Precision models of ARX-associated neurodevelopmental disorders

Genetic testing has greatly advanced diagnosis of early onset epilepsy. In spite of the expanding number of monogenetic disorders, such as epilepsy in patients with Aristaless-related homeobox gene (ARX) mutations, many of these rare diseases do not have a validated experimental model and thus suffer from limited understanding of the mechanisms of brain development and epileptogenesis. ARX is a transcription factor which is widely expressed in the developing and adult brain, skeletal muscle, pancreas, and testes. While the phenotypic expression of Arx mutations in the development of epilepsy vary in humans, generally large deletions or truncating mutations cause a brain malformation phenotype while missense mutations and polyalanine tract expansions cause non-brain malformation phenotypes. The most common Arx mutation associated with epilepsy occur in the first two polyalanine (pAla) domains (PA1 and PA2). Evidence from mouse and in vitro models suggests that expanded pAla tracts contribute to the cortical interneuron defects and development of epilepsy. The overarching hypothesis is that a threshold of pAla mutations in ARX cause specific interneuron dysfunction and mismigration leading to the neurologic phenotype. However, there is lack of human-specific evidence to corroborate findings in mouse models with human disease.







While mouse models have greatly advanced the field to its current state, complementary approaches are needed given marked evolutionary differences of brain development in mice as compared to humans. Human induced pluripotent stem cells (hiPSCs) are a critical advance that can potentially address these issues. hiPSCs can be differentiated to specific neural lineages and offer a unique opportunity to perform in vitro study of epileptogenesis during the patient’s life. Further, the ability to modify the genome of iPSCs with CRISPR/Cas9 technology will control for genetic background and allow us to address the impact of pAla repeat dosage on phenotypes related to epilepsy. Using ARX patient hiPSCs differentiated to 3D cortical spheroids comprised of excitatory and inhibitory interneurons, we find changes in interneuron number/morphology/migration, network activity)compared to controls. These data link decreased production of GABAergic interneurons with increased network activity. We will test the hypothesis that ARX patient interneurons have defects in proliferation, differentiation, and/or migration that lead to network changes, and ultimately, to increased epileptic activity in vivo.