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.

Fig. (1)

Schematic representation of the cloning and sub-cloning of α-synuclein fused with Venus (YFP). All other fusion transgenes were created in a similar fashion.

Fig. (2)

(a) Egg to adult development time, (b) Brood size, (c) Pharyngeal pumping rate and (d) Locomotion rate for the various transgenic worm strains tested. Error bars show the standard error of the mean. All trangenic strains were integrated lines, apart from C and SV which were both non-integrated lines showing high transmission (~80%) of the transgene to progeny.

Fig. (3)

Lifespans of (a) non-synuclein and (b) α-synuclein expressing transgenic worms, calculated as the percentage left alive on each day. I = integrated strain; NI = non-integrated SV strain (only fluorescent worms were selected for the lifespan analysis).

Fig. (4)

(a) Corrected net FRET signal of L4 and day 6 SC+SV worms and (b) YFP signal of L4 and day 6 NL5901 (SV) worms. Error bars show the standard error of the mean.

Fig. (5)

Confocal Microscopy: (a) unc-54::SC+SV worms (I) showing FRET signal with CFP excitation, (b) control unc-54::C worms (NI) showing the absence of FRET signal under identical conditions and (c) the head region of a young adult unc-54::SV+C worm (NI) showing diffuse CFP but aggregated YFP. The excitation and emission wavelengths used were 458/485 nm for CFP, and 514/545 nm for YFP. All photographs used the same gain settings and exposure times; scale bars =100µm. Each part [(a), (b) and (c)] shows images of the same worm, using CFP fluorescence [(a)i, (b)i, (c)i], FRET [(a)ii, b(ii)], CFP fluorescence with YFP excitation [(a)iv, (b)iv] or YFP fluorescence [(a)v, (b)v, (c)ii]. Differential interference contrast (DIC) images of the same worms are shown in panels (a)iii, (a)vi, (b)iii and (b)vi. I = integrated strain. NI = non-integrated strain.

Fig. (6)

Confocal microscopy of unc-54:: SC+SV L4 and adult worms (I). Body wall muscles of worms with Z-stacking for CFP (part a) or YFP (parts b-d): (a) L4 larval head imaged for CFP (458 nm laser, 485 nm emission filter); (b) the same L4 head region imaged for YFP (514 nm laser, 545 nm emission filter); (c) 1-day old adult showing small YFP-labelled granules; (d) 6-day adult showing larger and more intense or merged YFP-labelled granules. The YFP excitation and emission wavelengths for parts c and d were the same as for part b. All images used the same exposure time and gain settings; scale bar =100µm. I = integrated strain.