Abstracts

Rationale: Voltage-gated sodium channels (VGSCs) consist of a central pore-forming α-subunit and two non-pore-forming b subunits. Inherited, loss of function variants in SCN1B, the gene encoding VGSC b1, are linked to developmental and epileptic encephalopathies (DEEs). b1 subunits are multifunctional proteins that participate in cell adhesion, intracellular signal transduction, and modulation of VGSC α-subunit functional properties. How SCN1B variants impact brain development in DEE remains unclear. b1 subunits are substrates for regulated intramembrane proteolysis (RIP), whereby the b1 extracellular domain is cleaved by b-secretase 1 (BACE1), releasing it into the extracellular space, followed by γ-secretase cleavage of the C-terminal fragment, releasing the b1 intracellular domain (b1-ICD). While we have identified these cleavage mechanisms, we do not understand what signals initiate b1 cleavage. Further, while we know that b1-ICD translocates to the nucleus following γ-secretase cleavage, the machinery involved in its nuclear trafficking remains unknown.

Methods: We investigated the mechanisms regulating b1-ICD trafficking to the nucleus and its subsequent regulation of gene expression. Immunoprecipitation (IP) and western blotting were used to determine whether b1-ICD is in complex with the importin machinery, which has been implicated in nuclear trafficking of other molecules. Immunocytochemistry (ICC) was used to investigate b1-ICD localization to the nucleus in Chinese Hamster Lung (CHL) cells and primary mouse neurons. Finally, we used Cut&Run sequencing from CHL cells overexpressing the b1-ICD, as well as RT-PCR from wild type and Scn1b-null mouse somatosensory cortex, to investigate b1-ICD-mediated changes in gene expression.

Results: ICC staining of CHL cells treated with the importin inhibitors, Ivermectin or Importazole, showed that Ivermectin, but not Importazole, excluded the b1-ICD from the nucleus, suggesting that b1-ICD nuclear translocation may involve karyopherin α1 (KPNA1) but not karyopherin b1 (KPNB1). IP from mouse brain lysates supported these data, suggesting that b1 is in complex with the importin machinery in vivo. Staining of primary mouse cortical neurons treated with the proteosome inhibitor, MG132, provided additional evidence for b1-ICD nuclear localization in vivo. Finally, we found that the b1-ICD regulates mRNA expression of the VGSC α-subunit gene, Scn1a, as well as SrGAP3, which is involved in neuronal cytoskeletal reorganization, and RNF11, which contributes to neuroinflammatory responses.

Conclusions: This study demonstrates that, following RIP of VGSC b1, b1-ICD traffics to the nucleus via an importin-dependent mechanism. Our results suggest that KPNA1, but not KPNB1, is involved in b1-ICD translocation. Further, once b1-ICD reaches the nucleus, it can regulate gene expression, including Scn1a, which is also a critical target in DEE. Understanding these mechanisms is essential to understanding how pathogenic SCN1B variants affect brain developmental in DEE.

Funding: NIH R37 NS076752