Fig. 4: SIRT3 deficiency in MNs results in ALS-like phenotypes.
From: ALS motor neurons exhibit hallmark metabolic defects that are rescued by SIRT3 activation
a Western blot analysis of day 28 MNs derived from BJ-iPS and two isogenic SIRT3+/− (#6 and #17) clones confirmed reduction in SIRT3 protein, along with increased MnSOD (K68ac) and increased acetylation of mitochondrial proteins. b Densitometric analyses of western blot bands reveal 50% decrease in SIRT3 protein levels and increased MnSOD (K68ac) in both SIRT3+/− #6 and #17 versus healthy MNs. c qPCR measurements of CHOP and sXBP1 show significant upregulation of both ER stress transcripts in SIRT3+/− #6 and #17 relative to the isogenic BJ-iPS control. d Measurements of OCR using the MitoStress assay of day 28 MNs differentiated from BJ-iPS (shown in black), SIRT3+/− #6 (pink), and #17 (violet). e Measurements of basal respiration, ATP production, and spare respiration of day 28 MNs differentiated from BJ-iPS (shown in black), SIRT3+/− #6 (pink), and #17 (violet). f Measurements of ECAR using the Glycolysis Stress assay of day 28 MNs differentiated from BJ-iPS (shown in black), SIRT3+/− #6 (pink), and #17 (violet). g Measurements of basal acidification, glycolysis, and glycolytic capacity of day 28 MNs differentiated from BJ-iPS (shown in black), SIRT3+/− #6 (pink), and #17 (violet). h Quantification of ISL1+ MNs derived from both BJ-SIRT3+/− clones from days 25 to 35 revealed a progressive death phenotype as compared to its healthy control (BJ-iPS). i Representative images of ISL1+SMI32+ MNs derived from BJ-iPS, BJ-SIRT3+/− #6 and #17 iPSCs, showing cell body sizes (outlined in white dotted lines) from MNs at day 28, and at day 31. Scale bars, 50 μm. j Quantification of mean cell body size and number of primary neurites of both BJ-SIRT3+/− clones reveal deteriorating neuronal health from days 28 to 31. **p < 0.01, ***p < 0.001, ns non-significant; one-way ANOVA, Tukey’s multiple comparisons post hoc test.
