Abstracts

Rationale: DEPDC5 is a subunit of the GATOR1 complex (GAP activity toward RAGs complex 1), and together with NPRL2 and NPRL3, it inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway. Mutations in DEPDC5 are increasingly identified in a broad spectrum of focal epilepsies. Previous knockout animals failed to recapitulate the pathogenesis observed in human brains, and therefore new models are urgently needed for therapy development.  Methods: Here we use human embryonic stem cells (hESCs) and CRISPR/CAS9 genome editing to model DEPDC5 loss-of-function (LOF) in vitro. Our sgRNA is designed to target human DEPDC5 gene in exon 12.  To generate this cell model, hESCs are co-transfected with DEPDC5 CRISPR and GFP. Control cells are co-transfected with PX330 and GFP. We differentiate these stem cells into neurons using dual SMAD inhibition. Using CRISPR/CAS9 in utero electroporation (IUE) in the rat brain, we also establish an in vivo DEPDC5 focal somatic LOF model.  Results: hESC- derived neurons with DEPDC5 LOF are cytomegalic and have increased levels of phosphorylated S6 (pS6). Postnatal administration of Everolimus, a Rapamycin analog, reverses the cytomegaly. In vivo DEPDC5 knockout lead to regions of cortical abnormalities, with cytomegalic neurons and increased levels of pS6.Animals with focal DEPDC5 LOF are more susceptible to pentylenetetrazol (PTZ) - induced seizures and also develop spontaneous seizures.  Conclusions: Our hESC and rat models of DEPDC5 LOF recapitulate both neuronal cytoarchitecture and excitability abnormalities observed in patients, and therefore provide a novel platform to develop mechanistic-based therapeutics for DEPDC5-related focal epilepsies. These models should also yield conceptual insights broadly relevant to understanding focal cortical dysplasia and for establishing a logical molecular network underlying mTOR-based neurological diseases. Funding: NIH1 K08 NS099379-01, Pediatric Epilepsy Research Foundation (PERF)