Formation of toxic oligomeric alpha-synuclein species in living cells.
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.
Outeiro
Tiago Fleming
TF
Alzheimer's Research Unit, MassGeneral Institute for Neurodegenerative Disease, MGH Harvard Medical School, Charlestown, Massachusetts, United States of America. touteiro@fm.ul.pt
Putcha
Preeti
P
Tetzlaff
Julie E
JE
Spoelgen
Robert
R
Koker
Mirjam
M
Carvalho
Filipe
F
Hyman
Bradley T
BT
McLean
Pamela J
PJ
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
D018840:HSP70 Heat-Shock Proteins; D051844:alpha-Synuclein