Extended Data Fig. 9: Impact of conditional ATG5:GLUT2 deletion in GABAergic neurons on bodyweight and motor coordination in mice.
From: Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity
a, PCR for floxed (700 bp) and WT Atg5 (350 bp), floxed (326 bp) and WT Slc2a2 (235 bp). Double-floxed mice for Atg5 and Slc2a2 with additional hemizygosity for the Cre gene (235 bp + 326 bp) were defined as ATG5:GLUT2 cKO mice. b,c, Bodyweight over time (b) and at 8 weeks of age (c) of WT, ATG5 cKO, GLUT2 cKO and ATG5:GLUT2 cKO mice (N = 7 for WT, N = 8 for ATG5 cKO, N = 6 for GLUT2 cKO and N = 8 for ATG5:GLUT2 cKO mice). One-way ANOVA followed by Holm-Šidák multiple comparisons (WT vs ATG5 cKO: p = 0.011, WT vs GLUT2 cKO: p = 0.0005). d-f, Fluorescent analysis of GLUT2 levels in PCs of WT, GLUT2 cKO (d) and ATG5:GLUT2 cKO (e) at 3 M. Scale bar, 100 µm. N = 3 mice for WT, N = 1 for GLUT2 cKO and N = 3 for ATG5:GLUT2 cKO. One-way ANOVA followed by Holm-Šidák multiple comparisons (WT vs GLUT2 cKO: p < 0.0001, WT vs GLUT2 cKO: p < 0.0001). g, Immunoblot analysis of GLUT2 and ATG5 protein levels in WT, ATG5 cKO and ATG5:GLUT2 cKO cerebellar lysates from 1 independent experiment. h, 2-NBDG levels in WT (125 PCs from N = 5 mice) and GLUT2 cKO (85 PCs from N = 4) PCs. Two-tailed unpaired t-Test. i, Time spent on rotarod by WT and ATG5 cKO mice at 3 M. N = 11 for WT and N = 12 for ATG5 cKO mice. Two-tailed unpaired t-Test. j, Average number of slips of female WT and ATG5 cKO mice at 3 M when crossing 5 mm beam. N = 8 mice for WT, N = 6 for ATG5 cKO and N = 5 for ATG5:GLUT2 cKO. One-way ANOVA followed by Holm-Šidák multiple comparisons (WT vs ATG5 cKO: p = 0.041). k, Average number of slips of male WT and ATG5 cKO mice at 3 M when crossing 5 mm beam. N = 3 mice for WT, N = 4 for ATG5 cKO and N = 3 for ATG5:GLUT2 cKO. One-way ANOVA followed by Holm-Šidák multiple comparisons (WT vs ATG5 cKO: p = 0.004, ATG5 cKO vs ATG5:GLUT2 cKO: p = 0.019). l, Average number of slips of WT and ATG5 cKO mice at 12 M when crossing 25 mm and 12 mm beams. N = 5 mice for WT and N = 7 for ATG5 cKO. Two-way ANOVA followed by Holm-Šidák multiple comparisons (WT12 mm vs ATG5 cKO12 mm: p = 0.0002). 1 M, 3 M and 12 M indicate 1, 3 and 12 months, respectively m.g.v.-mean gray value. Squares in Fig. S9d,e 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.
