Abstracts

Rationale: Epilepsy is characterized by recurrent seizures due to neuronal hyperactivity. Approximately 75% of epilepsy begins during childhood, reflecting the heightened susceptibility of the developing brain to seizures. While 30% of cases have a known cause, the vast majority of patients have unknown etiology. Nevertheless, recent advances in genomic technologies such as array CGH or DNA sequencing 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 (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 cortical spheroids. Then, we generated cortical spheroids from ARX mutated patient iPSCs and from healthy control iPSCs. When we analyzed cortical spheroid growth at different time points, we detected a significant decrease in ARX cortical spheroid size at 21 DIV compared to controls. Moreover, the number of Ki67⁺ and Tuj1⁺ cells were decreased in ARX cortical spheroids compared to controls at 30 DIV, while the number of AC3⁺ cells was increased. Conclusions: These data suggest that ARX affects cell proliferation and neuronal differentiation during cortical spheroid 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: LGS Foundation