Fig. 5: Cerebellar GLUT2 is degraded by ATG5-dependent autophagy.

a, Immunoblot analysis of GLUT2 levels in WT cerebellum at different postnatal stages. b, Fluorescence-based analysis of GLUT2 levels in WT PCs. c, Quantitative analysis of fluorescence in b. N = 4 mice for each condition. 1 M versus 3 M: P = 0.012. Scale bars, 50 µm. d,e, LC3 puncta density in WT PCs. Scale bars, 20 µm; magnified image scale bars, 10 µm. N = 4 for 1 M and N = 3 for 3 M mice. 1 M versus 3 M P = 0.007. f,g, Immunoblot (f) and analysis (g) of GLUT2 levels in chloroquine (CQ)-treated (400 µM) cerebellar and cortical acute slices of 3 M WT animals. N = 6 for cerebellum and N = 4 for cortex. Cerebellum^Vehicle versus cerebellum^CQ: P = 0.031. h,i, Immunoblot (h) and analysis (i) of GLUT2 protein levels in control, CQ-treated (400 µM) and ACSF-treated (6 h) cerebellar slices of 3 M WT animals. N = 5 per condition. Statistical significance calculated by ratio-paired two-tailed t-test (vehicle versus ACSF: P = 0.043). j,k, LC3 overlap with GLUT2 in WT PC soma and dendrites, immunostained for GLUT2, LC3 and calbindin. Right: 3D reconstructions. Pink arrows indicate GLUT2⁺ autophagosomes. Scale bars, 20 µm; magnified regions, 5 µm; 3D reconstructions, 1 µm; 15 sections from N = 3 mice. l, Left: confocal images (left) and 3D reconstructions (right) of colocalization of GLUT2 with cathepsin D in WT PCs. Right: confocal images (left) and 3D reconstructions (right) of colocalization of GLUT2 with cathepsin D in ATG5 cKO PCs. Scale bars, 5 µm; 3D reconstruction scale bars, 1 µm. m, Colocalization analysis of GLUT2 with cathepsin D in WT and ATG5 cKO PCs. N = 3 mice per genotype. WT versus cKO: P = 0.01. n, Schematic of tandem–tagged mCherry-EGFP-GLUT2. o, mCherry-EGFP-GLUT2 ratios in WT and ATG5 cKO PCs. Scale bars, 50 µm; magnified image scale bars, 10 µm. p, Quantitative analysis of data in o. N = 4 mice per genotype. WT versus cKO P = 0.004. q, GLUT2 levels in PCs in 3 M WT and ATG5 cKO mice injected with either AAV-EGFP-ATG5 or AAV-EGFP. Scale bars, 20 µm. Insets: transduced PCs outlined by dashed circles. r, Quantitative analysis of data in q. cKO^EGFP: 17 PCs N = 3; cKO^ATG5-EGFP: 27 PCs N = 3; WT^ATG5-EGFP: 47 PCs N = 3, WT^EGFP: 36 PCs N = 5. WT^EGFP versus KO^EGFP: P = 0.001, WT^EGFP versus WT^ATG5-EGFP: P < 0.0001. In j and l, the colour-coded bar represents colocalization ratio, with the warm colours indicating strong colocalization. Squares in d, j and o indicate regions magnified. All graphs show the mean ± s.e.m. Statistical significance in c, e, m and p was determined by two-tailed unpaired t-test. Statistical significance in g and r was determined by two-way ANOVA followed by Holm–Sidak multiple-comparisons test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

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