Extended Data Fig. 7: α-Syn Neurotoxicity in Flies.
(a) Validation of an α-syn fly model where wild-type human α-syn transgene downstream of a modified UAS that significantly increases gene expression59 is expressed using the inducible pan-neuronal driver Elav-GS-GAL4 (‘Elav-GS>UAS-SNCA-WT, RU+’) through adulthood. Un-induced controls are ‘Elav-GS>UAS-SNCA-WT, RU-’. Head lysates of induced and uninduced flies were immunoblotted as indicated. The band intensity of a-syn is normalized to that of β-actin. n=3 independent experiments. Two-sided T Test with Welch’s correction. p=0.0238. (b) Climbing ability shown as Performance Index of flies with adult-onset induction and of un-induced controls at different ages. n=60 (RU-) and 61 (RU+) flies per genotype, 3 independent experiments. (c) The DA neuron number in the PPM1/2, PPL1, or PPL2 clusters per brain from flies as indicated at day 40 is counted and compared. n=6 fly brains per genotype. Two-sided Mann-Whitney Test. p=0.0022, 0.0065. The rest of the precise p values are in Source Data. (d) Head lysates of 40-day-old flies were immunoblotted with antibodies against DMiro and the loading control β-actin. The band intensity of DMiro is normalized to that of β-actin from the same blot. n=3 independent experiments. Two-sided Student T Test. p=0.0138. (e) Climbing time for 30-day-old flies. n=27, 26, 57, 26 flies (from left to right). One-Way Anova Post Hoc Tukey Test. p<0.0001. (f) Flying ability of 30-day-old flies. The percentage of flies scored as a ‘1’ (able to fly) is shown in solid black bars, and the percentage of flies scored as a ‘0’ (unable to fly) is shown in white bars. n=100. Chi-Square Test. p<0.0001. (e-f) Comparisons with ‘TH-GAL4’. (g-h) HEK293T cells transfected as indicated were lysed and immunoblotted. One-Way Anova Post Hoc Tukey Test. (g) The band intensity of Miro1 is normalized to that of GAPDH from the same blot. Data from each genotype are compared to those of ‘Control RNAi, EGFP’ (the left box) and asterisk is given directly on top of the box if it is significant. Data between ‘Control RNAi, EGFP-SNCA’ and ‘MIC60 RNAi, EGFP-SNCA’ (the right 2 boxes) are compared and asterisk is given on top of a line. n=5 transfections. p=0.0004, <0.0001. The rest of the precise p values are in Source Data. (h) The band intensity of Myc-Miro1 is normalized to that of β-actin from the same blot. Data from each genotype are compared to those of ‘Myc-Miro1, dMIC60-WT, EGFP’ (the left box) and asterisk is given directly on top of the box if it is significant. Data between ‘Myc-Miro1, dMIC60-WT, EGFP-SNCA’ and ‘Myc-Miro1, dMIC60-CS, EGFP-SNCA’ (the right 2 boxes) are compared and asterisk is given on top of a line. n=5 transfections. p=0.013, 0.0006. The rest of the precise p values are in Source Data. (a, d) Data are presented as Mean±S.E.M. with dots. (c, e, g, h) Boxes show 25th/75th percentiles, whiskers are the minimum and maximum, and middle line is median.
