Fig. 1: PITRM1−/− iPSC-derived neurons show the induction of UPRmt and enhanced mitophagy. | Molecular Psychiatry

Fig. 1: PITRM1−/− iPSC-derived neurons show the induction of UPRmt and enhanced mitophagy.

From: Loss of function of the mitochondrial peptidase PITRM1 induces proteotoxic stress and Alzheimer’s disease-like pathology in human cerebral organoids

Fig. 1

Control PITRM1+/+ (WT) and isogenic PITRM1−/− (KO) iPSCs were differentiated into cortical neurons. a Immunostaining of indicated differentiated iPSC cultures at DIV 35. Cells were stained for TBR1 (green) and β-III-tubulin (β-TUBIII, red). Nuclei were counterstained with DAPI (blue). Scale bar,50 μm. b Quantification of β-TUBIII/DAPI and TBR1/β-TUBIII-positive cells in differentiated iPSC cultures at DIV 35 (mean + SEM; n = 3). c Isogenic PITRM1+/+ and PITRM1−/− iPSC-derived neurons were stained with tetramethylrhodamine methylester (TMRM) and MitoTracker Green (MGreen) fluorescent dyes to determine mitochondrial membrane potential and mitochondrial mass, respectively. Representative images are shown. Scale bar, 10 μm. d Mitochondrial membrane potential (TMRM fluorescence) in neuronal soma and neurites (mean + SEM; *p < 0.05, two-tailed t test, n = 3). e Gene expression levels of mitochondrial stress response genes in PITRM1+/+ and PITRM1−/− iPSC-derived cortical neurons (mean + SEM; *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test, n = 5). f, g Representative western blots of the mitochondrial chaperones HSPA9 and HSP60 and the mitochondrial protease LONP1 in PITRM1+/+ and PITRM1−/− iPSC-derived cortical neurons. Quantification of protein levels relative to the loading control is shown in (g) (mean + SEM; *p < 0.05, two-tailed t test, n = 3). h Western blot analysis for LC3 in PITRM1+/+ and PITRM1−/− iPSC-derived neuronal cultures, untreated (−) or treated with 200 μM leupeptin and 20 mM NH4Cl for 4 h (+). i Quantification of LC3 flux normalized to WT (mean + SEM; *p < 0.05, two-tailed t test, n = 3). j Representative western blot of isolated mitochondria from PITRM1+/+ and PITRM1−/− iPSC-derived neurons probed against ubiquitin and VDAC as the loading control. k Quantification of mitochondrial protein ubiquitination levels in PITRM1+/+ and PITRM1−/− iPSC-derived neurons (mean + SEM; **p < 0.01, two-tailed t test, n = 3). I mtDNA content was measured as the mitochondrial (16S) to nuclear (RPLP0) DNA ratio by qRT-PCR (mean + SEM; *p < 0.05, two-tailed t test, n = 3).

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