Clozapine's Molecular Signature: Selective 5-HT2A Receptor Downregulation and Signalling in Rat Brain Cortical...

The superior clinical efficacy of clozapine (CLO) in treatment-resistant schizophrenia remains incompletely understood. To elucidate these mechanisms, we employed a multi-platform proteomic strategy-combining data-dependent acquisition (DDA) with fractionation, data-independent acquisition (DIA) and targeted parallel reaction monitoring (PRM)-to analyse chronic drug-induced proteomic changes in the prefrontal cortex (PFC) and remaining cerebral cortex (CX) of rats. This approach enabled unbiased analysis of over 6300 proteins per group in DDA and over 8700 in DIA. Consistent across all analytical methods, chronic CLO administration induced robust downregulation of the serotonin 5-HT2A receptor (5HTR2A) in both PFC and CX, whereas risperidone (RIS) exhibited no significant effect on 5-HTR2A levels at clinically relevant doses. Despite sharing high affinity for this receptor, CLO and RIS elicited markedly distinct signalling profiles. In the PFC, CLO inhibited serotonergic, G-protein (Gαq and Gβγ), calcium, and cytoskeletal signalling pathways, accompanied by strong downregulation of phospholipase C delta 4 (PLCδ4), an isoform implicated in suicidal behaviour. Concurrently, CLO upregulated calcium-independent phospholipase A2γ (iPLA2γ) and guanylyl cyclase C (GUCY2C), suggesting potential modulation of docosahexaenoic acid (DHA) signalling and cGMP-driven synaptic plasticity. In contrast, RIS predominantly activated Gαi, Gαz, and Gβγ signalling in the CX and enhanced mitochondrial function. RIS also upregulated GUCY2C. Both drugs differentially modulated protein networks associated with potential heteromeric complexes involving 5HTR2As, including interactions with 5-HT1A, 5-HT2C, dopamine D2, oxytocin and cannabinoid CB1 receptors, highlighting their impact on complex serotonergic modulation. Notably, despite CLO's suppression of Gαq signalling, compensatory upregulation of Gαi was not observed, indicating a net attenuation of 5HTR2A-mediated output rather than a simple G-protein coupling switch. These findings advance our understanding of serotonergic, metabolic and receptor-level adaptations induced by atypical antipsychotics and suggest that CLO's superior efficacy may derive from its unique ability to downregulate 5HTR2A and remodel signalling networks beyond classical dopaminergic mechanisms.