Figure 8
PMR1 is critical for αSyn neurotoxicity in nematodes and flies. (a) Survival of C. elegans dopaminergic neurons in WT or PMR-1-deficient (pmr-1(tm1840)) animals expressing GFP and α-Syn. Mean±S.E.M., n>250 individual animals; ***P<0.001. (b) Fluorescence-based quantification of cytoplasmic Ca2+ levels in WT or PMR-1-deficient (pmr-1(tm1840)) nematodes expressing the Ca2+ indicator GCaMP2.0 and α-Syn. Mean±S.E.M., n>150 dopaminergic neurons. ***P<0.001. (c and d) Survival of male (c) and female (d) WT flies and of flies either expressing human αSyn or an RNAi depleting SPoCk (the Drosophila homologue of PMR1) or both (driven by elav-GAL4) upon supplementation of food (10% sucrose) with 20 mM Mn2+. Means±S.E.M., n=12–20 with 35–40 flies per experiment; ***P<0.001. (e) Immunoblot analysis of brain lysats obtained from flies expressing human αSyn driven by elav-GAL4 with or without co-expression of an RNAi-depleting SPoCk using antibodies directed against human αSyn or Drosophila α-tubulin as loading control. (f) Climbing activity of female flies described in (d) after 24 h of Mn2+ treatment. Means±S.E.M., n=6–10 with 8 flies per experiment; ***P<0.001 and *P<0.05. (g and h) Total count of tyrosine hydroxylase (TH)-immunoreactive dopaminergic neurons (g) in the DM, PM and DL1 brain clusters of female flies expressing αSyn alone or in combination with an RNAi-depleting SPoCk after treatment with Mn2+ for 96 h. Representative confocal microscopy images of dissected brains immunostained for TH and for Bruchpilot (BRPNc82) to visualize brain structure are shown in (h). Neuronal counts were quantified by inspection of the individual planes of the z-stack. Means±S.E.M., n=5–10; **P<0.01 and *P<0.05
