A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson’s Disease

Author(s): Archana Nagarajan , Rakesh Bodhicharla , Jody Winter , Charumathi Anbalagan , Kevin Morgan , Mark Searle , Aamir Nazir , Ademola Adenle , April Fineberg , Declan Brady , Kelly Vere , Jo Richens , Paul O'Shea , David Bell , David de-Pomerai .

Journal Name: CNS & Neurological Disorders - Drug Targets

Volume 14 , Issue 8 , 2015

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Abstract:

The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson’s disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo.

Keywords: FRET, C. elegans, α-synuclein, aggregation, pollutants, fluorescent proteins.

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Article Details

VOLUME: 14
ISSUE: 8
Year: 2015
Page: [1054 - 1068]
Pages: 15
DOI: 10.2174/1871527314666150821110538
Price: $58

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