2003/11/01 by George K. Tofaris, Azam Razzaq, Bernardino Ghetti +2 · 28 citations
Medicine · Neuroscience · #Parkinson's Disease Mechanisms and Treatments #Neurological diseases and metabolism #Lysosomal Storage Disorders Research
paper · pdf · doi:10.1074/jbc.m308041200
Lewy bodies are intracellular fibrillar inclusions composed of α-synuclein. They constitute the pathological hallmark of Parkinson's disease, dementia with Lewy bodies, and other neurodegenerative diseases. Although the majority of Lewy bodies are stained for ubiquitin by immunohistochemistry, the substrate for this modification is poorly understood. Insoluble, urea-soluble α-synuclein was separated from soluble fractions and subjected to two-dimensional gel electrophoresis to further characterize pathogenic α-synuclein species from disease brains. By using this approach, we found that in sporadic Lewy body diseases a highly modified, disease-associated 22–24-kDa α-synuclein species is ubiquitinated. Conjugation of one, two, and, to a lesser extent, three ubiquitins was detected. This 22–24-kDa α-synuclein species represents partly phosphorylated protein. Furthermore, no generalized impairment of the proteolytic activity of the proteasome was detected in brain regions with Lewy body pathology. Because unmodified α-synuclein is degraded by the proteasome in a ubiquitin-independent manner, these data suggest that accumulation of modified 22–24-kDa α-synuclein is a disease-specific event which may overwhelm the proteolytic system, leading to aberrant ubiquitination. Accordingly, carboxyl-terminal-truncated α-synuclein, presumably the result of aberrant proteolysis, is found only in association with α-synuclein aggregates. Lewy bodies are intracellular fibrillar inclusions composed of α-synuclein. They constitute the pathological hallmark of Parkinson's disease, dementia with Lewy bodies, and other neurodegenerative diseases. Although the majority of Lewy bodies are stained for ubiquitin by immunohistochemistry, the substrate for this modification is poorly understood. Insoluble, urea-soluble α-synuclein was separated from soluble fractions and subjected to two-dimensional gel electrophoresis to further characterize pathogenic α-synuclein species from disease brains. By using this approach, we found that in sporadic Lewy body diseases a highly modified, disease-associated 22–24-kDa α-synuclein species is ubiquitinated. Conjugation of one, two, and, to a lesser extent, three ubiquitins was detected. This 22–24-kDa α-synuclein species represents partly phosphorylated protein. Furthermore, no generalized impairment of the proteolytic activity of the proteasome was detected in brain regions with Lewy body pathology. Because unmodified α-synuclein is degraded by the proteasome in a ubiquitin-independent manner, these data suggest that accumulation of modified 22–24-kDa α-synuclein is a disease-specific event which may overwhelm the proteolytic system, leading to aberrant ubiquitination. Accordingly, carboxyl-terminal-truncated α-synuclein, presumably the result of aberrant proteolysis, is found only in association with α-synuclein aggregates. Lewy bodies (LB) 1The abbreviations used are: LB, Lewy body; PD, Parkinson's disease; DLB, dementia with Lewy bodies; AD, Alzheimer's disease; SN, substantia nigra; 2-DE, two-dimensional gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride.1The abbreviations used are: LB, Lewy body; PD, Parkinson's disease; DLB, dementia with Lewy bodies; AD, Alzheimer's disease; SN, substantia nigra; 2-DE, two-dimensional gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride. are intracytoplasmic eosinophilic inclusions, which (ultrastructurally) are made of a core of granular and filamentous material surrounded by radially oriented filaments 10–15 nm in diameter (1Forno L.S. J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Crossref PubMed Scopus (1236) Google Scholar). The element of the LB fibril remained unknown until genetic studies in early onset autosomal-dominant Parkinson's disease (PD) led to the identification of two mutations in the α-synuclein gene (2Polymeropoulos M.H. Lavendan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanasiadou 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 (6600) Google Scholar, 3Kruger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schos L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3297) Google Scholar). This finding was followed by the identification of α-synuclein as the major component of the LB fibrils in sporadic PD and dementia with LB (DLB) (4Spillantini M.G. Schmidt M.L. Lee V.M-Y. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 338: 839-840Crossref Scopus (6111) Google Scholar, 5Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2396) Google Scholar). LB pathology and α-synuclein aggregation in neurons may contribute to their dysfunction and degeneration. Formation of α-synuclein fibrils has been studied extensively in in vitro systems using recombinant protein. However, the mechanism by which α-synuclein, a natively unfolded protein, accumulates in neurons to form insoluble fibrils with amyloid characteristics is largely unknown. It is feasible that in vivo post-translational modifications interfere with the function and/or degradation of α-synuclein or alter its biophysical properties in a way to facilitate aggregation. Alternatively, protein modifications may occur in an attempt to prevent aberrant interactions and/or inhibit further aggregation. Therefore, a detailed understanding of the extent to which disease-associated α-synuclein is modified may provide insights into cellular pathways that are activated during fibril formation. In this study, we used differential centrifugation and 2-dimensional gel electrophoresis (2-DE) to characterize LB-associated α-synuclein. This approach allows the separation of proteins on the basis of their solubility, charge, and molecular mass. By using this method, we show that in sporadic LB disorders, a disease-associated 22–24-kDa α-synuclein species is a substrate for mono-, di-, and trirather than poly-ubiquitination. This 22–24-kDa species gives a characteristic pattern on 2-DE, which is consistent with a highly modified form of the protein, and we identify phosphorylation as one of the modifications. Furthermore, we show that accumulation of ubiquitinated α-synuclein is not invariably associated with significant impairment of proteasome function. These data may help to unravel the role of the ubiquitin-proteasome pathway in LB formation. Human Brain Tissue—Brain tissue was obtained from the Neuropathology Department of the University of Indiana School of Medicine. The diagnoses of PD (n = 3, age of death = 70 ± 8 years, duration of disease = 6.3 ± 3 years), DLB (n = 3, age of death = 75 ± 1 years, duration of disease = 7.7 ± 2 years), and Alzheimer's disease (AD; n = 3, age of death = 81 ± 5 years, duration of disease = 12 ± 2 years) was established by standard neuropathological criteria. Immunohistochemistry using anti-α-synuclein and anti-ubiquitin antibodies was performed as described previously (5Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2396) Google Scholar). Four cases with no neurological disease were used as age-matched controls (age of death = 65.8 ± 9 years). Four brain regions were examined from each case: substantia nigra (SN), frontal, cingulate, and occipital cortices. Fractionation of Human Brain Tissue—Subcellular fractionation of human brain was performed based on a modification of a previously published protocol (6Culvenor J.G. McLean C.A. Cutt S. Campbell B.C. Maher F. Jakala P. Hartmann T. Beyreuther K. Masters C.L. Li Q.X. Am. J. Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Campell B.C.V. McLean C.A. Culvenor J.G. Gai W.P. Blumbergs P.C. Jakala P. Beyreuther K. Masters C.L. Li Q.-X. J. Neurochem. 2001; 76: 87-96Crossref PubMed Scopus (160) Google Scholar) as follows. Approximately 0.5 g of tissue was homogenized on ice in 10 volumes of TBS+ (50 mm Tris-HCl, pH 7.4, 175 mm NaCl, 5 mm EDTA, 0.1 mm PMSF, 1 mm N-ethylmaleimide, plus complete proteasome inhibitor mixture; Roche Diagnostics, Mannheim, Germany). After 5 min of centrifugation at 1,000 × g, the supernatant was ultracentrifuged for 30 min at 120,000 × g at 4 °C. The resulting supernatant represented the TBS+ soluble fraction. All subsequent centrifugation steps were performed at 120,000 × g for 20 min at 4 °C. The pellet was rinsed twice with TBS+ and extracted sequentially with 500 μl of TBS+ containing 1% Triton X-100 and TBS+, 1 M sucrose to remove myelin. The pellet was then extracted with 500 μl of RIPA buffer (50 mm Tris-HCl, pH 7.4, 175 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS). The extensively washed detergent-insoluble pellet was solubilized either in 8 m urea/5% SDS (and termed the urea-soluble fraction) or buffer A+ (10 mm Tris-HCl, pH 8.8, 7 m urea, 2 m thiourea, 5 mm MgCl2, 2% amidosulfobetaine-14) for 2-DE. The 1,000 × g pellet from the first step was subsequently extracted in the same way. Immunoblotting—20–30 μg of extract from each fraction was loaded per lane and separated on 12 or 15% SDS-polyacrylamide gel and transferred to nitrocellulose. The membranes were then incubated with either 3% BSA or 4% milk in TBS to block nonspecific binding. After washing, the membranes were incubated overnight with appropriate primary antibodies. Monoclonal antibodies Syn-1 (1:600; Transduction Laboratories, Lexington, KY), LB509 (1:5000; Zymed Laboratories Inc., San Francisco, CA), polyclonal antisera directed against ubiquitin (1: 800; Dako, Cambridgeshire, UK), carboxy terminus of α-synuclein (PER4; 1:1000), and phosphoserine 129 of α-synuclein (PS129, 1:1000; kind gift of Dr. T. Iwatsubo) were used. Bound antibodies were visualized with peroxidase-conjugated secondary antisera (1:2000; Dako) and enhanced chemiluminescence (PerkinElmer Life Sciences). Deglycosylation Assay—Urea-soluble fractions from human cases with LB disease were dialyzed overnight against 50 mm Tris-HCl, pH 7.2 to remove urea. 30 μl of dialysates were subjected to a deglycosylation assay using N-glycosidase, sialidase A, and endo-O-glycosidase following the manufacturer's instructions (ProZyme, San was used as Deglycosylation in was using 5 of which which was visualized with and as substrate Laboratories, 2-DE, were in μl of buffer μl were then into a of μl with buffer m urea, 2 m thiourea, 2% mm 1% and loaded pH by for a using an on the was subsequently performed at 500 for 1 for 1 and for 5 After were in SDS buffer mm Tris-HCl, 8 m urea, 1% for loaded 10 or using a S.E. and for min at 20 per then for at per were then and as for gel in Human brain regions were SN, frontal, cingulate, and occipital cortices. The were on ice with a each of the brain regions three were for each were and homogenized in three volumes of 50 mm pH The extract was at for 5 min at 4 °C. 50 μg of each was for the activity of the proteasome as described previously R. M.G. 2001; PubMed Scopus Google Scholar) using 20 of as The were from the and as min per of protein. and DLB cases with to were to to Immunohistochemistry with anti-α-synuclein antibodies LB, bodies, and Lewy in DLB and PD and In anti-ubiquitin antibodies stained only of the inclusions Fractionation of from Human the extent to which LB-associated α-synuclein is modified, we used a that is a modification of a previously protocol (6Culvenor J.G. McLean C.A. Cutt S. Campbell B.C. Maher F. Jakala P. Hartmann T. Beyreuther K. Masters C.L. Li Q.X. Am. J. Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Campell B.C.V. McLean C.A. Culvenor J.G. Gai W.P. Blumbergs P.C. Jakala P. Beyreuther K. Masters C.L. Li Q.-X. J. Neurochem. 2001; 76: 87-96Crossref PubMed Scopus (160) Google Scholar) and that allows the separation of of α-synuclein from soluble This is based differential fractionation that not the from the of and The of this approach was to identify which the of LB-associated of α-synuclein filamentous and The fractions were by α-synuclein was detected in and fractions from and and of from PD, DLB, and as in In these α-synuclein was detected by using Syn-1 as a of in with the molecular of protein in disease cases and controls (5Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2396) Google Scholar, R. M.G. Goedert M. PubMed Scopus Google Scholar). molecular α-synuclein were detected. In to in which urea-soluble were of α-synuclein, was found in urea-soluble from PD and DLB and molecular were the species in this fraction. molecular with recombinant protein, three were detected with Syn-1 which the first of the protein. to α-synuclein a 22–24-kDa as as α-synuclein These were examined using a of antibodies against and The and 22–24-kDa not the protein were detected with LB509 and directed against the carboxy terminus of the protein (5Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2396) Google Scholar, R. Crowther R.A. Lee V.M-Y. Trojanowski J.Q. T. Goedert M. 1999; PubMed Scopus Google Scholar). These that the is a protein, the and are composed of protein. Because a form of α-synuclein with a molecular of was in cases of with mutations in the gene H. M.G. A. R. Science. 2001; PubMed Scopus Google we the 22–24-kDa α-synuclein is by from sporadic LB disease with the same as used by H. M.G. A. R. Science. 2001; PubMed Scopus Google Scholar). found that these at the used by not alter the of the 22–24-kDa α-synuclein to the as detected with LB509 In a significant of proteins was detected with Deglycosylation of used as was detected by as a in the of the protein from 75 to 50 not Lewy an attempt to further the characteristics of disease-associated α-synuclein, urea-soluble fractions from disease cases and controls were subjected to further separation a pH from 3 to 10 proteins were separated to their molecular by 2-DE. the of α-synuclein in 2-DE, we loaded recombinant protein, which was found to at the molecular of as in electrophoresis and of In brain regions with LB, α-synuclein a characteristic pattern which of of molecular charge, as as molecular This pattern was detected in extracted α-synuclein was with the recombinant protein, we detected the protein at a form at a molecular and a 22–24-kDa The represents a modified disease-associated form of α-synuclein was not detected in and of of of or molecular separated the pH by an in This result was consistent with the finding of a 22–24-kDa using and and 22–24-kDa the majority of LB are stained with antibodies to the of the ubiquitinated in these inclusions is of each of ubiquitin the molecular of a protein on by and its that of ubiquitin from PD and DLB subjected to two-dimensional followed by with either ubiquitin or anti-α-synuclein antibodies. This that of the an ubiquitinated form of the protein ubiquitinated α-synuclein was detected in soluble fractions or brain regions not regions of were on of a molecular of and, to a lesser extent, with associated This pattern of molecular and is consistent with the of a of mono-, di-, and α-synuclein. two-dimensional α-synuclein has an molecular of and to the substrate for ubiquitination. the other we that the pattern of ubiquitinated α-synuclein was to that of the 22–24-kDa which was not for ubiquitin and with and with α-synuclein, the 22–24-kDa species the appropriate and that this is the α-synuclein substrate for These data show that in sporadic LB disease, a modified α-synuclein species of is a substrate for 22–24-kDa on pattern of which separated the pH not in their molecular that these of α-synuclein on was previously to LB-associated α-synuclein with of protein H. Hasegawa M. A. E. J. K. T. Nat. PubMed Scopus Google Scholar). that protein was only of the 22–24-kDa species and associated ubiquitinated protein were in LB 22–24-kDa α-synuclein was in brain regions containing Therefore, we to accumulation of modified 22–24-kDa α-synuclein in disease is associated with proteasome this we proteasome activity in regions from brain from PD, DLB, AD, and age-matched SN, which is to LB pathology in PD and DLB, cingulate, which are in DLB, and occipital which is of found a significant in proteasome function in the of PD and DLB with cases However, no significant was detected in cingulate, and occipital of PD, DLB, and cases further the proteolytic activity of the proteasome in and by tissue from DLB and in diseases is found was a in proteasome activity in brain regions with no significant was detected in DLB and In this study, we the first of LB-associated α-synuclein based on solubility, charge, and molecular mass. By using this approach, we in a modified 22–24-kDa α-synuclein is a substrate for Furthermore, the proteolytic activity of the proteasome in with LB-associated 22–24-kDa α-synuclein was not invariably of studies that ubiquitin and of the ubiquitin are found in LB, the of the substrate for Although we proteins in fractions ubiquitinated α-synuclein was extracted only with disease-associated insoluble and not in soluble In ubiquitinated α-synuclein a characteristic pattern of from to separated by and a of as by the of a ubiquitin data show that one or two and, to a lesser extent, three ubiquitin are on LB-associated α-synuclein. This pattern is to that of in filaments of which is M. Hasegawa M. K. M. K. Full Text PDF PubMed Scopus Google Scholar). It is established that for degradation to of at ubiquitins on a protein is Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). and previously that of unmodified α-synuclein not occur of the proteasome in R. M.G. 2001; PubMed Scopus Google Scholar, J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, K. C. K. F. PubMed Scopus Google Scholar, D. L. J. Neurochem. 2001; PubMed Scopus Google Scholar, L. PubMed Scopus Google Scholar). This result is in with the which show that the α-synuclein is not ubiquitinated. these data the that degradation is to a major mechanism for α-synuclein of LB-associated α-synuclein represents a disease-specific In this are two an of in their attempt to and/or proteins either the which or the which M. Science. PubMed Scopus Google Scholar). Alternatively, a to interactions of Neuropathol. 2001; PubMed Scopus Google Scholar). one of these is to In with a disease-specific pathway is the finding that is invariably associated with further post-translational modifications. Because of each of ubiquitin the molecular of a protein by and its that of ubiquitin of by and provide into the of ubiquitinated 2-DE, we show for the first that in sporadic LB a 22–24-kDa α-synuclein species and not protein is the substrate for This finding is in to a by M. H. T. K. H. Lee V.M-Y. Trojanowski J.Q. D. T. J. Full Text Full Text PDF PubMed Scopus Google Scholar) which that α-synuclein is This by the that that were using a and, are in filamentous α-synuclein. In filamentous and α-synuclein from early Furthermore, we and K. H. E. A. M J. T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. J. Neuropathol. Exp. Neurol. 1996; 55: PubMed Scopus Google Scholar, M. M. M. T. E. J. Neurochem. 1998; PubMed Scopus Google Scholar) found that α-synuclein with an molecular of at This to 22–24-kDa α-synuclein is the same as as ubiquitinated α-synuclein. However, is that α-synuclein ubiquitinated at of disease and is detected in found that of the 22–24-kDa α-synuclein is phosphorylated on only a molecular were with antibodies modifications other than phosphorylation for a in with a is or are a in was detected in recombinant protein. data may help to the of ubiquitinated α-synuclein inclusions in on α-synuclein to occur only in the of further post-translational modifications. Therefore, this cellular may a disease-specific pathway which is not activated in In this identification of the may provide insights into the cellular to fibril formation. a form of α-synuclein has been to in vitro by an H. M.G. A. R. Science. 2001; PubMed Scopus Google Scholar). to the of H. M.G. A. R. Science. 2001; PubMed Scopus Google we not of disease-associated α-synuclein in from sporadic LB It that a form of in LB-associated α-synuclein. of the 22–24-kDa form of α-synuclein the of this species in α-synuclein has been to a substrate for by to the understanding of the ubiquitin system, in an L. Full Text Full Text PDF PubMed Scopus Google Scholar). α-synuclein is a substrate for the proteasome has been a of J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, K. C. K. F. PubMed Scopus Google Scholar). previously that unmodified α-synuclein is degraded by the proteasome in an ubiquitin-independent R. M.G. 2001; PubMed Scopus Google Scholar). Because α-synuclein is a natively unfolded protein, is feasible that in unfolded α-synuclein the for and and the proteasome The is in with the finding that other unfolded proteins degraded by the proteasome in a ubiquitin-independent J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. S. M. J. J. 2001; PubMed Scopus Google Scholar). P. 2001; PubMed Scopus Google Scholar) has that proteasome activity is in the of PD Therefore, is feasible that accumulation of α-synuclein is associated with proteasome However, LB-associated α-synuclein is found the regions found significant in the proteolytic activity of the proteasome in the from PD and DLB cases not in other regions with LB as the and of DLB Furthermore, in the not LB pathology was associated with a significant in proteasome function. for the in the is that this brain is in the other in the of to is the of proteasome function in with However, the is to the the tissue was from early of the disease was to Because the are in the core of the is that in vivo regions on the which detected with are to modification of proteasome T. R. K. J. A. Sci. Sci. 2001; PubMed Scopus Google Scholar). These may prevent the and of the or the of to the proteasome than their the other modification of per may their and/or by the In this is feasible that accumulation of modified and presumably α-synuclein the proteolytic leading to aberrant ubiquitination. The in interfere with the of a that to further protein accumulation and formation. the proteolytic is by modified α-synuclein then α-synuclein as a result of aberrant proteolysis, found in association with Accordingly, we found that α-synuclein is from fractions containing α-synuclein not in of these that protein. Because in fibrils the and of α-synuclein in the (5Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2396) Google Scholar, H. H. T. M. J. Full Text Full Text PDF PubMed Scopus Google the of only carboxyl-terminal-truncated protein is to an of studies the of α-synuclein (6Culvenor J.G. McLean C.A. Cutt S. Campbell B.C. Maher F. Jakala P. Hartmann T. Beyreuther K. Masters C.L. Li Q.X. Am. J. Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Campell B.C.V. McLean C.A. Culvenor J.G. Gai W.P. Blumbergs P.C. Jakala P. Beyreuther K. Masters C.L. Li Q.-X. J. Neurochem. 2001; 76: 87-96Crossref PubMed Scopus (160) Google Scholar, M. S. T. K. Lee Trojanowski J.Q. T. Am. J. Pathol. 1998; Google Scholar). In to data α-synuclein protein was found in soluble and insoluble fractions and was in soluble fractions from disease and cases (6Culvenor J.G. McLean C.A. Cutt S. Campbell B.C. Maher F. Jakala P. Hartmann T. Beyreuther K. Masters C.L. Li Q.X. Am. J. Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Campell B.C.V. McLean C.A. Culvenor J.G. Gai W.P. Blumbergs P.C. Jakala P. Beyreuther K. Masters C.L. Li Q.-X. J. Neurochem. 2001; 76: 87-96Crossref PubMed Scopus (160) Google Scholar). is to Trojanowski J.Q. Lee V.M-Y. J. 2001; 276: Full Text Full Text PDF PubMed Scopus Google and a of proteolytic are during that M. S. T. K. Lee Trojanowski J.Q. T. Am. J. Pathol. 1998; Google Scholar) and (6Culvenor J.G. McLean C.A. Cutt S. Campbell B.C. Maher F. Jakala P. Hartmann T. Beyreuther K. Masters C.L. Li Q.X. Am. J. Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Campell B.C.V. McLean C.A. Culvenor J.G. Gai W.P. Blumbergs P.C. Jakala P. Beyreuther K. Masters C.L. Li Q.-X. J. Neurochem. 2001; 76: 87-96Crossref PubMed Scopus (160) Google Scholar). Therefore, the that α-synuclein is a soluble degradation are to that aberrant either by the proteasome or other a of associated with LB formation. The finding that α-synuclein is degraded by the proteasome at Science. PubMed Scopus Google Scholar) that the proteasome contribute to the of these In we that in PD and DLB, a modified 22–24-kDa α-synuclein is a substrate for which in the of a generalized significant impairment of the proteolytic activity of the Because unmodified α-synuclein is degraded by the proteasome in a ubiquitin-independent R. M.G. 2001; PubMed Scopus Google data suggest that accumulation of modified 22–24-kDa α-synuclein is a disease-specific event that may overwhelm the proteolytic system, presumably of and/or the core of the leading to aberrant and formation. the separation of α-synuclein on may in insights into unknown modifications and, cellular pathways that may in LB formation. R. Jakes for E. for on the M. Goedert for and T. for the kind gift of the