Fig. 7: GLUT2 deletion in ATG5 cKO mice mitigates PC neurodegeneration and improves their ataxic gait.
From: Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity
a, Generation of ATG5:GLUT2 cKO mice. Created with BioRender.com. b, 2-NBDG uptake in WT (125 cells, N = 5), ATG5 cKO (129 cells, N = 4) and ATG5:GLUT2 cKO (75 cells, N = 5) PCs. Scale bar, 50 µm. c, Quantitative analysis of data in b. WT versus ATG5 cKO, P = 0.0007; ATG5 cKO versus ATG5:GLUT2 cKO: P < 0.0001. d–f, Laconic FRET imaging (d) and its radiometric (e) and AUC analysis (f) in WT, ATG5 cKO and ATG5:GLUT2 cKO PCs, after 5-min perfusion with 5 mM pyruvate. WT (baseline 51 PCs, pyruvate 59 PCs from N = 3); ATG5 cKO (baseline 84 PCs, pyruvate 70 PCs from N = 4); ATG5:GLUT2 cKO (baseline 49 PCs, pyruvate 49 PCs from N = 3); GLUT2 cKO (baseline 29 PCs, pyruvate 29 PCs from N = 4). Scale bars, 20 µm. A coloured bar indicates the relative FRET signal. WT versus ATG5 cKO, P < 0.0001, ATG5 cKO versus ATG5:GLUT2 cKO: P = 0.032, ATG5:GLUT2 cKO versus GLUT2 cKO: P = 0.017. g, Levels of MG-modified proteins in PCs from WT (N = 7), ATG5 cKO (N = 5) and ATG5:GLUT2 cKO (N = 3) mice. Scale bars, 50 µm. h, Quantitative analysis of data in g. Statistical significance calculated by mixed-effects ANOVA followed by Holm–Sidak multiple-comparisons test. WT versus ATG5 cKO: P = 0.023, ATG5 cKO versus ATG5:GLUT2 cKO: P = 0.023. i, Top: Nissl-stained WT, ATG5 cKO and ATG5:GLUT2 cKO cerebellum. Scale bars, 200 µm. Bottom: magnified Nissl-stained cerebellum; scale bars, 50 µm. j, PC density in WT (N = 4), ATG5 cKO (N = 4) and ATG5:GLUT2 cKO (N = 5) cerebellum at 3 M. Statistical significance calculated by two-way ANOVA followed by Holm–Sidak multiple-comparisons test. ATG5 cKO versus ATG5:GLUT2 cKO: lobule VIII P = 0.006; lobule IX P = 0.004. WT versus ATG5 cKO, P < 0.0001. k, PC density in lobules VIII–IX in WT (N = 5), ATG5 cKO (N = 4) and ATG5:GLUT2 cKO (N = 5) mice. Scale bars, 200 µm. l, Quantitative analysis of data in k. WT versus ATG5 cKO: P < 0.0001, ATG5 cKO versus ATG5:GLUT2: P = 0.006, WT versus ATG5:GLUT2 cKO: P = 0.002. m, Kinematic analysis of WT, ATG5 cKO and ATG5:GLUT2 cKO mice walking on the 5-mm-wide beam. White arrowhead points to the slip in ATG5 cKO. n, Average number of slips in WT (N = 11), ATG5 cKO (N = 11) and ATG5:GLUT2 cKO (N = 6) mice at 3 M. Statistical significance calculated by two-way ANOVA followed by Holm–Sidak multiple-comparison (WT versus ATG5 cKO: P < 0.0001, ATG5 cKO versus ATG5:GLUT2 cKO: P < 0.0001). o–r, Angle, velocities and coordination of WT (N = 9; o), ATG5 cKO (N = 9; p) and ATG5:GLU T2 cKO (N = 6; q) mice walking on a 25-mm-wide beam and PCA analysis of these parameters (r). Red arrowheads in p indicate the points at which the relative positions of joints differ in ATG5 cKOs compared to WT and ATG5:GLUT2 cKOs. In r, individual mice are represented as coloured circles. Squares in i indicate regions magnified. All graphs show the mean ± s.e.m. Statistical significance in c, f and l was determined by one-way ANOVA followed by Holm–Sidak multiple-comparisons test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.
