Defective corticogenesis 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 in male children is caused by expanded runs of GCG repeats in the X-chromosome gene Aristaless-related homeobox (ARX), which encodes a homeotic transcription factor with a key role in mammalian corticogenesis (1-3). To ascertain cellular diversity and disease mechanisms associated to polyalanine elongations in ARX, we conducted single cell RNA sequencing (scRNA-seq) in the epileptogenic neocortex of the Arx polyalanine mouse (Arx(GCG)7/Y) (4). This is a DEE model that spontaneously develops severe tonic-clonic seizures in a phenotype that recapitulates the chronic epilepsy detected in ARX male patients (3). We detailed cell-specific gene expression patterns, unveiling how transcriptional changes in specific cell subpopulations are associated with a defective cell composition of the Arx(GCG)7/Y embryonic cortex, characterized by a lower proportion of neuronal precursors (NPs) and higher proportion of immature neurons (INs). in vivo BrdU pulse-chase and immunofluorescence studies confirmed that neurogenesis and corticogenesis are both damaged in Arx(GCG)7/Y neocortex. Enrichment analysis in NPs identified consistently altered pathways implicated in cell cycle, chromatin remodelling and RNA metabolism; whereas functions implicated in neuronal structure and synapse organization were widely damaged in INs. Finally, immunocytochemistry and morphometric analysis highlighted a defective neurite arborization and hypoconnectivity in Arx(GCG)7/Y cortical neurons. These results provide new insights into the cell types and neuronal functions perturbed in the Arx polyalanine neocortex disclosing a defective corticogenesis that could potentially underlie the DEE pathogenesis.