Nitric Oxide-Mediated S-Nitrosylation of TSC2 Drives mTOR dysregulation across Shank3 and Cntnap2 Models of Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by core behavioral symptoms. We previously showed that nitric oxide (NO) plays a key role in ASD. However, the precise molecular mechanism through which NO acts via its posttranslational modification, S-nitrosylation (SNO), in ASD remains largely unknown. Emerging evidence, including our previous studies, suggests that the mechanistic target of the rapamycin (mTOR) signaling pathway plays a critical role in ASD pathophysiology. Our SNO-proteome systems biology analysis showed the enrichment of the mTOR pathway. In this study, we deciphered a novel mechanism of the cross talk between NO and mTOR pathway using two well-established mouse models as well as clinical samples of children with ASD. To assess changes in the SNO-proteome, we used the SNOTRAP method, revealing increased S-nitrosylation of tuberous sclerosis complex 2 (TSC2) in Shank3^Δ4–22 and Cntnap2^(-/-) mutant mice. We proved that this modification led to the loss of TSC2 protein via ubiquitination, resulting in dysregulated mTOR signaling in both excitatory and inhibitory neurons. Pharmacological inhibition of neuronal nitric oxide synthase (nNOS) successfully prevented TSC2 S-nitrosylation, mTOR overactivation, and altered protein translation in ASD models, highlighting NO’s role in modulating mTOR function. To further validate the role of TSC2 S-nitrosylation in ASD, we generated a cysteine-to-serine mutation (C203S) in TSC2 to prevent its S-nitrosylation. Intracranial injection of the mutant TSC2 (C203S) in Shank3^Δ4–22 mice in the prefrontal cortex prevented ASD-like behaviors, confirming the pathogenic role of NO-mediated TSC2 modification. Critically, analysis of clinical samples from children with ASD, including those with SHANK3 mutations and idiopathic ASD, revealed reduced TSC2 levels and increased mTOR signaling activity, further validating our findings. Collectively, this study uncovers a novel molecular mechanism by which S-nitrosylation disrupts TSC2 function, leading to aberrant mTOR signaling and ASD-like phenotypes. By revealing a unique SNO-TSC2-mTOR axis, our work deciphers the novel nitric oxide-mediated mTOR activation and opens new avenues for targeted therapeutic strategies in ASD, including those carrying SHANK3 mutations.