Fig. 6: By-products of aerobic glycolysis cause neurodegeneration of PCs.
From: Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity
a, Schematic illustrating upregulated glycolytic by-products in ATG5 cKO cerebellum. AGEs, advanced glycation end products; ROS, reactive oxygen species. b, Fluorescence-based analysis of MG-modified proteins in WT and ATG5 cKO PCs. Scale bars, 50 µm; magnified image scale bars, 10 µm. c, Quantitative analysis of data in b. N = 4 mice per genotype. Statistical significance calculated by two-way ANOVA followed by Holm–Sidak multiple-comparisons test (WT3M versus cKO3M, P = 0.008). d,e, Immunoblot (d) and analysis (e) of MG-modified proteins in WT and ATG5 cerebellum at 3 M. N = 4 mice per genotype. P = 0.046. f, Fluorescence-based analysis of levels of d-serine in WT and ATG5 cKO PCs. Scale bars, 20 µm. g, Quantitative analysis of data in f. N = 4 for 1 M and N = 5 for 3 M mice. Statistical significance calculated by two-way ANOVA followed by Holm–Sidak multiple-comparisons test (WT1M versus cKO1M: P = 0.025, WT3M versus cKO3M: P = 0.031). h, Fluorescence-based analysis of PC density in OTCs cultured in control media (vehicle) or 5 µM d-serine. Scale bars, 100 µm. i, Quantitative analysis of data in h; 16 images for control, 21 images for d-serine, N = 3. P = 0.0003. j, Fluorescence-based analysis of PC density in OTCs treated with either vehicle or 100 nM LPA. Scale bars, 100 µm. k, Quantitative analysis of data in j; 14 images for control, 11 images for LPA, N = 3. P = 0.007. l, Fluorescence-based analysis of PC density in OTCs treated for 24 h with vehicle, RSL3 (1 µM) or RSL3 + Fer1 (10 µM). Scale bars, 100 µm. m, Quantitative analysis of data in l; 28 images for control, 30 images for RSL3, 25 images for RSL3 + Fer1, N = 4. Vehicle versus RSL3: P = 0.0007; RSL3 versus RSL3 + Fer1: P = 0.037. n, PC density in OTCs treated with vehicle, LPA (100 nM) or LPA + Fer1 (5 µM). 15 images for control, 17 images for LPA and 17 images for LPA + Fer1, N = 3. Vehicle versus LPA: P = 0.01, LPA versus LPA + Fer1: P = 0.0003. o, PC density in OTCs cultured in control media containing vehicle, d-Serine (10 µM) or d-serine + Fer1 (5 µM); 14 images for control, 22 images for d-serine, 17 images for d-serine + Fer1, N = 4. Vehicle versus d-serine: P = 0.0006, d-serine versus d-serine + Fer1: P = 0.045. p, GCaMP7f-based time-lapse imaging of WT, WTLPA (100 nM, 7 days; q and r), and ATG5 cKO (s and t) PCs at baseline (0 seconds) and after stimulation with 100 action potentials (APs) at 100 Hz (20 seconds). Scale bar, 50 µm. q–t, Quantitative analysis of data in p; 164 cells for WT, 166 cells for WTLPA, 142 cells for cKO, N = 3. WTUntreated versus WTLPA: P = 0.0002; WTUntreated versus cKO: P < 0.0001. Squares in Fig. b indicate regions magnified. All graphs show the mean ± s.e.m. Statistical significance in e, i, k, r and t was determined by two-tailed unpaired t-test. Statistical significance in m–o 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.
