Fig. 2: Inhibition of autophagy restores mitochondrial content in ADOA RGCs axons.
From: Inhibition of autophagy curtails visual loss in a model of autosomal dominant optic atrophy
a Representative z-projections of stacks of confocal images of mitoKeima fluorescence in RGCs 24 h after cotransfection with the indicated plasmids. Where indicated, empty vector (EV) cotransfected cells were treated for 2 h with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Bar, 10 µm. b MitoKeima ratio from three independent experiments as in (a) (EV, n = 60; Opa1, n = 60; Opa1K301A, n = 56; Opa1R905*, n = 60; Opa1Q297V, n = 59; EV + CCCP, n = 58 fields) **p = 0.079; ****p < 0.0001 vs. EV in one-way ANOVA/Tukey’s test. c Representative z-projections of stacks of confocal images of mtRFP (red) and YFP-LC3 (green, autophagosome-LC3, auto-LC3) fluorescence in primary Atg7fl/+ RGCs co-transfected as indicated. The cytoplasmic YFP-LC3 signal (cyto-LC3) is pseudocolored in grey for the sake of clarity. Boxed axonal regions and the soma region were magnified in the corresponding bottom panels. Asterisks: axonal hillocks. Bars, 20 μm. d Mitochondria-autophagosomes co-localization from three independent experiments as in (c) (EV: EV, n = 54; Opa1, n = 54; Opa1K301A, n = 50; Opa1R905*, n = 45; Opa1Q297V, n = 58 cells; Cre: EV, n = 45; Opa1, n = 57; Opa1K301A, n = 54; Opa1R905*, n = 51; Opa1Q297V, n = 54 cells). ****p < 0.0001 vs. EV in two-way ANOVA/Sidak’s test. e Mitochondrial content in axons from three independent experiments as in (c) (EV: EV, n = 54; Opa1, n = 54; Opa1K301A, n = 52; Opa1R905*, n = 53; Opa1Q297V, n = 57 cells; Cre: EV, n = 55; Opa1, n = 56; Opa1K301A, n = 55; Opa1R905*, n = 55; Opa1Q297V, n = 54 cells). ****p < 0.0001; **p = 0.003 vs. EV in two-way ANOVA/Sidak’s test. f Representative confocal images of Atg7fl/+ RGCs co-transfected with GFP (green) and the indicated plasmids and processed for TUNEL (red) after 24 h. Arrowheads: GFP+, TUNEL+ cells. Scale bar, 10 µm. g Percentage of TUNEL positivity from three independent experiments as in (f) (EV, n = 139; Cre, n = 168 GFP+ cells). ***p = 0.0002 vs. EV in parametric t test/Welch’s test. In box plots, centre line represents mean, bounds of boxes SEM, whiskers the 10th–90th percentiles; each dot represents independent experiments. Source data are provided as a Source Data file.
