Fig. 3: Hyper-acetylation of mitochondrial proteins in familial and sporadic ALS MNs.
From: ALS motor neurons exhibit hallmark metabolic defects that are rescued by SIRT3 activation
a Western blot analyses at day 28 revealed no significant changes in levels of mitochondrial proteins in both WT and ALS MNs. b Western blot analyses of iPSC-derived MNs at day 28 probing for SIRT3, total MnSOD, MnSOD with specific acetylation at lysine 68 (MnSOD K68ac) on whole-cell lysate as well as probing for acetyl-lysine proteins in purified mitochondrial extracts. c Quantitative analyses of Western blot bands where MnSOD(K68ac) levels were normalized to total MnSOD levels revealed between 4-fold to 5.5-fold increase in MnSOD(K68ac) in all the ALS iPSC-derived MNs compared to healthy MNs. d Immunohistochemistry of control and sporadic ALS patients (SALS) lumbar sections revealed increased MnSOD (K68ac) signals in SALS patients lumbar motor neurons (arrow). Lipofuscin is visible in large, healthy motor neurons, a function of normal cellular aging and unrelated to disease (*). Scale bars, 2.5 mm (left panel) and 100 μm (right panel). e Quantification of MnSOD (K68ac) signals in both control (n = 380) and SALS (n = 216) lumbar motor neurons demonstrates increased MnSOD (K68ac) signals in SALS patients. ***p < 0.001; Two-tailed t-test. f Densitometric analysis of acetyl-lysine signals normalized to TOMM20 revealed approximately threefold increase in acetylation of mitochondria proteins in all of our ALS iPSC-derived MNs. g, h Immunoprecipitation of Complex I subunits demonstrated increased acetyl-lysine signals in ALS MNs. i Complex I activity in healthy and ALS MNs was measured, which revealed significant decline of between 30 and 80% in the ALS MNs. *p < 0.05, **p < 0.01, ***p < 0.001, ns non-significant; one-way ANOVA, Tukey’s multiple comparisons post hoc test.
