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BRAFV600E Expression in Neural Progenitors Results in a Hyperexcitable Phenotype in Neocortical Pyramidal Neurons.

Somatic mutations have emerged as the likely cause of focal epilepsies associated with developmental malformations and epilepsy-associated glioneuronal tumors (GNT). Somatic BRAFV600E mutations in particular have been detected in the majority of low-grade neuroepithelial tumors (LNETS), and in neurons in focal cortical dysplasias adjacent to epilepsy associated tumors. Furthermore, conditional expression of an activating BRAF mutation in neocortex causes seizures in mice. In this study we characterized the cellular electrophysiology of layer 2/3 neocortical pyramidal neurons induced to express BRAFV600E from neural progenitor stages. In-utero electroporation of a piggyBac transposase plasmid system was used to introduce transgenes expressing BRAFwt, BRAFV600E, and/or eGFP and mRFP into radial glia progenitors in mouse embryonic cortex. Whole-cell patch-clamp recordings of pyramidal neurons in slices prepared from P15-P70 mice showed that BRAFV600E resulted in neurons with a distinct hyperexcitable phenotype characterized by depolarized resting membrane potentials, increased input resistances, lowered action potential (AP) thresholds, and increased AP firing frequencies. BRAFV600E expressing neurons also displayed increased hyperpolarization induced inward currents (Ih), and decreased sustained potassium currents. Neurons adjacent to BRAFV600E transgene expressing neurons, and neurons with genetically deleted TSC1 by CRISPR or induced to carry PIK3CAE545K transgenes did not show similar excitability phenotypes. Together these results indicate that BRAFV600E leads to a distinct hyperexcitable neuronal phenotype which could contribute to the development of seizures.