Title: Aggresomes Formed by α-Synuclein and Synphilin-1 Are Cytoprotective
Abstract: Lewy bodies (LBs), which are the hallmark pathologic features of Parkinson's disease and of dementia with LBs, have several morphologic and molecular similarities to aggresomes. Whether such cytoplasmic inclusions contribute to neuronal death or protect cells from the toxic effects of misfolded proteins remains controversial. In this report, the role of aggresomes in cell viability was addressed in the context of over-expressing α-synuclein and its interacting partner synphilin-1 using engineered 293T cells. Inhibition of proteasome activity elicited the formation of juxtanuclear aggregates with characteristics of aggresomes including immunoreactivity for vimentin, γ-tubulin, ubiquitin, proteasome subunit, and hsp70. As expected from the properties of aggresomes, the microtubule disrupting agents, vinblastin and nocodazole, markedly prevented the formation of these inclusions. Similar to LBs, the phosphorylated form of α-synuclein co-localized in these synphilin-1-containing aggresomes. Although the caspase inhibitor z-VAD-fmk significantly reduced the number of apoptotic cells, it had no impact on the percentage of aggresome-positive cells. Finally, quantitative analysis revealed aggresomes in 60% of nonapoptotic cells but only in 10% of apoptotic cells. Additionally, α-synuclein-induced apoptosis was not coupled with increased prevalence of aggresome-bearing cells. Taken together, these observations indicate a disconnection between aggresome formation and apoptosis, and support a protective role for these inclusions from the toxicity associated with the combined over-expression of α-synuclein and synphilin-1. Lewy bodies (LBs), which are the hallmark pathologic features of Parkinson's disease and of dementia with LBs, have several morphologic and molecular similarities to aggresomes. Whether such cytoplasmic inclusions contribute to neuronal death or protect cells from the toxic effects of misfolded proteins remains controversial. In this report, the role of aggresomes in cell viability was addressed in the context of over-expressing α-synuclein and its interacting partner synphilin-1 using engineered 293T cells. Inhibition of proteasome activity elicited the formation of juxtanuclear aggregates with characteristics of aggresomes including immunoreactivity for vimentin, γ-tubulin, ubiquitin, proteasome subunit, and hsp70. As expected from the properties of aggresomes, the microtubule disrupting agents, vinblastin and nocodazole, markedly prevented the formation of these inclusions. Similar to LBs, the phosphorylated form of α-synuclein co-localized in these synphilin-1-containing aggresomes. Although the caspase inhibitor z-VAD-fmk significantly reduced the number of apoptotic cells, it had no impact on the percentage of aggresome-positive cells. Finally, quantitative analysis revealed aggresomes in 60% of nonapoptotic cells but only in 10% of apoptotic cells. Additionally, α-synuclein-induced apoptosis was not coupled with increased prevalence of aggresome-bearing cells. Taken together, these observations indicate a disconnection between aggresome formation and apoptosis, and support a protective role for these inclusions from the toxicity associated with the combined over-expression of α-synuclein and synphilin-1. Parkinson's disease (PD) 1The abbreviations used are: PDParkinson's diseaseLBLewy bodyz-VAD-fmkbenzyloxycarbonyl-VAD-fluoromethyl ketonePBSphosphate-buffered salineDAPI4′,6-diamidino-2-phenylindoleEGFPenhanced green fluorescent proteinANOVAanalysis of variance. is a progressive, neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra and by cytoplasmic inclusions known as Lewy bodies (LBs). The LB is a well recognized pathologic feature in several additional age-related neurodegenerative disorders, including dementia with LBs and Alzheimer's disease (1Hashimoto M. Masliah E. Brain Pathol. 1999; 9: 707-720Crossref PubMed Scopus (221) Google Scholar). Although the molecular mechanisms that lead to the genesis of LBs have not been fully elucidated, several lines of evidence suggest that dysfunction of the ubiquitin-proteasome pathway is generally involved in the pathogenesis of PD (2McNaught K.S. Olanow C.W. Halliwell B. Isacson O. Jenner P. Nat. Rev. Neurosci. 2001; 2: 589-594Crossref PubMed Scopus (456) Google Scholar). For example, LBs are rich in ubiquitin and proteasome subunits (3Iwatsubo T. Yamaguchi H. Fujimuro M. Yokosawa H. Ihara Y. Trojanowski J.Q. Lee V.M. Am. J. Pathol. 1996; 148: 1517-1529PubMed Google Scholar, 4Ii K. Ito H. Tanaka K. Hirano A. J. Neuropathol. Exp. 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Furthermore, two more gene products associated with hereditary PD, namely, the E3 ubiquitin ligase parkin and ubiquitin C-terminal hydrolase L1, are both LB components (12Schlossmacher M.G. Frosch M.P. Gai W.P. Medina M. Sharma N. Forno L. Ochiishi T. Shimura H. Sharon R. Hattori N. Langston J.W. Mizuno Y. Hyman B.T. Selkoe D.J. Kosik K.S. Am. J. Pathol. 2002; 160: 1655-1667Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) and are functional enzymes in the ubiquitin-proteasome pathway. Parkinson's disease Lewy body benzyloxycarbonyl-VAD-fluoromethyl ketone phosphate-buffered saline 4′,6-diamidino-2-phenylindole enhanced green fluorescent protein analysis of variance. Whether LBs are cytotoxic or cytoprotective to neuronal cells remains debatable. These inclusions could potentially be deleterious, because the number of cortical LBs reportedly correlates with the severity of clinical symptoms in dementia with LBs (13Hurtig H.I. Trojanowski J.Q. Galvin J. Ewbank D. 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Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1786) Google Scholar), as well as inhibition of proteasomal activity have been associated with the formation of stable, multi-ubiquitinated aggregates termed aggresomes. These inclusions are characterized by their localization to the centrosome and by the redistribution of intermediate filaments. The function of aggresomes is thought to be the disposition of misfolded or otherwise damaged proteins that accumulate in cells because of oxidative stress, proteasomal impairment, or other cellular insults. Their formation requires an energy-dependent intracellular transport mechanism, because the intermediate state of aggresomes or micro-aggregates that form in the peripheral cytoplasm are transported to the centrosome through the microtubular cytoskeleton mediated by dynein/dynactin complexes (22Johnston J.A. Illing M.E. Kopito R.R. Cell Motil. Cytoskeleton. 2002; 53: 26-38Crossref PubMed Scopus (184) Google Scholar). Thus, aggresomes presumably form as a cellular defense mechanism against elevated concentrations of unwanted proteins (23Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1614) Google Scholar). However, a cytoprotective function for aggresome has not been directly demonstrated. Recently, several similarities between LBs and aggresomes have been observed. In cellular models, the aggresomes that result from parkin over-expression and proteasomal inhibition have both morphologic and immunocytochemical features of LBs, including the core and halo organization and the presence of vimentin, γ-tubulin, α-synuclein, synphilin-1, proteasome subunits, and chaperones (17Junn E. Lee S.S. Suhr U.T. Mouradian M.M. J. Biol. Chem. 2002; 277: 47870-47877Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Further, postmortem studies with Parkinson-affected brains have confirmed that LBs contain centrosome components including γ-tubulin, pericentrin, PA700, and P28 (24McNaught K.S. Shashidharan P. Perl D.P. Jenner P. Olanow C.W. Eur. J. Neurosci. 2002; 16: 2136-2148Crossref PubMed Scopus (233) Google Scholar). Thus, LBs seem to be formed through a mechanism similar to that of aggresomes. To date, two point mutations, A53T and A30P in the α-synuclein gene have been associated with autosomal dominant PD (9Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6734) Google Scholar, 25Kruger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. 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Neurochem. 2002; 83: 346-352Crossref PubMed Scopus (34) Google Scholar) that synphilin-1 is polyubiquitinated and subjected to proteasomal degradation. Upon proteasomal impairment, synphilin-1 is preferentially concentrated in perinuclear aggregates in 293T cells together with ubiquitin and α-synuclein. Additionally, synphilin-1 overexpression increases the vulnerability to the toxicity of proteasome inhibitors (44Lee G. Junn E. Tanaka M. Kim Y.M. Mouradian M.M. J. Neurochem. 2002; 83: 346-352Crossref PubMed Scopus (34) Google Scholar). In the present study, we addressed the question whether aggresomes are cytoprotective or cytotoxic. We first characterized aggresomes formed in synphilin-1-engineered cells challenged by proteasome inhibitors. We then examined the role of these inclusions in apoptotic cell death. We found that aggresome formation does not directly correlate with cell death. Rather, aggresomes seem to promote cell survival. Cells, Antibodies, and Reagents—293T cells (ATCC) were transfected with FLAG-synphilin-1 to isolate stable transformant lines (named Synph-293) and cultured in the presence of 1 mg/ml G418 (44Lee G. Junn E. Tanaka M. Kim Y.M. Mouradian M.M. J. Neurochem. 2002; 83: 346-352Crossref PubMed Scopus (34) Google Scholar). Synph-293 or 293T cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen) at 37 °C in 10% CO2. The following antibodies were used: FLAG Cy3-conjugated monoclonal (1:2000, Sigma), GTU-88 γ-tubulin monoclonal (1:1000, Sigma), V9 vimentin monoclonal (1:2000, Oncogene, San Diego, CA), 20S proteasome subunit rabbit polyclonal (1:200, Calbiochem, La Jolla, CA), Hsp70 rabbit polyclonal (1:200, Upstate Biotechnology, Waltham, MA), ubiquitin rabbit polyclonal (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), fluorescein-conjugated anti-mouse or anti-rabbit (1:400, Santa Cruz Biotechnology). An antibody raised against phosphorylated α-synuclein at Ser-129 was described previously (45Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell. Bio. 2002; 4: 160-164Crossref PubMed Scopus (162) Google Scholar). MG132 and lactacystin were purchased from Calbiochem, vinblastin sulfate and nocodazole from Sigma, and z-VAD-fmk from Promega (Madison, WI). cDNA Constructs and Transfection—An expression vector for FLAG-tagged synphilin-1 was cloned as described before (44Lee G. Junn E. Tanaka M. Kim Y.M. Mouradian M.M. J. Neurochem. 2002; 83: 346-352Crossref PubMed Scopus (34) Google Scholar). Wild-type, A53T, and A30P α-synuclein cloned previously (46Kanda S. Bishop J.F. Eglitis M.A. Yang Y. Mouradian M.M. Neuroscience. 2000; 97: 279-284Crossref PubMed Scopus (180) Google Scholar) were transferred to pcDNA3.1 by restriction enzyme cleavage. To generate α-synuclein cDNA with an S129A substitution, wild-type α-synuclein cDNA was used as template to introduce the T385G point mutation using the QuikChange site-directed mutagenesis kit (Stratagene). Cells were transfected with the calcium phosphate method using a commercially available kit (Clontech, Palo Alto, CA). Seven micrograms of DNA were suspended with calcium phosphate in HEPES-buffered saline per each 6-cm plate. After a 10-h incubation, the medium was removed, and cells were rinsed once with phosphate-buffered saline (PBS) and then incubated with fresh medium. As appropriate, 0.35 μg of pEGFP-C2 (Clontech) was mixed with DNA. Immunocytochemistry—Synph-293 cells grown on poly-lysine-coated glass coverslips were rinsed once with PBS and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature. After washing with PBS, cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min and treated with 1% fetal bovine serum in PBS for 20 min. Cells were incubated with primary antibody diluted with 1% fetal bovine serum in PBS for 2 h at 4 °C. Coverslips were rinsed with PBS five times and then incubated for 1 h with secondary antibody at 4 °C. After removing from the antibody solution, cells were incubated with 10 μm 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 2 min. And after washing with PBS three times for 5 min each, the coverslips were mounted with the anti-fading medium (Southern Biotechnology, Birmingham, AL) and visualized with a fluorescence microscope (Axiophot, Zeiss, Thornwood, NY). Quantification of Apoptotic Cells and Aggresome Formation—Cells transfected with the indicated cDNA(s) and enhanced green fluorescent protein (EGFP) were transferred to a glass coverslip coated with polylysine and cultured for 24 h. Cells were then treated with 10 μm MG132 or lactacystin. In certain experiments, 100 μm z-VAD-fmk, 1 μm of vinblastin, or 100 ng/ml of nocodazole were added as indicated. After incubation for 14 h, cells were fixed and immunostained using anti-FLAG Cy3-conjugated antibody, followed by staining with DAPI as described above. EGFP-expressing cells were visualized using a fluorescence microscope with a fluorescein isothiocyanate filter and were determined to be apoptotic based on DAPI staining that showed condensed or fragmented chromatin. Aggresome formation was detected as a single perinuclear anti-FLAG-positive inclusion and was confirmed by the indented shape of the nucleus. For each cell condition, more than 200 transfected cells from randomly selected fields were analyzed with a fluorescence microscope (Optiphot-2, Nikon) and IPLab software (Biovision Technologies, Exon, PA). Each experiment was repeated three times. Consistent with our previous report (44Lee G. Junn E. Tanaka M. Kim Y.M. Mouradian M.M. J. Neurochem. 2002; 83: 346-352Crossref PubMed Scopus (34) Google Scholar), 293T cells stably over-expressing synphilin-1 (Synph-293) developed perinuclear round structures immunoreactive to FLAG-synphilin-1 after treatment with the proteasome inhibitor MG132 for 14 h (Fig. 1). To confirm that these inclusions represented aggresomes and to demonstrate their similarities to LBs, further characterization was undertaken. (i) Vimentin immunoreactivity, which distributes diffusely throughout the cytoplasm under basal conditions (data not shown), localized to the halo of synphilin-1-positive perinuclear inclusions upon proteasome inhibition (Fig. 1a). (ii) γ-Tubulin, which is a normal component of the centrosome (24McNaught K.S. Shashidharan P. Perl D.P. Jenner P. Olanow C.W. Eur. J. Neurosci. 2002; 16: 2136-2148Crossref PubMed Scopus (233) Google Scholar, 47Zheng Y. Jung M.K. Oakley B.R. Cell. 1991; 65: 817-823Abstract Full Text PDF PubMed Scopus (350) Google Scholar) and is only weakly immunoreactive in untreated cells (data not shown), acquired an intense signal upon MG132 challenge (Fig. 1b). (iii) Synphilin-1 aggregates were immunoreactive to antibodies against ubiquitin (Fig. 1c), 20S proteasome subunit (Fig. 1d), and to Hsp-70 (Fig. 1e), all of which are components of LBs (4Ii K. Ito H. Tanaka K. Hirano A. J. Neuropathol. Exp. Neurol. 1997; 56: 125-131Crossref PubMed Scopus (200) Google Scholar, 28Auluck P.K. Chan H.Y. Trojanowski J.Q. Lee V.M. Bonini N.M. Science. 2002; 295: 865-868Crossref PubMed Scopus (1073) Google Scholar, 48Kuzuhara S. Mori H. Izumiyama N. Yoshimura M. Ihara Y. Acta Neuropathol. 1988; 75: 345-353Crossref PubMed Scopus (355) Google Scholar). In particular, the immunoreactivity to Hsp-70 localized intensely to the halo of the aggregates (Fig. 1e), whereas that of ubiquitin and 20S proteasome subunit distributed homogeneously in both the core and halo (Fig. 1, c and d). Finally, an antibody raised against phosphorylated α-synuclein at Ser-129 immunostained these inclusions (Fig. 1f), similar to LBs (45Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell. Bio. 2002; 4: 160-164Crossref PubMed Scopus (162) Google Scholar). Quantitative analysis indicated that only about 7% of Synph-293 cells harbored synphilin-1-positive aggresomes in the absence of a proteasome inhibitor, but that this percentage increased dramatically to 48% in the presence of MG132 (Fig. 2). Treatment with microtubule destabilizing agents, vinblastin or nocodazole, reduced the number of aggregate-containing cells, consistent with the properties of aggresomes (23Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1614) Google Scholar). Thus, aggresomes formed in Synph-293 cells are dependent upon the microtubule-mediated intracellular transport system. The relationship between aggresome formation and apoptotic cell death upon proteasomal inhibition was assessed next (Fig. 3). FLAG-synphilin-1-positive aggresomes co-localizing with ubiquitin were detected in both non-apoptotic as well as apoptotic cells identified by condensed DAPI-stained nuclei (Fig. 3a). Similarly, vimentin immunoreactive aggresomes were observed in some apoptotic and non-apoptotic cells (Fig. 3b). Thus, aggresomes can be detected even in relatively late stages of apoptosis. However, neither FLAG-synphilin-1 (Fig. 3a) nor vimentin immunoreactivity (Fig. 3b), along with DAPI staining, revealed a strong correlation between aggresomes and apoptotic nuclei. Some aggresome-bearing cells were found not to be apoptotic, whereas some aggresome-negative cells were clearly apoptotic. To begin quantifying the relationship between cell viability and aggresome formation in the context of synphilin-1 and α-synuclein over-expression, Synph-293 cells were transiently co-transfected with different cDNA isoforms of α-synuclein. In addition, EGFP was co-transfected to distinguish transfected cells from non-transfected ones. Approximately 30% of cells were fluorescent by EGFP in each transfection experiment. Green fluorescent positive cells were evaluated for apoptosis by DAPI staining and for aggresome content by FLAG immunoreactivity, as in Fig. 3a. The pathogenic mutants of α-synuclein, A53T and A30P, exacerbated cell death more than their wild-type counterpart, whereas the phosphorylation mutant S129A did not increase cell death (Fig. 4). The rank order of apoptotic cell death was: mock control < wild-type = S129A < A30P < A53T. Toxicity due to pathogenic α-synuclein mutants increased significantly by 2.0- to 2.5-fold, compared with mock control cells. On the other hand, aggresome formation in α-synuclein-transfected cells was only minimally higher than in empty vector transfected cells. The count of aggresome-bearing cells with A30P expression, for example, was merely 1.3-fold higher than mock control. These data suggest that aggresome formation does not necessarily correlate with apoptotic cell death. To further examine a possible connection between aggresome formation and apoptosis, these two parameters were measured in cells co-expressing wild-type α-synuclein and synphilin-1 after treatment with the caspase inhibitor z-VAD-fmk in the presence of MG132. Proteasome inhibition resulted in apoptotic morphology in about 19% of these cells. As expected, this percentage diminished significantly down to 10% in the presence of z-VAD-fmk (Fig. 5a). Aggresome count, on the other hand, which was about 50% in the presence of MG132 alone, was not affected by addition of the caspase inhibitor (Fig. 5b). These observations confirmed that the increased cell death due to co-expression of α-synuclein and synphilin-1 is dissociated from aggresome formation. To determine the role of aggresomes in cell death, formation of these inclusions in apoptotic cells was quantified directly. Synph-293 cells transfected with various α-synuclein isoforms were individually categorized based on apoptotic morphology and aggresome formation (Fig. 6). Among apoptotic cells, a much larger proportion was aggresome-negative compared with aggresome-positive cells (Fig. 6a). For example, about 20% of A53T α-synuclein-transfected cells were apoptotic but aggresome-negative, whereas only about 2% were apoptotic and aggresome-positive. Notably, the increase in cell death due to co-expression of synphilin-1 and α-synuclein derived exclusively from aggresome-negative cells, whereas cells bearing aggresomes did not seem to be susceptible to this form of cell death (Fig. 6a). On the other hand, among non-apoptotic cells over-expressing α-synuclein isoforms, aggresome-positive cells were slightly more prevalent than aggresome-negative cells (Fig. 6b). Among cells transfected with α-synuclein isoforms, aggresomes were present in 53–60% of non-apoptotic cells but only in 10–13% of apoptotic cells. Similar results were obtained when counting aggresomes based on vimentin immunostaining (data not shown). Taken together, we concluded that cells with aggresomes are more resistant to toxicity associated with the co-expression of α-synuclein and synphilin-1 than cells without aggresomes in the presence of a proteasome inhibitor. The present data highlight three aspects of inclusion body formation associated with synphilin-1 and α-synuclein overexpression and with proteasomal impairment. (i) These cytoplasmic inclusions represent aggresomes based both on morphological and molecular characteristics, including the round structure with a core and halo organization and the juxtanuclear localization, as well as on immunocytochemical evidence confirming the presence of centrosome comp