*4.2. Oxygen Consumption*

Oxygen consumption measurements in fibroblasts and iPSCs were performed in an Oroboros Oxygraph-2k and analyzed using DatLab4 software (Oroboros Instruments). Approximately one million fibroblasts or two million iPSCs were used in each chamber. An intact cell measurement protocol has been used. The experimental regime started with routine respiration (Cr), which is defined as respiration in cell-culture medium without additional substrates. After reaching steady-state respiratory flux, ATP synthase was inhibited with oligomycin (2 μg/mL), followed by uncoupling of oxidative phosphorylation by titration of FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) with 0.5 μM steps (CrU). Finally, respiration was inhibited by sequential addition of rotenone at 0.5 μM (to test for the e ffect of inhibiting complex I activity) (CRot) and antimycin A at 2.5 μM (for inhibiting complex III) (non-mitochondrial respiration, ROX). Values were normalized by number of cells, and ROX was subtracted. Basal respiration (Cr-ROX), maximum respiration (CrU-ROX), complex I contribution, CrU-(CRot-ROX), or complex II contribution (CRot-ROX) were calculated from a minimum of three independent experiments. Oxygen consumption measurements of induced neurons (iNs) were performed in a Seahorse XFe96 Analyzer and analyzed by Wave software (Agilent Technologies). Induction of iNs from iPSCs was performed in the seahorse microplate following a previously described protocol [**?** ] and measured at day 7, after puromycin selection. Before the experiment, growth medium was replaced by XF-Base Medium (Agilent Technologies) supplemented with 2 mM L-glutamine, 5 mM sodium pyruvate and 10 mM glucose (pH 7.4), and cells were kept 1h at 37 ◦C at atmospheric O2 and CO2. Oxygen consumption was determined at basal conditions (Cr) as well as after addition of the following drugs: 500 μM NV241 or DMSO as vehicle, FCCP titration (0.125, 1 or 2 μM), 2 μM rotenone (CRot) and 1 μg/mL antimycin A (ROX). After the experiment, Hoechst 33342 was added to the wells, and pictures were acquired in a fluorescence microscope to quantify DAPI surface. Values were normalized by DAPI surface and non-mitochondrial respiration (ROX) was subtracted. Basal respiration (Cr-ROX), maximum respiration (CrU-ROX), complex I contribution, CrU-(CRot-ROX), or complex II contribution (CRot-ROX) and e ffect of NV241 were calculated from three independent experiments.

#### *4.3. Protein Extraction and Western Blot*

Protein was extracted using cold RIPA bu ffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF) supplemented with protease inhibitor cocktail (Roche, 11873580001) and quantified using BCA Protein Assay Kit (Thermo

Fisher Scientific 23225). Fifty micrograms of protein was separated on a 12% SDS-PAGE gel and electrotransferred to Immobilon-P membranes (Millipore, IPVH00010). Primary antibodies used were Mitoprofile (Abcam ab110411; 1:1000), CS (Abcam ab96600; 1:10000) and GAPDH (Abcam ab8245; 1:6000). Appropriate secondary antibodies coupled to horseradish peroxidase were used, and peroxidase activity was tested using ECL (GE Healthcare, RPN2209). Quantification of WB was performed by densitometric analysis in Image J; values were normalized by total protein amount (GAPDH) and/or by mitochondrial mass (CS).

## *4.4. Lactic Acid Production*

Extracellular lactic acid concentration in the cell culture medium was determined using a lactate-dehydrogenase activity assay. First, 500,000 fibroblasts were plated on P100 culture dishes. The next day, growth medium was changed by fresh medium; exactly 24 h after medium was changed, 100 μL was removed, deproteinized and adjusted to pH 6–8. Samples were kept at −80 ◦C until assay. For the assay, 15 μL of the sample was incubated with 30 μL of NAD+ 15 mM, 5 μL of LDH 1 mg/mL (Roche, 10 127 230 001), 150 μL of assay buffer (consisting of 0.5 M glycine, 0.2 M hydrazine and 3.4 mM EDTA; pH 9.5) and adjusted to a final volume of 300 μL with bidistilled water. This reaction was incubated for 105 min at 37 ◦C before measuring absorbance at 340 nm. The lactate present in the sample together with NAD+ was transformed by lactate dehydrogenase in NADH, with concentration proportional to the increase in absorbance at 340 nm. A standard curve of lactate ranging from 4 to 0.25 mM was used to extrapolate the lactate concentration present in the sample. These values were normalized by the total amount of protein present in the culture measured with BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA, Ref 23225). For iPSCs, a similar protocol was used, but starting cell density was 80% confluent. The choice of measurement at 24 h after changing the medium was determined using previous kinetics data. At least three independent experiments were performed.

#### *4.5. Respiratory Chain Activity Determination by Spectrophotometry*

Measurements of the specific activities (SA) of respiratory chain complexes were determined following the protocol established in [**?** ]. Values were normalized with the specific activity (SA) of citrate synthase (CS). A minimum of three independent experiments was performed.

#### *4.6. NSC Generation and Neuronal Di*ff*erentiation*

For generation of NSC, the commercially available PSC Neural Induction Medium (ThermoFisher, Waltham, MA, USA, A1647801) was used, following manufacturer´s instructions. NSCs were routinely maintained in NEM medium (ThermoFisher, Waltham, MA, USA). For neuronal differentiation, 42,000 cells/cm<sup>2</sup> were seeded in GFR Matrigel-coated plates in NEM medium and maintained for 48 h. After this, growth medium was replaced with differentiation medium consisting of DMEMF12 (Thermo Fisher; Waltham, MA, USA, 11330-057) supplemented with 1x N2 (Thermo Fisher Scientific, Waltham, MA, USA, 1750200), 1x B27 (Thermo Fisher Scientific, Waltham, MA, USA, 17504044), 1x NEAA (Thermo Fisher Scientific, Waltham, MA, USA, 11140035) and 100 μM β-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA, 21985023). Culture medium was changed every other day for 6 weeks. For Patch Clamp and Calcium Imaging experiments, BrainPhys (Stemcell Technologies, Grenoble, France, 05792) was used instead of DMEMF12.

#### *4.7. Mutation Analysis and Heteroplasmy Quantification of mtDNA m.13513G*>*A Mutation*

For this purpose, the protocol described in Galera-Monge et al. [**?** ] was followed. Briefly, total DNA was extracted using a standard phenol-chloroform protocol. For mutation analysis, amplification by PCR of a mtDNA region containing the m.13513G>A position was carried out using the following primers: mt-20F: 5- ATCTGTACCCACGCCTTC 3- and mt-20R: 5- AGAGGGGTCAGGGTTGATTC 3-. Following PCR amplification, direct sequencing of amplicons was performed on both strands in an ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA) using a dye terminator cycle sequencing kit (Applera, Rockville, MD). Heteroplasmy quantification of m.13513G>A mutation was studied by RFLP. PCR amplification with the following primers: 13513F: 5-GACTGACTGACTGACAAGTCAACTAGGACTCATAATA3´ and 13513R: 5-CAGGCGTTTGTGTATGATATGTTTGCGGTTTCGATGACGTGG3´ was followed by digestion with restriction enzyme PflFI (New England Biolabs. Reference: R0595S) and quantified with the Agilent DNA 1000 Kit (Agilent, Santa Clara, CA, USA, 5067-1504) in an Agilent 2100 Bioanalyzer.
