Increased CD expression reduces α-syn-toxicity in C. elegans. a. RNAi knockdown of a C. elegans Ctsd ortholog worsens aggregation of human α-syn in vivo. Isogenic worm strain expressing α-syn::GFP alone (a) or with TOR-2 (b), in body wall muscle cells of C. elegans. The presence of TOR-2, a protein with chaperone activity, attenuates the misfolded α-syn protein (b). When worms expressing α-syn::GFP + TOR-2 are exposed to CD RNAi, the misfolded α-syn::GFP returns (c). d-f. Overexpression of CD protects DA neurons from α-syn-induced degeneration. Worm DA neurons degenerate as animals age. At the 7 d stage, most worms are missing anterior DA neurons of the CEP (cephalic) and/or ADE (anterior deirid) classes. d. Note the presence of 3 of 4 CEP DA neurons (arrows) and the absence of the 2 ADE neurons. e. Overexpression of CD protects worms from neurodegeneration whereby worms display all 4 CEP (arrows) and both ADE (arrowheads) neurons. f. RNAi knock down of asp-4 does not reduce tor-2 expression level. Semi-quantitative RT-PCR was performed by using primers to amplify cdk- 5 (loading control) and tor-2. For all strains analyzed, normal (non-RNAi) condition was used as a negative control, and tor-2 RNAi was used as a positive control.g. The percentage of worms exhibiting the wildtype neuronal complement of all 6 anterior DA neurons (30%) was significantly greater than animals without CD overexpression (15%). CD mutants (D295R and F229I), CB and CL, in transgenic worms overexpressing human cDNAs encoding these mutated CD or the representative lysosomal cysteine proteases, do not have the same effect as the wildtype CD in reducing α-syn toxicity. *p < 0.001 compared to α-syn alone, by Fisher Exact Test.