ARX polyalanine expansion mutations lead to migration impediment in the rostral cortex coupled with a developmental deficit of calbindin-positive cortical GABAergic interneurons.

The Aristaless-related homeobox gene (ARX) is indispensable for interneuron development. Patients with ARX polyalanine expansion mutations of the first two tracts (namely PA1 and PA2) suffer from intellectual disability of varying severity, with seizures a frequent comorbidity. The impact of PA1 and PA2 mutations on the brain development is unknown, hindering the search for therapeutic interventions. Here, we characterized the disturbances to cortical interneuron development in mice modeling the two most common ARX polyalanine expansion mutations in human. We found a consistent ∼40-50% reduction of calbindin-positive interneurons, but not Stt+ or Cr+ interneurons, within the cortex of newborn hemizygous mice (p=0.024) for both mutant strains compared to wildtype (p=0.011). We demonstrate that this was a consequence of calbindin precursor cells being arrested or delayed at the ventral subpallium en route of tangential migration. Ex-vivo assay validated this migration deficit in PA1 cells (p=0.0002) suggesting that the defect is contributed by intrinsic loss of Arx function within migrating cells. Both humans and mice with PA1 mutations present with severe clinical features, including intellectual disability and infantile spasms. Our data further demonstrated the pathogenic mechanism was robustly shared between PA1 and PA2 mutations, as previously reported including Arx protein reduction and overlapping transcriptome profiles within the developing mouse brains. Data from our study demonstrated that cortical calbindin interneuron development and migration is negatively affected by ARX polyalanine expansion mutations. Understanding the cellular pathogenesis contributing to disease manifestation is necessary to screen efficacy of potential therapeutic interventions.