Nearly 1% of the US population suffers from epilepsy (prevalence 5-8.4/1000), with a slightly higher prevalence in children. Despite this high frequency, the molecular and cellular basis for only a few types of epilepsy have been defined, while the basis for most remains unknown. Mutations in one gene, ARX, are of considerable interest as distinct mutations are associated with a spectrum of neurological disorders with epilepsy representing one of the few consistent features. ARX has 4 poly-alanine (pAla) tracts and expansions in the 1st or 2nd tract are consistently associated with epilepsy. pAla tract expansion mutations are a relatively newly described mutation type and are associated with a growing number of human developmental disorders, epilepsy being a component of several. How this mutation type results in human disorders and epilepsy in particular are not well understood. Our prior work has demonstrated that an expansion in the first pAla tract of ARX results in structural change in the protein and the resulting protein has differential effects on developing cortical interneuron- and projection neuron progenitor cells. In other studies, we have shown that the loss of Arx from each progenitor population accounts for specific components of the mouse and human phenotypes. In this multi-PI R01 proposal, building on our data from the past ten years, we seek to unite human stem cell models with mouse models to elucidate the pathobiology underlying ARX related epilepsy, and specifically the function of pAla tracts along with mutations in these tracts.
Aim 1 will evaluate the cellular impact of ARX pAla mutations in patient-derived spheroids.
Aim 2 will examine the role of ARX pAla mutations on cortical interneuron migration and network activity.
Aim 3 will determine the effects of Arx pAla expansion mutations on brain development and function. This project will utilize human induced pluripotent stem cell (hiPSC) and spheroid models and complement these with mouse embryonic stem cell lines and behavioral and physiological assays in mice. Together, these studies are expected to provide a greater understanding of how pAla tracts function in normal and abnormal brain development, contribute to our understanding of the pathogenesis of epilepsy, and generate valuable resources and mouse models to test potential therapeutic strategies for developmental epilepsies.
Neurodevelopmental disorders are frequently caused by underlying genetic mutations and is often intractable due to lack of understanding regarding the pathophysiologic effects of these genes. Polyalanine expansion mutations in the transcription factor ARX is associated with childhood epilepsy, cortical interneuron dysfunction, and intellectual disability. Using new technologies such as CRISPR/Cas9-based genome editing and human induced pluripotent stem cells (iPSC), we are now able to model aspects of ARX-associated neurodevelopmental disorders in human spheroid cultures that are isogenic to epilepsy patients, corroborate the phenotypes in mouse models, and use these results as a discovery tool to generate new hypotheses regarding the mechanisms of polyalanine expansion. The overarching goal of this project is to use human iPSC and mouse models to investigate the cellular, genetic, and functional mechanisms underlying neurodevelopmental disorders.
