Extended Data Fig. 2: Basal mitochondrial function is intact in ATG5 cKO cerebellum.
From: Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity
a,b, RNAscope in situ hybridization examples (a) and cell density analysis in the cervical and lumbar spinal cord segments (b) of WT and ATG5 cKO animals at 3 M for probes against VGAT, GlyT2 or VGluT2 mRNAs. Scale bar, 200 µm. N = 3 mice per genotype. Multiple t test with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 1%. c, Cerebellum and cortex of WT and ATG5 cKO mice at 3 M, immunostained for p62. Scale bar, 25 µm. See quantification in Fig. 1j (N = 4 for cKOs both ages and 3 M WT mice, N = 3 for 1 M WT mice). d-f, Cerebellum and cortex of WT and ATG5 cKO mice at 1 M (d) and 3 M (e), immunostained for NBR1. Scale bar, 25 µm. Fluorescence analysis of the area of NBR1 foci in PCs and cortical interneurons (f). N = 4 mice per genotype for ATG5 cKO1&3M and WT1M, and N = 3 for WT3M. Two-way ANOVA followed by Holm-Šidák multiple comparisons test (1 M cKOcerebellum vs cKOcortex: p = 0.003; 1M cKOcortex vs WTcortex: p = 0.002). g, Mitochondrial area in WT and ATG5 cKO PCs at 3 M. N = 4 mice for each genotype. Two-tailed unpaired t-Test. h, Mitochondrial cristae perimeter normalized to mitochondrial area in WT and ATG5 cKO PCs at 3 M. N = 4 mice for each genotype. Two-tailed unpaired t-Test. i-o, OCR rates during Seahorse XF Cell Mito Stress test in WT and ATG5 cKO primary cerebellar cells with injections of 1.5 µM Oligomycin A, 2 µM FCCP and 0.5 µM Rotenone/Antimycin A. N = 5 cultures per genotype with 26 wells with ± 20 000 cells. Two-tailed unpaired t-Test. p,q, mt-mKeima ratio in WT and ATG5 cKO PCs in OTCs at baseline or after 6 h incubation with 10 µM CCCP. Scale bar, 20 µm. N = 5 WT/N = 3 ATG5 cKO mice for control and N = 3 WT/ N = 4 ATG5 cKO for CCCP. Two-way ANOVA followed by Holm-Šidák multiple comparisons test (WTcontrol vs WTCCCP: p = 0.007, WTCCCP vs cKO CCCP p = 0.005). r, Example confocal images of WT and ATG5 cKO cerebellum, immunostained for TIM23 and BNIP3 from N = 1 experiment. Scale bar, 25 µm. s,t, BNIP3 protein levels in WT and ATG5 cKO cerebellar lysates. N = 3 per genotype. Two-tailed unpaired t-Test. 1 M and 3 M indicate 1 and 3 months of age, respectively. All graphs show mean ± SEM. n.s.—non-significant; * indicates P ≤ 0.05; ** indicates P ≤ 0.01; *** indicates P ≤ 0.001; **** indicates P ≤ 0.0001.
