Abstracts

Rationale: Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a genetic epilepsy syndrome characterized by seizures from sleep. Pathogenic mutations in the DEP domain-containing protein 5 (DEPDC5) gene are a significant cause of ADNFLE and are linked to both drug-resistance and an increased risk of sudden unexpected death in epilepsy (SUDEP). DEPDC5 encodes a subunit of the GATOR1 complex (GAP activity toward RAGs complex 1), part of the nutrient-sensing arm of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway. Together with NPRL2 and NPRL3, DEPDC5 functions to inhibit mTORC1 signaling when amino acids are scarce. To gain mechanistic insight into the role of DEPDC5 haploinsufficiency in altering neurodevelopment and metabolism in DEPDC5-associated epilepsies, we characterized neurons generated from induced pluripotent stem cells (iPSCs) from a patient with ADNFLE. Methods: iPSCs were derived from a patient with ADNFLE secondary to a nonsense mutation in DEPDC5 (Arg874X). In addition to wild-type controls, an isogenic control line was created using CRISPR/Cas9-mediated genomic editing via homology-directed repair to correct the DEPDC5 point mutation. iPSCs were differentiated into excitatory cortical neurons using either a dual-SMAD inhibition protocol or a rapid lentiviral-mediated Ngn2-induction protocol. Neuronal morphology was characterized with immunofluorescence whereas mTORC1 signaling and autophagy was assessed with a combination of immunofluorescence and immunoblotting. Results: Neurons expressing glutamatergic markers were created from control and DEPDC5^+/- iPSCs. DEPDC5^+/- neurons demonstrate increased cell size that is evident early in development and persists in mature neurons. mTORC1 hyperactivation was confirmed with elevated S6 phosphorylation, an increase which was further exacerbated in nutrient-limited media. Preliminary findings support impaired induction of autophagy in DEPDC5^+/- neurons likely due to mTORC1-mediated Ulk1 phosphorylation.     Conclusions: Mutations in DEPDC5 are a leading genetic cause of focal epilepsies. We utilized patient-derived iPSCs to create a neuronal model of DEPDC5 haploinsufficiency, demonstrating findings consistent with hyperactive mTORC1 signaling. Future studies will examine signaling activity throughout neuronal development and further evaluate neuronal morphology and dendritic arborization. Improving our understanding of how DEPDC5 haploinsufficiency contributes to epileptogenesis may suggest new drug targets and will provide fundamental knowledge about the pathogenic mechanisms driving the development of epilepsy. Funding: K08 NS083710