Reciprocal effects of alpha-synuclein overexpression and proteasome inhibition in neuronal cells and tissue.
Defects in the 20S/26S proteasome and conformational changes in alpha-synuclein (alpha-syn) are implicated in the development of sporadic and familial cases of PD. The objective of this study was to evaluate whether alpha-syn affects proteolysis by the proteasome and, reciprocally, whether proteasome inhibition affects alpha-syn solubility and localization. Although alpha-syn directly inhibited purified 20S proteasomes reversibly in vitro, its overexpression in neuroblastoma (SH-SY5Y and SK-N-BE), embryonic kidney (HEK293) cells, or mouse brain did not affect proteasome activity. Proteasome inhibition with MG132 and epoxomicin in SH-SY5Y cells failed to induce alpha-syn aggregation, although it increased membrane bound forms of endogenous and overexpressed wild-type, but not mutant, alpha-syn. Concomitantly this treatment generated cytoplasmic alpha-syn inclusions devoid of polyubiquitin in a small percentage of cells. The combination of proteasome inhibition with serum deprivation, which induced oxidative stress and autophagy, caused the appearance of high molecular weight alpha-syn species, such as those found in Lewy bodies. Our data suggest that high concentrations of alpha-syn do not affect proteasome function in vivo, whereas proteasome inhibition can modify synuclein solubility, most prominently under conditions of cell stress which occur during aging. These results have implications for the convergence of age-related oxidative stress and impaired protein degradation in neurodegeneration.
Dyllick-Brenzinger
Melanie
M
Department of Medicine, University of Toronto, Toronto, ON, M5S 3H2, Canada.
D'Souza
Cheryl A
CA
Dahlmann
Burkhardt
B
Kloetzel
Peter-Michael
PM
Tandon
Anurag
A
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007211:Indoles; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology*; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D007211:Indoles; D007381:Intermediate Filament Proteins; C430456:desmuslin; C007293:DAPI; D046988:Proteasome Endopeptidase Complex