Abstracts

Rationale: Dravet syndrome (DS) is a devastating pediatric epileptic encephalopathy (EE). Disease onset occurs within the first year of life and presents with drug resistant seizures, behavioral and developmental delays, and a high risk of sudden unexpected death in epilepsy (SUDEP). While the majority of DS patients have de novo mutations in SCN1A, encoding the voltage-gated sodium channel (VGSC) Nav1.1 α subunit, homozygous loss-of-function (LOF) mutations in SCN1B, encoding VGSC β1 and β1B, are also linked to DS. Scn1b null mice provide a model of DS. β1 is a multifunctional protein that participates in channel modulation, cell adhesion, cell signaling, neuronal pathfinding, and neuronal fasciculation. β1 is also a substrate for sequential cleavage by BACE1 and γ-secretase, resulting in the formation of an intracellular domain (ICD). We are investigating the biochemical mechanisms and downstream signaling of these cleavage events in vitro to better understand how modifications of β1 might affect protein function. Methods: We used expression of C-terminal V5-tagged WT and mutant β1 cDNA constructs to examine the biochemical reactions regulating β1 subunit cleavage by BACE1 and γ-secretase. Chinese Hamster Lung (CHL) cells were transfected with cDNAs encoding WT β1 and mutant β1. Cleavage of β1 was assayed by gel electrophoresis, followed by western blotting with an antibody directed against the V5 epitope. Results: CHL cells endogenously express BACE1 and presenilin-1, the catalytic subunit of γ-secretase, at levels that are sufficient to drive sequential cleavage of β1 in vitro. Cleavage can be blocked using inhibitors of BACE1 and γ-secretase, consistent with sequential cleavage. We analyzed two β1 DS patient LOF mutants, R89C and R125C, to determine if they are cleaved by these enzymes. We have shown that β1 molecules that carry these mutations are not detected at the plasma membrane, however they are cleaved by BACE1, suggesting that β1 cleavage may not occur on the plasma membrane, but on the membrane of an intracellular organelle. These data provide new insight as to the trafficking and half-life of β1 DS mutations within the cell.  Our previous work implicated β1 phosphorylation at the intracellular residue Y181 in downstream signaling. We examined the role of this residue in sequential cleavage using phosphorylation-null (β1Y181A and β1Y181F) and a phosphomimetic mutant (β1Y181E). β1Y181A and β1Y181E were sequentially cleaved in vitro, while β1Y181F was not. Treatment of cells with leptomycin B (LMB), which inhibits nuclear export by blocking exportin-1, results in nuclear co-localization of DAPI with the V5 tagged β1 ICD. In cells co-treated with the γ-secretase inhibitor, DAPT, which blocks generation of the β1 ICD, this co-localization is not detected, suggesting that β1 nuclear localization is dependent on ICD generation by sequential cleavage. Conclusions: These data confirm that β1 is sequentially cleaved in vitro by BACE1 and γ-secretase. Cleavage may be regulated by the tyrosine at position 181 and may occur on the membrane of an intracellular organelle. Preliminary evidence suggests the β1 ICD generated by sequential cleavage is translocated to the nucleus. We hypothesize this mechanism may underlie differential gene expression of VGSCs in brain and heart in Scn1b DS mice. Future work will examine transcriptional regulation by the β1 ICD in vivo to further understand the mechanism of SCN1B mutations in DS and DS-like EEs. Funding: NIH-T32 Systems and Integrative Physiology Training Grant GM008322-26, NIH R37NS076752 to LLI.