Extended Data Fig. 7: Age-related alterations in GLUT2 expression and degradation dynamics in the cerebellum.
From: Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity
a, GLUT2 protein levels in WT cerebellar lysates at different postnatal stages. N = 5 mice per condition. One-way ANOVA followed by Holm-Šidák multiple comparisons (p7 vs 12 M: p = 0.034, p7 vs 3 M: p = 0.024, p7 vs 12 M: p = 0.003). b, GLUT2 protein levels in PC dendrites. N = 4 mice for 1 M and N = 3 mice for 3 M. Two-tailed unpaired t-Test: p = 0.007. c,d, LC3 protein levels in DMSO- and CQ- treated cerebellar and cortical acute slices of WT animals at 3 M. LC3 II levels in (d) were normalized to DMSO- treated acute slices (set to 100%). N = 6 mice for cerebellum and N = 4 for cortex. Two-way ANOVA followed by Holm-Šidák multiple comparisons (cerebellumvehicle vs cerebellumCQ: p = 0.003, cortexvehicle vs cortexCQ: p = 0.027). e, Slc2a2 mRNA levels in 3 M WT cerebellar acute slices after 6 h in ACSF. mRNA levels were normalized to Gapdh. N = 6 mice per condition. One-tailed unpaired t-Test. f-h, GLUT2 levels in 3 M WT cerebellar slices after 0 h, 3.5 h and 7 h of incubation in control medium, CHX (60 µM), ACSF or ACSF + CHX. GLUT2 levels were normalized to 0 h. N = 4 for each condition and timepoint. For (g) two-way ANOVA followed by Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (control CHX 3.5h vs ACSFCHX 3.5h: p = 0.032; controlCHX 7h vs ACSFCHX 7h: p = 0.011, ACSF7h vs control7h: p = 0.015). For (h) GLUT2 levels were normalized to controlCHX 0h followed by two-way ANOVA followed by Holm-Šidák multiple comparisons (ACSFCHX 3,5h vs controlCHX 3.5h: p = 0.0008, ACSFCHX 7h vs control CHX 7h: p = 0.0002). i, Example confocal images of cerebellar slices from WT animals at 3 M, immunostained for Puromycin from N = 1 independent experiment. Scale bar, 50 µm. j,k, LC3 puncta density in cortical WT interneurons. Scale bar, 50 µm. N = 4 mice for 1 M and N = 3 for 3 M. Two-tailed unpaired t-Test. l-o, Fluorescence-based (l-n) and EM-based (o) analyses of lysosomal density and area in WT and ATG5 cKO PCs at 3 M. Scale bar, 50 µm. Image in (l) represents Cathepsin channel shown in Fig. 5l. N = 4 mice per genotype. Two-tailed unpaired t-Test. p,q, Analysis of mKeima -GLUT2 in PCs from WT and ATG5 cKO OTCs. Scale bar, 50 µm. N = 4 WT and N = 3 ATG5 cKO mice. Two-tailed unpaired t-Test: p = 0.041. 1 M and 3 M indicate 1 and 3 months, respectively. Squares in Fig. S7j,p indicate regions magnified. 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.
