A novel, high-efficiency cellular model of fibrillar alpha-synuclein inclusions and the examination of mutations that inhibit amyloid formation.
Intracytoplasmic alpha-synuclein (alpha-syn) amyloidogenic inclusions are a major pathological feature of Parkinson's disease, dementia with Lewy body disease and multiple systems atrophy. The mechanisms involved in the formation and inhibition of these aggregates are areas of intense investigation. The present study characterizes a novel cellular model for the study of alpha-syn aggregation, incorporating nucleation-dependent aggregation and a new function for calcium phosphate precipitation. Cultured cells were readily induced to develop large, cytoplasmic alpha-syn filamentous aggregates that were hyperphosphorylated, often ubiquitinated and thioflavin positive. These cellular aggregates formed in the majority of transfected cells and recruited approximately half of endogenously expressed alpha-syn. Using this system, we examined single-point mutations that inhibit alpha-syn amyloid formation in vitro. Three mutations (V66P, T72P and T75P) significantly hindered alpha-syn aggregation in this cell model. The T75P mutant, which could abrogate amyloid formation of wild-type alpha-syn in vitro, did not prevent wild-type alpha-syn cellular aggregates. These studies suggest that the propensity of alpha-syn to form cellular aggregates may be more pronounced than in isolated in vitro studies. This novel high-efficiency cellular model of alpha-syn aggregation is a valuable system that may be used to further understand alpha-syn aggregation and allow for the generation of future therapeutics.
Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104-6084, USA.
Elisa A
EA
Waxman
Benoit I
BI
Giasson
D000682:Amyloid / Q000378:metabolism* / Q000648:ultrastructure; D052160:Benzothiazoles; D002130:Calcium Phosphates / Q000494:pharmacology; D002461:Cell Line, Transformed; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D002479:Inclusion Bodies / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017354:Point Mutation / Q000235:genetics*; D013844:Thiazoles / Q000378:metabolism; D014162:Transfection / Q000379:methods; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism*
D000682:Amyloid; D052160:Benzothiazoles; D002130:Calcium Phosphates; D013844:Thiazoles; D051844:alpha-Synuclein; C009462:thioflavin T; C485838:calcium phosphate, monobasic, anhydrous