Phosphoproteomics identifies Rho-kinase-associated phospho-signaling and autophagy-associated alterations in the medial prefrontal cortex of a schizophrenia-related mouse model

Schizophrenia is a chronic, debilitating psychiatric disorder with high heritability. We have previously established Arhgap10 S490P/NHEJ (Arhgap10) mice, a schizophrenia-related mouse model carrying Japanese schizophrenia patient-derived ARHGAP10 variants. These mice exhibited activated Rho-kinase (ROCK) signaling, schizophrenia-related behavioral and neurobiological phenotypes that were ameliorated by the ROCK inhibitor fasudil. Although phospho-signaling is increasingly implicated in schizophrenia pathophysiology, comprehensive phosphoproteomic profiling in the brain of Arhgap10 mice has not yet been performed, leaving key downstream ROCK-dependent targets unidentified. Here, we conducted unbiased phosphoproteomic analysis of the medial prefrontal cortex (mPFC) of wild-type (WT) and Arhgap10 mice, treated with or without fasudil. We identified 15 phosphoproteins that were significantly upregulated in the mPFC of Arhgap10 mice compared with WT controls. Among these, phosphorylated microtubule-associated protein tau at serine 404 (pTau_S404), phosphorylated autophagy-related protein 9 A at serine 828 (pATG9A_S828), and phosphorylated serine/threonine-protein kinase WNK2 at threonine 282 (pWNK2_T282) were markedly reduced to control levels by fasudil treatment. Upstream kinase enrichment analysis, together with targeted validation, revealed alterations in the AKT/GSK3β signaling node as the context for these fasudil-responsive candidates. Since these phosphoproteins are linked to autophagy-related processes, we further examined autophagy-lysosome-associated readouts. Arhgap10 mice showed autophagy-associated alterations in the mPFC, while fasudil increased autophagosome- and autolysosome-associated vesicle puncta in primary cortical neurons. This study provides the first phosphoproteomic network map of the mPFC in the Arhgap10 mouse model of schizophrenia, identifying ROCK-responsive phosphoproteins along with autophagy-associated alterations and fasudil-responsive changes.