A Fluorescence Resonance Energy Transfer Assay For Monitoring α-Synclein Aggregation In A Caenorhabditis elegans Model For Parkinson's Disease.

Nagarajan, Archana and Bodhicharla, Rakesh and Winter, Jody and Anbalagan, Charumathi and Morgan, Kevin and Searle, Mark and Nazir, Aamir and Adenle, Ademola and Fineberg, April and Brady, Declan and Vere, Kelly and Richens, Jo and O'Shea, Paul and Bell, David and de-Pomerai, David (2015) A Fluorescence Resonance Energy Transfer Assay For Monitoring α-Synclein Aggregation In A Caenorhabditis elegans Model For Parkinson's Disease. CNS & neurological disorders drug targets. ISSN 1996-3181

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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.

Item Type:
Journal Article
Journal or Publication Title:
CNS & neurological disorders drug targets
Uncontrolled Keywords:
/dk/atira/pure/subjectarea/asjc/2700
Subjects:
ID Code:
126039
Deposited By:
Deposited On:
22 Jun 2018 09:18
Refereed?:
Yes
Published?:
Published
Last Modified:
01 Jan 2020 11:24