title: Effects of α-synuclein overexpression in transgenic Caenorhabditis elegans strains.
abstract: The neural protein α-synuclein aggregates both in vivo and in vitro to form insoluble fibrils that are involved in Parkinson's disease pathogenesis. We have generated α-synuclein/fluorescent-protein fusion constructs overexpressed in muscle cells of the nematode, Caenorhabdtis elegans. Green Fluorescent Protein (GFP) variants, Cerulean (C) or Venus (V), were fused to the C-terminus of human α-synuclein (S); the resultant fusion genes were designated SV and SC, plus a CV fusion as well as S, C and V singly. The aggregation behavior of the purified fusion proteins (expressed in E. coli) will be described elsewhere. These constructs were fused to a C. elegans unc-54 myosin promoter, and integrated transgenic lines generated by microinjection, λ-irradiation, and outcrossing of fluorescent progeny. All transgenic lines expressing α- synuclein showed significant reductions (p <0.05) in lifespan, motility and pharyngeal pumping, as compared to wildtype worms or lines expressing CFP and/or YFP only. We showed that CFP and YFP labels colocalised in granular inclusions throughout the body wall in transgenic lines expressing both SC and SV fusions (SC+SV), whereas SV+C worms displayed YFP-labelled inclusions on a diffuse CFP background. These findings implied that the α-synuclein moieties of these fusion proteins still aggregated together in vivo, whereas CFP or YFP moieties alone did not. This in turn suggested that Foerster Resonanace Energy Transfer (FRET) between CFP and YFP labels in α-synuclein aggregates could allow the extent of aggregation to be quantified. Accordingly, we also showed that net FRET signals increased 2- fold between L4 and adult SC+SV worms.
authors.affiliation: School of Biology, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Bodhicharla
authors.forename: Archana
authors.initials: A
authors.lastname: Nagarajan
authors.forename: Jody
authors.initials: J
authors.lastname: Winter
authors.forename: Ademola
authors.initials: A
authors.lastname: Adenle
authors.forename: Aamir
authors.initials: A
authors.lastname: Nazir
authors.forename: Declan
authors.initials: D
authors.lastname: Brady
authors.forename: Kelly
authors.initials: K
authors.lastname: Vere
authors.forename: Jo
authors.initials: J
authors.lastname: Richens
authors.forename: Paul
authors.initials: P
authors.lastname: O'Shea
authors.forename: David R
authors.initials: DR
authors.lastname: Bell
authors.forename: David
authors.initials: D
authors.lastname: de Pomerai
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000378:metabolism*; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008136:Longevity / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D009068:Movement; D016019:Survival Analysis; D051844:alpha-Synuclein / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology
chemical_list: D008164:Luminescent Proteins; D051844:alpha-Synuclein; D049452:Green Fluorescent Proteins