Abstracts

Rationale: Epilepsy is characterized by recurrent seizures due to neuronal hyperactivity. Approximately 75% of epilepsy begins during childhood. Recent advances in genomic technologies have identified de novo mutations in genes, such as aristaless-related homeobox (ARX) gene, in patients with epilepsy. ARX is implicated in a wide spectrum of X-linked neurological disorders. Poly-alanine repeat mutations in ARX have been found in patients with less severe phenotypes characterized by mental retardation with no apparent brain developmental abnormalities. Animal models have shown that Arx is critical for cortical interneuron development and migration. In addition, Arx polyalanine expansion modifies glutamatergic neurons excitability and morphology. Methods: To elucidate the impact of poly-alanine expansion in ARX gene in human, we have taken a promising new approach to generate 'epilepsy-in-a-dish' models from human induced pluripotent stem cells (iPSCs) using cortical and subpallium spheroids (hCS and hSS, respectively; a 3D model of human brain development in a dish). Results: We detected ARX expression during spheroid differentiation by quantitative PCR and in human pediatric cortex. We also found that ARX co-localized with the proliferation marker Ki67 in hCS and with the neuronal marker TUJ1 in both hCS and hSS. Then, we generated hCS and hSS from ARX mutated patient iPSCs and from healthy control iPSCs. When we analyzed hCS at 120 days in vitro (DIV), we detected a significant decrease of deep cortical neurons by single cell RNA-seq, as well as a decreased in neuronal activity by multielectroarray. Moreover, the number of Ki67+ cells were decreased in ARX hSS compared to controls at 30 DIV. Conclusions: These data suggest that ARX affects cell proliferation and neuronal differentiation during cortical development. Understanding the mechanisms by which ARX mutations contribute to epileptogenesis may help to define the underlying biology of epilepsy and ultimately impact treatment. Funding: Supported by a grant from the American Epilepsy Society.