Phosphoproteomic profiling reveals reversal of dysregulated kinase signaling by nitric oxide inhibition in the Shank3 mouse model of autism

Autism spectrum disorder (ASD) is a wide-ranging neurodevelopmental disorder characterized by behavioral deficits, including difficulties in social communication and interaction, restricted or repetitive behaviors, and limited interests. The SHANK3 gene encodes a scaffolding protein in the postsynaptic density of glutamatergic excitatory synapses and is one of the highest-confidence ASD risk genes. Our previous studies showed abnormally elevated levels of nitric oxide (NO) in Shank3 models of ASD, and inhibition of the neuronal NO synthase (nNOS) reversed ASD-like behavioral and synaptic phenotypes in these models. Protein phosphorylation is involved in all biological processes in the body, but it is still unknown how protein phosphorylation is affected by the NO signaling in ASD. We studied the global proteome and phosphoproteome of the cortical tissue of the Shank3 Δ4–22 mouse model of autism, followed by system biology analysis. First, we compared Shank3 KO to the WT mice, revealing altered phosphorylation states in proteins enriched in synaptic, developmental, and RNA metabolism biological processes. Following treatment with nNOS inhibitor, 7-nitroindazole , we observed reversal of the phosphorylation state in proteins participating in the same biological processes, indicating a link between NO signaling and protein phosphorylation patterns. Importantly, Shank3 mutation revealed altered phosphorylation in several ASD SFARI high-risk genes, and many of these changes were reversed by the NO inhibition. This work is the first to investigate the cross-talk between NO and kinase signaling in a Shank3 ASD model. Finally, it might provide new insights into the NO-mediated protein phosphorylation mechanisms and help identIfying new therapeutic targets for ASD.