Abstracts

Rationale: Dravet Syndrome (DS) is a pediatric epileptic encephalopathy that typically begins in the first year of life. DS presents with intractable seizures, ataxia, behavioral and developmental delay, and high risk of sudden unexpected death in epilepsy. Homozygous mutations in SCN1B, which encodes voltage-gated sodium channel (VGSCs) ß1 and ß1B subunits, are linked to DS and Scn1b null mice model DS. ß1 is multifunctional, modulating the gating and kinetics of the ion channel, cell adhesion, cell signaling, neuronal pathfinding and fasciculation. ß1 is a substrate for sequential cleavage by BACE1 and ?-secretase, resulting in the formation of an intracellular domain (ICD). We are investigating the biochemical mechanisms and downstream signaling of ß1 cleavage in vitro to understand how this signaling pathway, or loss thereof, may contribute to cell function or DS. Methods: We used exogenous expression of V5 tagged wild-type (WT) and mutant ß1 cDNA constructs to study the biochemical mechanisms regulating cleavage and its subsequent downstream signaling. ß1 cleavage was assayed by immunoblot with an antibody directed against the V5 tag or the ß1-ICD. Imaging was performed on a Nikon A1R confocal microscope. Results: Chinese hamster lung cells endogenously express BACE1 and ?-secretase at levels sufficient to drive ß1 sequential cleavage in vitro. Cleavage can be blocked using BACE1 and ?-secretase inhibitors in a manner consistent with sequential cleavage. Previous work showed ß1 is phosphorylated at tyrosine residue,181 by fyn kinase and this phosphorylation is involved in neurite outgrowth. ß1-mediated neurite outgrowth can be inhibited using ?-secretase inhibitors, suggesting ß1 cleavage may also be important in its regulation. To examine the role of Y181 in cleavage, we used residues that cannot be phosphorylated (ß1p.Y181A and ß1p.Y181F) and phosphomimetic (ß1p.Y181E) mutants. Cell surface biotinylation indicated that like WT, the mutants localize to the plasma membrane. Interestingly, ß1.pY181A, ß1p.Y181F, and ß1p.Y181E are cleaved in this system. The ß1-ICD is localized to the nucleus, suggesting the ICD generated is translocated to the nucleus independently of phosphorylation at the Y181 position. To identify potential gene targets regulated by the ß1-ICD, we will complete RNA-seq from WT vs. Scn1b null animals. Conclusions: We confirm ß1 is sequentially cleaved in vitro by BACE1 and ?-secretase. Phosphorylation at the Y181 position does not affect localization to the plasma membrane or cleavage, suggesting ß1-mediated neurite outgrowth may be regulated by multiple pathways. The ß1-ICD generated by cleavage is translocated to the nucleus in a Y181-phosphorylation independent manner. We hypothesize loss of this signaling pathway in Scn1b null mice may underlie differential gene expression of VGSCs in brain and heart. RNA-seq was utilized to identify gene target candidates potentially regulated by the ß1-ICD. Funding: NIH-T32 Systems and Integrative Physiology Training Grant GM008322-26, NIH R37NS076752 to LLI