Fig. 1: Autophagy loss increases PC vulnerability, independent of its role in protein and mitochondria quality control.

a,b, [¹⁸F]FDG-PET (a) and [¹⁸F]UCB-H and [¹⁸F]MNI1126-PET (b) imaging in WT and ATG5 cKO mice (WT 3 months (3 M): N = 7; 12 months (12 M): N = 6; cKO: N = 6 mice per group). T-map with voxel-wise comparison (two-tailed t-test, corrected for multiple testing) is shown to the right. Significant changes (P < 0.05) in cKO mice are indicated in red (higher) and blue (lower). c, Pearson correlation between [¹⁸F]UCB-H- and [¹⁸F]FDG-PET signals for ATG5 cKO mice (two-tailed t-test corrected for multiple testing). d, Nissl-stained WT and ATG5 cKO cerebellum at 3 M. Scale bars, 2 mm. e–g, PC density in WT and ATG5 cKO cerebellum, immunostained for calbindin in 1 M (e) and 12 M (f) mice. g, Quantification of PC density. INs, interneurons. Scale bars, 100 µm; magnified image scale bars, 25 µm. 1 M and 12 M: N = 3 mice; 3 M: N = 4. WT^3M versus cKO^3M: P = 0.015; WT^12M versus cKO^12M: P < 0.0001. h, Interneuron density in WT and ATG5 cKO cerebellum. 1 M and 12 M: N = 3 mice; 3 M: N = 4. WT^12M versus cKO^12M: P = 0.002. i, Cerebellum and cortex from WT and ATG5 cKO mice, immunostained for p62. Scale bars, 25 µm. j, p62 foci area in PCs and cortical interneurons of WT and ATG5 cKO animals. N = 4 mice for cKO both ages and 3 M WT; N = 3 mice for 1 M WT. P < 0.0001. k, Cerebellum (left) and cortex (right) from WT and ATG5 cKO mice at 3 M, immunostained for p62 and ubiquitin. Scale bars, 25 µm; magnified image scale bars, 5 µm. l, Percentage of ubiquitin-positive p62 puncta in the cortex and cerebellum of WT and ATG5 cKO mice at 3 M. N = 4 mice per genotype. cKO^Cerebellum versus cKO^Cortex P = 0.001; WT^Cortex versus cKO^Cortex P = 0.002. m–p, EM analysis of mature and immature autophagosomes (AVs) in WT and ATG5 cKO PCs. m, Magenta arrowheads mark mature AVs or a mitophagy event. Yellow arrowheads indicate immature AVs. Scale bars, 1 µm. N = 4 mice per genotype. n, Magnified images of mature and immature AVs shown in m. o,p, Quantitative analysis of mature (o) and immature (p) AVs; mature AVs: P < 0.0001, immature AVs, P < 0.0001. Statistical significance calculated by unpaired two-tailed t-test. q,r, EM images (q) and quantitative analysis (r) of mitochondrial density in WT and ATG5 cKO PCs at 3 M. Scale bars, 0.5 µm. N = 4 mice per genotype. Statistical significance calculated by unpaired two-tailed t-test. s, Analysis of mitochondrial turnover in mitoTimer-expressing WT and ATG5 cKO PCs at 3 M. Scale bars, 20 µm; magnified image scale bars, 1 µm. t, Quantitative analysis of data in s. N = 4 WT/3 cKO. 1 M, 3 M and 12 M indicate 1, 3 and 12 months of age, respectively. Squares in f, m and s indicate regions magnified. All graphs show the mean ± s.e.m. Statistical significance in g, h, j, l and t was determined by two-way analysis of variance (ANOVA) followed by Holm–Sidak multiple-comparisons test. NS, not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

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