Amyloid aggregation of the abundant neural protein alpha-synuclein contributes to cellular toxicity in Parkinson.s disease. We have created transgenic nematodes carrying fusion constructs encoding human alpha-synuclein tagged with YFP and/or CFP as a fluorescent marker. Using the
unc-54 myosin promoter, a synuclein-YFP (
unc-54::SV) fusion construct is abundantly expressed in the body wall muscles of Caenorhabditis elegans. Similar constructs, i.e. (i)
unc-54::V, (ii)
unc-54::S +
unc-54::V, and (iii)
unc-54::SC +
unc-54::SV, were also injected. Permanent integrated lines were successfully generated for
unc-54::V, (ii)
unc-54::S +
unc-54::V, and (iii)
unc-54::SC +
unc-54::SV, using gamma irradiation but we were unable to get integrated
unc-54::SV transgenic worms. A non-integrated
unc-54::SV strain was found to be radiation sensitive when compared with wild type N2 worms and
unc-54::V. In order to investigate further, two different lines of
unc-54::SV and
unc-54::S +
unc-54::V were irradiated at different dose rates to determine radiation sensitivity. Comparing these transgenic worms with wild type N2 worms, we found there was a drastic effect of gamma irradiation on the frequency of sterility in the F1 generation as compared to F0. we have shown that
unc-54::SV,
unc-54::S +
unc-54::V and
unc-54::SC+
unc-54::SV worms have a significantly shorter life span than either N2 or
unc-54::V strain worms. We also found that the locomotion and pharyngeal pumping rates were significantly reduced in transgenic as compared to N2 worms; however brood size and developmental timing were found to be similar in all these transgenic strains and in wild type N2 worms. The radiation-sensitivity, shortened life span, lower pharyngeal pumping rates and slower movement of these transgenic synuclein-expressing worms suggest that the presumed aggregation of the synuclein fusion protein does indeed cause cellular toxicity. Permanent integrated transgenic lines were obtained for
unc-54::SC +
unc-54::SV,
unc-54::C +
unc-54::V and
unc-54::CV which will be used for assessing earlier events during aggregation using fluorescence-based techniques such as FRET and anisotropy.