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• W1769600480 abstract "Article15 October 2013Open Access Source Data Endonuclease G mediates α-synuclein cytotoxicity during Parkinson's disease Sabrina Büttner Sabrina Büttner Institute of Molecular Biosciences, University of Graz, Graz, Austria Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Lukas Habernig Lukas Habernig Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Filomena Broeskamp Filomena Broeskamp Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Doris Ruli Doris Ruli Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author F Nora Vögtle F Nora Vögtle Institut für Biochemie und Molekularbiologie, ZBMZ, University of Freiburg, Freiburg, Germany Search for more papers by this author Manolis Vlachos Manolis Vlachos Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece Search for more papers by this author Francesca Macchi Francesca Macchi Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium Search for more papers by this author Victoria Küttner Victoria Küttner Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany Search for more papers by this author Didac Carmona-Gutierrez Didac Carmona-Gutierrez Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Tobias Eisenberg Tobias Eisenberg Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Julia Ring Julia Ring Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Maria Markaki Maria Markaki Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece Search for more papers by this author Asli Aras Taskin Asli Aras Taskin Institut für Biochemie und Molekularbiologie, ZBMZ, University of Freiburg, Freiburg, Germany Faculty of Biology and Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Stefan Benke Stefan Benke Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Christoph Ruckenstuhl Christoph Ruckenstuhl Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Ralf Braun Ralf Braun Cell Biology, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Chris Van den Haute Chris Van den Haute Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium Search for more papers by this author Tine Bammens Tine Bammens Functional Biology, KU Leuven, Leuven, Belgium Search for more papers by this author Anke van der Perren Anke van der Perren Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium Search for more papers by this author Kai-Uwe Fröhlich Kai-Uwe Fröhlich Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Joris Winderickx Joris Winderickx Functional Biology, KU Leuven, Leuven, Belgium Search for more papers by this author Guido Kroemer Guido Kroemer INSERM, U848, Villejuif, France Metabolomics Platform, Institut Gustave Roussy, Villejuif, France Centre de Recherche des Cordeliers, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Veerle Baekelandt Veerle Baekelandt Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece Search for more papers by this author Gabor G Kovacs Gabor G Kovacs Institute of Neurology, Medical University of Vienna, Vienna, Austria Search for more papers by this author Jörn Dengjel Jörn Dengjel Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany Search for more papers by this author Chris Meisinger Chris Meisinger Institut für Biochemie und Molekularbiologie, ZBMZ, University of Freiburg, Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Stephan J Sigrist Corresponding Author Stephan J Sigrist Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Frank Madeo Corresponding Author Frank Madeo Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Sabrina Büttner Sabrina Büttner Institute of Molecular Biosciences, University of Graz, Graz, Austria Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Lukas Habernig Lukas Habernig Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Filomena Broeskamp Filomena Broeskamp Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Doris Ruli Doris Ruli Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author F Nora Vögtle F Nora Vögtle Institut für Biochemie und Molekularbiologie, ZBMZ, University of Freiburg, Freiburg, Germany Search for more papers by this author Manolis Vlachos Manolis Vlachos Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece Search for more papers by this author Francesca Macchi Francesca Macchi Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium Search for more papers by this author Victoria Küttner Victoria Küttner Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany Search for more papers by this author Didac Carmona-Gutierrez Didac Carmona-Gutierrez Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Tobias Eisenberg Tobias Eisenberg Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Julia Ring Julia Ring Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Maria Markaki Maria Markaki Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece Search for more papers by this author Asli Aras Taskin Asli Aras Taskin Institut für Biochemie und Molekularbiologie, ZBMZ, University of Freiburg, Freiburg, Germany Faculty of Biology and Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Stefan Benke Stefan Benke Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Christoph Ruckenstuhl Christoph Ruckenstuhl Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Ralf Braun Ralf Braun Cell Biology, University of Bayreuth, Bayreuth, Germany Search for more papers by this author Chris Van den Haute Chris Van den Haute Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium Search for more papers by this author Tine Bammens Tine Bammens Functional Biology, KU Leuven, Leuven, Belgium Search for more papers by this author Anke van der Perren Anke van der Perren Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium Search for more papers by this author Kai-Uwe Fröhlich Kai-Uwe Fröhlich Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Joris Winderickx Joris Winderickx Functional Biology, KU Leuven, Leuven, Belgium Search for more papers by this author Guido Kroemer Guido Kroemer INSERM, U848, Villejuif, France Metabolomics Platform, Institut Gustave Roussy, Villejuif, France Centre de Recherche des Cordeliers, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Veerle Baekelandt Veerle Baekelandt Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece Search for more papers by this author Gabor G Kovacs Gabor G Kovacs Institute of Neurology, Medical University of Vienna, Vienna, Austria Search for more papers by this author Jörn Dengjel Jörn Dengjel Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany Search for more papers by this author Chris Meisinger Chris Meisinger Institut für Biochemie und Molekularbiologie, ZBMZ, University of Freiburg, Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Stephan J Sigrist Corresponding Author Stephan J Sigrist Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Frank Madeo Corresponding Author Frank Madeo Institute of Molecular Biosciences, University of Graz, Graz, Austria Search for more papers by this author Author Information Sabrina Büttner1,2, Lukas Habernig1, Filomena Broeskamp2, Doris Ruli1, F Nora Vögtle3, Manolis Vlachos4, Francesca Macchi5, Victoria Küttner6, Didac Carmona-Gutierrez1, Tobias Eisenberg1, Julia Ring1, Maria Markaki4, Asli Aras Taskin3,7, Stefan Benke1, Christoph Ruckenstuhl1, Ralf Braun8, Chris Van den Haute5, Tine Bammens9, Anke van der Perren5, Kai-Uwe Fröhlich1, Joris Winderickx9, Guido Kroemer10,11,12,13,14, Veerle Baekelandt5, Nektarios Tavernarakis4, Gabor G Kovacs15, Jörn Dengjel6, Chris Meisinger3,16, Stephan J Sigrist 2 and Frank Madeo 1 1Institute of Molecular Biosciences, University of Graz, Graz, Austria 2Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany 3Institut für Biochemie und Molekularbiologie, ZBMZ, University of Freiburg, Freiburg, Germany 4Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece 5Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium 6Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany 7Faculty of Biology and Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany 8Cell Biology, University of Bayreuth, Bayreuth, Germany 9Functional Biology, KU Leuven, Leuven, Belgium 10INSERM, U848, Villejuif, France 11Metabolomics Platform, Institut Gustave Roussy, Villejuif, France 12Centre de Recherche des Cordeliers, Paris, France 13Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France 14Université Paris Descartes, Sorbonne Paris Cité, Paris, France 15Institute of Neurology, Medical University of Vienna, Vienna, Austria 16BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany *Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany. Tel.:+49 3083856940; Fax:+49 3083856938; E-mail: [email protected] or Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, Graz 8010, Austria. Tel.:+43 3163808878; Fax:+43 3163809898; E-mail: [email protected] The EMBO Journal (2013)32:3041-3054https://doi.org/10.1038/emboj.2013.228 There is a Have you seen? (November 2013) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity. Introduction The pathogenesis of Parkinson's disease (PD), one of the most prevalent neurodegenerative disorders, is influenced by a complex and largely elusive interplay between genetic and environmental factors. Death of dopaminergic neurons in the substantia nigra and accumulation of intracellular inclusions (Lewy bodies) constitute basic pathological features of PD. α-synuclein, a protein prominently expressed in the central nervous system, has been identified as the main component of the insoluble filaments forming Lewy bodies (Spillantini et al, 1997). In cultured human dopaminergic neurons, accumulation of α-synuclein may result in apoptosis mediated by reactive oxygen species (ROS) (Xu et al, 2002). Numerous studies using yeast, nematodes, flies, transgenic mice and cultured human cells indicate that α-synuclein functions in lipid metabolism and vesicular trafficking (Moore et al, 2005; Cooper et al, 2006). Additional evidence implicates mitochondrial (dys)function as a crucial factor in the pathogenesis of PD in general and α-synuclein toxicity in particular (Moore et al, 2005). In fact, several environmental toxins like rotenone or paraquat can accelerate the development of PD by interfering with mitochondrial function (Uversky, 2007). Furthermore, several proteins associated with familial PD, including parkin, DJ-1 and PINK1, are functionally linked to mitochondria (Moore et al, 2005; Abou-Sleiman et al, 2006). We previously reported that the death of yeast cells induced by expression of human α-synuclein strictly depends on functional, respiring mitochondria (Büttner et al, 2008). Here, we identify the mitochondrial pro-apoptotic nuclease endonuclease G (EndoG) as a crucial determinant of α-synuclein-inflicted cellular demise in yeast, nematodes, flies and neuroblastoma cells and show nuclear translocation of EndoG in dopaminergic neurons of PD-patient brain tissue samples. Results Yeast EndoG mediates α-synuclein-induced cell death α-Synuclein has been demonstrated to physically interact with mitochondrial membranes, thereby interfering with mitochondrial function (Li et al, 2007; Cole et al, 2008; Chinta et al, 2010). These studies directly link the toxic consequences of α-synuclein to organelles that are pivotal determinants in cell death execution and constitute the major source of cellular ROS (Zamzami and Kroemer, 2001). Thus, we hypothesized that one or several mitochondrial factors may constitute the executor of α-synuclein-triggered neuronal cell death. To further explore the connection between α-synuclein and mitochondria, we took advantage of budding yeast (Saccharomyces cerevisiae), which is amenable to mitochondrial manipulation (Madeo et al, 1999). Consistent with data obtained in higher model systems, we found that a small portion of α-synuclein localized to purified mitochondria of yeast cells heterologously expressing human α-synuclein (Figure 1A). Immunoblotting using an antibody directed against the cytosolic 3-phosphoglycerat kinase Pgk1p excluded cytosolic contamination of the mitochondrial fractions (Supplementary Figure S1A). Mitochondrially located α-synuclein was lost upon proteinase K digest, indicating attachment to the outer mitochondrial membrane (Figure 1A). Automated quantification of ROS production was used to determine the precise contribution of known mitochondrial cell death mediators to α-synuclein cytotoxicity (Supplementary Figure S1B). Deletion of several genes involved in the regulation of mitochondrial dynamics, mitophagy and mitochondrial phospholipid metabolism (Supplementary Figure S1B) or deletion of the mitochondrial apoptosis-inducing factor AIF1 (Büttner et al, 2008) had no effect on α-synuclein-induced cell killing. Instead, deletion of yeast EndoG (NUC1) strongly suppressed α-synuclein-induced ROS overproduction and death (Figure 1B and C; Supplementary Figure S1B). Re-introduction of Nuc1p into Δnuc1 cells could re-install α-synuclein toxicity, while a point mutation within the active nuclease site of Nuc1p partly inhibited this complementation (Supplementary Figure S2). Although the pro-apoptotic mitochondrial nuclease EndoG has been associated with cellular degeneration in age-dependent muscle atrophy (Leeuwenburgh et al, 2005) and cerebral ischemia (Lee et al, 2005), thus far no links between EndoG and PD have been described. Figure 1.EndoG mediates yeast cell death upon α-synuclein expression. (A) Immunoblot analysis of mitochondria isolated from wild-type yeast cells expressing human α-synuclein (αSyn) or harbouring respective vector control (Ctrl.). Purified mitochondria were subjected to proteinase K (Prot. K) digest as indicated. Blots were probed with antibodies directed against FLAG epitope to detect FLAG-tagged αSyn, the NADH-cytochrome b5 reductase Mcr1p as a marker of the outer and inner mitochondrial membrane, and the mitochondrial matrix chaperone Mge1. (B, C) Clonogenic survival (B) and ROS production measured by assessing the ROS-driven conversion of dihydroethidium into ethidium, DHE→Eth (C) of wild-type (WT) and EndoG-deficient (Δnuc1) yeast cells upon galactose-induced expression of αSyn for 24 h (ROS production) or 36 h (survival). Means±s.e.m., n=8. ***P<0.001. (D) Nuclear translocation of Nuc1pFLAG in cells overexpressing Nuc1pFLAG with or without co-expression of αSyn under the control of a galactose promoter for 24 h quantified using immunoblot analysis. The Nuc1pFLAG signal was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in whole protein extracts and to histone H3 in isolated nuclei. The ratio of Nuc1pFLAG in cells expressing αSyn and cells harbouring the respective vector control was plotted. Representative blots are shown in Supplementary Figure S3. Means±s.e.m., n=6. *P<0.01. (E, F) Flow cytometric quantification of DNA fragmentation (E) and representative micrographs (F) using TUNEL staining of WT and Δnuc1 cells upon galactose-induced expression of αSyn for 36 h. Means±s.e.m., n=6. ***P<0.001. Scale bar represents 10 μm. (G) Immunoblot analysis of αSyn expression in wild-type and Δnuc1 cells using antibodies directed against FLAG epitope to detect FLAG-tagged α-synuclein and against GAPDH as a loading control. (H, I) Quantification of ROS production (DHE→Eth) (H) and clonogenic survival (I) of yeast cells expressing yeast EndoG (Nuc1p) under a galactose-inducible promoter or human α-synuclein under a methionine-repressible promoter or co-expressing both proteins. Of note, the growth conditions and expression vectors used here differ from those used in (B, C and E). Mid-exponential cells grown on glucose media were shifted to media containing 0.5% galactose and 1.5% glucose (instead of 1.5% galactose and 0.5% glucose) and subjected to determination of ROS production and survival after 24 or 36 h of growth, respectively. Expression vectors and growth conditions in which both proteins alone showed minor toxicity were applied (low galactose concentrations and no complete methionine depletion for low-level expression of Nuc1p and αSyn). Means±s.e.m., n=9–12. ***P<0.001. Download figure Download PowerPoint Upon cell death induction, EndoG can translocate from mitochondria (its normal location) to the nucleus, where it mediates DNA fragmentation and eventually cell death (Li et al, 2001; Parrish et al, 2001, 2003; Büttner et al, 2007). Immunoblot analyses of subcellular fractions revealed that α-synuclein induced the mitochondria-nuclear translocation of the yeast orthologue of EndoG, Nuc1p (Figure 1D; Supplementary Figure S3A and B). Deletion of yeast EndoG significantly reduced nuclear DNA fragmentation induced by α-synuclein (Figure 1E and F). Expression levels of α-synuclein were unaffected by the absence of NUC1 (Figure 1G). Furthermore, enhanced levels of yeast EndoG due to overexpression exacerbated α-synuclein-driven ROS production and cell death (Figure 1H and I). Of note, in contrast to the experimental set-up used in Figure 1B and C, where high level expression of α-synuclein is driven by a galactose promoter, we applied expression vectors and growth conditions in which both proteins alone exhibited none or minor toxicity to visualize synergistic effects of Nuc1p and α-synuclein. The pathway for α-synuclein-mediated cytotoxicity involves modulators of the permeability transition pore and the karyopherin Kap123p Yeast EndoG physically interacts with proteins that have been suggested to modulate the activity of the mitochondrial permeability transition pore (PTP) and depends on the adenine nucleotide translocator (ANT) to execute death (Büttner et al, 2007). Consistently, we identified regulators of the PTP as facilitators of α-synuclein cytotoxicity while analysing the contribution of various mitochondrial proteins to α-synuclein-mediated ROS production as described above (Supplementary Figure S1B). Yeast cells that lack the voltage-dependent anion channel (POR1), cyclophilin D (CPR3) or the three isoforms of the adenine-nucleotide translocator (AAC1/2/3, only viable in a W303 background) were all protected against α-synuclein-induced death and ROS production, although none of these yeast mutants affected the expression level of α-synuclein (Figure 2A–C; for unnormalized, absolute values of colony-forming units (CFUs), please see Supplementary Figure S4). In addition, yeast cells devoid of the karyopherin Kap123p, which functions in nuclear protein import and interacts with yeast EndoG, were not only protected against EndoG-mediated cell death (Büttner et al, 2007) but against α-synuclein cytotoxicity as well (Figure 2D and E). The absence of Kap123p largely inhibited α-synuclein-facilitated ROS overproduction (Figure 2E) and cell killing (Figure 2D), but did not compromise α-synuclein expression (Figure 2F). Figure 2.α-Synuclein cytotoxicity involves components of the permeability transition pore and the karyopherin Kap123p. (A, B) Clonogenic survival (A) and ROS production (DHE→Eth) (B) upon galactose-induced expression of αSyn in indicated deletion mutants and isogenic wild-type (WT) yeast cells for 24 h (DHE) and 36 h (survival). Means±s.e.m., n=8. ***P<0.001; **P<0.01; *P<0.05 compared to αSyn toxicity in the corresponding wild-type cells. Survival was normalized to isogenic vector control cells. For absolute values (colony-forming units), see Supplementary Figure S4. (C) Immunoblot analysis of αSyn expression in indicated deletion mutants and the corresponding wild-type cells using antibodies directed against FLAG epitope to detect FLAG-tagged αSyn and against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. (D, E) Survival determined by clonogenicity (D) and quantification of ROS production (DHE→Eth) (E) of wild-type (WT; BY4741) and Δkap123 cells upon galactose-induced expression of αSyn for 24 h (ROS) or 36 h (survival), and the corresponding vector control cells. Means±s.e.m., n=8. ***P<0.001; *P<0.05. (F) Immunoblot analysis of αSyn expression in wild-type and Δkap123 cells using antibodies directed against FLAG epitope to detect FLAG-tagged αSyn and against GAPDH as a loading control. Download figure Download PowerPoint To test whether the amelioration of α-synuclein toxicity observed in deletion mutants of the PTP modulators occurs with a concomitant reduction in apoptotic EndoG release from mitochondria, we generated strains harbouring an endogeneously HA-tagged NUC1 version (Nuc1pHA). Isolation of mitochondria and subsequent immunoblot analysis of Nuc1pHA content demonstrated that the deletion of POR1 and of all three isoforms of AAC prominently inhibited α-synuclein-triggered mitochondrial release of EndoG (Figure 3A–F). While the overall expression of Nuc1pHA analysed by immunoblotting of total cell extracts seemed largely unaffected by α-synuclein, the deletion of the AAC genes (and to a lesser extent the deletion of POR1) provoked an increase in total cellular Nuc1pHA content (Figure 3C and F). Deletion of CPR3 did not affect the release of EndoG but instead caused a decrease in mitochondrial as well as total cellular Nuc1pHA levels per se (Figure 3D–F). Figure 3.α-Synuclein-induced mitochondrial release of EndoG requires regulators of the PTP. (A–C) Densitometric quantification (A) of the mitochondrial Nuc1pHA signal via immunoblot analysis and representative blots of isolated mitochondria (B) and total cell extracts (C) from wild-type cells and Δpor1 cells harbouring endogeneously HA-tagged NUC1 upon expression of αSyn for 36 h. Blots were probed with antibodies directed against HA epitope to detect Nuc1pHA, against Por1p, against the ADP/ATP translocator Aac1p as a mitochondrial loading control and against GAPDH. Mitochondrial Nuc1pHA signals were normalized to Aac1p, and this normalized mitochondrial Nuc1pHA content is shown as a ratio of αSyn and control cells. Means±s.e.m., n=8. **P<0.001. (D–F) Densitometric quantification (D) of the mitochondrial Nuc1pHA signal via immunoblot analysis of mitochondria isolated from W303 wild-type cells, Δcpr3 cells and cells deleted in all three isoforms of AAC (Δaac123) harbouring endogeneously HA-tagged NUC1. Representative blots of isolated mitochondria (E) and of total cell extract (F) are shown. Blots were probed with antibodies directed against HA epitope to detect Nuc1pHA, against Por1p as a mitochondrial loading control and against GAPDH. Means±s.e.m., n=4–8. *P<0.05.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3 [embj2013228-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint α-Synuclein deregulates specific proteins at the mitochondrial outer membrane To further elucidate the mechanism underlying α-synuclein-induced mitochondrial cell death in general and the release of EndoG in particular, we searched for α-synuclein-triggered alterations within the mitochondrial protein composition using different approaches. Examining mitochondria isolated from cells expressing α-synuclein or harbouring respective vector control on SDS–PAGE followed by immunodecoration with antibodies directed against various mitochondrial proteins, we could not detect any changes in the levels of typical proteins located within the mitochondrial matrix, the intermembrane space, or the inner mitochondrial membrane (a selection of 30 proteins analysed via immunodecoration is shown in Supplementary Figure S5A). In addition, no alterations regarding the assembly of the supramolecular structures formed by respiratory complexes III and IV (Schägger and Pfeiffer, 2000) could by detected using blue native gel electrophoresis (BN-PAGE) (Supplementary Figure S5B). However, a protein found at the outer mitochondrial membrane, namely the cell integrity pathway protein Zeo1p, was specifically downregulated upon α-synuclein expression, while the levels of additional outer mitochondrial membrane proteins such as components of the TOM (translocase of the outer membrane) complex were unaffected (Figure 4A). Performing mass spectrometry-based quantitative proteomics using a ‘stable isotope labelling by amino acids in cell culture’ (SILAC) approach to analyse mitochondrial fractions for proteomic changes triggered by α-synuclein, we identified several proteins to be deregulated upon expression of α-synuclein (Supplementary Table S1), among them again Zeo1p. Interestingly, while all of the identified proteins have been demonstrated to co-purify with mitochondria, the majority is not exclusively mitochondrial but has been shown to display cytosolic and/or plasma membrane localization, as well (Huh et al, 2003; Sickmann et al, 2003; Brandina et al, 2006; Reinders et al, 2007). Thus, the expression of α-synuclein and its attachment to the outer mitochondrial membrane might influence the association of several proteins to mitochondria, thereby triggering alterations that subsequently allow PTP-dependent release of EndoG. To test whether one or several of the proteins identified to be deregulated upon α-synuclein expression is causally involved in its cytotoxicity, we quantified α-synuclein-driven ROS production in respective deletion mutants if viable (selected candidates are presented in Figure 4B; complete results are shown in Supplementary Table S1 and Supplementary Figure S6). While t" @default.
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• W1769600480 title "Endonuclease G mediates α-synuclein cytotoxicity during Parkinson's disease" @default.
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• W1769600480 doi "https://doi.org/10.1038/emboj.2013.228" @default.
• W1769600480 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3844953" @default.
• W1769600480 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/24129513" @default.
• W1769600480 hasPublicationYear "2013" @default.

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