Cell-produced α-synuclein oligomers are targeted to, and impair, the 26S proteasome

Neurobiology of Aging 31 (2010) 953–968

Cell-produced ␣-synuclein oligomers are targeted to, and impair, the 26S proteasome Evangelia Emmanouilidou a , Leonidas Stefanis a,b , Kostas Vekrellis a,∗ a

Division of Basic Neurosciences, Biomedical Research Foundation of the Academy of Athens (BRFAA), Soranou Efesiou 4, 11527 Athens, Greece b Second Department of Neurology, University of Athens Medical School, Athens, Greece Received 23 January 2008; received in revised form 15 May 2008; accepted 11 July 2008 Available online 20 August 2008

Abstract Proteasomal dysfunction may play a role in neurodegenerative conditions and protein aggregation. Overexpression in neuronal cells of ␣-synuclein, a molecule linked to Parkinson’s Disease, may lead to proteasomal dysfunction. Using PC12 cells stably expressing wild-type or mutant ␣-synuclein and gel filtration, we demonstrate that soluble, intermediate size oligomers of ␣-synuclein co-elute with the 26S proteasome. These soluble oligomers associate with the 26S proteasome and are significantly increased following treatment with proteasomal, but not lysosomal, inhibitors, indicating specific degradation of these particular species by the 26S proteasome. Importantly, expression of ␣-synuclein resulted in a significant inhibition of all proteasomal activities without affecting the levels or assembly of the 26S proteasome. Pharmacological dissociation of ␣-synuclein oligomers restored proteasomal function and reduced polyubiquitinated protein load in intact cells. Our findings suggest a model where only a subset of specific soluble cell-derived ␣-synuclein oligomers is targeted to the 26S proteasome for degradation, and simultaneously inhibit its function, likely by impeding access of other proteasomal substrates. © 2008 Elsevier Inc. All rights reserved. Keywords: ␣-Synuclein; Oligomers; Aggregation; Neurodegeneration; Parkinson’s Disease; 26S proteasome

1. Introduction Accumulation of misfolded, aggregated proteins is a common pathological feature in several neurodegenerative diseases. One such protein is ␣-synuclein (␣S), a natively unfolded protein, which accumulates in an aggregated conformation in cytoplasmic inclusion bodies, known as Lewy Bodies, in Parkinson’s Disease (PD) and other “synucleinopathies” (Baba et al., 1998; Dickson et al., 1999; Kahle et al., 2001). Fibrillar aggregates are formed in vitro by the oligomerization of monomeric ␣S and it has been proposed Abbreviations: PD, Parkinson’s Disease; ␣S, ␣-synuclein; UPS, ubiquitin–proteasome system; PrP, prion protein; WT, wild-type; CTL, control; CT, chymotrypsin; T, trypsin; C, caspase; SEC, size exclusion chromatography; HMW, high molecular weight; LMW, low molecular weight; pPrF, pooled proteasome fractions; Vo , void volume; CR, Congo Red; PSI, ZIE[O-tBu]-Ala-Leu-al; GA, geldanamycin; Tg, transgenic. ∗ Corresponding author. Tel.: +30 210 6597498; fax: +30 210 6597545. E-mail address: [email protected] (K. Vekrellis). 0197-4580/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2008.07.008

that intermediate soluble ␣S oligomers are critical in PD pathogenesis. In support of this idea, point mutations linked to early-onset familial PD lead to an increase of such species (Conway et al., 2000, 2001; Vekrellis et al., 2004); whether such species are indeed toxic and the nature of their effects in a cellular context remain unresolved. The ubiquitin–proteasome system (UPS) is a major system for intracellular protein degradation, a complex and tightly regulated process (Golberg, 2003). Evidence suggests that dysfunction of protein degradation through this pathway may be involved in the pathogenesis of PD and other neurodegenerative conditions (Snyder et al., 2003; Lindersson et al., 2004, reviewed by Lang-Rollin et al., 2003). Expression of mutant or, even wild-type ␣S can selectively cause proteasomal dysfunction in neuronal cell culture systems (Stefanis et al., 2001a; Petrucelli et al., 2002; Tanaka et al., 2001; Snyder et al., 2003; Smith et al., 2005). In vitro studies have supported this notion, by showing that ␣S can interact with the proteasome and inhibit its function. Aggregated recombinant

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␣S had a much stronger effect on proteasomal function when compared to the monomer (Snyder et al., 2003; Lindersson et al., 2004). However, a well-executed study failed to find evidence for UPS dysfunction in PC12 cells overexpressing mutant ␣S or ␣S transgenic mice (Martin-Clemente et al., 2004), casting some doubt on the association of aberrant ␣S with proteasomal dysfunction. Furthermore, the mechanisms through which the putative inhibitory effects of ␣S on proteasomal function occur, and the species involved, have not been deciphered in a cellular context. The relationship of UPS dysfunction to protein aggregation, in particular as it relates to neurodegeneration, is more general, and was addressed in the seminal study of Bence et al. (2001). It was demonstrated that protein aggregation and inclusion formation induced by mutant Huntingtin caused UPS dysfunction in a cellular context, although the mechanisms through which this occurs are unclear. Recently, Kristiansen et al. (2007) showed that only oligomeric PrP could inhibit the 26S proteasome. Accordingly, Tseng et al. (2007) demonstrated that oligomeric amyloid-beta caused significant impairment of proteasomal activity in an Alzheimer’s disease mouse model. Various models have been proposed, including sequestration of UPS components by the aggregating proteins, “clogging” of the proteasome by aggregated proteins and indirect effects, such as those mediated through energy depletion and caspase activation (Hoglinger et al., 2003; Vekrellis and Stefanis, 2005). Furthermore, the species involved are unknown, and may include formed inclusions, fibrillar or prefibrillar forms (Lashuel et al., 2002). We have undertaken the present study to address some of these uncertainties, and in particular to examine in a cellular context the species of ␣S that are responsible for UPS dysfunction and the mechanisms through which they exert their effects. We have also shed further light on the manner of degradation of ␣S, which has been quite controversial.

2. Materials and methods 2.1. Reagents All reagents were obtained from Sigma unless otherwise specified. The peptides for the determination of proteasome activities, Z-LLE-AMC, Suc-LLVY-AMC, and Z-ARR-AMC, as well as epoxomycin, PSI and G418 were from Calbiochem. Recombinant ␣S was from Chemicon. Geldanamycin was purchased from Sigma. Lipofectamine 2000 transfection reagent and SimplyBlue Safestain were from Invitrogen. Lactacystin and purified 20S proteasome were from Biomol. DTBP was obtained from Pierce. 2.2. Cell lines and cell culture The clones stably expressing empty vector, wild-type (WT) and mutant A53T ␣S have been previously described

(Stefanis et al., 2001a,b). In a similar fashion, we have generated PC12 cells stably expressing EGFP. PC12 cells expressing empty vector, EGFP, WT and A53T ␣S were grown on rat tail collagen coated plates as previously described (Stefanis et al., 2001a). For size exclusion chromatography, cells from each condition were grown on 150mm dishes until approximately 80% confluent. 2.3. Mice For our experiments, we used double transgenic (Tg) C57BI/C3H mice expressing human A53T ␣S under the control of the prion promoter, or control wild type mice derived from matings of single Tg mice. The generation and phenotype of these mice has been previously described by Giasson et al. (2002). The mice were purchased from Jackson Laboratory (Bar Harbor, Main) and were housed in the animal facility of the Biomedical Research Foundation of the Academy of Athens (BRFAA) in a room with a controlled light–dark cycle (12 h light–12 h dark) and free access to food and water. Mice were sacrificed by cervical dislocation. Brains were harvested, dissected to obtain the region of interest and immediately frozen. All mice were processed in a similar manner. Tissue was stored at −80 ◦ C until further use. Genotyping was performed by quantitative Southern dot blot analysis with a 32 P-labeled oligonucleotide-primed ␣S DNA probe as described previously (Giasson et al., 2002). All efforts were made to minimize animal suffering and to reduce the number of the animals used, according to the European Communities Council Directive (86/609/EEC) guidelines for the care and use of laboratory animals. All animal experiments were approved by the Institutional Animal Care and Use Committee of BRFAA. 2.4. Size exclusion chromatography (SEC) For SEC, cells were treated as described previously (Hendil et al., 1998, 2002) with some modifications. Briefly, cells were harvested in PBS, washed once with PBS and homogenized by sonication for 3 × 15 s in 1 ml buffer A containing 25 mM Tris–HCl (pH 7.6), 5 mM DTT, 2 mM MgCl2 , 2 mM ATP and 0.1 mM EDTA. After centrifugation (10,000 × g, 5 min), the supernatant was filtered through a 0.45 ␮m filter (Nalgene), supplemented with glycerol to 10% final concentration and injected into a Superose 6 10/300 GL column (Amersham Pharmacia) which had been equilibrated with buffer B consisting of 25 mM Tris–HCl (pH 7.6), 0.1 mM ATP, 5 mM DTT, 1 mM MgCl2 , 100 mM NaCl, 0.1 mM EDTA, and 10% glycerol. Elution was performed at a constant flow rate of 0.25 ml/min in buffer B and fractions of 250 ␮l were collected. Mouse cortex was dissected and homogenized in 6 volumes of buffer C containing 25 mM Tris-Cl (pH 7.6), 1 mM DDT, 2 mM MgCl2 , 2 mM ATP, 0.1 mM EDTA, 1% glycerol and protease inhibitors using a Dounce homogenizer. The homogenate was initially centrifuged at 70 × g for 10 min

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at 4 ◦ C. The supernatant was supplemented with buffer C to 1 ml and centrifuged again at 14,000 × g for 20 min at 4 ◦ C. The supernatant was directly injected into Superose 6 10/300 GL column and eluted as described above. Congo Red was prepared in DMSO and added to cell lysates at a final concentration of 100 ␮M. Cell lysates were treated with CR for 45 min at 4 ◦ C with constant shaking before SEC (Sanchez et al., 2003; Heiser et al., 2000). Geldanamycin was prepared in DMSO and added to cells in culture in a final concentration of 0.8 ␮M for 48 h. Ammonium chloride was prepared as a 2 M stock solution and added to the cells in a final concentration of 20 mM for 24 h. 2.5. Proteasome activity assays The three enzymatic activities of the proteasome, chymotrypsin (CT)-like, trypsin (T)-like and caspase (C)-like were assayed by measuring the hydrolysis of the fluorogenic synthetic peptides, Suc-LLVY-AMC, Z-ARR-AMC, and Z-LLE-AMC, respectively (Rodgers and Dean, 2003; Akaishi et al., 1995). HMW proteasome fractions, corresponding to 1 ␮g of protein, were incubated at 37 ◦ C for 10 min in a reaction buffer containing 50 mM Tris–HCl (pH 7.6), 5 mM DTT, 10 mM ATP, 50 mM MgCl2 , and 100 ␮M of fluorogenic substrate. Reactions were terminated by addition of 5% SDS. Fluorescence was measured at 437 nm using a PerkinElmer LS-55 luminescence spectrophotometer. All fluorescence values were corrected by subtracting the fluorescence measured in the presence of the specific proteasome inhibitors epoxomycin (1 ␮M) or PSI (100 nM). Quantification of proteasome activity was achieved by integrating the area specified by the graph of the fluorescence in the proteasome fractions from each cell lysate using Image J software. 2.6. RAIDD-Flag construct transfection Transient overexpression of human RAIDD-Flag (Jabado et al., 2004) in PC12 cells was achieved by Lipofectaminemediated transfection according to the manufacturer’s recommendations. Briefly, transfections were performed in 4 × 10 cm culture dishes with 4 × 106 cells per dish with 20 ␮g of plasmid DNA. All transfections were performed for 16 h in Optimem medium and complete growth medium was added the next day. Samples for SEC were harvested 48 h post transfection. 2.7. Immunoprecipitation Following SEC of CTL or A53T-expressing cell lysates, pooled HMW fractions rich in 26S proteasome were immunoprecipitated for ␣S using the monoclonal Syn-1 antibody. Immunoprecipitation was carried out in a buffer containing 20 mM Tris–HCl (pH 7.6), 5 mM ATP, 10% glycerol, 0.2% Nonidet P-40 and complete protease inhibitor cocktail mixture (Roche). Immunoprecipitated complexes were eluted from agarose G plus beads (Calbiochem) by

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heating at 85 ◦ C for 3 mins in non-reducing Laemmli buffer supplemented with 0.02 M iodoacetamide (Chondrogianni et al., 2005). For cross-linking experiments cells were washed once in cross-linking buffer (20 mM Hepes (pH 7.4), 0.15 M NaCl) before treatment with 2 mM DTBP in cross-linking buffer at 25 ◦ C for 30 min (Tanaka et al., 2000). 2.8. Electrophoresis and Western blot analysis Fractions containing the 26S proteasome were pooled and concentrated 50 fold by ultrafiltration using a 300 kDa cut-off filtering device (Vivascience). 4% or 8% acrylamide native gels were also used for the analysis of proteasome and ␣S species in the HMW fractions (Hoffman and Rechsteiner, 1994). Non-denaturing gels consisted of 90 mM Tris–HCl (pH 8.8), 80 mM boric acid, and 80 ␮M EDTA. Native gel electrophoresis was performed at 4 ◦ C in TBE buffer at 50 V. Denaturing gel electrophoresis was carried out in 12% SDS PAGE gels in Tris-Glycine buffer. Immunoblotting was performed using the following antibodies: anti-␣S (mouse monoclonal from Santa Cruz, Syn-211, rabbit polyclonal from Santa Cruz, C-20 or Syn-1 mouse monoclonal from BD), anti-19S regulator ATPase subunit Rpt6 (mouse monoclonal Biomol), anti-20S proteasome subunit ␤5 (rabbit polyclonal Biomol), anti-ubiquitin (mouse monoclonal, Chemicon), anti-ERK 2 (rabbit polyclonal, Santa Cruz,), antiGFP (mouse monoclonal, Santa Cruz), anti-Hsp70 (mouse monoclonal, Stressgen), anti-c-jun (rabbit polyclonal, Santa Cruz), anti-␤-actin (mouse monoclonal, Sigma), anti-Flag (mouse monoclonal M2, Sigma), anti-␥-tubulin (mouse monoclonal, Sigma). All immunoblots represent one of at least three experiments. Quantification of bands on Western immunoblots was performed using Gel Analyser software (Biosure, Greece). Differences in protein expression levels were quantified after standarization of all values using the appropriate loading controls (20S ␤5, ␥-tubulin, ERK). All statistical analyses were performed using the Student’s t-test, p values of < 0.05 were considered significant.

3. Results 3.1. Isolation of active 26S proteasomes from cell homogenates The 26S proteasome is a 2.4 MDa protease complex that selectively degrades proteins modified by polyubiquitin chains. The 26S proteasome is composed of two 700 kDa multisubunit complexes: the 20S proteasome, which serves as the proteolytic core of the complex, and 19S, an ATPase regulatory complex responsible for the binding, modification, and delivery of substrates to the proteolytic chamber (Golberg, 2003). To assess more accurately the effect of cellproduced ␣S on proteasome function, we used size exclusion chromatography (SEC) to isolate fractions containing active 26S proteasomes from A53T or empty vector expressing

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Fig. 1. Separation of the 26S proteasome from cell homogenates by gel filtration. PC12 cells were homogenized by sonication and soluble proteins were loaded into a Superose 6 HR 10/30 column. Fractions were analyzed by SDS PAGE. (a) Chromatographic profile of lysates. (b) The proteasome-containing fractions were examined for CT-like peptidase activity that corresponds to assembled 26S proteasome. (c) Immunoblotting with antibodies against the Rpt6 subunit of the 19S complex and the ␤5 subunit of the 20S particle showed that 26S proteasome eluted in fractions 32–50. L = cell lysate. The molecular mass markers for gel filtration were: blue dextran (Vo ), thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), carbonic anhydrase (29 kDa).

PC12 cells. SDS PAGE and immunoblotting analysis of fractionated cell homogenates with antibodies against the 19S Rpt6 subunit and the 20S ␤5 subunit revealed that the 19S and 20S complexes co-eluted in fractions 32-50. Enzymatic chymotrypsin-like proteasomal activity was detected using the synthetic fluorogenic substrate Suc-LLVY-AMC in fractions 32-50, corresponding to molecular weights above 1.5 MDa, indicating that these fractions contain assembled, active 26S proteasome (Fig. 1a–c). Assays in the absence of ATP showed no activity, confirming isolation of the ATPdependent 26S proteasome (data not shown). 3.2. A53T αS overexpression impairs the activity of the 26S proteasome To investigate the effect of ␣S overexpression on proteasomal enzymatic activities, cell homogenates from A53T and empty vector (CTL) expressing PC12 cells were fractionated by SEC and the chymotrypsin, trypsin and caspase hydrolyzing activities of the fractions containing 26S proteasomes were determined by measuring the hydrolysis of the fluorogenic synthetic peptides, Suc-LLVY-AMC, Z-ARR-AMC, and Z-LLE-AMC, respectively. The CT-like activity of A53T expressing cells was lower than that of empty vector control cells (25.6 ± 3.0% decrease, n = 4, mean ± S.D.) consistent with our previous result with the cruder assay in the total cell lysates (Stefanis et al., 2001a) (Fig. 2a). Similar results were observed with both the T-like and the C-like activities as illustrated in Fig. 2b (19.4 ± 5.8% and 23.7 ± 2.9% decrease, respectively, n = 4, mean ± S.D.). Proteasomal dys-

function in the mutant ␣S expressing cells was further verified by examining the levels of polyubiquitinated proteins and cjun, a specific 26S proteasome substrate (Treier et al., 1994; Jariel-Encontre et al., 1995), by immunoblotting. The levels of polyubiquitinated proteins and of c-jun were significantly elevated in the lysate (6.1 ± 2.3 fold, n = 3, mean ± S.D.) and proteasome-containing fractions of the A53T ␣S expressing cell line compared to CTL cells (Supplementary Fig. 1 and Fig. 6d), indicative of proteasomal dysfunction. We verified that the decrease in proteasomal activity observed in A53T expressing cells was not due to decreased proteasome levels, as the levels of the Rpt6 subunit of the 19S cap and the ␤5 subunit of the 20S complex were similar across the two lines (Fig. 2c). Furthermore, proteasome assembly, as assessed by levels of the ␤5 subunit in HMW fractions containing the peak proteasomal enzymatic activity on non-denaturing gel electrophoresis, was unaltered in A53T expressing cells (Fig. 2d). Similar results were obtained using an antibody to the 19S Rpt6 subunit (not shown). Therefore, ␣S overexpression does not alter the levels or assembly of the 26S proteasome complexes. 3.3. A53T αS co-elutes in the 26S proteasome-containing fractions Although the degradation pathway of ␣S remains under investigation, a number of studies, using mostly recombinant ␣S, have suggested a direct link of ␣S with the 20S complex of the proteasome (Ghee et al., 2000; Snyder et al., 2003; Lindersson et al., 2004). To investigate the mechanism

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Fig. 2. Overexpression of A53T ␣S inhibits the enzymatic activity of the 26S proteasome. (a) Following gel filtration, the fractions containing the 26S proteasome were assayed for proteasome peptidase activities as described in Section 2. One representative experiment for the CT-like activity is shown. Fluorescence is measured in arbitrary units. (b) Quantification analysis of the CT-like, the T-like and the C-like proteasome activities, comparing A53T cells with CTL cells (n = 4, mean ± S.D., in each case enzymatic activity of CTL cells is considered 100%). Asterisks indicate statistical significant differences in enzymatic activities (independent t-test, * p < 0.0005). (c) CTL and A53T cell lysates were immunoblotted with antibodies against the Rpt6 subunit of the 19S cap and the ␤5 subunit of the 20S complex. ERK-2 immunoreactivity was used as loading control. A representative immunoblot is depicted. (d) After gel filtration, the fraction possessing the peak proteasomal activity from each one of CTL and A53T cells was electrophoresed on a 4% native acrylamide gel. Immunoblot analysis using the 20S ␤5 antibody was then performed. Purified 20S proteasome was included as marker.

through which mutant ␣S affects proteasomal activity, we first examined whether it is also present in the HMW fractions that contain whole 26S proteasome. To this end, SEC fractions rich in active 26S proteasome from A53T ␣S expressing cells were analyzed for the presence of ␣S and 26S proteasome using the Syn-1 and 19S Rpt6 antibodies, respectively. As depicted in Fig. 3a, ␣S co-eluted in the 26S proteasomecontaining fractions in A53T expressing cells. In addition, proteasome-containing fractions from CTL and A53T cells were pooled and filtered through a 300 kDa cut-off filter. The concentrate and the eluate were then subjected to SDS PAGE and analyzed for the presence of ␣S and 26S proteasome. As expected, the levels of the ␤5 proteasomal subunit were enriched in the concentrates, and were not different between CTL and A53T expressing lines. Importantly, as depicted in Fig. 3b, ␣S co-eluted in the pool of concentrated 26S proteasome-containing fractions in A53T expressing cells, and was absent from the eluates with a cut-off of 300 kDa. ␣S in these fractions appeared only at the level of 16–17 kDa on these SDS PAGE gels. No HMW species were observed, even after prolonged exposures. Immunoprecipitation of ␣S from pooled HMW proteasomal fractions using Syn-1 antibody did not co-precipitate with ubiquitin, even when lactacystin was

used (see below), indicating that the ␣S that co-elutes with the 26S proteasome is not ubiquitinated (data not shown). The levels of ␣S present in the proteasome-containing fractions were quantified using known amounts of human recombinant ␣S. Proteasome-containing fractions were pooled and concentrated through a 100 kDa filter. After SDS PAGE and immunoblotting with the Syn-1 antibody we estimated that the amount of ␣S that co-elutes with the 26S proteasome is 0.5% of the total ␣S in the lysates. Taken together these data indicate that a small amount of nonubiquitinated A53T ␣S, in a monomeric form on SDS PAGE gels, co-elutes in HMW fractions with the 26S proteasome. To confirm that the presence of ␣S in 26S proteasomecontaining fractions was not an artifact of mere protein overexpression we analyzed in the same manner 26S proteasome-containing fractions from EGFP expressing PC12 cells. Although the protein was expressed at high levels in the lysates of these cells it was absent from the HMW proteasome fractions even after concentration (Fig. 3c and d). As expected, GFP exclusively eluted in LMW fractions 62–76 (Fig. 3c). We were also interested to determine whether co-elution of ␣S with the 26S proteasome reflected the result of overexpressing an aggregation-prone protein. To

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Fig. 3. ␣S co-elutes with the 26S proteasome (a) Representative immunoblot showing the elution profile of ␣S in HMW FPLC fractions that contain the 26S proteasome. Starting lysate and fractions were analyzed by SDS PAGE and immunoblotting with antibodies against Rpt6 proteasomal subunit and ␣S. (b) The FPLC fractions containing the 26S proteasome were concentrated by ultrafiltration using a 300 kDa-cut-off filter. The starting lysate (lys), the concentrate (conc) containing proteins >300 kDa, and the eluate (el) containing proteins <300 kDa, were analyzed by SDS PAGE and immunoblotting with antibodies against ␣S and the ␤5 proteasomal subunit. A representative immunoblot is depicted. (c) PC12 cells stably expressing EGFP were similarly subjected to lysis, fractionation by SEC, SDS PAGE analysis of SEC fractions, and immunoblotting with anti-GFP antibody. (d) Proteasome-containing fractions from EGFP expressing cells were pooled and concentrated through a 300 kDa-cut-off filter. Following SDS PAGE analysis, membranes were immunoblotted with antibodies against EGFP and the ␤5 subunit. (e) PC12 cells overexpressing RAIDD-Flag were lysed and fractionated by FPLC as described in Section 2. HMW proteasome fractions as well as LMW fractions were analyzed by SDS PAGE and immunoblotting with antibodies against Flag and the 19S Rpt6 subunit.

this end, we analyzed 26S proteasome-containing fractions from PC12 cells overexpressing the human caspase 2 adaptor, RAIDD, a known aggregation-prone protein (Jabado et al., 2004). Again, although RAIDD was highly expressed in the cell lysates it was only present in LMW, and not in the proteasome-containing fractions following SEC fractionation (Fig. 3e). 3.4. A53T αS present in 26S proteasome fractions is oligomeric We wished to further examine the nature of the conformational state of A53T ␣S in the 26S proteasomal fractions. Even though the band corresponding to ␣S in these fractions migrated as a monomer on SDS PAGE, the possibility existed that it may in fact contain oligomeric species of ␣S that collapse upon SDS treatment. There is a precedent for such ␣S species (Dixon et al., 2005). We therefore analyzed the migration pattern of ␣S on native gels, and compared it to that of recombinant ␣S. Using concentrates derived from 26S proteasome-containing HMW fractions, we did not detect any species of ␣S on a 4% native gel where the proteasome complexes are detected. In contrast, species of ␣S were apparent, using the Syn-1 antibody, on an 8% native gel, where they migrated between 150 and 450 kDa, higher than recombinant ␣S run on the same gel (Fig. 4a). The presence of ␣S

oligomeric species in the 26S containing HMW fractions was also confirmed using monoclonal antibody 211 (Fig. 4b). To assess in a more functional manner the nature of these particular species of ␣S, we used the anti-amyloidogenic compound Congo Red (CR), which has been shown to disrupt preformed oligomeric/aggregated forms of various proteins, including mutant Huntingtin (Carter and Chou, 1998; Sanchez et al., 2003). To this end, we analyzed the state of these particular ␣S species before and after CR treatment. As shown in Fig. 4a and b, CR treatment significantly reduced the amount of the ␣S species in 26S proteasome-containing fractions. To investigate whether CR treatment affected other higher assembly oligomers of ␣S we also analyzed void volume fractions not containing the 26S proteasome from A53T ␣S expressing cells. We found that higher (>700 kDa) ␣S soluble oligomers were present in such fractions and were also disrupted by CR treatment (Fig. 4c). In contrast, ␣S in LMW fractions did not decrease, and even showed a tendency to increase, likely due to the conversion of HMW oligomeric species to the monomer following CR treatment (Fig. 4d). To exclude the possibility that these results might reflect non-specific effects of CR treatment, we analyzed SEC proteasomal fractions of cell lysates treated with or without CR by highly sensitive Coomasie Blue staining and found that protein patterns were identical (Supplementary Fig. 2a). Furthermore, the accumulated polyubiquitin species in the proteasome-containing

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Fig. 4. Congo Red (CR) disrupts ␣S oligomers present in high MW proteasome fractions. (a and b) FPLC fractions containing the 26S proteasome with or without CR treatment were concentrated by ultrafiltration and analyzed on an 8% native gel for the presence of ␣S species, using immunoblotting with two different ␣S monoclonal antibodies, Syn-1, BD Biosciences (a) and Santa Cruz 211 (b). Markers for non-denaturing electrophoresis: apoferritin (443 kDa), alcohol dehydrogenase (150 kDa). (c) CR also disrupted ␣S species present in the Vo fractions as shown after non-denaturing electrophoresis (4% native gel) and immunoblotting with a ␣S antibody (BD Biosciences). (d) Representative immunoblot of low molecular weight LMW fractions (68 and 70) treated with or without CR. CR treatment slightly increased the levels of monomeric ␣S species.

fractions do not change following treatment with CR at 4 ◦ C (Supplementary Fig. 2b). Under these conditions, the proteasome is inactive, and would not be expected to degrade polyubiquitinated proteins, even though the offending ␣S is removed (Fig. 4). Therefore, the lack of difference in polyubiquitinated proteins minus or plus CR merely reflects the fact that CR does not disrupt preformed polyubiquitinated proteins. These data support a specific effect of CR on oligomeric species of ␣S, including the species co-eluting with the 26S proteasome. Taken together, these data indicate that a specific subset of ␣S oligomeric species co-elute with the 26S proteasome, and that they can be disrupted by CR treatment. 3.5. Congo Red treatment ameliorates the inhibitory effect of αS on proteasome activity A number of studies now support the idea that protein aggregation can lead to proteasomal dysfunction. However, how this dysfunction is mediated is unclear. A matter of particular contention is which particular species of aggregated proteins are responsible for UPS dysfunction. Having found that CR disrupted specific oligomeric species of ␣S that coeluted with the proteasome, and removed them from these fractions, we wished to examine whether these particular

species of ␣S within these fractions were directly responsible for the inhibitory effect on the 26S proteasome, by measuring CT-like proteasomal activity with or without CR treatment prior to SEC. We found that CR treatment of the lysates almost completely ameliorated the inhibitory effect of A53T ␣S expression on proteasome activity by increasing the CT-like activity by 27.7% ± 7.0% (n = 3, mean ± S.D.) relative to control empty vector expressing cells (Fig. 5a and b). We further ensured that CR did not influence the assembly of the 26S proteasome, as proteasome-containing fractions before and after CR treatment showed a similar immunolabeling pattern for the 20S ␤5 subunit on a 4% non-denaturing gel (Fig. 5c). We conclude that CR restores proteasomal function by removing the specific oligomeric species of ␣S that co-elute with the 26S proteasome. 3.6. Pharmacological induction of chaperones also disrupts αS oligomers present in proteasomal fractions and reduces the load of polyubiquitinated proteins in intact cells Since, in our hands, CR proved to be membraneimpermeable and toxic at high concentrations in PC12 cells (data not shown), we have used an alternative strategy to

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Fig. 5. Congo Red ameliorates the inhibitory effect of ␣S on proteasome activity. (a) CT-like activity measurement of FPLC proteasome fractions from CTL and A53T cells after treatment with CR. One representative experiment is shown. (b) Quantitative analysis of the activity graphs showed that CR application caused a significant increase of 27.7 ± 7.0% (n = 4, mean ± S.D., independent t-test, * p < 0.005) compared with CTL cells, whose activity in each experiment was considered as 100%. (c) The concentrated FPLC proteasome fractions with or without CR treatment were analyzed in a 4% native gel and immunoblotted with a ␤5 antibody.

modulate specifically ␣S oligomeric species in intact cells, to ensure that the results achieved with CR do not merely reflect post-lysate conditions. To this end, we utilized geldanamycin (GA), a known pharmacological regulator of heat

shock proteins, which has been reported to prevent ␣S aggregation and toxicity (McLean et al., 2004; Klucken et al., 2004). As expected, 48 h of GA treatment induced Hsp70 in intact A53T expressing cells in a dose-dependent manner

Fig. 6. Geldanamycin disrupts ␣S oligomers in the HMW proteasome fractions and restores proteasome activity. (a) A53T cells treated with various concentrations of GA were lysed and analyzed by SDS PAGE and immunoblotting with antibodies against Hsp70 and ␣S. ERK is used as loading control. (b) CTL and A53T cells were treated with GA as described in Section 2. Following SEC, the HMW proteasome fractions were concentrated and analyzed in an 8% native gel and immunoblotted with ␣S antibody. (c) CT-like activity measurement of FPLC proteasome fractions from CTL and A53T cells after treatment with GA. One representative experiment is shown. (d) Representative immunoblot of HMW proteasome fractions from CTL and A53T cells after treatment with GA. GA reduces the amount of polyubiquitinated proteins present in these fractions. 20S ␤5 subunit is shown as loading control.

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without affecting the total levels of ␣S (Fig. 6a). Most importantly, GA-treated A53T cells showed significantly reduced levels of ␣S oligomers associating with pooled proteasome fractions when compared to control or untreated cells under native electrophoresis conditions (Fig. 6b). We further wished to examine whether removal of the proteasome-associated oligomers by GA had an effect on proteasomal function. To this end we examined proteasome enriched fractions for CT-like enzymatic activity and polyubiquitinated protein load following treatment of the cells with the compound. As depicted in Fig. 6c, GA-treated cells exhibited restored proteasomal function reflected by a significant increase in CTlike activity (17.5 ± 4.6%, n = 3, mean ± S.D.) and a decrease in polyubiquitinated species compared to untreated cells (Fig. 6d). GA treatment of CTL cells did not show any significant effect on CT-like proteasomal activity (not shown). To further prove that the effect of GA was specific to the ␣S

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oligomers we simultaneously treated cells with PSI and GA. We found that although GA did not affect the levels of polyubiquitinated proteins in the proteasome-containing fractions it specifically reduced the ␣S oligomeric burden in the same fractions (Supplementary Fig. 3). These data strongly suggests that the observed proteasomal impairment is functionally linked to the presence of specific ␣S oligomers. 3.7. WT αS also co-elutes in the 26S proteasome-containing fractions and impairs its activity Multiplications of the locus encoding for ␣S are tightly linked to PD state suggesting that altered amounts of wildtype ␣S contribute to the pathophysiology of neuronal loss (Singleton et al., 2003; Farrer et al., 2004; Chartier-Harlin et al., 2004). We therefore decided to examine the effect of WT ␣S expression on proteasomal activity using fractions

Fig. 7. Proteasomal inhibitors increase the amount of ␣S species present in high MW proteasome fractions. (a) Lactacystin (10 ␮M, 16 h) or PSI (2.5 ␮M, 16 h) or vehicle was applied to A53T cells, and the CT-like activity in FPLC proteasome fractions was measured. (b) The FPLC proteasome fractions from A53T cells with or without lactacystin or PSI were pooled and concentrated through a 300 kDa filter. Subsequent analysis on an 8% native gel and immunoblotting with an anti-␣S antibody (Syn-1, BD Biosciences) was performed. (c) Lysates and concentrated proteasome fractions of lactacystin or vehicle (DMSO)-treated cultures were subjected to SDS PAGE electrophoresis and immunoblotting using anti-␣S and anti-␤5 antibodies. (d) Lysates, void volume fractions (Vo ) and concentrated proteasome fractions of PSI- or vehicle (DMSO)-treated cultures were subjected to SDS PAGE electrophoresis and immunoblotting using anti-␣S anti-␤5 and anti-␥-tubulin antibodies. The bar graph represents quantification of the increase in ␣S expression levels (mean ± S.D.) from 3 individual experiments. Asterisk indicates statistical significant increase in ␣S expression in pPrF (independent t-test, * p < 0.005).

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obtained from SEC of lysates from PC12 cells stably overexpressing human WT ␣S. Interestingly, we found that, like A53T, WT ␣S expression also impaired all three proteasomal activities compared to empty vector control cells (Supplementary Fig. 4b and c). Most importantly, examination of proteasome-rich fractions for the presence of WT ␣S and proteasomal subunits (19S Rpt6 and 20S ␤5, not shown) by immunoblotting revealed that WT ␣S co-eluted with the HMW proteasome-containing fractions (Supplementary Fig 4a). Consistent with these results, we observed an accumulation in polyubiquitinated proteins in the WT ␣S expressing cells compared to CTL cells (Supplementary Fig 4d). 3.8. The specific soluble αS oligomers that co-elute with the 26S proteasome are degraded by it Our results with CR and GA suggested that a functional interaction between ␣S and the 26S proteasome does exist, and that it could mediate the proteasomal impairment induced by ␣S. It has been proposed that aggregated or semi-aggregated proteins that are themselves substrates of the proteasome, due to their aberrant conformation, impede access of other substrates leading to proteasomal dysfunction (“clogging” hypothesis). To test whether the oligomeric ␣S species present in HMW fractions are destined for proteasomal degradation, we treated our cultures with the selective proteasomal inhibitors lactacystin and PSI, which, as expected, led to complete loss of 26S proteasomal activity

(Fig. 7a). These treatments also led to a substantial increase of the soluble ␣S oligomeric species that co-eluted with the 26S proteasome on non-denaturing gels (Fig. 7b), or on SDS PAGE gels (5.9 ± 1.4 fold increase, n = 3, mean ± S.D.). In contrast, there was no effect of the proteasomal inhibitors on the overall levels of ␣S in the lysates or in the Vo fractions (0.9 ± 0.3 and 1.3 ± 0.2 fold difference, respectively, Fig. 7c and d). We conclude that the specific oligomeric species of ␣S present in proteasomal fractions are themselves substrates for proteasomal degradation. 3.9. Inhibition of the lysosomal degradation pathway does not significantly affect the levels of the αS oligomers that co-elute with the 26S proteasome A number of studies have demonstrated that ␣S oligomers can be degraded by the lysosome/autophagosome system. To investigate the effect of lysosomal inhibition on the status of the proteasome-associated ␣S oligomers, we treated A53T expressing cells with NH4 Cl, a known inhibitor of the lysosome (Cuervo et al., 2004). Lysotracker staining of the cultures confirmed that for the time and dose used (16 h, 20 mM) NH4 Cl inhibited lysosomal acidification without significantly affecting proteasome activity (data not shown). NH4 Cl resulted in only a small, non-specific, increase of ␣S levels in the lysates, proteasomal fractions, and void volume fractions (1.5 ± 0.6, 1.8 ± 0.2, 1.6 ± 0.8, respectively, n = 3, mean ± S.D.) arguing that the lysosome does not play a major

Fig. 8. Lysosomal inhibition does not affect the degradation of ␣S oligomers present in proteasome fractions. (a) Lysates, pPrF and Vo fractions from CTL and A53T cells after ammonium chloride treatment (20 mM, 16 h) were analyzed by SDS PAGE and immunoblotting with an anti-␣S antibody. ␤5 proteasomal subunit and ␥-tubulin are shown as loading controls. The bar graph represents quantification of the increase in ␣S expression levels (mean ± S.D.) from 3 individual experiments. Asterisk indicates statistical significant difference of ␣S expression in pPrF (independent t-test, * p < 0.005). (b) The same pPrF were also analyzed on an 8% native gel and immunoblotted using the anti-␣S antibody.

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role in the specific degradation of the select ␣S species coeluting with the proteasome (Fig. 8a and b). Our data suggest that the select ␣S oligomers that co-elute with the proteasome are not specifically degraded by the lysosomal system. Rather, lysosomes, as we have previously suggested (Cuervo et al., 2004), may play a more general role in degrading ␣S. 3.10. αS present in high MW proteasomal fractions interacts with the 26S proteasome In order to examine whether the presence of ␣S species within the 26S proteasomal fractions signified a covalent interaction, as has been proposed by others in in vitro studies (Snyder et al., 2003; Lindersson et al., 2004; Ghee et al., 2000, 2005), we pooled HMW fractions containing the 26S proteasome from CTL and A53T cells and performed immunoprecipitation with the Syn-1 or an irrelevant antibody under non-denaturing conditions to prevent the disassembly of the proteasome. The presence of putative proteasomal proteins were investigated by immunoblotting using monoclonal antibodies to the 20S ␤5 subunit, the 19S Rpt6 subunit, the 19S Rpt5 subunit (Tbp1) and the 19S Rpn7 subunit. Under these conditions, ␣S was successfully immunoprecipitated from the proteasome-containing fractions but failed to coprecipitate with subunits of the 19S and 20S complexes. We failed to show any direct interaction of ␣S and the 26S

Fig. 9. ␣S present in high MW proteasomal fractions co-immunoprecipitate with the 26S complex. CTL and A53T cells were treated with DMSO or lactacystin (10 ␮M, 16 h), lysed and processed for immunoprecipitation as described in Section 2. Supernatants (sup) or pellets (ip) were collected and all samples were run on an SDS PAGE gel and immunoblotted using the Syn1 antibody or the 19S Rpt6 antibody. The location of bands corresponding to 19S Rpt6 subunit and ␣S is indicated by arrows.

proteasome even after performing similar immunoprecipitation experiments after cross-linking (not shown). In contrast, when proteasomal fractions were enriched in ␣S oligomeric species following treatment of the cells with the proteasome inhibitor lactacystin, we detected the Rpt6 subunit of 19S complex in the Syn-1 immunoprecipitates (Fig. 9). Therefore, ␣S and the 26S proteasome physically interact under these experimental conditions.

Fig. 10. A53T Tg mouse cortices accumulate soluble ␣S oligomers in the 26S proteasome fractions. (a) Homogenates (lys) and pooled HMW proteasome fractions (pPrF) from Ctl and Tg cortex were analyzed by SDS PAGE and immunoblotting using an anti-␣S antibody. ␤5 subunit is shown as loading control. (b) The same proteasome fractions were also analyzed in 8% native gel and immunoblotted with the anti-␣S Syn-1 antibody (left panel) and the polyclonal C-20 (right panel) (c) Quantification (mean ± S.D.) of CT-like proteasome activity in Ctl and Tg cortex (n = 3). Asterisk indicates statistical significant decrease in Tg cortices compared with age-matched Ctl cortices (independent t-test, * p < 0.05). (d) Ctl and Tg cortex homogenates were analyzed by SDS PAGE and immunoblotting with antibodies against Rpt6 and ␤5 proteasomal subunits. ERK is shown to confirm equal loading.

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3.11. Soluble oligomeric αS is present in the 26S proteasome fractions of A53T Tg mouse cortex Our data indicate that specific cell-derived soluble ␣S oligomers impair the 26S proteasome. To validate the pathological significance of our findings in PC12 cells, we expanded our analysis of proteasome-associated soluble oligomeric ␣S species to the A53T ␣S Tg mouse brain (Giasson et al., 2002). These mice exhibit an age-dependent accumulation of aberrant ␣S species and motor impairment (Giasson et al., 2002). The cortex of such mice was selected as the area to study since it has relatively modest pathology, and therefore any phenomena observed would not be attributed to frank neuronal degeneration, as may occur in the other brain regions. Cortices from 18-month-old double Tg and naive control mice were homogenized and fractionated by SEC as described in Section 2. Part of the homogenate was used to verify ␣S expression using the Syn-1 and C-20 antibodies. As expected, monomeric and oligomeric ␣S were readily detected in the cortex of Tg mice (Fig. 10a). Denaturing and non-denaturing gel electrophoresis and immunoblotting with the Syn-1 and C-20 ␣S specific antibodies further revealed the presence of soluble ␣S oligomers in the proteasomecontaining fractions of the A53T Tg mouse cortex (Fig. 10a and b). These oligomers on native gels appeared similar in nature to those we detected in our PC12 cells (Figs. 4a and 10b). On SDS PAGE gels, however, in addition to a band at around 16 kDa, similar to the one seen in PC12 cells, we also detected HMW species (Fig. 10a). It has to be stressed that, although the induction of the ␣S monomer achieved in the Tg mouse cortex was quite modest, there was a clear all-or-none induction of ␣S oligomeric species in the fractions co-eluting with the proteasome. As expected, A53T expression did not affect the levels of the 26S proteasome compared to control mice (Fig. 10a and d). To specifically test whether enzymatic proteasomal activity was decreased in A53T mouse cortex we used proteasome-rich fractions from cortical tissue that was immediately snap frozen (and stored for a week at −80 ◦ C) before being assessed. This is critical in order to retain functional proteasomes. We found that proteasome-rich fractions of Tg cortical mouse tissue showed significantly reduced levels of proteasomal activity (21.0 ± 4.9%, n = 3, mean ± S.D.), when compared to non-Tg controls (Fig. 10c).

4. Discussion Proteasomal dysfunction and ␣S have both been implicated in the pathogenesis of PD. We and others have demonstrated that mutant ␣S can reduce the net proteasomal activity in living cells (Stefanis et al., 2001a; Petrucelli et al., 2002; Tanaka et al., 2001; Smith et al., 2005). Several studies employing recombinant ␣S have suggested that the soluble oligomeric intermediates of ␣S may be the cause of cellular dysfunction and demise (Gosavi et al., 2002;

Kayed et al., 2003; Volles and Lansbury, 2003). In in vitro studies aggregated states of recombinant ␣S preferentially bound to and inhibited proteasomal function (Snyder et al., 2003; Lindersson et al., 2004). Despite this evidence, the physiologic causal relationship of ␣S aggregation state and proteasomal dysfunction in cells has remained unclear. We demonstrate here for the first time that specific soluble cellderived mutant ␣S oligomers of intermediate size impair the function of the 26S proteasome. Similar species were also found in the cortex of A53T Tg mice which also exhibited reduced proteasomal activity adding further physiological relevance to our findings. These findings are important not only in the context of PD, but also in neurodegeneration in general, as aggregate-prone proteins linked to neurodegenerative diseases appear to impact proteasomal function, but the mechanisms and the particular species involved remain elusive. Since the impact of ␣S on the UPS remains a subject of ongoing debate (Martin-Clemente et al., 2004), to address this issue accurately it is critical to assess proteasomal activity in purified proteasome complexes rather than analyzing crude cell lysates. Other proteinases present in cell lysates can degrade the commonly used proteasome substrates under standard assay conditions (Rivett et al., 2002; Rodgers and Dean, 2003). To avoid such masking of proteasomal activities, we used SEC to isolate functional proteasomes and increase the accuracy of proteasomal activity measurement. All three proteasome hydrolytic activities measured in this fashion were significantly reduced in cells where A53T ␣S was expressed. This is in contrast to an in vitro study which reported that ␣S results in a marked inhibition only of the chymotrypsin-like hydrolytic activity of the proteasome (Lindersson et al., 2004). This discrepancy most likely reflects methodological differences and especially the use of recombinant ␣S. Indeed, we have observed that the aggregation propensity and the range of oligomeric species produced during in vitro aggregation of recombinant ␣S vary greatly (unpublished data). We had previously reported that PC12 cells stably overexpressing WT human ␣S did not exhibit an overt reduction in proteasomal activity (Stefanis et al., 2001a). However, in our current experiments, assessment of proteasomal activity in purified proteasome complexes from such cells demonstrated a reduction of all three proteasomal enzymatic activities, and immunoblot assessment showed an accumulation of polyubiquitinated proteins. We also found that soluble WT ␣S co-elutes in the proteasome-containing fractions. Therefore, WT ␣S may affect proteasomal function in certain situations. This effect appears controversial (Tanaka et al., 2001; Smith et al., 2005; Snyder et al., 2003) and is perhaps related to the different propensity of WT ␣S to form proteasome-targeted oligomers, to expression levels, or to other unknown methodological variables. Importantly, experiments we performed in differentiated SHSY5Y cells that are inducibly expressing either WT or A53T ␣S or ␤-galactosidase again demonstrated a decrease of proteasomal activity in both WT and

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A53T-overexpressing cells, but not in ␤-gal-controls (data not shown). Following the confirmation of our earlier results with A53T ␣S using this more refined methodology, it was important to investigate whether a topological frame existed between ␣S and the proteasome-containing fractions. Indeed, we found that mutant ␣S co-elutes in fractions containing active 26S proteasome. This co-elution is specific to ␣S and not an artifact of protein overexpression since overexpressed GFP and RAIDD, a generic and an aggregation-prone mammalian protein, respectively, do not co-elute with the proteasomal fractions. Previously (Stefanis et al., 2001a), we reported that stable A53T ␣S expression in PC12 cells leads to a number of morphological and functional abnormalities in the absence of ␣S inclusions or oligomers on SDS PAGE. Still, the possibility existed that soluble ␣S oligomers that collapsed into a monomeric form on SDS PAGE gels might be responsible for the proteasomal inhibition observed in our study. We indeed found such ␣S species using non-denaturing gels, co-eluting with the 26S proteasome. Importantly, similar species were also found in the cortex of Tg mice expressing A53T ␣S, and 26S proteasomal activity was decreased in this area of Tg compared to control mice, adding further physiological relevance to our findings. To directly assess a role of oligomerization in proteasomal inhibition we determined whether CR could restore A53T-induced proteasomal inhibition. In agreement with its ␤-sheet disruptive capacity, CR reversed the inhibitory effect of A53T ␣S on the 26S proteasome, and removed the soluble ␣S oligomers present in the proteasome-containing fractions. These soluble oligomeric species of ␣S appeared to range between ∼150 and ∼450 kDa. Our data are consistent with those of Sanchez et al. (2003), who demonstrated that the preferential binding of CR to ␤-sheet amyloids could specifically inhibit oligomerization and disrupt preformed oligomers in an expanded polyglutamine model of Huntington’s disease. These results suggest that only a small specific subset of ␣S soluble oligomers, migrating at 150–450 kDa on native gels collapsing onto a monomeric state upon SDS treatment, co-localize with and impair the function of the 26S proteasome. We have further buttressed these findings by applying geldanamycin, a known inducer of the chaperone Hsp70, to intact cells. Chaperones can prevent protein oligomerization and provide protection in various models of neurodegenerative diseases (Evans et al., 2006; De Los Rios et al., 2006; Chan et al., 2000; Cummings et al., 1998). Pharmacological or molecular induction of Hsp70 inhibits ␣S aggregation both in vivo and in vitro (Klucken et al., 2004; McLean et al., 2004) and prevents ␣S toxicity in Drosophila (Auluck et al., 2002, 2005). Further arguing for a specific effect of GA on oligomeric ␣S species, Outeiro et al. (2008), recently demonstrated that molecular Hsp70 induction reduced HMW ␣S oligomers and prevented ␣S toxicity in living cells. Similarly, in our experiments, GA treatment reduced the soluble ␣S oligomeric species found in the proteasome-

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rich fractions, without affecting total ␣S levels, restored proteasomal activity and decreased the polyubiquitinated protein load in the same fractions. The anti-oligomeric effect of GA is specific to ␣S oligomerization since co-applocation of GA with PSI does not affect the protein polyubiquitination but still reduces ␣S oligomer levels. Therefore, specific elimination of soluble ␣S oligomers functionally associating with the proteasome in a cellular context also restored proteasomal activity, indicating that these species are responsible for proteasomal dysfunction. Further supporting a functional interaction between these oligomeric species and the 26S proteasome, proteasomal inhibition led to a marked and specific induction of these species, whereas other ␣S species remain unaltered. Therefore, the same species that are involved in the inhibition of the proteasome are themselves specific substrates of the proteasome. This process does not depend on ubiquitination of these ␣S species. We were able to detect a direct interaction between the 26S proteasome and oligomeric ␣S by co-immunoprecipitation only following enrichment of such oligomeric ␣S species in the proteasome-containing fractions. The instability of the ␣S-proteasome interaction under IP conditions, the poor accessibility of proteasomeassociated ␣S species to the antibodies utilized, or the limiting small amounts of the particular conformations of ␣S interacting with the proteasome may all play a role in the difficulty in detecting a physical association between ␣S and the proteasome in the absence of a proteasomal inhibitor. Other researchers (Holmberg et al., 2004) also failed to detect a direct covalent interaction by immunoprecipitation between mutant huntingtin and the proteasome, although FRET imaging convincingly demonstrated an association. It is possible that ␣S oligomers could directly inhibit the proteolytic active sites of the 20S proteasome ␤ subunits (i.e. ␤1, ␤2 and ␤5 subunits). However, this mechanism would require unfolding and insertion of ␣S oligomers into the catalytic chamber through the narrow (∼2 nm) open-gated channel of the 26S proteasome (Pickart and Cohen, 2004). Alternatively, ␣S oligomers may interfere with basic processes performed by the 19S complex such as unfolding and translocation of protein substrates and gate opening of the 20S cylinder. Our data clearly show that the presence of ␣S oligomers in proteasome fractions does not affect 26S complex assembly therefore excluding any significant disturbance of the ATP binding site. Given the transient nature of 19S–substrate complexes, we can still detect a small amount of 19S Rpt6 ATPase being bound to ␣S in the proteasome fractions which implies that protein binding in the 19S complex can still occur. Misfolded polypeptides can be selectively recognized by the 26S proteasome due to the ability of certain 19S ATPases to function as molecular chaperones (Benaroudj et al., 2003). The fact that the ␣S oligomeric species in proteasome-containing fractions lack polyubiquitination suggests that these species are targeted to the 26S proteasome by means of their aberrant conformation. To allow protein unfolding and translocation, two interrelated

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processes, the 19S ATPases must obtain a specific conformation (Smith et al., 2007). The bulky ␣S oligomers targeted to the proteasome for degradation may greatly obstruct this ability due to steric hindrance. Non-translocatable ␣S may then reduce the numbers of the substrates that normally interact with the 19S ATPase ring or prevent unfolding and degradation of other substrates as suggested for oligomeric PrP (Kristiansen et al., 2007). In a number of studies ␣S levels accumulate in cells upon proteasome inhibition, suggesting that the UPS normally degrades ␣S (Bennett et al., 1999; Sawada et al., 2004; Liu et al., 2002; Tofaris et al., 2001; Webb et al., 2003). We and others, however, using stable cell lines or cells normally expressing ␣S, were unable to replicate this finding (Petrucelli et al., 2002; Ancolio et al., 2000; Paxinou et al., 2001; Rideout et al., 2001; Rideout and Stefanis, 2002). These discrepancies have been hard to resolve, but may be due, in part, to the presence or absence of epitope tags, the transient or stable ␣S expression and cell-specific effects. In our present work, proteasomal inhibition did not alter overall levels of mutant ␣S, and only led to an increase of the specific oligomers of ␣S that co-elute with the proteasome. We and others have provided evidence that lysosomal pathways are involved in the degradation of monomeric or oligomeric ␣S (Webb et al., 2003; Paxinou et al., 2001; Cuervo et al., 2004; Lee et al., 2004). Lysosomal inhibition in the current studies caused a mild, non-specific, increase in all species of ␣S examined, including those in Vo fractions. Therefore, lysosomes may have a role in degrading these particular species, but this is non-specific, as it applies to all ␣S species examined; clearly the major specific degradation pathway for the proteasome-associated species is via the proteasomes. The lysosomes may play a more substantial role in degrading other, non-proteasome-associated ␣S oligomers, but this was not tested specifically in this study. In conjunction, ours and others’ results indicate that the route of ␣S degradation depends critically on its particular conformational state. In conclusion, our data indicate that intermediate soluble oligomeric species of ␣S are targeted to and inhibit the 26S proteasome, providing support for a functional interaction between proteasome and aggregate-prone proteins.

Conflicts of interest There are no actual or potential conflicts of interest for all authors.

Acknowledgments This work was supported in part by grants from the Parkinson’s Disease Foundation to KV and by NIH grant R21 NS0556 to LS and KV.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. neurobiolaging.2008.07.008.

References Akaishi, T., Yokosawa, H., Sawada, H., 1995. Regulatory subunit complex dissociated from 26S proteasome, isolation and characterization. Biochim. Biophys. Acta 1245, 331–338. Ancolio, K., Alves da Costa, C., Ueda, K., Checler, F., 2000. Alpha-synuclein and the Parkinson’s disease-related mutant Ala53Thr-alpha-synuclein do not undergo proteasomal degradation in HEK293 and neuronal cells. Neurosci. Lett. 285, 79–82. Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M., Bonini, N.M., 2002. Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson’s disease. Science 295, 865–868. Auluck, P.K., Meulener, M.C., Bonini, N.M., 2005. Mechanisms of suppression of {alpha}-synuclein neurotoxicity by geldanamycin in Drosophila. J. Biol. Chem. 280, 2873–2878. Baba, M., Nakajo, S., Tu, P.-H., Tomita, T., Kakaya, K., Lee, V.M., Trojanowski, J.-Q., Iwatsubo, T., 1998. Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. Am. J. Pathol. 152, 879–884. Benaroudj, N., Zwickl, P., Seemüller, E., Baumeister, W., Goldberg, A.L., 2003. ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. Mol. Cell 11, 69–78. Bence, N.F., Sampat, R.M., Kopito, R.R., 2001. Impairment of the ubiquitin–proteasome system by protein aggregation. Science 292, 1552–1555. Bennett, M.C., Bishop, J.F., Leng, Y., Chock, P.B., Chase, T.N., Mouradian, M.M., 1999. Degradation of alpha-synuclein by proteasome. J. Biol. Chem. 274, 33855–33858. Carter, D.B., Chou, K.C., 1998. A model for structure-dependent binding of Congo red to Alzheimer beta-amyloid fibrils. Neurobiol. Aging 19, 37–40. Chan, H.Y., Warrick, J.M., Gray-Board, G.L., Paulson, H.L., Bonini, N.M., 2000. Mechanisms of chaperone suppression of polyglutamine disease, selectivity, synergy and modulation of protein solubility in Drosophila. Hum. Mol. Genet. 9, 2811–2820. Chartier-Harlin, M.C., Kachergus, J., Roumier, C., Mouroux, V., Douay, X., Lincoln, S., Levecque, C., Larvor, L., Andrieux, J., Hulihan, M., Waucquier, N., Defebvre, L., Amouyel, P., Farrer, M., Destée, A., 2004. Alpha-synuclein locus duplication as a cause of familial Parkinson’s disease. Lancet 364, 1167–1169. Chondrogianni, N., Tzavelas, C., Pemberton, A.J., Nezis, I.P., Rivett, A.J., Gonos, E.-S., 2005. Overexpression of proteasome ␤5 subunit increases the amount of assembled proteasome and confers ameliorated response to oxidative stress and higher survival rates. J. Biol. Chem. 280, 11840–11850. Conway, K.A., Lee, S.J., Rochet, J.C., Ding, T.T., Williamson, R.E., Lansbury, P.T., 2000. Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson’s disease, implications for pathogenesis and therapy. Proc. Natl. Acad. Sci. U.S.A. 97, 571–576. Conway, K.A., Rochet, J.C., Bieganski, R.M., Lansbury, P.T., 2001. Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alphasynuclein adduct. Science 294, 1346–1349. Cuervo, A.-M., Stefanis, L., Fredenburg, R., Lansbury, P.T., Sulzer, D., 2004. Impaired degradation of mutant a-synuclein by chaperone-mediated autophagy. Science 305, 1292–1295. Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T., Zoghbi, H.Y., 1998. Chaperone suppression of aggregation and altered

E. Emmanouilidou et al. / Neurobiology of Aging 31 (2010) 953–968 subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet. 19, 148–154. Dickson, D.-W., Liu, W., Hardy, J., Farrer, M., Mehta, N., Uitti, R., Mark, M., Zimmerman, T., Golbe, L., Sage, J., Sima, A., D’Amato, C., Albin, R., Gilman, S., Yen, S.H., 1999. Widespread alterations of alpha-synuclein in multiple system atrophy. Am. J. Pathol. 155, 1241– 2125. Dixon, C., Mathias, N., Zweig, R.-M., Davis, D.-A., Gross, D.-S., 2005. Alpha-synuclein targets the plasma membrane via the secretory pathway and induces toxicity in yeast. Genetics 170, 47–59. De Los Rios, P., Ben-Zvi, A., Slutsky, O., Azem, A., Goloubinoff, P., 2006. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling. PNAS 103, 6166–6171. Evans, C.G., Wisen, S., Gestwicki, J.E., 2006. Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro. J. Biol. Chem. 281, 33182–33191. Farrer, M., Kachergus, J., Forno, L., Lincoln, S., Wang, D.S., Hulihan, M., Maraganore, D., Gwinn-Hardy, K., Wszolek, Z., Dickson, D., Langston, J.W., 2004. Comparison of kindreds with parkinsonism and alphasynuclein genomic multiplications. Ann. Neurol. 55, 174–179. Ghee, M., Fournier, A., Mallet, J., 2000. Rat alpha-synuclein interacts with Tat binding protein 1, a component of the 26S proteasomal complex. J. Neurochem. 75, 2221–2224. Ghee, M., Melki, R., Mishot, N., Mallet, J., 2005. PA700, the regulatory complex of the 26S proteasome, interferes with a-synuclein assembly. FEBS J. 272, 4023–4033. Giasson, B.I., Duda, J.E., Quinn, S.M., Zhang, B., Trojanowski, J.Q., Lee, V.M., 2002. Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron 34, 521–533. Golberg, A.L., 2003. Protein degradation and protection against misfolded or damaged proteins. Nature 426, 895–899. Gosavi, N., Lee, H.-J., Lee, J.-S., Patel, S., Lee, S.-J., 2002. Golgi fragmentation occurs in the cells with prefibrillar alpha-synuclein aggregates and precedes the formation of fibrillar inclusion. J. Biol. Chem. 277, 48984–48992. Heiser, V., Scherzinger, E., Boeddrich, A., Nordhoff, E., Lurz, R., Schugardt, N., Lehrach, H., Wanker, E.-E., 2000. Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules, implications for Huntington’s disease therapy. Proc. Natl. Acad. Sci. U.S.A. 92, 6739–6744. Hendil, K.-B., Khan, S., Tanaka, K., 1998. Simultaneous binding of PA28 and PA700 activators to 20S proteasomes. Biochem. J. 332, 749–754. Hendil, K.-B., Hartmann-Petersen, R., Tanaka, K., 2002. 26S proteasomes function as stable entities. J. Mol. Biol. 315, 627–636. Hoffman, L., Rechsteiner, M., 1994. Activation of the multicatalytic protease. J. Biol. Chem. 269, 16890–16895. Hoglinger, G.U., Carrard, G., Michel, P.P., Medja, F., Lombes, A., Ruberg, M., Friguet, B., Hirsch, E.C., 2003. Dysfunction of mitochondrial complex I and the proteasome, interactions between two biochemical deficits in a cellular model of Parkinson’s disease. J. Neurochem. 86, 12097–21307. Holmberg, C.I., Staniszewski, K.E., Mensah, K.N., Matouschek, A., Morimoto, R.I., 2004. Inefficient degradation of truncated polyglutamine proteins by the proteasome. EMBO J. 23, 4307–4318. Jabado, O., Wang, Q., Rideout, H.J., Yeasmin, M., Guo, K.X., Vekrellis, K., Papantonis, S., Angelastro, J.M., Troy, C.M., Stefanis, L., 2004. RAIDD aggregation facilitates apoptotic death of PC12 cells and sympathetic neurons. Cell Death Diff. 11, 618–630. Jariel-Encontre, I., Pariat, M., Martin, F., Carillo, S., Salvat, C., Piechaczyk, M., 1995. Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26 S proteasome. J. Biol. Chem. 270, 11623–11627. Kahle, P.-J., Neumann, N., Ozmen, L., Muller, V., Odoy, S., Okamoto, N., Jacob sen, H., Iwatsubo, T., Trojanowski, J.-Q., Takahashi, H., Wakabayashi, K., Bogdanovic, N., Riederer, P., Kretzschmar, H.A., Haass, C., 2001. Selective insolubility of alpha-synuclein in human Lewy body

967

diseases is recapitulated in a transgenic mouse model. Am. J. Pathol. 159, 2215–2225. Kayed, R., Head, E., Thompson, J.-L., McIntire, T.-M., Milton, S.-C., Cotman, C.-W., Glabe, C.-G., 2003. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489. Klucken, J., Shin, Y., Masliah, E., Hyman, B.T., McLean, P.J., 2004. Hsp70 reduces alpha-synuclein aggregation and toxicity. J Biol Chem. 279, 25497–254502. Kristiansen, M., Deriziotis, P., Dimcheff, D.E., Jackson, G.S., Ovaa, H., Naumann, H., Clarke, A.R., van Leeuwen, F.W., Menendez-Benito, V., Dantuma, N.P., Portis, J.L., Collinge, J., Tabrizi, S.J., 2007. Diseaseassociated prion protein oligomers inhibit the 26S proteasome. Mol. Cell. 26, 175–188. Lang-Rollin, I., Rideout, H., Stefanis, L., 2003. Ubiquitinated inclusions and neuronal cell death. Histol. Histopathol. 18, 509–517. Lashuel, H.A., Petre, B.M., Wall, J., Simon, M., Nowak, R.J., Walz, T., Lansbury, P.T., 2002. a-synuclein, especially the Parkinson’s diseaseassociated mutants, forms pore-like annular and tubular protofibrils. J. Mol. Biol. 322, 1089–1102. Lee, H.J., Khoshaghideh, F., Patel, S., Lee, S.J., 2004. Clearance of alpha-synuclein oligomeric intermediates via the lysosomal degradation pathway. J. Neurosci. 24, 1888–1896. Lindersson, E., Beedholm, R., Hojrup, P., Moos, T., Gai, W., Hendil, K.B., Jensen, P.H., 2004. Proteasomal inhibition by alpha-synuclein filaments and oligomers. J. Biol. Chem. 279, 12924–12934. Liu, Y., Fallon, L., Lashuel, H.-A., Liu, Z., Lansbury Jr., P.T., 2002. The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson’s disease susceptibility. Cell 111, 209–218. Martin-Clemente, B., Alvarez-Castelao, B., Mayo, I., Sierra, A.B., Diaz, V., Milan, M., Farinas, I., Gomez-Isla, T., Ferrer, I., Castano, J.G., 2004. ␣-Synuclein expresion levels do not significantly affect proteasome function and expression in mice and stably transfected PC12 cell lines. J. Biol. Chem. 279, 52984–52990. McLean, P.J., Klucken, J., Shin, Y., Hyman, B.T., 2004. Geldanamycin induces Hsp70 and prevents alpha-synuclein aggregation and toxicity in vitro. Biochem. Biophys. Res. Commun. 321, 665–669. Outeiro, T.F., Putcha, P., Tetzlaff, J.E., Spoelgen, R., Koker, M., Carvalho, F., Hyman, B.T., McLean, P.J., 2008. Formation of toxic oligomeric ␣-synuclein species in living cells. PLOS One 3, e1867. Paxinou, E., Chen, Q., Weisse, M., Giasson, B.-I., Norris, E.-H., Rueter, S.M., Trojanowski, J.-Q., Lee, V.M., Ischiropoulos, H., 2001. Induction of alpha-synuclein aggregation by intracellular nitrative insult. J. Neurosci. 21, 8053–8061. Petrucelli, L., O’Farrell, C., Lockhart, P.J., Baptista, M., Kehoe, K., Vink, L., Choi, P., Wolozin, B., Farrer, M., Hardy, J., Cookson, M.R., 2002. Parkin protects against the toxicity associated with mutant alpha-synuclein, proteasome dysfunction selectively affects catecholaminergic neurons. Neuron 36, 1007–1019. Pickart, C.M., Cohen, R.E., 2004. Proteasomes and their kin, proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187. Rideout, H.J., Larsen, K.E., Sulzer, D., Stefanis, L., 2001. Proteasomal inhibition leads to formation of ubiquitin/␣-synuclein immunoreactive inclusions in PC12 cells. J. Neurochem. 78, 899–908. Rideout, H.J., Stefanis, L., 2002. Proteasomal inhibition-induced inclusion formation and death in cortical neurons require transcription and ubiquitination. Mol. Cell Neurosci. 21, 223–238. Rivett, J.A., Bose, S., Pemberton, A.J., Brooks, P., Onion, D., Shirley, D., Stratford, F.L.L., Forti, K., 2002. Assays of proteasome activity in relation to aging. Exp. Gerontol. 37, 1217–1222. Rodgers, K.J., Dean, R.T., 2003. Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates. Int. J. Biochem. Cell Biol. 35, 716–727. Sanchez, I., Mahlke, C., Yuan, J., 2003. Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders. Nature 421, 373–379.

968

E. Emmanouilidou et al. / Neurobiology of Aging 31 (2010) 953–968

Sawada, H., Kohno, R., Kihara, T., Izumi, Y., Sakka, N., Ibi, M., Nakanishi, M., Nakamizo, T., Yamakawa, K., Shibasaki, H., Yamamoto, N., Akaike, A., Inden, M., Kitamura, Y., Taniguchi, T., Shimohama, S., 2004. Proteasome mediates dopaminergic neuronal degeneration and its inhibition causes a-synuclein inclusions. J. Biol. Chem. 279, 10710–10719. Singleton, A.B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R., Lincoln, S., Crawley, A., Hanson, M., Maraganore, D., Adler, C., Cookson, M.R., Muenter, M., Baptista, M., Miller, D., Blancato, J., Hardy, J., GwinnHardy, K., 2003. alpha-Synuclein locus triplication causes Parkinson’s disease. Science 302, 841. Smith, D.M., Chang, S.C., Park, S., Finley, D., Cheng, Y., Goldberg, A.L., 2007. Docking of the proteasomal ATPases’ carboxyl termini in the 20S proteasome’s a ring opens the gate for substrate entry. Mol. Cell 27, 731–744. Smith, W.W., Jiang, H., Pei, Z., Tanaka, Y., Morita, H., Sawa, A., Dawson, V.L., Dawson, T.M., Ross, C.A., 2005. Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutantalphasynuclein-inducedtoxicity. Hum. Mol. Genet. 14, 3801–3811. Snyder, H., Mensah, K., Theisler, C., Lee, J., Matouschek, A., Wolozin, B., 2003. Aggregated and monomeric alpha-synuclein bind to the S6’ proteasomal protein and inhibit proteasomal function. J. Biol. Chem. 278, 11753–11759. Stefanis, L., Larsen, K.E., Rideout, H.J., Sulzer, D., Greene, L.A., 2001a. Expression of A53T mutant but not wild-type a-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death. J. Neurosci. 21, 9549–9560. Stefanis, L., Kholodilov, N., Rideout, H.J., Burke, R.E., Greene, L.A., 2001b. Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells. J. Neurochem. 76, 1165–1176.

Tanaka, E., Sawada, M.T., Morinaga, C., Yokosawa, H., Sawada, H., 2000. Isolation and characterization of a novel 530-kDa protein complex (PC530. capable of associating with the 20S proteasome from Starfish oocytes. Arch. Biochem. Biophys. 374, 181–188. Tanaka, Y., Engelender, S., Igarashi, S., Rao, R.K., Wanner, T., Tanzi, R.E., Sawada, A., Dawson, V.L., Dawson, T.M., Ross, C.A., 2001. Inducible expression of mutant alpha-synuclein decreases proteasome activity and increases sensitivity to mitochondria-dependent apoptosis. Hum. Mol. Genet. 10, 919–926. Tofaris, G.K., Layfield, R., Spillantini, M.G., 2001. Alpha-synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome. FEBS Lett. 509, 22–26. Treier, M., Staszewski, L.M., Bohmann, D., 1994. Ubiquitin-dependent c-jun degradation in vivo is mediated by the delta domain. Cell 78, 787–798. Tseng, B.P., Green, K.N., Chan, J.L., Blurton-Jones, M., Laferla, F.M., 2007. Abeta inhibits the proteasome and enhances amyloid and tau accumulation. Neurobiol. Aging, 2007, doi:10.1016/j. neurobiolaging.2007.04.014. Vekrellis, K., Rideout, H.J., Stefanis, L., 2004. Neurobiology of a-synuclein. Mol. Neurobiol. 30, 1–21. Vekrellis, K., Stefanis, L., 2005. In: Keller, J.N., Stefanis, L. (Eds.), Ubiquitin and the proteasome in neurodegeneration. Kluwer Academic/Plenum, NY. Volles, M.J., Lansbury Jr., P.T., 2003. Zeroing in on the pathogenic form of alpha-synuclein and its mechanism of neurotoxicity in Parkinson’s disease. Biochemistry 42, 7871–7878. Webb, J.L., Ravikumar, B., Atkins, J., Skepper, J.N., Rubinstein, D.C., 2003. Alpha-synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 278, 25009–32501.