Abstracts

Rationale: Inherited, homozygous loss-of-function variants in SCN1B, which encodes the voltage-gated sodium channel (VGSC) β1/β1B subunits, are linked to early infantile developmental and epileptic encephalopathies, including Dravet syndrome. VGSC β1 subunits undergo regulated intramembrane proteolysis, in which sequential cleavage of the β1 subunit by BACE1 and γ-secretase releases a β1 extracellular domain and an intracellular domain (β1-ICD) that has been shown to translocate to the nucleus and regulate transcription. The purpose of this work was to examine the role of both wild type (WT) β1 and patient variant β1-R89C as a transcriptional regulator of SCN1A (Na_V1.1) expression.

Methods: Somatosensory cortex, hippocampus, brainstem, cerebellum, whole brain, and heart ventricle were dissected from postnatal day 15 – 17 Scn1b null and WT mice, followed by RNA isolation and RT-qPCR to examine Na⁺ channel gene expression in vivo. To investigate whether patient variant SCN1B-p.R89C undergoes cleavage similarly to WT β1, cleavage assays were performed in Chinese hamster lung (CHL) cell lines that overexpress bicistronic β1-V5-2A-eGFP or β1-p.R89C-V5-2A-eGFP cDNA constructs. CHLs were treated with γ-secretase inhibitors Avagacestat (10 μM) or L-685,458 (10 μM) for 24h, followed by western blotting to examine V5 (β1) expression. RNA isolation and RT-qPCR were used to examine SCN1A expression changes in SCN1B-p.R89C patient-derived iPSC cortical neurons and cardiac myocytes.

Results: RT-qPCR experiments demonstrated that Scn1a (Na_V1.1) gene expression was significantly downregulated in Scn1b null mouse somatosensory cortex (p < 0.001) and heart ventricle (p < 0.05) compared to WT littermates. This reduction in Scn1b null cortex was specific to Scn1a, as Scn2a, Scn3a, Scn4a, Scn5a, Scn8a, and Scn9a were not significantly different from WT cortex. No changes in Scn1a expression were detected between genotypes in the mouse cerebellum, hippocampus, or brainstem (p > 0.05). Cleavage assays in β1-V5-2A-eGFP or β1-p.R89C-V5-2A-eGFP CHLs demonstrated that treatment with Avagacestat or L-685,458 led to an accumulation of the β1-C-terminal fragment, indicating that patient variant β1-p.R89C undergoes cleavage in a heterologous system. RT-qPCR analyses showed that SCN1A expression is significantly downregulated in iPSC cortical neurons (p < 0.05) derived from a SCN1B-R89C patient with a severe DEE phenotype, however, SCN1A expression was not changed in an unrelated SCN1B-R89C patient with a less severe phenotype. iPSC-derived cardiac myocytes from the patient with the severe phenotype also had significantly reduced SCN1A expression compared to control (p < 0.001).