title: Formation of toxic oligomeric alpha-synuclein species in living cells.
abstract: BACKGROUND
  Misfolding, oligomerization, and fibrillization of alpha-synuclein are thought to be central events in the onset and progression of Parkinson's disease (PD) and related disorders. Although fibrillar alpha-synuclein is a major component of Lewy bodies (LBs), recent data implicate prefibrillar, oligomeric intermediates as the toxic species. However, to date, oligomeric species have not been identified in living cells.
  
  
  RESULTS
  Here we used bimolecular fluorescence complementation (BiFC) to directly visualize alpha-synuclein oligomerization in living cells, allowing us to study the initial events leading to alpha-synuclein oligomerization, the precursor to aggregate formation. This novel assay provides us with a tool with which to investigate how manipulations affecting alpha-synuclein aggregation affect the process over time. Stabilization of alpha-synuclein oligomers via BiFC results in increased cytotoxicity, which can be rescued by Hsp70 in a process that reduces the formation of alpha-synuclein oligomers. Introduction of PD-associated mutations in alpha-synuclein did not affect oligomer formation but the biochemical properties of the mutant alpha-synuclein oligomers differ from those of wild type alpha-synuclein.
  
  
  CONCLUSIONS
  This novel application of the BiFC assay to the study of the molecular basis of neurodegenerative disorders enabled the direct visualization of alpha-synuclein oligomeric species in living cells and its modulation by Hsp70, constituting a novel important tool in the search for therapeutics for synucleinopathies.
authors.affiliation: Alzheimer's Research Unit, MassGeneral Institute for Neurodegenerative Disease, MGH Harvard Medical School, Charlestown, Massachusetts, United States of America. touteiro@fm.ul.pt
authors.forename: Tiago Fleming
authors.initials: TF
authors.lastname: Outeiro
authors.forename: Preeti
authors.initials: P
authors.lastname: Putcha
authors.forename: Julie E
authors.initials: JE
authors.lastname: Tetzlaff
authors.forename: Robert
authors.initials: R
authors.lastname: Spoelgen
authors.forename: Mirjam
authors.initials: M
authors.lastname: Koker
authors.forename: Filipe
authors.initials: F
authors.lastname: Carvalho
authors.forename: Bradley T
authors.initials: BT
authors.lastname: Hyman
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: McLean
mesh_terms: D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D016631:Lewy Bodies / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D010300:Parkinson Disease / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D018840:HSP70 Heat-Shock Proteins; D051844:alpha-Synuclein