430. Investigating cellular phenotypes and genotypes of a preclinical model relevant to schizophrenia using single-cell RNA sequencing

Abstract

Abstract Background Schizophrenia is a highly heritable neurodevelopmental disorder characterised by disruptions in cortical circuitry and excitatory-inhibitory balance. Converging genetic and post-mortem evidence implicates early dysfunction of GABAergic interneurons, and transcription factor ARX plays a critical role in interneuron differentiation and migration. Mutations in ARX are associated with a spectrum of neurodevelopmental disorders, including epilepsy, autism and schizophrenia. Our laboratory identified a rare missense variant (ARX R264Q) in a female patient with schizophrenia. To investigate the developmental impact of this mutation on phenotypes relevant to schizophrenia a mouse model of the mutation that was developed using CRISPR-cas9 technology. The mouse shows behavioural and electrophysiological phenotypes relevant to schizophrenia, including disrupted sensorimotor processing and reversal learning, and social deficits coupled with altered gamma power during the aforementioned tasks. Aims & Objectives To understand how this mutation could lead to the above behavioural and electrophysiological phenotypes this study now aimed to characterise cell-type-specific transcriptional changes induced by the ArxR264Q mutation during early postnatal brain development using single-cell RNA sequencing (scRNA-seq). We sought to (i) identify transcriptionally distinct brain cell populations from WT and ArxR264Q mice, (ii) assess differential gene expression between mutant and wild-type mice within specific cell types, and (iii) define disrupted biological pathways that the identified differentially expressed genes are enriched in. We hypothesised that ArxR264Q mice would exhibit disrupted gene expression in pathways related to neuronal development, with pronounced effects in GABAergic interneuron populations. Method Whole brains from wild-type and ArxR264Q mutant mice were collected at postnatal days 3-4 and processed for multiplexed scRNA-seq using 10x Genomics technology. Data were analysed bioinformatically using Cellranger and Seurat. Cells were clustered into transcriptionally distinct populations and annotated using GPT-4o annotations. Differential gene expression was assessed within each cell type between ArxR264Q mutants and wildtypes, followed by gene set enrichment analyses to identify affected biological pathways that those differentially expressed genes act on. Results Distinct subtypes were identified in neuronal, glial and progenitor populations. Differential expression analyses revealed prominent transcriptional alterations in GABAergic interneurons, layer 4-5 excitatory neurons, developing GABAergic interneurons, and developing excitatory neurons. Enriched pathways included neurogenesis, cell migration, cell adhesion, and neuronal differentiation. Key genes implicated in interneuron migration and GABAergic function, including Cxcl12, Slc6a13, and Slc32a1, were differentially expressed in mutant mice. Additional dysregulation of developmental regulators such as Sox9, Nrp1, and Sema3a suggested broader effects on neuronal fate specification and circuit assembly. Discussion & Conclusions These findings demonstrate that the ArxR264Q mutation induces early, cell-specific transcriptional dysregulation affecting pathways critical for cortical interneuron development. This work provides mechanistic insight into how a schizophrenia-associated ARX variant may perturb early neurodevelopmental trajectories, potentially contributing to later behavioural abnormalities relevant to schizophrenia such as disrupted social behaviour, delayed reversal learning and abnormal acoustic startle, as well as disrupted neural network firing during these tasks. Future work will screen the key genes identified from this study for potentially druggable candidate targets for treating interneuronopathies commonly found in neurodevelopmental disorders.