title: A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson's Disease.
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.
authors: Centre for Organelle Research, University of Stavanger, Stavanger, Norway. archana.nagarajan@uis.no.
authors: Archana
authors: A
authors: Nagarajan
authors: Rakesh
authors: R
authors: Bodhicharla
authors: Jody
authors: J
authors: Winter
authors: Charumathi
authors: C
authors: Anbalagan
authors: Kevin
authors: K
authors: Morgan
authors: Mark
authors: M
authors: Searle
authors: Aamir
authors: A
authors: Nazir
authors: Ademola
authors: A
authors: Adenle
authors: April
authors: A
authors: Fineberg
authors: Declan
authors: D
authors: Brady
authors: Kelly
authors: K
authors: Vere
authors: Jo
authors: J
authors: Richens
authors: Paul
authors: P
authors: O'Shea
authors: David
authors: D
authors: Bell
authors: David
authors: D
authors: de-Pomerai
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D017173:Caenorhabditis elegans; D002942:Circular Dichroism; D004926:Escherichia coli; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D020734:Parkinsonian Disorders / Q000378:metabolism*; D066329:Protein Aggregates / Q000502:physiology; D066263:Protein Aggregation, Pathological / Q000378:metabolism; D034622:RNA Interference; D051844:alpha-Synuclein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018840:HSP70 Heat-Shock Proteins; D008164:Luminescent Proteins; D066329:Protein Aggregates; C497604:SNCA protein, human; D051844:alpha-Synuclein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins