JNK-regulated phosphoproteome links synaptic and metabolic pathways to mood regulation

c-Jun N-terminal kinases (JNKs) are implicated in both neurodegeneration and mood regulation, including anxiety and depressive-like behaviours. Yet the consequences of JNK inhibition in vivo on protein phosphorylation in the brain remain largely unknown. This study aimed to (1) determine how chronic JNK inhibition altered proteome-wide phosphorylation in hippocampus and nucleus accumbens, regions central to affective processing, and (2) determine which JNK-regulated phosphoproteins were associated with the anxiolytic response, representing potential drivers. Mice underwent intracerebral (ICV) infusion with DJNKI-1 or control TAT peptide for six weeks, after which behaviours were assessed and phosphoproteomic profiling performed. JNK inhibition reduced anxiety-like behaviour and significantly altered 163 and 97 phosphosites in the hippocampus and nucleus accumbens, respectively. JNK-regulated phosphoproteins were enriched for regulators of cytoskeleton organization and synaptic function. GSK3 signalling was inhibited by DJNKI-1, leading to extensive depletion of phosphorylation on GSK3 motifs within the hippocampus and nucleus accumbens. These affected proteins involved in adhesion, cytoskeleton, proteostasis and synaptic activity. Moreover, several energy metabolism proteins exhibited phosphorylation changes on sites that control their enzymatic activity. The predicted net effect is a metabolic shift from oxidative phosphorylation to anaerobic glycolysis. Network analysis revealed enhanced phosphoproteome connectivity in mice displaying low anxiety-like behaviour, with spectrin-α/β, syntaxin-1b, CRMP2 and MAPT emerging as central hubs. Notably, claudin-11, an oligodendrocyte-specific, tight junction protein, was identified as a novel phospho-target that was highly reduced upon DJNKI-1 treatment. Together, these findings highlight potential molecular markers of anxiolytic response and suggest synaptic and metabolic interplay in mood regulation.