Inhibition of autophagy curtails visual loss in a model of autosomal dominant optic atrophy

Abstract

In autosomal dominant optic atrophy (ADOA), caused by mutations in the mitochondrial cristae biogenesis and fusion protein optic atrophy 1 (Opa1), retinal ganglion cell (RGC) dysfunction and visual loss occur by unknown mechanisms. Here, we show a role for autophagy in ADOA pathogenesis. In RGCs expressing mutated Opa1, active 5' AMP-activated protein kinase (AMPK) and its autophagy effector ULK1 accumulate at axonal hillocks. This AMPK activation triggers localized hillock autophagosome accumulation and mitophagy, ultimately resulting in reduced axonal mitochondrial content that is restored by genetic inhibition of AMPK and autophagy. In C. elegans, deletion of AMPK or of key autophagy and mitophagy genes normalizes the axonal mitochondrial content that is reduced upon mitochondrial dysfunction. In conditional, RGC specific Opa1-deficient mice, depletion of the essential autophagy gene Atg7 normalizes the excess autophagy and corrects the visual defects caused by Opa1 ablation. Thus, our data identify AMPK and autophagy as targetable components of ADOA pathogenesis.

Fig. 1. Mitochondrial content is reduced in axons of ADOA RGCs.

a Representative z-projections of stacks of confocal images of mtRFP (red) in primary RGCs co-transfected with the indicated plasmids and after 24 h fixed and immunostained with β-tubulin III (βtub III, grey). Axons and somas were magnified in the bottom panels. EV, empty vector. Bars, 20 μm. b Quantification of mitochondrial length from four independent experiments as in (a) (EV, n = 86; Opa1, n = 113; Opa1^K301A, n = 82; Opa1^R905*, n = 71; Opa1^Q297V, n = 114 cells). ****p < 0.0001 vs. EV in one-way ANOVA/Tukey’s test. c Quantification of mitochondrial content in axons from four independent experiments as in (a) (EV, n = 58; Opa1, n = 65; Opa1^K301A, n = 69; Opa1^R905*, n = 54; Opa1^Q297V, n = 64 cells). *p = 0.018 Opa1, p = 0.027 Opa1^Q297V vs. EV; ****p < 0.0001 vs. EV in one-way ANOVA/Bonferroni’s test. d Representative z-projections of stacks of confocal images acquired 24 h after transfection of primary RGCs co-transfected with mtRFP (red) and YFP-LC3 (green, autophagosome-LC3, auto-LC3) and the indicated plasmids. The cytoplasmic YFP-LC3 signal (cyto-LC3) is pseudocolored in grey for the sake of clarity. The region corresponding to the soma was magnified in the inset. Bars, 20 μm. e Quantification of soma mitochondria and autophagosome distribution towards the axonal hillock in 4 independent experiments as in (d) (EV, n = 65 mitochondria and 49 autophagosomes; Opa1, n = 69 mitochondria and autophagosomes; Opa1^K301A, n = 58 mitochondria and 47 autophagosomes; Opa1^R905*, n = 69 mitochondria and autophagosomes; Opa1^Q297V, n = 70 mitochondria and autophagosomes) **p = 0.0023 Opa1^K301A vs. EV; p = 0.0084 Opa1^R905* vs. EV; ****p < 0.0001 vs. EV in one-way ANOVA/Tukey’s test. f Quantification of mitochondria and autophagosome co-localization in six independent experiments as in (d). (EV, n = 86; Opa1, n = 86; Opa1^K301A, n = 101; Opa1^R905*, n = 82; Opa1^Q297V, n = 91 cells). *p = 0.0153 Opa1^K301A, p = 0.043 Opa1^R905* vs. EV in one-way ANOVA/Tukey’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.

Fig. 2. Inhibition of autophagy restores mitochondrial content in ADOA RGCs axons.

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; Opa1^K301A, n = 56; Opa1^R905*, n = 60; Opa1^Q297V, 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 Atg7^fl/+ 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; Opa1^K301A, n = 50; Opa1^R905*, n = 45; Opa1^Q297V, n = 58 cells; Cre: EV, n = 45; Opa1, n = 57; Opa1^K301A, n = 54; Opa1^R905*, n = 51; Opa1^Q297V, 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; Opa1^K301A, n = 52; Opa1^R905*, n = 53; Opa1^Q297V, n = 57 cells; Cre: EV, n = 55; Opa1, n = 56; Opa1^K301A, n = 55; Opa1^R905*, n = 55; Opa1^Q297V, n = 54 cells). ****p < 0.0001; **p = 0.003 vs. EV in two-way ANOVA/Sidak’s test. f Representative confocal images of Atg7^fl/+ 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.

Fig. 3. Peri-hillock AMPK activation reduces axonal mitochondrial content in ADOA RGCs.

a Representative z-projections of stacks of confocal images of phosphorylated-AMPK (p-AMPK, red) and GFP (blue) fluorescence in primary RGCs co-transfected as indicated and after 24 h immunostained for p-AMPK. The vector from the axonal hillock to the opposite side of the soma indicates the line used to measure p-AMPK fluorescence intensity in (b). Asterisks: axon. Bars, 20 μm. b Average ± SEM of p-AMPK fluorescence intensity along the vector drawn in the soma of RGCs co-transfected with GFP and the indicated plasmids in four independent experiments as in (a). EV, n = 73; Opa1, n = 69; Opa1^K301A, n = 75; Opa1^R905*, n = 73; Opa1^Q297V, n = 71 cells. c Average ± SEM ULK1 fluorescence intensity along the vector drawn in the soma of RGCs co-transfected with GFP and the indicated plasmids in three independent experiments as in (a) except that cells where immunostained for ULK1. EV, n = 21; Opa1^K301A, n = 26 cells. d Representative z-projections of stacks of confocal images of mtRFP (red) and YFP-LC3 (green, autophagosome-LC3, auto-LC3) in primary 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. Bars, 20 μm. e, f Quantification of autophagosomes (e) and mitochondria (f) distribution in somas from three independent experiments as in (d). In (e), for EV: EV, n = 54; Opa1, n = 53; Opa1^K301A, n = 51; Opa1^R905*, n = 52; Opa1^Q297V, n = 52 cells; for AMPK^DN: EV, n = 46; Opa1, n = 48; Opa1^K301A, n = 60; Opa1^R905*, n = 55; Opa1^Q297V, n = 51 cells. In (f) for EV: EV, n = 54; Opa1, n = 53; Opa1^K301A, n = 51; Opa1^R905*, n = 51; Opa1^Q297V, n = 52 cells; for AMPK^DN: EV, n = 46; Opa1, n = 48; Opa1^K301A, n = 60; Opa1^R905*, n = 55; Opa1^Q297V, n = 51 cells. ****p < 0.0001 vs. EV in two-way ANOVA/Sidak’s test. g Quantification of mitochondrial content in RGCs axons from three independent experiments as in (d). For EV: EV, n = 53; Opa1, n = 46; Opa1^K301A, n = 51; Opa1^R905*, n = 47; Opa1^Q297V, n = 52 cells; for AMPK^DN: EV, n = 59; Opa1, n = 53; Opa1^K301A, n = 59; Opa1^R905*, n = 50; Opa1^Q297V, n = 52 cells. ****p < 0.0001 vs. EV in two-way ANOVA/Sidak’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.

Fig. 4. Inhibition of autophagy restores mitochondrial content in axons of C. elegans carrying mitochondrial dysfunction.

a Confocal images of mtGFP (green) and cytoplasmic mCherry (red) in GABA motor neurons of the indicated transgenic nematodes. Bar, 20 μm. b Quantification of mitochondrial content in axons in n = 13 animals from three independent experiments as in (a). ****p < 0.0001 in one-way ANOVA/Tukey’s test. c Confocal images of mtGFP (green) and cytoplasmic mCherry (red) in GABA motor neurons of transgenic nematodes treated as indicated. Upper panels: magnified soma region. PQ, paraquat. Bars, 20 μm. d Quantification of mitochondrial content in axons in n = 25 animals from three independent experiments as in (c). **p = 0.0012 in one-way ANOVA/Tukey’s test. e Confocal images of mtGFP (green) and cytoplasmic mCherry (red) in GABA motor neurons of transgenic nematodes treated as indicated. Bar, 20 μm. f Quantification of mitochondrial content in axons in n = 25 animals from three independent experiments as in (e). ****p < 0.0001 in one-way ANOVA/Tukey’s test. g Confocal images of mtGFP (green) and cytoplasmic mCherry (red) in GABA motor neurons of transgenic nematodes treated as indicated. Bar, 20 μm. h Quantification of mitochondrial content in axons in n = 25 animals from three independent experiments as in (g). ****p < 0.0001 in one-way ANOVA/Tukey’s test. i Confocal images of mtGFP (green) and cytoplasmic mCherry (red) in GABAergic motor neurons of the indicated transgenic nematodes expressing Opa1^K301A. Bar, 20 μm. j Quantification of mitochondrial content in axons in n = 25 animals from three independent experiments as in (i). ****p < 0.0001 in one-way ANOVA/Tukey’s test. k Quantification of defecation motor program duration recorded in n = 10 transgenic nematodes from three independent experiments at the indicated days of life. ****p < 0.0001 in one-way ANOVA/Tukey test. In box plots, centre line represents mean, bounds of boxes SEM, whiskers the 10th–90th percentiles; each dot represents an individual nematode form the indicated N of independent experiments. Source data are provided as a Source Data file.

Fig. 5. Genetic autophagy inhibition curtails the visual defect of an ADOA mouse model.

a Average ± SEM visual acuity of 4-month-old mice of the indicated genotype in the optokinetic test. Mice were subjected to visual stimuli at the indicated temporal and spatial frequencies. Visual acuity is proportional to the percentage of correct answers to the stimulus. WT, n = 10; Opa1^ΔRGC/ΔRGC, n = 10; Opa1^ΔRGC/ΔRGCAtg7^ΔRGC/+, n = 8 mice. ****p < 0.0001 vs. WT in two-way ANOVA/Tukey’s test. b A schematic of the Y-shaped pool used for the vision guided forced-swimming test. The two screens (gray and vertically striped) used as visual cues and the submerged platform are shown. c Average ± SEM visual acuity of 4-month-old mice of the indicated genotype in the vision guided forced-swimming test. WT, n = 9; Opa1^ΔRGC/ΔRGC, n = 5; Opa1^ΔRGC/ΔRGCAtg7^ΔRGC/+, n = 7 mice. *p = 0.05, ***p = 0.0001 vs. WT in two-way ANOVA/Tukey test. d Representative z-projections of stacks of confocal images of the fluorescence of OPA1 (red), LC3B (green), and DAPI (blue) of retinal slices of 4-month-old mice of the indicated genotype. The soma of RGCs are circled with white lines in right panels. Bars, 20 μm. GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer. e Quantification of RGCs autophagosomes number in three independent experiments as in (d). WT, n = 163; Opa1^ΔRGC/ΔRGC, n = 155; Opa1^ΔRGC/ΔRGCAtg7^ΔRGC/+, n = 156 cells. ***p = 0.0002 vs. WT in one-way ANOVA/Bonferroni’s test. f Quantification of mitochondria and autophagosomes accumulation in axonal hillocks of RGCs in three independent experiments as in (d). For mitochondria, WT, n = 165; Opa1^ΔRGC/ΔRGC, n = 155; Opa1^ΔRGC/ΔRGCAtg7^ΔRGC/+, n = 156; for autophagosomes, WT, n = 164; Opa1^ΔRGC/ΔRGC, n = 155; Opa1^ΔRGC/ΔRGCAtg7^ΔRGC/+, n = 155 cells. **p = 0.003 ***p = 0.001 vs. WT in one-way ANOVA/Tukey’s test. g Quantification of anti-LC3B antibody signal in sorted mtYFP⁺ RGCs from five animals of each indicated genotype. *p = 0.012 vs. WT in one-way ANOVA/Kruskal–Walli’s test. In box plots, centre line represents mean, bounds of boxes SEM, whiskers the 10^th–90^th percentiles; each dot represents an individual nematode form the indicated N of independent experiments. Source data are provided as a Source Data file.