CELL TYPE-SPECIFIC TRANSCRIPTIONAL CHANGES FOLLOWING CHRONIC TREATMENT OF CLOZAPINE IN MOUSE PREFRONTAL CORTEX

Abstract Background Schizophrenia is a major psychotic disorder with complex genetic underpinnings. Clozapine is one of the representative antipsychotics for its unique and superior efficacy. The current understandings of clozapine’ s mechanism do not fully explain the favorable therapeutic properties of clozapine, in which the differential gene activation related to the pathophysiology of schizophrenia may be involved. Therefore, studying the transcriptomic changes induced by clozapine could contribute to the advancement of schizophrenia treatments. Aims & Objectives We aimed to investigate cell type-specific clozapine-induced transcriptional changes in mouse prefrontal cortex through single-cell RNA sequencing (scRNA-seq). The goal of this study was to provide important insights into the mechanism of action of clozapine and examine its associations with genes related to the etiology of schizophrenia. Method In this study, clozapine was systemically injected into male C58BL/6 mice for 21 consecutive days, and single-cell RNA sequencing was applied to their prefrontal cortical tissue samples. Differentially expressed genes (DEGs) for each cell type, including microglia, astrocytes, neurons, endothelial cells, and vascular smooth muscle cells, were identified through single-cell RNA sequencing. Pathways and intercellular interactions associated with these DEGs were then analyzed. Additionally, a comparative analysis with human databases was conducted, using the toxicogenomics database (CTD), genetic loci implicated in schizophrenia, and genes with evidence of interaction with clozapine. Results Clozapine-induced changes in gene expression were most prominent in microglia, and the cell population clusters within microglia were altered by clozapine exposure. Pathway analyses highlighted TNF-α signaling via NF-κ B, IFN-γ response, IFN-α response, and p53 pathway as particularly engaged by clozapine. Cell-cell interaction analysis demonstrated the clozapine-induced interactive changes among microglia, endothelial cells, and vascular smooth muscle cells. Comparative analysis with human data also revealed enriched clozapine-induced DEGs in microglia. As a result, eleven genes, including Nfkb1, Nfe2l2, Egr1, Fos, and Atp6v1b2, were elucidated to have convergent evidence. Discussion & Conclusion By using scRNA-seq, we identified discrete cell types and their alterations in the mouse prefrontal cortex. While microglia are mainly impacted by clozapine exposure, other cell types also display distinct changes. Single-cell RNA sequencing could provide important insights into the mechanisms of action of clozapine, a representative antipsychotic with superior efficacy, which has links to the genetic background of pathogenetic and therapeutic mechanisms in schizophrenia.