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The following mass transitions were recorded (fragmentor voltage [FV] and collision energies [CE] in parentheses): sildenafil: m/z 475.2 → 58.0 (FV: 220 V, CE: 68 eV, quantifier), m/z 475.2 → 100.0 (FV: 220 V, CE: 28 eV), m/z 475.2 → 283.1 (FV: 220 V, CE: 44 eV); d3-sildenafil: m/z 478.2 → 60.9 (FV: 220 V, CE: 68 eV, quantifier), m/z 478.2 → 103.0 (FV: 220 V, CE: 28 eV), m/z 478.2 → 283.1 (FV: 220 V, CE: 44 eV). Peak areas were determined with MassHunter Software (Agilent Technologies) and sildenafil was directly quantified via its internal standard d3-sildenafil that was concentrated to 8 nM in the samples. Heteroplasmy levels and mtDNA haplotypes in the whole exome sequencing (WES) datasets were determined using the software MToolBox v.1.146,147 Results were visualized in the IGV viewer v2.16.148 Heteroplasmy levels of the MT-ATP6 mutations in fibroblasts, iPSCs, and NPCs were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, as described before.27 Genomic DNA was isolated using the Nucleo-Spin Tissue kit (Macherey-Nagel). We used restriction enzyme StuI (NEB, R0187, 10,000 units/ml) for the m.9185T>C mutation (wild-type: 24+90 pb, mutation: 114 pb), HpaII (NEB, R0171, 10,000 units/ml) for m.8993T>C and m.8993T>G (wild type: 25+155 bp; mutant: 180 bp), XbaI (NEB, R0145, 10,000 units/ml) for m.9176 T>G (wild type: 24+155 bp, mutant: 179 bp). The percentage of cleaved versus uncleaved fragments was determined by capillary electrophoresis and laser detection of the FAM-labelled RFLP-fragments using the 3,500 Series Genetic Analyzer (Applied Biosystems, RRID:SCR_021901) and normalized to a standard curve of known degrees of heteroplasmy. cMRI was performed for the six patients with LS carrying MT-ATP6. For Patient 1, imaging was carried out at 16.5 years of age and showed areas of increased T2-signal intensity in the Putamen and Nucleus caudatus on both sides (Figure 6E) and at the perisylvian gray matter (Figure S12H). For patient 2, imaging was carried out at 2.2 years of age and showed lesional areas in the Putamen and Nucleus caudatus in both T2-weighted images (Figure 6E) and Fluid Attenuated Inversion Recovery (FLAIR) images (Figure S12H). For patient 3, imaging was carried out at 5 years of age (Figure 6E) and at 14 years of age (Figure S12H). Areas with increased T2-signal intensity at 5 years of age (Figure 6E) had spontaneously resolved at 14 years of age (Figure S12H). In patient 4, FLAIR imaging was performed at 35 years of age and areas of increased signal intensity at the Nuclei caudati on both sides (Figure 6E) and enlargement of the internal and external liquor spaces due to diffused brain atrophy (Figure S12H). For patient 5, imaging was performed at 4.5 months of age and showed T2-weighted images delayed myelination, enlarged external cerebral spinal fluid spaces, and a general frontal brain atrophy (Figure 6E). For patient 6, imaging was performed at 7.5 years of age and showed areas of increased T2-signal intensity in the Putamen, Pallidum, and Nucleus caudatus on both sides (Figure 6E) and in the cortical gray matter (Figure S12H). CellTiter-Glo assay (Promega) was used to determine cell viability based on cellular ATP concentration.

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For permeabilization, we incubated the fixed cells with a blocking solution containing 10 % normal donkey serum (DNS) (Merck Millipore) and 1% Triton X-100 (Sigma-Aldrich) in DPBS with 0.05 % Tween 20 (Sigma-Aldrich) for 1 h at RT. We diluted primary antibodies in blocking solution and incubated them overnight at 4 oC on a shaker. Next, the primary antibody was removed, and the wells were rinsed three times with DPBS. Corresponding secondary antibodies (all Alexa Fluor, 1:2,000, Thermo Fisher Scientific) together with 1:2,500 Hoechst 33342 (Thermo Fisher Scientific) were diluted in blocking solution and added to the wells for 1 h at RT on a shaker. Finally, the staining solution was removed, and the wells were rinsed with DPBS three times following mounting of the coverslips on microscopic slides.

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We acquired the images of 2D cultures using the fluorescence microscope ZEISS Axio Observer Apotome 3 (Zeiss) in combination with the ZEN Microscopy Software (Zeiss) and further processed with ImageJ. For staining cortical brain organoids, we used 4 % PFA (Thermo Fisher Scientific) in DPBS for 1 h at RT and sliced them using the Vibratome Microm HM 650 V (Thermo Fisher Scientific). The samples were placed in a 3 % LB agar (Sigma-Aldrich) solution in DPBS and put in a cool place until the agar was solidified. The agar shape was freed from its mould and attached on the carrier plate of the vibratome and placed in cold DPBS. The cutting procedure was performed at an amplitude of 1.0 mm, a frequency of 60 Hz and a velocity of 13 mm/s.

S3 Fig. Western blot of p-ERK1/2 in the LV myocardium.

The organoids were sliced with a thickness of 100 μm and gently transferred to SuperFrost Plus glass slides (VWR) for staining. Blocking was carried out for 1 h at RT with a blocking solution containing 1 x DPBS, 10 % donkey serum (Sigma-Aldrich), 0.1 % Tween-20 (Sigma-Aldrich), and 1 % Triton-X (Merck). Primary antibodies were dissolved in blocking solution and applied on the slides and incubated at 4 °C overnight. The next day, the slides were rinsed three times for 10 min in sildenafil chewable tablets 1 x DPBS and then exposed to secondary antibodies in blocking solution at a dilution of 1:300 counterstained with 1:2,500 Hoechst 3342 (Invitrogen). The slides were incubated for 1 h at RT protected from light and subsequently rinsed three times for 10 min with 1 x DPBS. NPCs were isolated using Accutase and seeded onto Geltrex-coated 96-well plates at a density of 1.5 x 105 cells/cm2 and incubated in NPC medium overnight at 37 °C and 5 % CO2. The day after, NPCs were treated with DMSO, FCCP+AA or sildenafil dilution series in 100 μl and incubated for 16 h at 37 °C and 5 % CO2. 50 μl Cell Titer-Glo reagent solution was added to each well and placed on a plate shaker (1 min, 100 rpm) and incubated at ambient temperature for 10 min to ensure cell lysis. 100 μl of the solution was transferred onto a new white wall, white bottom 96-well plate, and luminescence was measured using an EnSight multimode plate reader (Revvity). For proliferation, NPCs were seeded in a coated 96-well plate (30,000 cells/well) and incubated at 37 °C and 5 % CO2. Medium glucose concentration was either 21 mM or 4.5 mM, sildenafil 50 mg tab or glucose was substituted with galactose. For toxicity analysis, the medium was additionally supplemented with Incuyte dye red (Sartorius) to indicate dead cells. Supplemented medium was changed every other day. Over the course of the experiment, the well plates were placed in the Cellcyte X (Cytena) microscope with a 10 x objective. Four pictures of each well were taken every three hours. Pipeline setup and analysis were done with the software CELLCYTE Studio (Cytena). Channels were “enhanced contour” for confluency and “red channel” (600 ms, 9 dB) for toxicity. Pictures taken with the “enhanced contour” channel were analyzed with the software-specific protocol “Cell Confluence” with the following settings: 58 a.u. Pictures taken with “red channel” were analyzed with the software-specific protocol “object count” with the following settings: 30 a.u. Analysis results were exported to Excel and further processed with R studio.

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We fixed NPCs and neurons grown on Matrigel-coated coverslips with 4 % PFA (Thermo Fisher Scientific) in DPBS for 20 min at RT and washed two times with DPBS. For permeabilization, we incubated the fixed cells with a blocking solution containing 10 % normal donkey serum (DNS) (Merck Millipore) and 1% Triton X-100 (Sigma-Aldrich) in DPBS with 0.05 % Tween 20 (Sigma-Aldrich) for 1 h at RT. We diluted primary antibodies in blocking solution and incubated them overnight at 4 oC on a shaker. Next, the primary antibody was removed, and the wells were rinsed three times with DPBS. Corresponding secondary antibodies (all Alexa Fluor, 1:2,000, Thermo Fisher Scientific) together with 1:2,500 Hoechst 33342 (Thermo Fisher Scientific) were diluted in blocking solution and added to the wells for 1 h at RT on a shaker. Finally, the staining solution was removed, and the wells were rinsed with DPBS three times following mounting of the coverslips on microscopic slides. We acquired the images of 2D cultures using the fluorescence microscope ZEISS Axio Observer Apotome 3 (Zeiss) in combination with the ZEN Microscopy Software (Zeiss) and further processed with ImageJ. For staining cortical brain organoids, we used 4 % PFA (Thermo Fisher Scientific) in DPBS for 1 h at RT and sliced them using the Vibratome Microm HM 650 V (Thermo Fisher Scientific).

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Afterwards, the insert membranes were carefully cut out of the holder with a scalpel and transferred to the centre of glass slides, with the cell side facing upwards. These were then covered with approximately 5 µl mounting medium and a cover glass, which was framed with transparent nail varnish after drying. Glass slides were imaged using the Operetta CLS High Content Imaging System. The samples were placed in a 3 % LB agar (Sigma-Aldrich) solution in DPBS and put in a cool place until the agar was solidified. The agar shape was freed from its mould and attached on the carrier plate of the vibratome and placed in cold DPBS. The cutting procedure was performed at an amplitude of 1.0 mm, a frequency of 60 Hz and a velocity of 13 mm/s.

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The organoids were sliced with a thickness of 100 μm and gently transferred to SuperFrost Plus glass slides (VWR) for staining.

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The day after, NPCs were treated with DMSO, FCCP+AA or sildenafil dilution series in 100 μl and incubated for 16 h at 37 °C and 5 % CO2. 50 μl Cell Titer-Glo reagent solution was added to each well and placed on a plate shaker (1 min, 100 rpm) and incubated at ambient temperature for 10 min to ensure cell lysis. 100 μl of the solution was transferred onto a new white wall, white bottom 96-well plate, and luminescence was measured using an EnSight multimode plate reader (Revvity). For proliferation, NPCs were seeded in a coated 96-well plate (30,000 cells/well) and incubated at 37 °C and 5 % CO2. Medium glucose concentration was either 21 mM or 4.5 mM, sildenafil 50 mg tab or glucose was substituted with galactose.

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For toxicity analysis, the medium was additionally supplemented with Incuyte dye red (Sartorius) to indicate dead cells. Supplemented medium was changed every other day. Over the course of the experiment, the well plates were placed in the Cellcyte X (Cytena) microscope with a 10 x objective. Four pictures of each well were taken every three hours. Pipeline setup and analysis were done with the software CELLCYTE Studio (Cytena).

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Channels were “enhanced contour” for confluency and “red channel” (600 ms, 9 dB) for toxicity. Pictures taken with the “enhanced contour” channel were analyzed with the software-specific protocol “Cell Confluence” with the following settings: 58 a.u. Pictures taken with “red channel” were analyzed with the software-specific protocol “object count” with the following settings: 30 a.u. Analysis results were exported to Excel and further processed with R studio. We fixed NPCs and neurons grown on Matrigel-coated coverslips with 4 % PFA (Thermo Fisher Scientific) in DPBS for 20 min at RT and washed two times with DPBS. Blocking was carried out for 1 h at RT with a blocking solution containing 1 x DPBS, 10 % donkey serum (Sigma-Aldrich), 0.1 % Tween-20 (Sigma-Aldrich), and 1 % Triton-X (Merck). Primary antibodies were dissolved in blocking solution and applied on the slides and incubated at 4 °C overnight.

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The next day, the slides were rinsed three times for 10 min in sildenafil chewable tablets 1 x DPBS and then exposed to secondary antibodies in blocking solution at a dilution of 1:300 counterstained with 1:2,500 Hoechst 3342 (Invitrogen). The slides were incubated for 1 h at RT protected from light and subsequently rinsed three times for 10 min with 1 x DPBS.

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Finally, coverslips were mounted using Pro-Long Glass Antifade Mountant (Invitrogen) on microscopic slides. Images of brain organoids were acquired using the Eclipse 90i upright widefield microscope (Nikon Microscope solutions) equipped with the imaging software NIS-Elements Advanced Research 3.2 (Nikon). Large images (7 x 7 stitches) were taken with a dry 20 x objective (Plan Apo VC 20 x / 0.75 air DIC N2 ∞/0.17 WD 1.0, Nikon Microscope Solutions) and three different filter channels (DAPI, FITC, TRITC). Specific structures of interest within the slices were imaged with the Confocal laser scanning microscope C1 (Nikon Microscope Solutions) and a dry 20 x objective (Plan Apo VC 20 x / 0.75 air DIC N2 ∞/0.17 WD 1.0, Nikon Microscope Solutions). Images were taken using the imaging software EZ-C1 Silver Version 3.91 at z-stack settings (1.1 μm step size, 10-30 steps). Primary antibodies used for NPCs and organoids include rabbit anti-PAX-6 (BioLegend, 1:200), mouse anti-ß-Tubullin III (TUJ1, Sigma-Aldrich, 1:2000), mouse anti-Nestin (Merck/Sigma-Aldrich, 1:200). Secondary antibodies used include donkey anti-rabbit 488 (Invitrogen, 1:300), donkey anti-rabbit 647 (Invitrogen, 1:1000) and donkey anti-mouse Cy3 (Sigma-Aldrich, 1:300).

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Tight junction proteins within BCECs were detected and visualized via immunofluorescence staining. For each step, 50 μl solution/insert was used.

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After washing three times with DPBS, fixated cells were incubated in permeabilization solution (0.1% Triton X-100 in DPBS) for 15 min, washed twice with DPBS for 5 min each and subsequently, unspecific binding sites were blocked by incubation for at least 30 min in blocking solution (3% BSA in DPBS, 0.1% Tween 20).

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ERK and calcineurin were activated in the RV as well as in the LV, and were inhibited by sildenafil treatment

Primary antibodies used for NPCs and organoids include rabbit anti-PAX-6 (BioLegend, 1:200), mouse anti-ß-Tubullin III (TUJ1, Sigma-Aldrich, 1:2000), mouse anti-Nestin (Merck/Sigma-Aldrich, 1:200). Secondary antibodies used include donkey anti-rabbit 488 (Invitrogen, 1:300), donkey anti-rabbit 647 (Invitrogen, 1:1000) and donkey anti-mouse Cy3 (Sigma-Aldrich, 1:300). Tight junction proteins within BCECs were detected and visualized via immunofluorescence staining. For each step, 50 μl solution/insert was used. After washing three times with DPBS, fixated cells were incubated in permeabilization solution (0.1% Triton X-100 in DPBS) for 15 min, washed twice with DPBS for 5 min each and subsequently, unspecific binding sites were blocked by incubation for at least 30 min in blocking solution (3% BSA in DPBS, 0.1% Tween 20).

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Primary antibodies were diluted in blocking solution and were applied to the inserts and incubated overnight at 4°C. Unbound antibodies were removed by washing twice for 5 min with DPBS. Next, cells were incubated with secondary antibodies, diluted in blocking solution, for 1 h at room temperature in the dark. After two additional washing steps, nuclear counterstaining was performed with 1 μg/ml Hoechst 33258 in DPBS. Cells were again washed twice with DPBS. Primary antibodies were diluted in blocking solution and were applied to the inserts and incubated overnight at 4°C. Unbound antibodies were removed by washing twice for 5 min with DPBS. Next, cells were incubated with secondary antibodies, diluted in blocking solution, for 1 h at room temperature in the dark.

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Tissue preparation

cMRI was performed for the six patients with LS carrying MT-ATP6. For Patient 1, imaging was carried out at 16.5 years of age and showed areas of increased T2-signal intensity in the Putamen and Nucleus caudatus on both sides (Figure 6E) and at the perisylvian gray matter (Figure S12H). For patient 2, imaging was carried out at 2.2 years of age and showed lesional areas in the Putamen and Nucleus caudatus in both T2-weighted images (Figure 6E) and Fluid Attenuated Inversion Recovery (FLAIR) images (Figure S12H). For patient 3, imaging was carried out at 5 years of age (Figure 6E) and at 14 years of age (Figure S12H). Areas with increased T2-signal intensity at 5 years of age (Figure 6E) had spontaneously resolved at 14 years of age (Figure S12H).

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In patient 4, FLAIR imaging was performed at 35 years of age and areas of increased signal intensity at the Nuclei caudati on both sides (Figure 6E) and enlargement of the internal and external liquor spaces due to diffused brain atrophy (Figure S12H). For patient 5, imaging was performed at 4.5 months of age and showed T2-weighted images delayed myelination, enlarged external cerebral spinal fluid spaces, and a general frontal brain atrophy (Figure 6E). For patient 6, imaging was performed at 7.5 years of age and showed areas of increased T2-signal intensity in the Putamen, Pallidum, and Nucleus caudatus on both sides (Figure 6E) and in the cortical gray matter (Figure S12H). CellTiter-Glo assay (Promega) was used to determine cell viability based on cellular ATP concentration. NPCs were isolated using Accutase and seeded onto Geltrex-coated 96-well plates at a density of 1.5 x 105 cells/cm2 and incubated in NPC medium overnight at 37 °C and 5 % CO2. After two additional washing steps, nuclear counterstaining was performed with 1 μg/ml Hoechst 33258 in DPBS. Cells were again washed twice with DPBS. Afterwards, the insert membranes were carefully cut out of the holder with a scalpel and transferred to the centre of glass slides, with the cell side facing upwards. These were then covered with approximately 5 µl mounting medium and a cover glass, which was framed with transparent nail varnish after drying. Glass slides were imaged using the Operetta CLS High Content Imaging System.

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