Activation of <scp>GPR35</scp> in the Anterior Cingulate Cortex Alleviates Neuropathic Pain and Depression‐Related Behavior

BACKGROUND: Neuropathic pain (NP) is frequently accompanied by anxiety and depression, and current treatments do not adequately address this comorbidity. The anterior cingulate cortex (ACC) plays a central role in sensory and emotional processing. However, the molecular pathways that connect these functions remain unclear. G protein-coupled receptor 35 (GPR35), an orphan receptor enriched in neurons, has been implicated in neuroinflammation and pain signaling. However, its specific involvement in NP and associated affective disturbances has not been fully elucidated. METHODS: Peripheral blood GPR35 expression was measured in human patients with NP and healthy controls. In mice, chronic constriction injury (CCI) was used to induce NP. Lentiviral knockdown or overexpression of GPR35 was performed in ACC cells. Behavioral assays were used to assess mechanical and thermal sensitivity, locomotor and anxiety metrics, cognitive performance, and depression-related behaviors. Molecular analyses included western blotting, RT-qPCR, immunofluorescence, RNA sequencing, and co-immunoprecipitation. Additional Nr4a1 knockdown and L-kynurenine (L-Kyna, a GPR35 agonist) administration were used to validate pathway involvement. RESULTS: Patients with NP had higher circulating GPR35 levels, which positively correlated with pain intensity. CCI induced a time-dependent increase in GPR35 expression in the ACC of mice, accompanied by hypersensitivity and emotional disturbances. GPR35 knockdown in the ACC worsens mechanical and thermal hypersensitivity, impairs cognition, increases depression-related behaviors, and amplifies microglial activation and pro-inflammatory cytokine production. GPR35 overexpression reversed these effects by reducing hypersensitivity, improving affective behaviors, and restoring the inflammatory balance. Transcriptomic and biochemical analyses identified Nr4a1 as a key downstream effector of GPR35, and Nr4a1 knockdown eliminated the protective effects of GPR35 overexpression. GPR35 primarily regulated the PI3K/AKT pathway. Treatment with L-Kyna reduced pain hypersensitivity, improved depression-related behaviors, and decreased neuroinflammation in CCI mice. CONCLUSIONS: GPR35 is an essential regulator of NP and pain-related affective disturbances in the ACC. Its effects are mediated through the Nr4a1-dependent activation of the PI3K/AKT pathway and suppression of neuroinflammation. The pharmacological activation of GPR35 using L-Kyna provides analgesic and antidepressant benefits, highlighting GPR35 as a promising therapeutic target for NP and its emotional comorbidities.