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We dissected brain organoids on days 70–74 to prepare cortical brain organoid slices (cBOSs),69 which we grew until day 129 and then treated with sildenafil for 24 h before applying acute metabolic stress (2 min of glucose deprivation and inhibition of glycolysis [GLY] and OXPHOS) (Figure 4A). The calcium response to metabolic stress was more pronounced and premature in LS cBOS compared with control cBOS, suggesting increased susceptibility to metabolic imbalance (Figures 4B and 4C). Pre-treatment with sildenafil in LS cBOS reduced their calcium load after metabolic stress and also their peak calcium amplitude (Figures 4B and 4C). Hence, sildenafil might prevent excessive decompensation in LS neuronal cells under acute metabolic stress. To investigate the interplay between calcium and bioenergetics in treated LS neural cells, we applied a mathematical model70 that coupled cytosolic calcium dynamics with mitochondrial function, incorporating F1F0 ATPase activity (F1F0), GLY, the adenine nucleotide translocator (ANT), NADH production via the aspartate-glutamate carrier (AGC), and NADH oxidation in ETC (o). Based on our previous calcium modeling,71,72,73 we integrated additional fluxes through the plasma membrane calcium ATPase (PMCA), the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and IP3 receptors (IP3Rs) (Table S2). Following global sensitivity analysis70 (Figure S9A), the model recapitulated the pattern of calcium traces measured in LS cBOS (Figure S9B). Combining a high rate of F1F0 with a low rate of IP3R (Figure S9C) reproduced the reduction of calcium peak in sildenafil-treated LS cBOS under stress (Figures 4B and 4C) and key features observed in sildenafil-treated LS NPCs, including MMP reduction (Figures 1D and 1E), slight NADH reduction (Figure S1D), and ATP increase (Figure 1G).

Parameter	Description	Typical Value
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Tmax (time to peak)	Time to reach maximum plasma concentration	30-120 minutes
Half-life	Duration of drug activity	4-5 hours
Metabolism	Main route	Liver (CYP3A4 enzyme)
Excretion	How the drug leaves the body	Mainly feces, some urine

Other combinations failed to recapitulate sildenafil effects (Figures S9D and S9E). The modeling thus suggested that sildenafil’s action may be mimicked by increasing CV activity together with decreasing calcium release from the endoplasmic reticulum (ER), in turn improving cellular bioenergetics. In the absence of extracellular calcium, pre-treatment with sildenafil in LS NPCs increased the thapsigargin-induced cytoplasmic calcium signal (Figures 4D and 4E), possibly indicating higher ER calcium storage or modulation of mitochondrial calcium homeostasis.74,75 A faster decline of calcium in sildenafil-treated cells (Figure 4D) could imply reduced calcium release from the ER through IP3R, as suggested by the model, or enhanced cytosolic calcium efflux to the extracellular space through the sodium-calcium exchanger (NCX) or the PMCA because of increased cytosolic ATP levels by sildenafil or augmented uptake into mitochondria. To indirectly address mitochondrial calcium, we investigated big-conductance calcium-activated potassium (BKCa) channels, which are localized in the plasma and mitochondrial membrane76 and have been implicated in sildenafil’s action.77,78 As previously shown,79 human BKCa KO cells80 displayed increased MMP compared with wild type (WT) (Figures 4F and 4G). Sildenafil normalized MMP when BKCa KO cells were incubated in DMSO (Figure 4F) but not when they were acutely exposed to the CV inhibitor oligomycin (Figure 4G). The results suggest that BKCa channels may contribute to the action of sildenafil on MMP when cells lack a functional CV and that residual CV activity is necessary for the restorative effect of sildenafil, as indicated by the model.

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In all the other cases, sildenafil was well tolerated (Table S4), leading to improvement in motor function and development (Tables 1 and S3). These findings raised the possibility that MMP normalization by sildenafil may not be due to direct mitochondrial uncoupling. To test this, we employed NPCs carrying a variant in the nuclear gene SURF125 (Figure S10H). The MMP of SURF1 mutant NPCs was depolarized compared with that of isogenic control NPCs (Figure 4H). Nonetheless, sildenafil still restored MMP (Figure 4H). Hence, sildenafil might also be beneficial in other forms of LS where MMP is depolarized. We applied chemical manipulation of the PDE5 pathway to prove its involvement in the MMP rescue of LS NPCs (Figure 4I). The cGMP analog 8-Br-cGMP recapitulated the MMP amelioration seen with sildenafil (Figure 4J). Inhibition of the downstream target PRKG1 with KT5823 blunted the effect of sildenafil on MMP normalization (Figure 4K).

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We quantified the effect of sildenafil on neuronal outgrowth81 of dopaminergic neurons (Figure S10C). In agreement with previous findings,25,30 MT-ATP6 mutant neurons and SURF1 mutant neurons exhibited reduced neurite length (Figures 4N, 4O, and S10D).

• Sildenafil was initially developed to treat hypertension and angina.
• The typical prescribed dose is up to 100mg, but only under medical supervision.
• Recreational misuse of high doses can result in serious health issues.
• Educate yourself about the potential risks and proper use of sildenafil.
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Sildenafil promoted neurite outgrowth in LS neurons (Figures 4N, 4O, and S10D).

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Upon placing LS mice in metabolic chambers (Figure S11A), we observed improved oxygen consumption and carbon dioxide production with sildenafil (Figure 5C), possibly indicating enhanced metabolic fitness. Cardiac bradyarrhythmia and dysfunction were also ameliorated (Figure S11B). Consistent with previous observations,14,87,88 LS mice showed cleaved caspase-3 staining in the cerebellum and brain stem regions associated with increased Iba1 staining,89 suggestive of cell death and microglial activation (Figures 5D and 5E). The number of cells positive for caspase-3 or Iba1 decreased in sildenafil-treated LS mice (Figures 5D and 5E). The loss of Purkinje cells of the cerebellum was also ameliorated (Figures S11C and S11D).

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The expression of Prkg1, which is particularly evident in normal cerebellar Purkinje neurons,15 was reduced in LS mice and rescued by sildenafil (Figures 5D and 5E). Next, we employed SURF1 KO pigs, which exhibit severe neurodevelopmental impairment.11 SURF1 KO piglets were treated immediately at birth (n = 3 animals with 2.1 mg/kg/day, n = 4 animals with 0.5 mg/kg/day). Sildenafil improved animal responsiveness, mobility, and suckling behavior, leading to lifespan extension once the unstable perinatal phase was overcome (Figure 5F). Some LS pigs survived beyond day 261 despite reduced body weight (98 kg vs. 140–170 kg in age-matched controls).

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Sildenafil increased the body temperature of LS pigs, suggesting an effect on systemic metabolism (Figures 5G and S11F). The expression of NPC-associated doublecortin (DCX) was normalized by sildenafil (Figures 5H and S11G), indicating neurodevelopmental amelioration. CIV activity was also restored (Figure 5I). Reduced PRKG1 levels in the basal ganglia of LS pigs increased with treatment in most animals (Figures 5J and S11G). By adapting the Newcastle Pediatric Mitochondrial Disease Scale (NPMDS), we observed improvement in multiple domains of neuromotor integrity (Figures 5K and S11E). This improvement was specific to LS neurons (carrying MT-ATP6, SURF1, or NDUFS4 variants) (Figure 4P) and was not observed in control neurons (Figure S10E). PRKG1 signal was reduced in LS neurons (Figure S10F), and PRKG1 knockdown in control neurons recapitulated the neurite growth defects seen in LS neurons (Figure 4Q). Because PRKG1 is involved in both calcium homeostasis and neurite outgrowth,82,83,84,85,86 its dysregulation in LS might underscore its role as a therapeutic target that could contribute to the sildenafil response (Figure S10G). To address the therapeutic potential of sildenafil in vivo, we employed the germline Ndufs4 KO mouse.14,15 Sildenafil was added to the drinking water of the animals starting on day 25. The treatment extended the lifespan of Ndufs4 KO mice (Figure 5A) and alleviated muscle weakness and ataxia by partially correcting defective energy sildenafil 100 mg tab expenditure (Figure 5B). Upon placing LS mice in metabolic chambers (Figure S11A), we observed improved oxygen consumption and carbon dioxide production with sildenafil (Figure 5C), possibly indicating enhanced metabolic fitness. Cardiac bradyarrhythmia and dysfunction were also ameliorated (Figure S11B). Consistent with previous observations,14,87,88 LS mice showed cleaved caspase-3 staining in the cerebellum and brain stem regions associated with increased Iba1 staining,89 suggestive of cell death and microglial activation (Figures 5D and 5E).

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To test this, we employed NPCs carrying a variant in the nuclear gene SURF125 (Figure S10H). The MMP of SURF1 mutant NPCs was depolarized compared with that of isogenic control NPCs (Figure 4H). Nonetheless, sildenafil still restored MMP (Figure 4H). Hence, sildenafil might also be beneficial in other forms of LS where MMP is depolarized. We applied chemical manipulation of the PDE5 pathway to prove its involvement in the MMP rescue of LS NPCs (Figure 4I).

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The cGMP analog 8-Br-cGMP recapitulated the MMP amelioration seen with sildenafil (Figure 4J). Inhibition of the downstream target PRKG1 with KT5823 blunted the effect of sildenafil on MMP normalization (Figure 4K). We quantified the effect of sildenafil on neuronal outgrowth81 of dopaminergic neurons (Figure S10C). In agreement with previous findings,25,30 MT-ATP6 mutant neurons and SURF1 mutant neurons exhibited reduced neurite length (Figures 4N, 4O, and S10D). Sildenafil promoted neurite outgrowth in LS neurons (Figures 4N, 4O, and S10D).

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This improvement was specific to LS neurons (carrying MT-ATP6, SURF1, or NDUFS4 variants) (Figure 4P) and was not observed in control neurons (Figure S10E). PRKG1 signal was reduced in LS neurons (Figure S10F), and PRKG1 knockdown in control neurons recapitulated the neurite growth defects seen in LS neurons (Figure 4Q). Because PRKG1 is involved in both calcium homeostasis and neurite outgrowth,82,83,84,85,86 its dysregulation in LS might underscore its role as a therapeutic target that could contribute to the sildenafil response (Figure S10G). To address the therapeutic potential of sildenafil in vivo, we employed the germline Ndufs4 KO mouse.14,15 Sildenafil was added to the drinking water of the animals starting on day 25. The treatment extended the lifespan of Ndufs4 KO mice (Figure 5A) and alleviated muscle weakness and ataxia by partially correcting defective energy sildenafil 100 mg tab expenditure (Figure 5B). The number of cells positive for caspase-3 or Iba1 decreased in sildenafil-treated LS mice (Figures 5D and 5E).

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We dissected brain organoids on days 70–74 to prepare cortical brain organoid slices (cBOSs),69 which we grew until day 129 and then treated with sildenafil for 24 h before applying acute metabolic stress (2 min of glucose deprivation and inhibition of glycolysis [GLY] and OXPHOS) (Figure 4A). The calcium response to metabolic stress was more pronounced and premature in LS cBOS compared with control cBOS, suggesting increased susceptibility to metabolic imbalance (Figures 4B and 4C). Pre-treatment with sildenafil in LS cBOS reduced their calcium load after metabolic stress and also their peak calcium amplitude (Figures 4B and 4C). Hence, sildenafil might prevent excessive decompensation in LS neuronal cells under acute metabolic stress. To investigate the interplay between calcium and bioenergetics in treated LS neural cells, we applied a mathematical model70 that coupled cytosolic calcium dynamics with mitochondrial function, incorporating F1F0 ATPase activity (F1F0), GLY, the adenine nucleotide translocator (ANT), NADH production via the aspartate-glutamate carrier (AGC), and NADH oxidation in ETC (o).

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Based on our previous calcium modeling,71,72,73 we integrated additional fluxes through the plasma membrane calcium ATPase (PMCA), the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and IP3 receptors (IP3Rs) (Table S2). Following global sensitivity analysis70 (Figure S9A), the model recapitulated the pattern of calcium traces measured in LS cBOS (Figure S9B). Combining a high rate of F1F0 with a low rate of IP3R (Figure S9C) reproduced the reduction of calcium peak in sildenafil-treated LS cBOS under stress (Figures 4B and 4C) and key features observed in sildenafil-treated LS NPCs, including MMP reduction (Figures 1D and 1E), slight NADH reduction (Figure S1D), and ATP increase (Figure 1G). Other combinations failed to recapitulate sildenafil effects (Figures S9D and S9E). The modeling thus suggested that sildenafil’s action may be mimicked by increasing CV activity together with decreasing calcium release from the endoplasmic reticulum (ER), in turn improving cellular bioenergetics.

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In the absence of extracellular calcium, pre-treatment with sildenafil in LS NPCs increased the thapsigargin-induced cytoplasmic calcium signal (Figures 4D and 4E), possibly indicating higher ER calcium storage or modulation of mitochondrial calcium homeostasis.74,75 A faster decline of calcium in sildenafil-treated cells (Figure 4D) could imply reduced calcium release from the ER through IP3R, as suggested by the model, or enhanced cytosolic calcium efflux to the extracellular space through the sodium-calcium exchanger (NCX) or the PMCA because of increased cytosolic ATP levels by sildenafil or augmented uptake into mitochondria. To indirectly address mitochondrial calcium, we investigated big-conductance calcium-activated potassium (BKCa) channels, which are localized in the plasma and mitochondrial membrane76 and have been implicated in sildenafil’s action.77,78 As previously shown,79 human BKCa KO cells80 displayed increased MMP compared with wild type (WT) (Figures 4F and 4G). Sildenafil normalized MMP when BKCa KO cells were incubated in DMSO (Figure 4F) but not when they were acutely exposed to the CV inhibitor oligomycin (Figure 4G). The results suggest that BKCa channels may contribute to the action of sildenafil on MMP when cells lack a functional CV and that residual CV activity is necessary for the restorative effect of sildenafil, as indicated by the model. These findings raised the possibility that MMP normalization by sildenafil may not be due to direct mitochondrial uncoupling. The loss of Purkinje cells of the cerebellum was also ameliorated (Figures S11C and S11D). The expression of Prkg1, which is particularly evident in normal cerebellar Purkinje neurons,15 was reduced in LS mice and rescued by sildenafil (Figures 5D and 5E). Next, we employed SURF1 KO pigs, which exhibit severe neurodevelopmental impairment.11 SURF1 KO piglets were treated immediately at birth (n = 3 animals with 2.1 mg/kg/day, n = 4 animals with 0.5 mg/kg/day). Sildenafil improved animal responsiveness, mobility, and suckling behavior, leading to lifespan extension once the unstable perinatal phase was overcome (Figure 5F). Some LS pigs survived beyond day 261 despite reduced body weight (98 kg vs. 140–170 kg in age-matched controls). Sildenafil increased the body temperature of LS pigs, suggesting an effect on systemic metabolism (Figures 5G and S11F).

• Side effects at high doses like 100g can include vision impairment and hearing loss.
• Sildenafil can cause headaches, dizziness, or temporary vision changes.
• Patients taking medications for blood pressure should consult their doctor beforehand.
• The drug's effect lasts around 4-6 hours, but duration varies.
• Misuse or overdose increases the risk of serious health complications.

The expression of NPC-associated doublecortin (DCX) was normalized by sildenafil (Figures 5H and S11G), indicating neurodevelopmental amelioration.

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CIV activity was also restored (Figure 5I). Reduced PRKG1 levels in the basal ganglia of LS pigs increased with treatment in most animals (Figures 5J and S11G). By adapting the Newcastle Pediatric Mitochondrial Disease Scale (NPMDS), we observed improvement in multiple domains of neuromotor integrity (Figures 5K and S11E). Collectively, the in vivo data highlighted the potential clinical effectiveness of sildenafil and underscored PRKG1 as one of its mechanistic targets. In some LS pigs treated with high sildenafil doses, we noticed microhemorrhages (Figure S11H). We further assessed toxicity using LS iPSC-derived cardiomyocytes (Figure S12A). The findings are in agreement with the higher mortality rate seen in children with PAH treated with high sildenafil doses.40 We then probed the blood-brain barrier (BBB) permeability of sildenafil using an iPSC-based model90 (Figure 6A). Similar to control brain capillary endothelial cells (BCECs), LS BCECs showed correct morphology and marker expression (Figure 6B) as well as monolayer integrity and transendothelial electrical resistance (TEER) (Figures S12F and S12G). The permeability of sildenafil and sildenafil citrate (the active pharmacological substance applied orally in clinical applications) in LS BCECs was similar to that of control BCECs (Figure 6C). The results are consistent with permeability values of sildenafil reported in other settings91 and imply that sildenafil can effectively cross the BBB in LS. Because sildenafil can be used safely in pediatric conditions,39,40,41,42 we initiated off-label treatment on an individual basis with sildenafil in six LS patients carrying MT-ATP6 variants (Tables 1 and S3). All patients showed cranial magnetic resonance imaging (cMRI) signs consistent with LS lesions (Figures 6E and S12H).

Frequently asked questions

Collectively, the in vivo data highlighted the potential clinical effectiveness of sildenafil and underscored PRKG1 as one of its mechanistic targets. In some LS pigs treated with high sildenafil doses, we noticed microhemorrhages (Figure S11H). We further assessed toxicity using LS iPSC-derived cardiomyocytes (Figure S12A). The findings are in agreement with the higher mortality rate seen in children with PAH treated with high sildenafil doses.40 We then probed the blood-brain barrier (BBB) permeability of sildenafil using an iPSC-based model90 (Figure 6A). Similar to control brain capillary endothelial cells (BCECs), LS BCECs showed correct morphology and marker expression (Figure 6B) as well as monolayer integrity and transendothelial electrical resistance (TEER) (Figures S12F and S12G).

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The permeability of sildenafil and sildenafil citrate (the active pharmacological substance applied orally in clinical applications) in LS BCECs was similar to that of control BCECs (Figure 6C). The results are consistent with permeability values of sildenafil reported in other settings91 and imply that sildenafil can effectively cross the BBB in LS. Because sildenafil can be used safely in pediatric conditions,39,40,41,42 we initiated off-label treatment on an individual basis with sildenafil in six LS patients carrying MT-ATP6 variants (Tables 1 and S3). All patients showed cranial magnetic resonance imaging (cMRI) signs consistent with LS lesions (Figures 6E and S12H). From patient 1, we also obtained iPSCs (line ATP6_7), which were used to generate NPCs and brain organoids (Figure S10H). From patient 1, we also obtained iPSCs (line ATP6_7), which were used to generate NPCs and brain organoids (Figure S10H). When selecting the sildenafil dosage, we referred to the trial STARTS-1 (Sildenafil in Treatment-Naive Children, Aged 1 to 17 Years, With Pulmonary Arterial Hypertension)41 and the trial STARTS-240 in children with PAH.

• Sildenafil can cause side effects such as headaches, flushing, or nasal congestion.
• At high doses, the risk of side effects and adverse reactions increases significantly.
• Combining sildenafil with nitrates can cause life-threatening blood pressure drops.
• The medication is available by prescription in various formulations and strengths.
• Proper dosing and medical supervision are essential for safe usage of sildenafil 100g.

Given the potential toxic effects of high dosages, we only considered low (0.66–1.49 mg/kg/day) or medium dosages (1.50–3.00 mg/kg/day). Two patients/guardians opted for low dosages and four for medium dosages. To monitor the occurrence of adverse reactions, we asked patients/guardians to respond to a questionnaire on side effects after 6 months of sildenafil use (Table S4).

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When selecting the sildenafil dosage, we referred to the trial STARTS-1 (Sildenafil in Treatment-Naive Children, Aged 1 to 17 Years, With Pulmonary Arterial Hypertension)41 and the trial STARTS-240 in children with PAH. Given the potential toxic effects of high dosages, we only considered low (0.66–1.49 mg/kg/day) or medium dosages (1.50–3.00 mg/kg/day). Two patients/guardians opted for low dosages and four for medium dosages. To monitor the occurrence of adverse reactions, we asked patients/guardians to respond to a questionnaire on side effects after 6 months of sildenafil use (Table S4). In patient 3, sildenafil had to be discontinued due to a rash, even though the frequent episodes of sudden muscle weakness were resolved during sildenafil medication and returned once the drug was discontinued (Tables 1 and S4). In patient 3, sildenafil had to be discontinued due to a rash, even though the frequent episodes of sudden muscle weakness were resolved during sildenafil medication and returned once the drug was discontinued (Tables 1 and S4). In all the other cases, sildenafil was well tolerated (Table S4), leading to improvement in motor function and development (Tables 1 and S3).