Single-cell transcriptomic analysis of neocortex in Arx mouse model of developmental and epileptic encephalopathy caused by polyalanine elongations

Developmental and Epileptic Encephalopathy (DEE) is a pediatric epilepsy characterized by abundant epileptiform activity resistant to traditional anti-epileptic therapies. A severe form of DEE, affecting only male children, is caused by expanded runs of consecutive GCN repeats in Aristaless-related homeobox (ARX) gene. This is an X-chromosome gene encoding a bifunctional transcription factor with a key role in mammalian corticogenesis. This Arx knockin mutant develops severe tonic-clonic seizures in a phenotype that recapitulates the chronic epilepsy associated to the c.304ins (GCG)7 mutation detected in DEE male patients. We examined the cellular diversity and the transcriptome landscapes of the epileptogenic neocortex in the Arx polyalanine mouse Arx(GCG)7/Y at the embryonic day 15.5 compared to the male control one by a single-cell RNASeq approach (scRNASeq). We identified 18 unique cell clusters, subsequently categorized into six major neuronal cell types (radial glia, immature neurons, SVZ neurons, layer V/VI neurons, layer I neurons and interneurons), two non-neuronal cell types (endothelial cells and microglia) and two remain unknown. scRNASeq data revealed an altered cell composition in the diseased neocortex with lower proportion of radial glia cells (RGCs) and higher proportion of immature neurons (INs). Analysis of differentially expressed genes (DEGs) involved in cell cycle and immunofluorescence studies upon in vivo BrdU pulse-chase assay confirmed that neurogenesis and corticogenesis are both damaged in Arx(GCG)7/Y developing cortex. Enrichment analysis showed altered pathways in RGC and IN populations implicating in chromatin remodelling and RNA metabolism, neuronal motility and structure, and synapse organization. Concerning the alterations in morphology-related genes, immunocytochemistry followed by morphometric analysis revealed a defective neurite arborization with hypoconnectivity in Arx(GCG)7/Y primary cortical neurons. Taken together, our scRNAseq and functional studies disclose a complex cell-type-specific dysregulation of cortical projections and neuronal morphology that potentially underlies DEE pathogenesis.