Fig 2: Deregulation of NDUFS4 AS impairs mitochondrial respiratory chain complex I assembly and activity. (A) Western blot analysis of mitochondria-enriched fractions from NUGC4 and NUGC4-IQGAP1KO cells, using antibodies against different subunits of mitochondrial respiratory chain complexes. Complex I: NDUFS1, NDUFAF2; Complex II: SDHA (Succinate Dehydrogenase Complex Flavoprotein Subunit A); Complex III: UQCRC2 (Ubiquinol-Cytochrome C Reductase Core Protein 2); Complex IV: NDUFA4 (NDUFA4 mitochondrial complex associated), COX4 (Cytochrome c oxidase subunit 4); Complex IV: ATP5A (ATP synthase lipid-binding protein). HSP60 levels were used for normalization and quantitation is shown in Figure S3A. Numbers indicate MW in kDa. (B) Schematic representation of Complex I and its intermediates detected in NUGC4 and NUGC4-IQGAP1KO cells as presented in (C). N, N-module; S1, NDUFS1; S4, NDUFS4. The Q/P subcomplex of Complex I is represented by the black area. (C) BNGE analysis of mitochondrial respiratory chain complexes from NUGC4 and NUGC4-IQGAP1KO cells. Crude mitochondria fractions were solubilized in digitonin, which maintains the interaction of complex I (CI) with complex III and IV (supercomplexes, SC), or with n-Dodecyl-B-D-Maltoside (DDM), which allows the efficient extraction of CI as monomer. Note that the migration pattern of CI monomers differs depending on the detergent used for the solubilisation (44). The blots were stained against NDUFS4 (left), detected only in fully assembled CI, and NDUFS1 (right), detected in CI and its N-module-containing intermediates. Complex V levels, detected with an anti-ATP5A antibody, were used to monitor loading. The arrow indicates the NDUFS1-containing intermediate which accumulates only in NUGC4-IQGAP1KO cells. The graphs show the relative abundance of NDUFS1-containing CI monomers (up) or N-module intermediates (down) normalized against ATP5A. The mean of 3 independent experiments (DDM solubilisation) is presented. (D) Optical Density (OD) at 450nm, reflecting dye reduction due to mitochondrial complex I activity (NADH→NAD+ oxidation), was plotted against time. Left: Lysates from NUGC4 and NUGC4-IQGAP1KO cells transfected with pcDNA3.1 and NUGC4-IQGAP1KO cells transfected with cMyc-IQGAP1 expressing plasmid. Right: Lysates from HFE145 vs HFE145-IQGAP1KO cells. The curve corresponds to the mean of 3 (NUGC4) or 2 (HFE145) independent experiments. The diagram on the top of each plot shows the mean slope, reflecting mean mitochondrial complex I activity, expressed relatively to the parental cell line (NUGC4 or HFE145). Error bars represent ± SEM. One-way ANOVA analysis was performed to reveal statistically significant differences between NUGC4 cell lines. Two-tailed unpaired t-test analysis was performed to compare HFE145 cell lines. *, P < 0.05; ***, P < 0.001; ns, not significant.

Fig 3: PITRM1−/− iPSC-derived neurons show the induction of UPRmt and enhanced mitophagy.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).

Fig 4: IQGAP1 regulates AS and expression of the mitochondrial respiratory chain complex I subunit NDUFS4. (A) Sashimi plot for the visualization of the NDUFS4 AS event derived from aligned RNA seq data from NUGC4 and NUGC4-IQGAP1KO cells. Exon-exon junction reads are indicated by the numbers above the line. The chromosomal location is indicated below. At the bottom, a schematic shows the exons and the introns involved or surrounding the alternative event. The box in red indicates the alternative exon 2. (B) Schematic showing the predicted impact that the alternative splicing event has on the NDUFS4 protein. ETC, Electron Transport Chain; PTC, Premature Termination Codon. (C) The number of reads of the RNA seq data were plotted against the dPsi values for all events between control and KO cells. We highlight the 2 most confident AS changes affecting NDUFS4 and AIMP1 (Aminoacyl tRNA Synthetase Complex Interacting Multifunctional Protein 1). (D) RT-qPCR analysis of NDUFS4 exon 2 inclusion versus skipping, using isoform-specific primers. Biological triplicates were used for each cell line. (E) RT-qPCR analysis of the same samples as in (D) to monitor NDUFS4 expression, using a set of primers that recognize both the inclusion and exclusion isoforms. GAPDH levels were used for the normalization. (F) Western blot analysis of NDUFS4 levels in whole cell and mitochondria-enriched fractions from NUGC4 parental and KO cells. The mitochondrial matrix marker HSP60 was used as loading control. An experiment representative of 4 independent experiments is presented (see also Figure S2A for lysate preparations and Figure S3A for quantification), where NDUFS4 levels are expressed relatively to the control (NUGC4). (G) Same as (D) for NUGC4-IQGAP1KO cells transfected with pcDNA3.1 or cMyc-IQGAP1 expressing plasmid. The mean of the 3 independent experiments is presented. (H) Same as (E) for NUGC4-IQGAP1KO cells transfected with pcDNA3.1 or cMyc-IQGAP1 expressing plasmid. The mean of the 3 independent experiments is shown. (I) NDUFS4 levels in whole cell lysates of NUGC4 and NUGC4-IQGAP1KOcells transfected with pcDNA3.1 and NUGC4-IQGAP1KO cells transfected with cMyc-IQGAP1 expressing plasmid were analyzed by Western blot. HSP60 and β-actin were used as loading controls. Anti-IQGAP1 and anti-myc immunoblotting was used for the detection of endogenous and exogenous IQGAP1. The diagram on the right presents the mean of 3 independent expeiments. NDUFS4 levels (normalized against HSP60) are expressed relatively to the control (NUGC4). Error bars represent ± SEM. P values were calculated with two-tailed unpaired t-test anlaysis. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Numbers indicate MW in kDa.