Figure 6.

EndoG is critical for α-synuclein neurotoxicity in flies. (A, B) Survival of female (A) and male (B) wild-type flies and of flies expressing either human αSyn or an RNAi specific for EndoG or both (driven by nsyb-GAL4 for pan-neuronal expression) upon supplementation of food (10% sucrose) with 20 mM Mn²⁺. Means±s.e.m., n=11–15 with 35–40 flies per experiment. ***P<0.001 and **P<0.01. (C) Climbing activity of female flies described in (A) after 36 h of Mn²⁺ treatment. Means±s.e.m., n=6 with 8 flies per experiment. **P<0.01. (D) Quantification of EndoG mRNA levels in brains (head extracts) of wild-type flies and of flies expressing either an RNAi depleting EndoG alone or in combination with human αSyn (driven by nsyb-GAL4) by Q-PCR normalized to mRNA levels of α-tubulin. Means±s.e.m., n=6–8. **P<0.01. (E) Survival of male wild-type flies and of flies expressing either αSyn or EndoG alone or co-expressing both proteins after supplementation of food with 20 mM Mn²⁺. Data represent means±s.e.m., n=8–12 with 35–40 flies per experiment. ***P<0.001, **P<0.01. (F, G) Total count of tyrosine hydroxylase (TH)-immunoreactive dopaminergic neurons (F) in the DM, PM and DL1 brain clusters of female flies expressing αSyn alone or in combination with an RNAi depleting EndoG after treatment with Mn²⁺ for 96 h. Representative confocal microscopy images of dissected brains immunostained for TH and for Bruchpilot (BRP^Nc82) to visualize brain structure are shown in (G). Neuronal counts were quantified by inspection of the individual planes of the z-stack. Means±s.e.m., n=5–8. ***P<0.001, *P<0.05. Scale bar represents 40 μm.