Formation of toxic oligomeric alpha-synuclein species in living cells

• Published in: PloS one 2008-04, Vol.3 (4), p.e1867
• Main Authors: Outeiro, Tiago Fleming, Putcha, Preeti, Tetzlaff, Julie E, Spoelgen, Robert, Koker, Mirjam, Carvalho, Filipe, Hyman, Bradley T, McLean, Pamela J
• Format: Article
• Language: English
• Subjects: alpha-Synuclein - chemistry; alpha-Synuclein - genetics; alpha-Synuclein - metabolism; Animals; Biochemistry/Protein Folding; Biotechnology; Biotechnology/Bioengineering; Cell Biology/Neuronal and Glial Cell Biology; Cell Line, Tumor; Cells (biology); CHO Cells; Cricetinae; Cricetulus; Cytotoxicity; Data processing; Disorders; Drosophila; Fibrillogenesis; Fluorescence; Gaussia; Heat shock proteins; HSP70 Heat-Shock Proteins - chemistry; Hsp70 protein; Humans; Insects; Intermediates; Lewy bodies; Lewy Bodies - metabolism; Medical schools; Microscopy; Microscopy, Fluorescence - methods; Movement disorders; Mutation; Neurodegeneration; Neurodegenerative diseases; Neurological Disorders/Cognitive Neurology and Dementia; Neurological Disorders/Movement Disorders; Neurotoxicity; Oligomerization; Oligomers; Parkinson Disease - metabolism; Parkinson's disease; Pathogenesis; Protein Folding; Protein Structure, Tertiary; Species; Synuclein; Toxicity
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0001867

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. 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. 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.