Title:Effects of α-Synuclein Overexpression in Transgenic Caenorhabditis elegans Strains
VOLUME: 11 ISSUE: 8
Author(s):Rakesh Bodhicharla, Archana Nagarajan, Jody Winter, Ademola Adenle, Aamir Nazir, Declan Brady, Kelly Vere, Jo Richens, Paul O’Shea, David R. Bell and David de Pomerai
Affiliation:School of Biology, University of Nottingham, University Park, Nottingham. NG7 2RD, UK.
Keywords:Caenorhabditis elegans, CFP and YFP reporters, FRET, protein aggregation, α-synuclein, transgenic strains, fibrils, C-terminus, pharyngeal, SC+SV worms.
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.
