Aberrant protein S-nitrosylation disrupts axonal development and metabolic homeostasis in a Cntnap2 mouse model of autism.

Protein S-nitrosylation (SNO) is a nitric oxide (·NO)-dependent post-translational modification that regulates various biological functions. Dysregulated SNO is involved in several brain disorders; however, its role in autism spectrum disorder (ASD) remains largely unexplored. This could be due to the transient nature and low abundance of SNO-modified proteins, which complicates their detection. Here, we employed SNOTRAP, a high-throughput mass spectrometry-based approach that captures, labels, and quantifies S-nitrosylated peptides at specific cysteines, to profile the cortical SNO-proteome in the Cntnap2 mouse model of ASD. Quantitative analysis identified significant alterations in S-nitrosylation across 76 peptides from 69 proteins. Among these, MAP1B, a key regulator of microtubule dynamics during axon formation, emerged as a prominent target, exhibiting exclusive S-nitrosylation at Cys1305 in the Cntnap2-/- cortex but not in WT. Consistently, Cntnap2-/- neurons displayed shortened axons, a phenotype that was rescued by pharmacological inhibition of ·NO signaling, linking aberrant S-nitrosylation to structural neuronal deficits. Beyond cytoskeletal regulation, pathway enrichment analysis revealed a significant influence of altered S-nitrosylation on metabolic networks, including glycolysis, the TCA cycle, pyruvate metabolism, and amino acid biosynthesis. Remarkably, many metabolic enzymes exhibited reduced or absent S-nitrosylation at specific cysteine residues, indicating a pathological shift in the SNO landscape. Consistent with these molecular changes, the Cntnap2-/- cortex exhibited an elevated NADH/NAD+ ratio, reflecting altered redox balance and thus dysregulated cellular metabolism, which was normalized upon inhibition of ·NO. Together, these findings highlight S-nitrosylation as a convergent regulatory mechanism acting across multiple neuronal domains, ultimately impacting synaptic function and ASD-related phenotypes.