Abstracts

Rationale: Recently, de novo mutations in SPTAN1, a highly conserved gene encoding alpha-II spectrin, were found in infants displaying an Infantile epileptic encephalopathy (IEE) associated with infantile spasm and developmental delay. SPTAN1 consists of 57 exons and encodes alpha-II spectrin, the primary alpha-spectrin subunit in the nervous system. However, the function of SPTAN1 in mammalian brain and how its mutations lead to epilepsy are largely unknown.. In addition, studying the function of SPTAN1 has been difficult because Sptan1-/- mice die before E16 with cardiac and neural tube malformations, and Sptan1-/+ mice have no phenotypes. Moreover, spectrins are membrane proteins that provide a membrane-cytoskeleton interface and mediate cell-cell interaction, adhesion and mechanical stress adaption, functions not easily recapitulated by in vitro cell culture. Methods: Here, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas) genome editing method, we developed an in vivo system that is highly efficient and specific to study the loss-of-function of IEEs genes in rodent forebrains. To study how pathogenic mutations in SPTAN1 lead to IEE, we constructed dominant-negative (DN) overexpression (OE) constructs harboring specific patient mutations and overexpressed them in rodent forebrains. In addition, we used innovative in vivo synaptic reporters (FingRs) to assess excitatory and inhibitory synapses formed onto mutant neurons, and confirmed the functional implications with electrophysiological recordings. Finally, we combined our rodent model with human iPSCs derived from patient fibroblast cells, an innovative approach that will be invaluable for studying early human neurodevelopment and serve as an entry point for future high-throughput drug screening Results: We show that Sptan1 regulates critical aspects of neurodevelopment including axonal initial segment (AIS) formation, polarity establishment, process outgrowth and clustering of postsynaptic proteins. Our electrophysiology studies strongly support that disinhibition is critical in epileptogenesis of SPTAN1 epileptic encephalopathy. In addition, we show that iPSCs-derived excitatory and inhibitory neurons display the same phenotypes observed in vivo animal experiments. Conclusions: In conclusion, we show that Sptan1 plays important roles in neurodevelopment, and that our novel in vivo and in vitro platforms can be applied to study other IEEs genes. Funding: NIH T32 Training Grant Pediatric Epilepsy Research Foundation