**2. Results**

#### *2.1. Diminished Respiration in LS Fibroblasts Caused by a Decrease of Mitochondrial Mass*

As a first step to test mitochondrial function in human LS cells, we subjected LS fibroblasts harboring the *MT-ND5* m.13513G>A mutation, with a mutant load of 55%, and control fibroblasts to high-resolution respirometry on an Oroboros Oxygraph-2k. Measurements revealed that basal respiration (Cr-ROX), maximal respiratory capacity (CrU-ROX) and complex I contribution to respiration, CrU-(CRot-ROX), were lower in patient as compared to control fibroblasts (Figure **??**A). However, albeit lowered to a similar extent, no significant difference was detected in complex II contribution to respiration (CRot-ROX) (Figure **??**A). To further understand if the respiratory deficiency was provoked by diminished mitochondrial mass or quantity of RC complexes, a Western blot against subunits of complexes I-V and citrate synthase (CS) as a marker of mitochondrial mass was performed. A diminished mitochondrial mass was indeed observed in Leigh fibroblasts, accounting for the defect in complexes I, III+V and IV (Figure **??**B). A defect in complexes I, III+V and IV was detected when normalizing with GAPDH but not when normalizing with both CS and GAPDH (Figure **??**B). Moreover, analysis of enzymatic activities of respiratory complexes highlighted a normal function of complexes II, III and IV (CS normalized) but a decreased activity of CS in LS fibroblasts (Figure **??**C), supporting a decrease in mitochondrial mass as the main contribution to the lower levels of respiration. Finally, extracellular lactate measurements demonstrated that LS fibroblasts produced more than twice the lactate generated by control fibroblasts (Figure **??**D), which is consistent with the decreased respiration of LS fibroblasts.

**Figure 1.** Decreased mitochondrial mass and respiration in Leigh syndrome (LS) fibroblasts. (**A**) Oxygen consumption measured in Oroboros Oxygraph-2k. All data are displayed as a percentage of control. (**B**) Western blot assay against mitoprofile, citrate synthase (CS) and GAPDH (left). Quantification of the Western blot, normalized with GAPDH as a marker of the total protein (right, top panel) or GAPDH and citrate synthase (CS) as a marker of mitochondrial mass (right, bottom panel). All data are displayed as a percentage of control. (**C**) Spectrophotometric measurements of the activity of electron transfer chain (ETC) complexes (left) and citrate synthase (CS) (right); SA: specific activity. (**D**) Extracellular lactate production normalized by total protein. (\* *p*-value < 0.05; \*\* *p*-value < 0.01; \*\*\* *p*-value < 0.001)

#### *2.2. Generation of the Control iPSc Line N44SV.1*

Since LS models are scarce, generation of LS iPSCs and their differentiation into neural lineages would allow to shed light on the pathological mechanisms causing this disease.

To model LS, we used a previously obtained iPSC line, LND554SV.4, with the mutation m.13513G>A at a percentage of 45% [**?** ]. This line was derived from the same patient fibroblasts

where we detected diminished respiration. As a control, we generated the iPSC line (N44SV.1) from normal human dermal fibroblasts (NHDFs) using Sendai virus. We could confirm by sequencing that it, in contrast to the LS iPSC line, lacked the m.13513G>A mutation in the mtDNA (See Supplementary Figure S1A). Moreover, the N44SV.1 line displayed hES-like colonies positive for the pluripotency marker alkaline phosphatase (see Supplementary Figure S1B,C). N44SV.1 also showed high levels of mRNAs of pluripotency-related genes *OCT4*, *SOX2*, *CRIPTO*, *NANOG* and *REX1* in comparison with the original fibroblasts (see Supplementary Figure S1D). At the protein level, we detected by immunofluorescence the presence of pluripotent surface proteins SSEA3, SSEA4, Tra-1-81 and Tra-1-60 and the pluripotent transcription factors OCT4, SOX2 and NANOG (see Supplementary Figure S1E). The ability to di fferentiate into the three germ layers was tested using an embryoid body (EB)-based methodology. Endoderm (positive for AFP), mesoderm (positive for SMA) and neuroectoderm cells (positive for TUJ1) were observed (see Supplementary Figure S1F). Moreover, N44SV.1 presented a complete clearance of the Sendai viruses used for inducing reprogramming (see Supplementary Figure S1G) and a normal karyotype (see Supplementary Figure S1H). Finally, DNA fingerprinting analysis revealed genetic identity of N44SV.1 with control NHDFs (see Supplementary Figure S1I). Importantly, we did not detect any di fference in the reprogramming process or in the pluripotency markers between LND554SV.4 [**?** ] and the control iPSC line, N44SV.1.

#### *2.3. LS iPSCs Manifest a Decreased Basal Respiration and a Combined RC Deficiency*

Afterwards, we analyzed metabolic function of LS iPSCs by performing a mitochondrial characterization. Oxygen consumption measurements revealed that basal respiration (Cr-ROX) was diminished in LS iPSCs, while maximum respiration (CrU-ROX) and complex I contribution, CrU-(CRot-ROX), were not significantly decreased. Complex II contribution (CRot-ROX) was unaltered as compared to control iPSCs (Figure **??**A). Except for a significant increase in CS, analysis of mitochondrial mass and protein content of RC complexes by Western blot demonstrated no major di fferences between the patient and the control (Figure **??**B). However, activity measurements showed a prominent defect in the activity of complexes I and III of LS iPSC (Figure **??**C). Similar to fibroblasts, lactate levels were increased in LS iPSCs as compared to control iPSCs (Figure **??**D).

These findings show that hyperlactacidemia (a molecular marker of mitochondrial dysfunction commonly found in LS) can be detected as increased lactate levels in both fibroblasts and iPSCs derived from LS patient fibroblasts.

#### *2.4. Similar Proliferation and Di*ff*erentiation Capacity of LS iPSC-Derived NSCs*

In order to assess pathology in cells more relevant to the disease, we derived neural stem cells (NSCs) from control and LS iPSCs. NSCs were e fficiently derived from both patient and control iPSCs and stained positive for the NSC marker nestin (Figure **??**A). The m.13513G>A mutation was retained in patient NSCs at a percentage of 19.26% while absent in control NSCs (Figure **??**B). No di fferences were observed in the percentages of EdU+ cells between control and patient NSCs (Figure **??**C,D), indicating a similar proliferative rate. Di fferentiation capacity was tested at 3 weeks of culture in di fferentiation media. At that time point, control NSCs had developed a network of neuronal cells with clustering of somas and interconnecting neurites (Figure **??**E). The tendency to cluster was less strong in patient NSCs, which showed a less organized network. Immunocytochemistry for the neuronal marker MAP2 and the astrocytic marker GFAP revealed that most of the cells, both in control and patient, were MAP2+, and only a few of them were GFAP+ (Figure **??**F). In order to study the subtype of neurons present in the culture, we allowed NSCs to di fferentiate for 6 weeks and analyzed by immunofluorescence the presence of the glutamatergic marker KGA and the GABAergic marker GAD65/67. Both control and patient NSCs generated MAP2+ or TUJ1+ cells, which co-stained with KGA or GAD65/67, without any obvious di fferences between groups (Figure **??**G,H), proving the presence of both glutamatergic and GABAergic neurons in our cultures.

#### *2.5. Cell death and Complex I deficiency in LS iPSC-Derived Neurons*

In order to specifically analyze neuronal properties, we used a previously published protocol [**?** ] to induce a pure population of neurons (iPSC-iNs) from control and LS iPSCs. To analyze appearance of neuronal networks, sparse cytoplasmic lentiviral GFP labeling was used. Initially, neuronal networks derived from control and patient iPSCs appeared similar (Figure **??**A). However, after prolonged culture on mouse astrocytes, pronounced neuronal death was observed in cultures from patient iPSCs at day 21; this was further aggravated at day 42 (Figure **??**B).

**Figure 2.** LS induced pluripotent stem cells (iPSCs) manifest a decreased basal respiration and a combined respiratory chain (RC) deficiency. (**A**) Oxygen consumption measured in Oroboros Oxygraph-2k. All data are displayed as a percentage of control. (**B**) Western blot assay against mitoprofile, citrate synthase (CS) and GAPDH (left). Quantification of the Western blot, normalized with GAPDH as a marker of the total protein (right, top panel) or GAPDH and citrate synthase (CS) as a marker of mitochondrial mass (right, bottom panel). All data are displayed as a percentage of control. (**C**) Spectrophotometric measurements of the activity of ETC complexes (left) and citrate synthase (CS) (right); SA: specific activity. (**D**) Extracellular lactate production normalized by total protein. (\* *p*-value < 0.05; \*\* *p*-value < 0.01; \*\*\* *p*-value < 0.001)

**Figure 3.** LS neural stem cells (NSCs) manifested similar proliferative and differentiation capacity. (**A**) Immunofluorescence analysis of the neural stem cell marker nestin, manifesting an efficient generation of NSCs from iPSCs; scale bar: 100 μm. (**B**) Electropherograms showing the mutation m.13513G>A in patient NSCs and its absence in control NSCs (left) and heteroplasmy levels of m.13513G>A mutation by RFLP followed by Agilent quantification. (**C**) Proliferation assay of NSCs with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). Scale bar: 15 μm. (**D**) Quantification of EdU (percentage of EdU+/Hoechst+). (**E**) Bright field images (4× and 10×) of neural populations obtained after differentiation of NSCs. (**F**) Immunofluorescence analysis of MAP2, a marker of mature neurons, and GFAP, a marker of astrocytes, in the neural populations obtained after differentiation of NSCs in N2B27 for 3 weeks (**G**–**H**) Immunofluorescence analysis of the GABAergic marker GAD 65/67 and glutamatergic marker KGA together with neuronal markers (Tuj1 and MAP2).

Measurement of oxygen consumption was performed on a Seahorse Analyzer directly on attached iNs without replating to avoid cell death. A complex I deficiency was detected in patient iNs evidenced by a decrease of the basal respiration (Cr-ROX), maximum respiratory capacity (CrU-ROX) and complex I contribution, CrU-(CRot-ROX), to maximal respiratory capacity (Figure **??**C,D). Complex II contribution (CRot-ROX) was not different in patient iNs as compared to control (Figure **??**C,D). Treatment with the cell-permeable succinate prodrug NV241 resulted in a similar increase in routine respiration in both patient and control iNs, however the response in patient iNs vehicle (DMSO) control also displayed increased respiration which rendered the NV241 response data in the patient iNs inconclusive (Figure **??**C). Further, the treatment with the succinate prodrug increased complex II contribution to maximal uncoupled respiration in control iNs (Figure **??**E). In patient iNs, the succinate prodrug induced a similar level of complex II contribution to maximal uncoupled respiration, but the difference to vehicle (DMSO) control did not reach significance (Figure **??**E).

**Figure 4.** Respiratory defect and neurodegeneration of patient iPSC-derived neurons. (**A**) iN generation from iPSCs using lentiviral vectors for NgN2, rtTA and GFP showing no alterations in derivation of iNs from the patient. (**B**) iNs co-cultured with mouse astrocytes showing a marked neurodegeneration in the patient in comparison with the control both at days 21 and 42. (**C**) Oxygen consumption plots of the different treatments (Control/Patient and NV241/DMSO). (**D**) Quantification of oxygen consumption measured in a Seahorse XFe96 Analyzer. All data are normalized with the control. (**E**) Quantification of the contributions of complexes I and II to the maximum respiration, in percentages. (\* *p*-value < 0.05)

#### *2.6. LS iPSC-Derived Neurons are Functional*

In order to test whether the alteration in mitochondrial function has an effect on neuronal function, electrophysiological properties of LS neurons obtained, after a differentiation period of six weeks, were analyzed using whole-cell patch clamp. Neurons differentiated from both control and patient iPSC-derived NSCs were able to fire action potentials (APs) upon current injection (Figure **??**A), and the number of elicited APs in each current step injected was not different between groups (Figure **??**B). Moreover, the maximal number of elicited APs was similar (Figure **??**C), and the application of ramps of currents triggered trains of APs in patient neurons in the same way as in controls (Figure **??**D). Furthermore, both control and patient neurons had depolarizing inward Na<sup>+</sup> currents blocked by TTX (Figure **??**E) and repolarizing outward K<sup>+</sup> currents blocked by TTX + TEA (Figure **??**F). In conclusion, no abnormalities were detected in intrinsic properties or AP characteristics of patient neurons as compared to control.

**Figure 5.** LS iPSC-derived neurons are electrophysiologically functional. (**A**) Neurons derived from control (black) and patient (orange) neural stem cells were able to generate action potentials (APs) upon current injection. (**B**) Graph showing the injected current versus the number of APs elicited (*n* = 8). (**C**) Bar diagram of the maximal number of APs induced (*n* = 8, n.s.). (**D**) Voltage traces show that current injection (ramp from 0–300 pA) induces trains of APs; \* denotes the expanded AP17. (**E**,**F**) Left: Current traces of the fast inward current peak (**E**) and the sustained outward current (**F**) activated by step depolarizations from a holding potential of −70 mV in the absence and presence of 1 mM TTX (**E**) or 1 mM TTX and 10 mM TEA (**F**); \* denotes the fast inward current peak (**E**) and the sustained outward current (**F**). Right: Voltage–current plot of the inward current peak (**E**) and sustained outward current (**F**).

#### *2.7. Disturbed Calcium Regulation in LS iPSC-Derived Neurons*

Given the role of the mitochondria in regulating intracellular calcium we wanted to assess calcium dynamics in LS neurons. Intracellular calcium concentrations were analyzed by live cell calcium imaging in NSC-derived neurons, after 6 weeks of differentiation. KCl was added in order to stimulate neuronal activity, and the number of evoked cells (responding to KCl) was drastically diminished in patient neurons as compared to controls (see Supplementary Video S1 (Control) and Supplementary Video S2 (Patient)). Moreover, patient evoked cells showed a very different response, compared to controls, with increase in the width of peaks (time from basal to basal level) and time between peaks (Figure **??**A). Quantification confirmed the increase in the width of peaks (Figure **??**B), which could be explained by a slower increase in cytoplasmic Ca2+ upon a depolarizing stimulus or a decrease in the calcium buffering capacity. In order to understand the specific step affected, peaks were measured as time to peak (from basal to maximum level) and time to decay (from maximum to basal level). Although both parameters were increased in patient cells, time to decay was more affected, indicating a Ca2+ buffering defect (Figure **??**B). Time between peaks was also assessed and showed an increase of the refractory period in patient neurons (Figure **??**C). All these results together indicate a dysregulation in calcium homeostasis caused by the m.13513G>A mutation.

**Figure 6.** Calcium dysregulation in LS iPSC-derived neurons. (**A**) Representative plots of calcium imaging displaying a different response to KCl in LS iPSC-derived neurons. FRU: fluorescence relative units. (**B**) Quantification of the width of the peaks (from basal to basal), time to peak (from basal to peak) and time to decay (from peak to basal). (**C**) Quantification of the time between peaks. (\*\*\* *p*-value < 0.001)
