Abstract

Article14 September 2010free access Inhibition of mitochondrial fusion by α-synuclein is rescued by PINK1, Parkin and DJ-1 Frits Kamp Corresponding Author Frits Kamp DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Nicole Exner Nicole Exner DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Anne Kathrin Lutz Anne Kathrin Lutz Adolf-Butenandt-Institute, Neurobiochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Nora Wender Nora Wender European Neuroscience Institute Goettingen and DFG Research Center for Molecular Physiology of the Brain (CMPB), Goettingen, Germany Search for more papers by this author Jan Hegermann Jan Hegermann European Neuroscience Institute Goettingen and DFG Research Center for Molecular Physiology of the Brain (CMPB), Goettingen, Germany Search for more papers by this author Bettina Brunner Bettina Brunner DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Brigitte Nuscher Brigitte Nuscher DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Tim Bartels Tim Bartels Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Armin Giese Armin Giese Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Klaus Beyer Klaus Beyer Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Department of Chemistry, The University of Arizona, Tucson, AZ, USA Search for more papers by this author Stefan Eimer Stefan Eimer European Neuroscience Institute Goettingen and DFG Research Center for Molecular Physiology of the Brain (CMPB), Goettingen, Germany Search for more papers by this author Konstanze F Winklhofer Konstanze F Winklhofer Adolf-Butenandt-Institute, Neurobiochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Christian Haass Corresponding Author Christian Haass DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Frits Kamp Corresponding Author Frits Kamp DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Nicole Exner Nicole Exner DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Anne Kathrin Lutz Anne Kathrin Lutz Adolf-Butenandt-Institute, Neurobiochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Nora Wender Nora Wender European Neuroscience Institute Goettingen and DFG Research Center for Molecular Physiology of the Brain (CMPB), Goettingen, Germany Search for more papers by this author Jan Hegermann Jan Hegermann European Neuroscience Institute Goettingen and DFG Research Center for Molecular Physiology of the Brain (CMPB), Goettingen, Germany Search for more papers by this author Bettina Brunner Bettina Brunner DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Brigitte Nuscher Brigitte Nuscher DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Tim Bartels Tim Bartels Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Armin Giese Armin Giese Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Klaus Beyer Klaus Beyer Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Department of Chemistry, The University of Arizona, Tucson, AZ, USA Search for more papers by this author Stefan Eimer Stefan Eimer European Neuroscience Institute Goettingen and DFG Research Center for Molecular Physiology of the Brain (CMPB), Goettingen, Germany Search for more papers by this author Konstanze F Winklhofer Konstanze F Winklhofer Adolf-Butenandt-Institute, Neurobiochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Christian Haass Corresponding Author Christian Haass DZNE-German Center for Neurodegenerative Diseases, Munich, Germany Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Author Information Frits Kamp 1,2,‡, Nicole Exner1,2,‡, Anne Kathrin Lutz3,‡, Nora Wender4, Jan Hegermann4, Bettina Brunner1,2, Brigitte Nuscher1,2, Tim Bartels2, Armin Giese5, Klaus Beyer2,6, Stefan Eimer4, Konstanze F Winklhofer3 and Christian Haass 1,2 1DZNE-German Center for Neurodegenerative Diseases, Munich, Germany 2Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany 3Adolf-Butenandt-Institute, Neurobiochemistry, Ludwig-Maximilians-University, Munich, Germany 4European Neuroscience Institute Goettingen and DFG Research Center for Molecular Physiology of the Brain (CMPB), Goettingen, Germany 5Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany 6Department of Chemistry, The University of Arizona, Tucson, AZ, USA ‡These authors contributed equally to this work *Corresponding authors. DZNE-German Center for Neurodegenerative Diseases, Adolf Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Schillerstrasse 44, 80336, Munich, Germany. Tel.: +49 89 2180 75472; Fax: +49 89 2180 75415; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2010)29:3571-3589https://doi.org/10.1038/emboj.2010.223 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 Aggregation of α-synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA-mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin Δ1–79 or by DJ-1 C106A. Introduction A characteristic feature of Parkinson's disease (PD) is the intracellular deposition of Lewy bodies, which are predominantly composed of α-synuclein (αS). This 140 amino acid protein is widely distributed throughout the brain and expressed at high levels in neurons where it can reach concentrations of 0.5–1% of total protein (i.e. 30–60 μM) (Iwai et al, 1995; Spillantini et al, 1997; Bodner et al, 2009). In Lewy bodies, αS is arranged in fibrils with a β-sheet like structure (Der-Sarkissian et al, 2003; Chen et al, 2007). It is assumed that the pathogenicity of αS is associated with aggregation of the protein, which involves formation of small neurotoxic oligomers that eventually mature to larger insoluble deposits (Lee et al, 2004a; Haass and Selkoe, 2007; Kramer and Schulz-Schaeffer, 2007; Kostka et al, 2008; Kayed et al, 2009). A similar cascade of protein aggregation and precipitation is causative for the onset of other neurodegenerative diseases, such as Alzheimer's disease (Dobson, 2003; Haass and Selkoe, 2007). A remarkable property of αS is its structural flexibility (Davidson et al, 1998; Beyer, 2007; Uversky, 2007). The protein is essentially unstructured in dilute aqueous solution (Uversky, 2002), whereas α-helical folding occurs upon binding to lipid surfaces. The NMR-derived structure of SDS-micelle-bound αS revealed two anti-parallel aligned amphipathic α-helices, the ‘N-helix’ spanning residues 3 through 37 and the ‘C-helix’ spanning residues 45 through 92. The C-terminal domain, which contains approximately 40 amino acids of which 14 are negatively charged and 2 positively charged, remains unstructured (Lee et al, 2004b; Ulmer and Bax, 2005; Ulmer et al, 2005). The structure of membrane-bound αS cannot be resolved by NMR as the rotation of vesicles is too slow. However, other biophysical techniques including electron spin resonance and circular dichroism (CD) revealed that α-helical folding also occurs for the N-terminal region when αS binds to membranes (Nuscher et al, 2004; Beyer, 2007; Jao et al, 2008; Drescher et al, 2008a). However, whether membrane-bound αS assumes a single extended α-helix, a broken helix or multiple structures (including oligomers) is unclear (Drescher et al, 2008a, 2009b; Bodner et al, 2009; Ferreon et al, 2009; Perlmutter et al, 2009; Trexler and Rhoades, 2009). It has also been reported that αS binds to synaptic vesicles (Maroteaux et al, 1988; Jensen et al, 1998; Abeliovich et al, 2000; Kahle et al, 2000; Murphy et al, 2000; Cabin et al, 2002; Chandra et al, 2004, 2005; Jo et al, 2004; Yavich et al, 2004; Larsen et al, 2006; Ben Gedalya et al, 2009) as well as to mitochondria (Martin et al, 2006; Nakamura et al, 2008; Shavali et al, 2008). Biophysical studies from our laboratory revealed that binding of αS to highly curved bilayers leads to a stabilization of defects in the lipid packing (Nuscher et al, 2004; Cornell and Taneva, 2006; Kamp and Beyer, 2006). This motivated us to investigate whether αS could have an impact on membrane fusion. So far little is known about the biological consequences of binding of αS to intracellular membranes. Studies in yeast revealed that overexpression of αS leads to cellular toxicity by interfering with vesicular transport between the endoplasmic reticulum and the Golgi complex (Cooper et al, 2006). Fragmentation of the Golgi apparatus was also reported in neurons containing Pale bodies, pathological deposits known as early stages of Lewy bodies (Gosavi et al, 2002; Fujita et al, 2006; Lee et al, 2006). Moreover, functional impairment of mitochondria was caused by expression of wild type or mutant αS (Hsu et al, 2000; Orth et al, 2003; Smith et al, 2005; Parihar et al, 2008, 2009). Although one of the well-described biochemical properties of αS is membrane binding associated with a structural switch, the biological function of the membrane-associated variant is unclear. Here, we demonstrate for the first time that αS inhibits fusion of model membranes. Biophysical studies led us to investigate the consequences of enhanced αS levels on membrane fusion in vivo. Life imaging in cultured cells and Caenorhabditis elegans demonstrates that expression of αS induces mitochondrial fragmentation, whereas downregulation of αS leads to elongation of mitochondria. Strikingly, the mitochondrial phenotype caused by expression of αS could be rescued by coexpression of three recessive PD-associated genes, PINK1, parkin and DJ-1, but not the corresponding familial PD-associated mutants PINK1 G309D, and parkin Δ1–79 or by the synthetic mutant DJ-1 C106A (Waak et al, 2009). Results αS inhibits membrane fusion in vitro We tested the effect of αS in several ‘classic’ fusion assays using protein-free model membranes. In our first protocol, we used small unilamellar vesicles (SUVs) consisting of dipalmitoyl-phosphatidylcholine (DPPC), which are known to fuse below the chain-melting temperature Tm, increasing their diameter from 30 to 70 nm (Schullery et al, 1980a, 1980b; Gaber and Sheridan, 1982). This spontaneous fusion is a very slow process (Supplementary Figure S1). Trace amounts of non-ionic detergent C12E8 accelerate the fusion of DPPC-SUV, particularly at temperatures just below Tm (the gel to liquid–crystalline phase transition of bilayers of DPPC occurs at Tm=41°C). We measured vesicle fusion by following changes in the static light scattering. At 36°C a suspension of DPPC-SUV reached maximal light scattering values within 10 min after detergent addition (Figure 1A). Fusion was suppressed when the experiment was performed in the presence of increasing amounts of αS and was blocked completely at lipid/αS ratios ⩽200 mole/mole (3 μM αS), that is at concentrations where αS binding to vesicles saturates (Nuscher et al, 2004). We also applied dynamic light scattering (DLS) experiments, which demonstrated the increase in diameter of fusing vesicles (Supplementary Figure S2). To confirm that the increase in light scattering of fusing vesicles was not an aggregation artifact, we used two fluorescent membrane fusion assays. In a lipid-mixing assay, NBD fluorescence of ‘donor’ vesicles was completely quenched prior to addition of detergent. Upon fusion of donor vesicles and vesicles without fluorescent probes, lipid mixing abolishes the quenching effect. Membrane fusion, monitored by this technique could be reduced by increasing amounts of αS (Figure 1B). Alternatively, in a contents-mixing assay we repeated the C12E8-induced fusion of DPPC-SUV by mixing equal amounts of vesicles with trapped Tb3+-citrate with vesicles containing dipicholinic acid (DPA). Formation of the Tb3+–DPA complex led to a strong increase in fluorescence (Figure 1C). Almost no fluorescence increase was observed after addition of αS at a lipid/protein molar ratio 200:1, indicating that fusion was effectively inhibited. Together, these independent experiments support the inhibition of membrane fusion by αS. Figure 1.αS inhibits membrane fusion in vitro. (A) Fusion of DPPC-SUV was monitored by the increase in static light scattering upon addition of an aliquot of C12E8. Increasing amounts of αS inhibit membrane fusion (blue lines). Lipid concentration 600 μM, T=36°C. (B) Lipid-mixing assay of DPPC-SUV. αS inhibited fusion completely at lipid/αS=200 mole/mole (purple line). T=35°C. Pink line: no fusion occurred at 45°C, that is at temperature above Tm. (C) Contents-mixing assay carried out in the presence and absence of αS (lipid/αS=200 mole/mole, green line). T=30°C. (D) An N-terminal fragment mutant of αS, αS(1–116) (blue line), lacking the negatively charged C-terminal domain was capable of completely suppressing the fusion of DPPC-SUV, like wt-αS (dark blue line); whereas peptides comprised of the C-terminal fragment, αS(116–140), or the central domain of αS, αS(41–65), failed to inhibit fusion (blue lines). Lipid/protein=100 mole/mole. T=25°C. (E) Comparison of inhibition of membrane fusion by αS with cytochrome c, lysozyme and Apolipoprotein A-I (ApoA-I). For all proteins: lipid/protein=200 mole/mole. T=36°C. (F) Ca2+-induced fusion of POPS-SUV. Fusion was initiated by adding an aliquot of CaCl2 and monitored by the lipid-mixing assay. αS, added 2 min after the addition of Ca2+ (arrow), blocked fusion almost completely (lipid/αS=200 mole/mole). Control experiments: cytochrome c (lipid/protein=20 mole/mole) or poly-lysine (lipid/protein 200 mole/mole) was added instead of αS. T=25°C. (G) SUV of a mixture of lipids with reported optimal fusion potential (DOPC/DOPE/BBSM/cholesterol, 35:30:15:20 molar ratio) (Haque et al, 2001). Fusion was initiated by addition of 4% PEG and followed using the lipid-mixing assay. Total lipid concentration was 300 μM. When the experiment was repeated with αS, fusion was slowed. T=37°C. (H) Spontaneous rapid fusion of SUV composed of lipids with opposite charges (POPS-SUV and PC+-SUV). Lipid-mixing assay performed in stop-flow fluorimetry. Lipid concentration was 60 μM. αS (1.2 μM) inhibited the fusion. When POPS was replaced by POPC (uncharged) no fusion occurred, as expected. T=25°C. Download figure Download PowerPoint Considering the domain structure of lipid-bound αS, the question arises whether its anti-fusogenic behaviour is due to stabilization of packing defects in the bilayer (Kamp and Beyer, 2006), or rather to membrane repulsion caused by the negatively charged C-terminal domain. To distinguish between these two possibilities, the fusion assay was repeated using an αS mutant lacking the last 24 amino acids of the C-terminus. This fragment (αS1–116) was still capable of completely suppressing membrane fusion (Figure 1D). On the contrary, a peptide composed of 25 amino acids of the C-terminus of αS (αS116–140), as well as a peptide comprised of 25 amino acids of the centre region of αS (αS41–65), was not capable of inhibiting membrane fusion (Figure 1D). We also compared the anti-fusogenic effect of αS with other membrane-binding proteins. Cytochrome c and lysozyme have similar molecular weights as αS. Both are globular proteins with a net positive charge known to bind to membrane surfaces. Cytochrome c and lysozyme did not significantly slow down the fusion of DPPC-SUV (Figure 1E). Exchangeable apolipoproteins have structural similarities to αS and share stabilization of lipid packing because of the binding of a ‘sided’ helix to the lipid surface (Derksen et al, 1996; Nuscher et al, 2004; Cornell and Taneva, 2006; Beyer, 2007). Interestingly, apolipoprotein A-I (ApoA-I) blocked the fusion completely, just like αS (Figure 1E). These findings indicate that the folding and membrane interaction of the N-terminal domain rather than the negative charges of the C-terminal domain are responsible for the anti-fusogenic effect of αS. To test whether the effect of αS also applies to vesicles composed of other lipids, we investigated fusion of vesicles composed of negatively charged palmitoyl-oleoyl-phosphatidylserine (POPS), triggered by Ca2+-ions (Wilschut et al, 1980). Fusion was initiated by adding CaCl2 and was complete within 10 min after the addition (Figure 1F). When αS was added after the addition of Ca2+, the fusion rate was reduced >10 times. Again, we compared the anti-fusogenic effect of αS with other membrane-binding proteins. In this case, we used cytochrome c and poly-lysine. Poly-lysine, like cytochrome c, is expected to bind to negatively charged membranes (Zhang and Rowe, 1994). Cytochome c had no effect even at a 10-fold higher molar concentration than αS. Poly-lysine reduced the fusion rate about five times (Figure 1F). Having established the suppression of membrane fusion by αS in classic fusion assays of vesicles composed of only one kind of lipid, we wondered whether αS would also suppress fusion of membranes of lipid mixtures mimicking compositions of biological membranes. The effect of αS on polyethyleneglycol (PEG)-mediated fusion of vesicles composed of a lipid mixture with reported optimal fusion potential (Haque et al, 2001) is shown in Figure 1G. The suppression of fusion by αS was significant, although higher amounts of αS were required compared with the DPPC-SUV and POPS-SUV, probably because of a lower affinity of αS to the membranes comprised of the chosen lipid mixture. Finally, rapid spontaneous fusion can be achieved upon mixing of vesicles with opposite interfacial net charges (Pantazatos and MacDonald, 1999; Lei and MacDonald, 2003). In this assay, fusion was complete after about 3 sec (Figure 1H). Again fusion was reduced by αS. Taken together, these findings demonstrate that αS selectively blocks membrane fusion in a number of independent in vitro fusion assay systems. αS impairs mitochondrial fusion in cultured cells Mitochondria change their morphology because of continuous fusion and fission (Detmer and Chan, 2007; Westermann, 2008). In addition, mitochondrial morphology and function is affected by loss of parkin or PINK1 function, which are both associated with familial PD (Kitada et al, 1998; Valente et al, 2004; Exner et al, 2007; Dagda et al, 2009; Lutz et al, 2009; Morais et al, 2009; Sandebring et al, 2009). To prove whether enhanced levels of αS, as observed in sporadic PD (Sharon et al, 2003; Chiba-Falek et al, 2006; Grundemann et al, 2008) as well as in familial PD associated with a triplication of the αS gene (Singleton et al, 2003), influence the balance between mitochondrial fission and fusion, we overexpressed αS in neuronal SH-SY5Y cells. This was particularly interesting as αS has been reported to bind to intracellular membranes including mitochondria (Nakamura et al, 2008; Shavali et al, 2008). Changes in mitochondrial morphology were monitored by imaging of cells, transfected with mito-GFP. When wild-type αS was overexpressed in SH-SY5Y cells, increased mitochondrial fragmentation was observed (Figure 2A). Quantification of the relative amounts of cells with fragmented mitochondria revealed that upon overexpression of αS, the number of cells that display fragmented mitochondria increased from 34% under control conditions to 46% (Figure 2B). Expression of similar amounts of mutant αS-A30P or A53T led to fragmentation of mitochondria to the same extent as the wild-type protein (Figures 2A–C). This is consistent with the finding that mutants of αS also bind to model membranes (Nuscher et al, 2004; Ramakrishnan et al, 2006; Giannakis et al, 2008; Karpinar et al, 2009; Perlmutter et al, 2009). β-Synuclein (βS) shares a number of biological and biophysical properties with αS, including binding to lipid surfaces (Nuscher et al, 2004; Beyer, 2007). We therefore investigated if βS may also affect mitochondrial fusion/fission. Indeed, both orthologs lead to the formation of fragmented mitochondria (Figures 2D–F), suggesting a redundant function of αS and βS. Figure 2.Mitochondrial fragmentation imaged in SH-SY5Y cells expressing αS. (A) Images of fluorescently labelled mitochondria. The panels display representative individual cells either control transfected (co) or transfected with wild-type αS (αS-wt), αS A30P or αS A53T. Scale bars=10 μm. (B) Statistical analyses of mitochondrial morphology of cells from the experiments shown in (A). Approximately 250 cells of each experiment were counted, and the relative amount of transfected cells with altered mitochondrial morphology (i.e. fragmentation) was determined. (C) Expression levels of αS were analysed by western blotting using β-actin as loading control (v, vector). (D) Images of fluorescently labelled mitochondria. The panels display representative individual cells either untransfected (co) or transfected with αS-V5 (αS) or β-synuclein-V5 (βS). Scale bars=10 μm. (E) Statistical analyses of mitochondrial morphology of cells from the experiments shown in (D). Approximately 300 cells of each experiment were counted, and the relative amount of transfected cells with altered mitochondrial morphology (i.e. fragmentation) was determined. Error bars indicate s.d. (F) Expression levels of αS and βS were analysed by western blotting with a V5-antibody using β-actin as loading control. *P⩽0.05, **P⩽0.01. Download figure Download PowerPoint To further address the question whether the increase in mitochondrial fragmentation observed in αS-expressing SH-SY5Y cells is due to alterations in mitochondrial fusion, we performed a PEG fusion assay (Niemann et al, 2005; Malka et al, 2007) (Figure 3). A first set of cells was transiently cotransfected with mito-GFP and αS or empty vector as a control. Another set of cells was cotransfected with mito-DsRed and αS or vector. At 8 h after transfection, both sets of cells were mixed and plated on coverslips. After 16 h, fusion of cocultured cells was induced by a 90-s treatment with PEG and fused cells were further incubated in the presence of cycloheximide for 5 h. Mitochondria of fused cells were analysed by confocal microscopy. Mitochondrial fusion is indicated by extensive colocalization of mito-GFP and mito-DsRed in control cells (Figures 3A and B). In contrast, upon overexpression of αS colocalization was dramatically reduced demonstrating that αS blocks mitochondrial fusion. Figure 3.αS decreases fusion of differentially labelled mitochondrial populations. Cells expressing mito-GFP or mito-DsRed were fused with PEG in the presence or absence of exogenous αS. (A) Confocal images of representative polykaryons are shown. Fusion was monitored by the extent of mito-GFP and mito-DsRed colocalization. Scale bars=15 μm. Upper panel: vector transfected (control); lower panel: αS transfected. (B) Quantification of mitochondrial fusion in αS and control cells. Each dot represents one measured value. Mean values are indicated by horizontal bars. Asterisks indicate significant differences in the percentage of cell hybrids with fused mitochondria compared with the vector control. Expression controls are provided in Supplementary Figure S3. Download figure Download PowerPoint Mitochondria are known to fragment in stress situations (Westermann, 2008; Cho et al, 2010). To exclude that the changes in mitochondrial phenotype caused by αS overexpression are due to a secondary stress response, we performed control experiments to prove whether mitochondrial function was impaired by the expression of αS. The membrane potential in SH-SY5Y cells expressing mito-GFP was evaluated by TMRM fluorescence intensity of the mitochondria. There was no difference in TMRM fluorescence intensity when we compared the vector control cells with cells expressing αS (Figures 4A and B). In addition, no reduction of ATP production was observed in cells expressing wt-αS, αS A30P or αS A53T compared with the control-transfected cells (Figures 4C and D). Figure 4.Mitochondrial function is not impaired by low level expression of αS. (A) SH-SY5Y cells were cotransfected with mito-GFP and vector (control) or αS. Living cells were stained with TMRM and colocalization was determined by overlay. (B) Quantification of TMRM fluorescence intensity. For each condition, n=30 pictures as shown in (A) were quantified. (C) Steady-state cellular ATP levels were measured in SH-SY5Y cells transfected with either vector (control), αS wt, αS A30P or αS A53T. (D) Expression levels of αS were analysed by western blotting using calnexin as loading control. Error bars indicate s.d. Download figure Download PowerPoint αS enhances age-dependent mitochondrial fragmentation in C. elegans In line with our observations in cultured cells, when expressed in C. elegans body wall muscles (BWMs) wt-αS led to dramatic alterations of mitochondrial morphology and also to mitochondrial fragmentation (Figure 5). As C. elegans BWMs contain a highly stereotyped planar arrangement of mitochondria (Figure 5A) they are particularly suited for the analysis of mitochondrial morphology. To visualize mitochondria, we used the transmembrane domain of the outer mitochondrial membrane protein TOM70 fused to CFP (Labrousse et al, 1999). Moderate expression of αS led to the formation of extremely thin and highly interconnected mitochondria (Figures 5B and D) in about 20–40% of the transgenic BWMs. However, the majority, 50–70% of αS-expressing BWMs contained highly fragmented mitochondria that are roundish in their appearance (Figures 5C and D) in all independent transgenic strains analysed. Strikingly, a similar mitochondrial fragmentation was observed in aged 7-day-old worms in the absence of exogenous αS expression (Figure 5E), suggesting that mitochondrial fragmentation also happens during the normal ageing process of the BWM tissue. C. elegans BWMs are particularly susceptible to ageing and have been shown to gradually and progressively deteriorate with age (Herndon et al, 2002). C. elegans mean life span is about 12–18 days. After reaching adulthood, C. elegans hermaphrodites lay all their eggs within approximately 3 days and then persist through a post-reproductive period were senescent decline is evident (Herndon et al, 2002). As C. elegans animals still grow after reaching adulthood, aged BWMs were bigger in size (Figure 5E). Interestingly, ectopic expression of αS accelerated the mitochondrial aging phenotype (Figures 5E and F). Figure 5.αS expression leads to mitochondrial fragmentation in C. elegans muscles and neurons. (A) In wild-type muscles without expression of αS, mitochondria are forming regular tubular structures. (B, C) Expression of human αS leads to changes in mitochondrial morphology, which can be classified into two categories: (B) very thin and highly interconnected tubules and (C) fragmented vesicular mitochondria. Sca