMAD3 or SMAD4 to DNA. Of three putative forkhead-binding elements identified in the murine Fshb promoter, only the most proximal is absolutely required for activin A induction of reporter activity in homologous cells. Additionally, mutations to the minimal SMAD-binding element adjacent to the proximal forkhead-binding element abrogate activin A or FOXL2/SMAD3 induction of reporter activity. In contrast, a mutation that impairs an adjacent PBX1/PREP1 (pre-B cell leukemia transcription factor 1-PBX/knotted-1 homeobox-1) binding site does not alter activin A-stimulated promoter activity in homologous cells. Collectively, these and previous data suggest a model in which activins stimulate formation of FOXL2-SMAD2/3/4 complexes, which bind to the proximal murine Fshb promoter to stimulate its transcription. Within these complexes, FOXL2 and SMAD3 or SMAD4 bind to adjacent cis-elements, with SMAD3 brokering the physical interaction with FOXL2. Because this composite response element is highly conserved, this suggests a general mechanism whereby activins may regulate and/or modulate Fshb transcription in mammals. PMID:21622537

  2. Tobacco/Nicotine and Endogenous Brain Opioids

    PubMed Central

    Xue, Yue; Domino, Edward F.

    2008-01-01

    Smoking is a major public health problem with devastating health consequences. Although many cigarette smokers are able to quit, equal numbers of others cannot! Standard medications to assist in smoking cessation, such as nicotine replacement therapies and bupropion, are ineffective in many remaining smokers. Recent developments in the neurobiology of nicotine dependence have identified several neurotransmitter systems that may contribute to the process of smoking maintenance and relapse. These include: especially dopamine, but also norepinephrine, 5-hydroxytryptamine, acetylcholine, endogenous opioids, gamma-aminobutyric acid (GABA), glutamate, and endocannabinoids. The present review examines the limited contribution of the endogenous opioid system to the complex effects of nicotine/tobacco smoking. PMID:18215788

  3. An endogenous model of the credit network

    NASA Astrophysics Data System (ADS)

    He, Jianmin; Sui, Xin; Li, Shouwei

    2016-01-01

    In this paper, an endogenous credit network model of firm-bank agents is constructed. The model describes the endogenous formation of firm-firm, firm-bank and bank-bank credit relationships. By means of simulations, the model is capable of showing some obvious similarities with empirical evidence found by other scholars: the upper-tail of firm size distribution can be well fitted with a power-law; the bank size distribution can be lognormally distributed with a power-law tail; the bank in-degrees of the interbank credit network as well as the firm-bank credit network fall into two-power-law distributions.

  4. Animal spirits, competitive markets, and endogenous growth

    NASA Astrophysics Data System (ADS)

    Miyazaki, Kenji

    2013-10-01

    This paper uses a simple model with an endogenous discount rate and linear technology to investigate whether a competitive equilibrium has a higher balanced growth path (BGP) than the social planning solution and whether the BGP is determinate or indeterminate. The implications are as follows. To start with, people with an instinct to compare themselves with others possess an endogenous discount rate. In turn, this instinct affects the economic growth rate in a competitive market economy. The competitive market economy also sometimes achieves higher economic growth than a social planning economy. However, the outcomes of market economy occasionally fluctuate because of the presence of the self-fulfilling prophecy or animal spirits.

  5. Murine Typhus, Reunion, France, 2011–2013

    PubMed Central

    Camuset, Guillaume; Socolovschi, Cristina; Moiton, Marie-Pierre; Kuli, Barbara; Foucher, Aurélie; Poubeau, Patrice; Borgherini, Gianandrea; Wartel, Guillaume; Audin, Héla; Raoult, Didier; Filleul, Laurent; Parola, Philippe; Pagès, Fréderic

    2015-01-01

    Murine typhus case was initially identified in Reunion, France, in 2012 in a tourist. Our investigation confirmed 8 autochthonous cases that occurred during January 2011–January 2013 in Reunion. Murine typhus should be considered in local patients and in travelers returning from Reunion who have fevers of unknown origin. PMID:25625653

  6. Characterization of mouse cellular deoxyribonucleic acid homologous to Abelson murine leukemia virus-specific sequences.

    PubMed Central

    Dale, B; Ozanne, B

    1981-01-01

    The genome of Abelson murine leukemia virus (A-MuLV) consists of sequences derived from both BALB/c mouse deoxyribonucleic acid and the genome of Moloney murine leukemia virus. Using deoxyribonucleic acid linear intermediates as a source of retroviral deoxyribonucleic acid, we isolated a recombinant plasmid which contained 1.9 kilobases of the 3.5-kilobase mouse-derived sequences found in A-MuLV (A-MuLV-specific sequences). We used this clone, designated pSA-17, as a probe restriction enzyme and Southern blot analyses to examine the arrangement of homologous sequences in BALB/c deoxyribonucleic acid (endogenous Abelson sequences). The endogenous Abelson sequences within the mouse genome were interrupted by noncoding regions, suggesting that a rearrangement of the cell sequences was required to produce the sequence found in the virus. Endogenous Abelson sequences were arranged similarly in mice that were susceptible to A-MuLV tumors and in mice that were resistant to A-MuLV tumors. An examination of three BALB/c plasmacytomas and a BALB/c early B-cell tumor likewise revealed no alteration in the arrangement of the endogenous Abelson sequences. Homology to pSA-17 was also observed in deoxyribonucleic acids prepared from rat, hamster, chicken, and human cells. An isolate of A-MuLV which encoded a 160,000-dalton transforming protein (P160) contained 700 more base pairs of mouse sequences than the standard A-MuLV isolate, which encoded a 120,000-dalton transforming protein (P120). Images PMID:9279386

  7. Mouse annexin V genomic organization includes an endogenous retrovirus.

    PubMed Central

    Rodriguez-Garcia, M I; Morgan, R O; Fernandez, M R; Bances, P; Fernandez, M P

    1999-01-01

    Mouse annexin V genomic clones were characterized by restriction analysis, Southern blotting and DNA sequencing. The entire gene spans close to 50 kb of the mouse genome and contains 14 exons ranging in size from 31 bp for exon 2 to 482 bp for exon 13 up to the polyadenylation site. Intron sizes range from 111 bp for intron 1b to more than 17 kb for intron 2. Non-coding exon 1 is present in two alternative forms separated by approx. 7.4 kb, and the two promoters associated with exons 1a and 1b are quite distinct. The upstream promoter has a TATA box and may direct the limited, tissue-specific expression of mRNA transcripts containing exon 1a. The downstream, TATA-less promoter has high G+C content, and exon 1b predominates among abundantly expressed mRNA species. The conservation of certain cis-elements, including Sp1, AP2, gamma-IRE and NF-IL6, in orthologous species of annexin V genes points to their possible role in trans-acting protein factor binding and gene regulation. Primer-extension analysis revealed multiple origins for transcription, with principal start sites 100-150 bp upstream of the ATG start codon in exon 2. Intron 4 was longer than that previously identified in the orthologous rat gene due to the integration of an apparently complete copy of the murine endogenous retrovirus element, MuERV-L. Phylogenetic analysis of annexin V from 12 species and the presence of neighbouring loci with paralogous counterparts linked to annexin VI pointed to the common ancestry of these genes via chromosomal duplication more than 600 million years ago. PMID:9854034

  8. Transplanted human bone marrow progenitor subtypes stimulate endogenous islet regeneration and revascularization.

    PubMed

    Bell, Gillian I; Broughton, Heather C; Levac, Krysta D; Allan, David A; Xenocostas, Anargyros; Hess, David A

    2012-01-01

    Transplanted murine bone marrow (BM) progenitor cells recruit to the injured pancreas and induce endogenous beta cell proliferation to improve islet function. To enrich for analogous human progenitor cell types that stimulate islet regeneration, we purified human BM based on high-aldehyde dehydrogenase activity (ALDH(hi)), an enzymatic function conserved in hematopoietic, endothelial, and mesenchymal progenitor lineages. We investigated the contributions of ALDH(hi) mixed progenitor cells or culture-expanded, ALDH-purified multipotent stromal cell (MSC) subsets to activate endogenous programs for islet regeneration after transplantation into streptozotocin-treated NOD/SCID mice. Intravenous injection of uncultured BM ALDH(hi) cells improved systemic hyperglycemia and augmented insulin secretion by increasing islet size and vascularization, without increasing total islet number. Augmented proliferation within regenerated endogenous islets and associated vascular endothelium indicated the induction of islet-specific proliferative and pro-angiogenic programs. Although cultured MSC from independent human BM samples showed variable capacity to improve islet function, and prolonged expansion diminished hyperglycemic recovery, transplantation of ALDH-purified regenerative MSC reduced hyperglycemia and augmented total beta cell mass by stimulating the formation of small beta cell clusters associated with the ductal epithelium, without evidence of increased islet vascularization or Ngn3(+) endocrine precursor activation. Thus, endogenous islet recovery after progenitor cell transplantation can occur via distinct regenerative mechanisms modulated by subtypes of progenitor cells administered. Further, understanding of how these islet regenerative and pro-angiogenic programs are activated by specific progenitor subsets may provide new approaches for combination cellular therapies to combat diabetes. PMID:21417581

  9. Feedback Response to Selective Depletion of Endogenous Carbon Monoxide in the Blood.

    PubMed

    Kitagishi, Hiroaki; Minegishi, Saika; Yumura, Aki; Negi, Shigeru; Taketani, Shigeru; Amagase, Yoko; Mizukawa, Yumiko; Urushidani, Tetsuro; Sugiura, Yukio; Kano, Koji

    2016-04-27

    The physiological roles of endogenous carbon monoxide (CO) have not been fully understood because of the difficulty in preparing a loss-of-function phenotype of this molecule. Here, we have utilized in vivo CO receptors, hemoCDs, which are the supramolecular 1:1 inclusion complexes of meso-tetrakis(4-sulfonatophenyl)porphinatoiron(II) with per-O-methylated β-cyclodextrin dimers. Three types of hemoCDs (hemoCD1, hemoCD2, and hemoCD3) that exhibit different CO-affinities have been tested as CO-depleting agents in vivo. Intraperitoneally administered hemoCD bound endogenous CO within the murine circulation, and was excreted in the urine along with CO in an affinity-dependent manner. The sufficient administration of hemoCD that has higher CO-affinity than hemoglobin (Hb) produced a pseudoknockdown state of CO in the mouse in which heme oxygenase-1 (HO-1) was markedly induced in the liver, causing the acceleration of endogenous CO production to maintain constant CO-Hb levels in the blood. The contents of free hemin and bilirubin in the blood plasma of the treated mice significantly increased upon removal of endogenous CO by hemoCD. Thus, a homeostatic feedback model for the CO/HO-1 system was proposed as follows: HemoCD primarily removes CO from cell-free CO-Hb. The resulting oxy-Hb is quickly oxidized to met-Hb by oxidant(s) such as hydrogen peroxide in the blood plasma. The met-Hb readily releases free hemin that directly induces HO-1 in the liver, which metabolizes the hemin into iron, biliverdin, and CO. The newly produced CO binds to ferrous Hb to form CO-Hb as an oxidation-resistant state. Overall, the present system revealed the regulatory role of CO for maintaining the ferrous/ferric balance of Hb in the blood. PMID:27057920

  10. Essays on Policy Evaluation with Endogenous Adoption

    ERIC Educational Resources Information Center

    Gentile, Elisabetta

    2011-01-01

    Over the last decade, experimental and quasi-experimental methods have been favored by researchers in empirical economics, as they provide unbiased causal estimates. However, when implementing a program, it is often not possible to randomly assign subjects to treatment, leading to a possible endogeneity bias. This dissertation consists of two…