MMP normalization occurred similarly in LS NPCs carrying different MT-ATP6 variants with overall MMP depolarization of ∼16% (Figure 1E). Sildenafil did not modify the MMP in control NPCs (Figures S3I and S3J). Bioenergetically, sildenafil did not lead to a measurable improvement in CV activity (Figure 1F) or in CV assembly structure43,44 (Figure S4C). The protein levels of known mitochondrial modulators were also not significantly impacted (Figures S4D and S4E). Nonetheless, sildenafil increased intracellular ATP in LS NPCs (Figure 1G) and restored mtDNA copy number (Figure 1H). The NAD+/NADH ratio was also normalized (Figures 1I and S1D).
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The glycolytic signature, suggested to be an indicator of brain organoid stress,68 was not altered, indicating that neither MT-ATP6 variants nor sildenafil posed additional stress (Figure S8E). Altogether, LS disrupted brain organoid development by impairing early neuronal organization of radial glia and progenitor populations, and sildenafil specifically affected those populations. We examined the functional consequences of sildenafil in LS neural cells. Given the reported calcium dysregulation26,27 and the known deterioration of LS patients upon metabolic decompensation,3 we induced acute metabolic stress in LS brain organoids to monitor their intracellular calcium response. 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). Because MT-ATP6 defects may disrupt mitochondrial cristae,45 we analyzed the cristae junction regulator MIC60 with stimulated emission depletion (STED) microscopy.46 We inspected the MIC60 labeling pattern of 4,000 STED images with a machine-learning approach by training a neural network classifier to distinguish control NPCs (P[healthy]-score = 1) from LS NPCs (P[healthy]-score = 0). Using images not used for training, we detected altered MIC60 localization in LS NPCs that were partially reversed by sildenafil (Figures 1J and S3K). Manual quantification of approximately 300 STED images yielded similar results (Figure S3L) and highlighted a peripheral MIC60 distribution pattern in LS NPCs that was restored by sildenafil (Figures S3M and S3N). Hence, sildenafil improved mitochondrial phenotypes in LS neural cells.
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The omics design identified (1) the disease signature by comparing DMSO-treated LS NPCs to DMSO-treated control NPCs and (2) the sildenafil signature by comparing sildenafil-treated LS NPCs to DMSO-treated LS NPCs (Figure S1G). The disease signature included biological processes (BPs) related to nervous system and axon development and cellular components (CCs) implicated in the mitochondrial inner membrane and neuronal cell body in transcriptomics (Figures S5A and S5B), and mitochondrial translation and mitochondrial membrane in proteomics (Figures S5F, S5G, and S5I). The sildenafil signature affected similar pathways, including axon and nervous system development (Figure 2A) and mitochondrial inner membrane and neuronal cell body in transcriptomics (Figure S5C) and electron transport chain (ETC) in proteomics (Figures 2B and S5H). Genes restored by sildenafil (Figures S5D and S5E) included neuron-specific calcium sensor CABP147 (Table S1) and neurodevelopment-associated genes HOXA548,49 and DBX150 (Figure S5E; Table S1). Restored proteins included BAG4, protecting against ATP depletion-induced cell death,51 downregulated in LS NPCs (Table S1) and upregulated after treatment (Figure S5J). No significant transcriptional changes were observed in sildenafil-treated control NPCs (Figure S5K). Metabolomics identified metabolites important for brain energy metabolism, including creatine52,53 and cysteine,54 that were altered by the disease (Figure S4F) and normalized by sildenafil (Figure S4G; Table S1). Creatinine, a non-enzymatic breakdown product of creatine, was not normalized, possibly reflecting an increased use of creatine. The disease and sildenafil signatures also impacted the glutathione metabolism pathway (Figures S6A and S6B). Multi-omics integration55 revealed 796 molecules shared between the integrated signatures for disease and sildenafil, with 724 exhibiting opposite regulation. Hence, sildenafil reversed 33.5% of dysregulated molecules (724 sildenafil syrup over 2,160). The integrated signatures impacted signaling pathways (e.g., Notch and WNT) and axon guidance (Figures S6C and S6D).
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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). Reversed BPs included one group related to mitochondrial respiration and one related to morphogenesis and axon development (Figure 2C). The multi-omics map of the sildenafil rescue signature (including only transcriptomics and metabolomics, as proteomics did not show sufficiently significant changes) highlighted a network comprising metabolites (e.g., biotin and pantothenic acid) and genes related to ETC (e.g., NDUFA6) and development (e.g., NOTCH1, BRD4, and STAT3) (Figure 2D).
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The omics design identified (1) the disease signature by comparing DMSO-treated LS NPCs to DMSO-treated control NPCs and (2) the sildenafil signature by comparing sildenafil-treated LS NPCs to DMSO-treated LS NPCs (Figure S1G). The disease signature included biological processes (BPs) related to nervous system and axon development and cellular components (CCs) implicated in the mitochondrial inner membrane and neuronal cell body in transcriptomics (Figures S5A and S5B), and mitochondrial translation and mitochondrial membrane in proteomics (Figures S5F, S5G, and S5I). The sildenafil signature affected similar pathways, including axon and nervous system development (Figure 2A) and mitochondrial inner membrane and neuronal cell body in transcriptomics (Figure S5C) and electron transport chain (ETC) in proteomics (Figures 2B and S5H). Genes restored by sildenafil (Figures S5D and S5E) included neuron-specific calcium sensor CABP147 (Table S1) and neurodevelopment-associated genes HOXA548,49 and DBX150 (Figure S5E; Table S1). Restored proteins included BAG4, protecting against ATP depletion-induced cell death,51 downregulated in LS NPCs (Table S1) and upregulated after treatment (Figure S5J).
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No significant transcriptional changes were observed in sildenafil-treated control NPCs (Figure S5K). Metabolomics identified metabolites important for brain energy metabolism, including creatine52,53 and cysteine,54 that were altered by the disease (Figure S4F) and normalized by sildenafil (Figure S4G; Table S1). Creatinine, a non-enzymatic breakdown product of creatine, was not normalized, possibly reflecting an increased use of creatine. The disease and sildenafil signatures also impacted the glutathione metabolism pathway (Figures S6A and S6B). Multi-omics integration55 revealed 796 molecules shared between the integrated signatures for disease and sildenafil, with 724 exhibiting opposite regulation. We identified 20 putative sildenafil targets whose gene expression was altered in LS NPCs compared with control NPCs and restored in LS NPCs treated with sildenafil (Figure 2E).
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The top responsive gene was HOXA5 (logFC_diseased = −0.02, logFC_treated = +5.2). The HOXA5 hub was involved in extracellular matrix organization in LS NPCs but became associated with differentiation and neurodevelopment upon treatment (Figures 2F and 2G). The HOXA5 network may thus be dysregulated in LS, and its modulation by sildenafil might contribute to healthy neurodevelopment. Another key responder was PRKG1, a master regulator and downstream target of PDE5,56 which was highlighted by the multi-omics map of the sildenafil rescue signature (Figure 2D). To monitor the expression of putative sildenafil-responsive genes, we treated LS NPCs grown in physiologically low glucose with 1 or 10 μM sildenafil for 6 and 24 h (Figures S6E and S6F). The treatments modulated the expression of sildenafil targets over time, including proliferation-related genes BRD4, STAT3, and NOTCH157,58; synapsis-associated NRG159; neuroinflammation-associated P2RX460; and glucose metabolism-related SLC37A4.61 We used cortical brain organoids to explore the impact of sildenafil on human neurodevelopment. In agreement with previous studies in LS brain organoids,25,29,30 MT-ATP6 variants impaired neurogenic zone formation (Figure 3A) and altered the ratio of early neurons to neural progenitors (Figure 3B). Two different protocols for generating cortical brain organoids showed defective growth rates (Figure S7A).
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Hence, sildenafil reversed 33.5% of dysregulated molecules (724 sildenafil syrup over 2,160). The integrated signatures impacted signaling pathways (e.g., Notch and WNT) and axon guidance (Figures S6C and S6D). Reversed BPs included one group related to mitochondrial respiration and one related to morphogenesis and axon development (Figure 2C). The multi-omics map of the sildenafil rescue signature (including only transcriptomics and metabolomics, as proteomics did not show sufficiently significant changes) highlighted a network comprising metabolites (e.g., biotin and pantothenic acid) and genes related to ETC (e.g., NDUFA6) and development (e.g., NOTCH1, BRD4, and STAT3) (Figure 2D). We identified 20 putative sildenafil targets whose gene expression was altered in LS NPCs compared with control NPCs and restored in LS NPCs treated with sildenafil (Figure 2E).
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The top responsive gene was HOXA5 (logFC_diseased = −0.02, logFC_treated = +5.2). The HOXA5 hub was involved in extracellular matrix organization in LS NPCs but became associated with differentiation and neurodevelopment upon treatment (Figures 2F and 2G). The HOXA5 network may thus be dysregulated in LS, and its modulation by sildenafil might contribute to healthy neurodevelopment. Another key responder was PRKG1, a master regulator and downstream target of PDE5,56 which was highlighted by the multi-omics map of the sildenafil rescue signature (Figure 2D). To monitor the expression of putative sildenafil-responsive genes, we treated LS NPCs grown in physiologically low glucose with 1 or 10 μM sildenafil for 6 and 24 h (Figures S6E and S6F). One protocol62 showed size defects in LS organoids after 50 days in culture (Figure S7B); another protocol,63 allowing initial homogeneous organoid shape, resulted in earlier growth defects in LS organoids that became less pronounced over time (Figure S7B). The results suggest that MT-ATP6 variants might affect neural progenitor development. We treated LS brain organoids with sildenafil for either 24 h (acute paradigm) or 45 days (chronic paradigm) (Figure 3C). MT-ATP6 variants disrupted BPs related to synapses and neuronal projections (Figure S7E) and CCs related to neuronal cell bodies and synapses (Figure S7F).
- Storage of sildenafil should be in a cool, dry place away from direct sunlight.
- Common side effects include dizziness, upset stomach, or vision changes.
- The dosage of sildenafil should be tailored to individual health needs.
- Sildenafil can interact with other medications, affecting its safety and efficacy.
- Always seek medical guidance before using sildenafil, especially at high doses.
The acute sildenafil signature modulated pathways related to embryonic development and Wingless-related integration site (WNT) signaling (Figure 3F) and corrected gene defects (Figures S7C and S7D), including WDR45B, which was downregulated in LS brain organoids (Table S1) and upregulated by sildenafil (Figure S7D), and whose variants are associated with neurodevelopmental disorders.64 Sildenafil also rescued the ratio of early neurons to neural progenitors (Figure 3D) and upregulated DBX1 (Figure 3E).
- 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.
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To dissect the cell populations affected by MT-ATP6 variants and chronic sildenafil, we performed single-nucleus RNA sequencing sildenafil from canada (snRNA-seq) (Figure 3C). Unsupervised clustering highlighted 9 clusters (Figure S8A): clusters 0 and 3 for radial glia, clusters 4 and 8 for progenitors, cluster 6 for proliferating progenitors, cluster 5 for immature neurons, cluster 2 for FOXG1-positive neurons, cluster 1 for FOXG1-negative neurons, and cluster 7 for other cell types (Figure S8B).
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MMP normalization occurred similarly in LS NPCs carrying different MT-ATP6 variants with overall MMP depolarization of ∼16% (Figure 1E). Sildenafil did not modify the MMP in control NPCs (Figures S3I and S3J). Bioenergetically, sildenafil did not lead to a measurable improvement in CV activity (Figure 1F) or in CV assembly structure43,44 (Figure S4C). The protein levels of known mitochondrial modulators were also not significantly impacted (Figures S4D and S4E). Nonetheless, sildenafil increased intracellular ATP in LS NPCs (Figure 1G) and restored mtDNA copy number (Figure 1H).
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The NAD+/NADH ratio was also normalized (Figures 1I and S1D). Because MT-ATP6 defects may disrupt mitochondrial cristae,45 we analyzed the cristae junction regulator MIC60 with stimulated emission depletion (STED) microscopy.46 We inspected the MIC60 labeling pattern of 4,000 STED images with a machine-learning approach by training a neural network classifier to distinguish control NPCs (P[healthy]-score = 1) from LS NPCs (P[healthy]-score = 0). Using images not used for training, we detected altered MIC60 localization in LS NPCs that were partially reversed by sildenafil (Figures 1J and S3K). Manual quantification of approximately 300 STED images yielded similar results (Figure S3L) and highlighted a peripheral MIC60 distribution pattern in LS NPCs that was restored by sildenafil (Figures S3M and S3N). Hence, sildenafil improved mitochondrial phenotypes in LS neural cells. This annotation revealed that MT-ATP6 variants impaired neuronal commitment, with alterations in radial glia, progenitors, and FOXG1-positive neurons (Figures 3G and 3H).
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This annotation revealed that MT-ATP6 variants impaired neuronal commitment, with alterations in radial glia, progenitors, and FOXG1-positive neurons (Figures 3G and 3H). The disease signature involved the downregulation in the progenitor population of PRKG1 and NLGN1, a regulator for synapse development65 (Figure 3I), and the downregulation in the neuronal population of genes impacting neurite outgrowth, such as STMN266 (Figure S8D; Table S1). The sildenafil signature upregulated neuronal outgrowth-associated genes, such as RGS667 (Figures S8C and S8D; Table S1). The effect of sildenafil was mostly evident in radial glia and progenitors (Figure S8C; Table S1). Whereas STMN2 was mainly present in FOXG1-positive neurons and immature neurons, RGS6 was found mainly in radial glia, and PRKG1 in radial glia and progenitors (Figures 3J and S8D). The disease signature involved the downregulation in the progenitor population of PRKG1 and NLGN1, a regulator for synapse development65 (Figure 3I), and the downregulation in the neuronal population of genes impacting neurite outgrowth, such as STMN266 (Figure S8D; Table S1). The sildenafil signature upregulated neuronal outgrowth-associated genes, such as RGS667 (Figures S8C and S8D; Table S1). The effect of sildenafil was mostly evident in radial glia and progenitors (Figure S8C; Table S1). Whereas STMN2 was mainly present in FOXG1-positive neurons and immature neurons, RGS6 was found mainly in radial glia, and PRKG1 in radial glia and progenitors (Figures 3J and S8D). The glycolytic signature, suggested to be an indicator of brain organoid stress,68 was not altered, indicating that neither MT-ATP6 variants nor sildenafil posed additional stress (Figure S8E). Altogether, LS disrupted brain organoid development by impairing early neuronal organization of radial glia and progenitor populations, and sildenafil specifically affected those populations.
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The treatments modulated the expression of sildenafil targets over time, including proliferation-related genes BRD4, STAT3, and NOTCH157,58; synapsis-associated NRG159; neuroinflammation-associated P2RX460; and glucose metabolism-related SLC37A4.61 We used cortical brain organoids to explore the impact of sildenafil on human neurodevelopment. In agreement with previous studies in LS brain organoids,25,29,30 MT-ATP6 variants impaired neurogenic zone formation (Figure 3A) and altered the ratio of early neurons to neural progenitors (Figure 3B). Two different protocols for generating cortical brain organoids showed defective growth rates (Figure S7A). One protocol62 showed size defects in LS organoids after 50 days in culture (Figure S7B); another protocol,63 allowing initial homogeneous organoid shape, resulted in earlier growth defects in LS organoids that became less pronounced over time (Figure S7B). The results suggest that MT-ATP6 variants might affect neural progenitor development.
Dosage adjustments
We treated LS brain organoids with sildenafil for either 24 h (acute paradigm) or 45 days (chronic paradigm) (Figure 3C). MT-ATP6 variants disrupted BPs related to synapses and neuronal projections (Figure S7E) and CCs related to neuronal cell bodies and synapses (Figure S7F). The acute sildenafil signature modulated pathways related to embryonic development and Wingless-related integration site (WNT) signaling (Figure 3F) and corrected gene defects (Figures S7C and S7D), including WDR45B, which was downregulated in LS brain organoids (Table S1) and upregulated by sildenafil (Figure S7D), and whose variants are associated with neurodevelopmental disorders.64 Sildenafil also rescued the ratio of early neurons to neural progenitors (Figure 3D) and upregulated DBX1 (Figure 3E). To dissect the cell populations affected by MT-ATP6 variants and chronic sildenafil, we performed single-nucleus RNA sequencing sildenafil from canada (snRNA-seq) (Figure 3C). Unsupervised clustering highlighted 9 clusters (Figure S8A): clusters 0 and 3 for radial glia, clusters 4 and 8 for progenitors, cluster 6 for proliferating progenitors, cluster 5 for immature neurons, cluster 2 for FOXG1-positive neurons, cluster 1 for FOXG1-negative neurons, and cluster 7 for other cell types (Figure S8B). We examined the functional consequences of sildenafil in LS neural cells. Given the reported calcium dysregulation26,27 and the known deterioration of LS patients upon metabolic decompensation,3 we induced acute metabolic stress in LS brain organoids to monitor their intracellular calcium response. 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).