Abstract
Chen C, Ziobro J, Robinson-Cooper L, Hodges SL, Chen Y, Edokobi N, Lopez-Santiago L, Habig K, Moore C, Minton J, Bramson S, Scheuing C, Daddo N, Štěrbová K, Weckhuysen S, Parent JM, Isom LL. Brain Commun. 2023;5(6):fcad283. Voltage-gated sodium channel β1 subunits are essential proteins that regulate excitability. They modulate sodium and potassium currents, function as cell adhesion molecules and regulate gene transcription following regulated intramembrane proteolysis. Biallelic pathogenic variants in SCN1B, encoding β1, are linked to developmental and epileptic encephalopathy 52, with clinical features overlapping Dravet syndrome. A recessive variant, SCN1B-c.265C > T, predicting SCN1B-p.R89C, was homozygous in 2 children of a nonconsanguineous family. One child was diagnosed with Dravet syndrome, while the other had a milder phenotype. We identified an unrelated biallelic SCN1B-c.265C > T patient with a clinically more severe phenotype than Dravet syndrome. We used CRISPR/Cas9 to knock-in SCN1B-p.R89C to the mouse Scn1b locus (Scn1bR89/C89). We then rederived the line on the C57BL/6J background to allow comparisons between Scn1bR89/R89 and Scn1bC89/C89 littermates with Scn1b+/+ and Scn1b−/− mice, which are congenic on C57BL/6J, to determine whether the SCN1B-c.265C > T variant results in loss-of-function. Scn1bC89/C89 mice have normal body weights and ∼20% premature mortality, compared with severely reduced body weight and 100% mortality in Scn1b−/− mice. β1-p.R89C polypeptides are expressed in brain at comparable levels to wild type. In heterologous cells, β1-p.R89C localizes to the plasma membrane and undergoes regulated intramembrane proteolysis similar to wild type. Heterologous expression of β1-p.R89C results in sodium channel α subunit subtype specific effects on sodium current. mRNA abundance of Scn2a, Scn3a, Scn5a and Scn1b was increased in Scn1bC89/C89 somatosensory cortex, with no changes in Scn1a. In contrast, Scn1b−/− mouse somatosensory cortex is haploinsufficient for Scn1a, suggesting an additive mechanism for the severity of the null model via disrupted regulation of another Dravet syndrome gene. Scn1bC89/C89 mice are more susceptible to hyperthermia-induced seizures at postnatal Day 15 compared with Scn1bR89/R89 littermates. EEG recordings detected epileptic discharges in young adult Scn1bC89/C89 mice that coincided with convulsive seizures and myoclonic jerks. We compared seizure frequency and duration in a subset of adult Scn1bC89/C89 mice that had been exposed to hyperthermia at postnatal Day 15 versus a subset that were not hyperthermia exposed. No differences in spontaneous seizures were detected between groups. For both groups, the spontaneous seizure pattern was diurnal, occurring with higher frequency during the dark cycle. This work suggests that the SCN1B-c.265C > T variant does not result in complete loss-of-function. Scn1bC89/C89 mice more accurately model SCN1B-linked variants with incomplete loss-of-function compared with Scn1b−/− mice, which model complete loss-of-function, and thus add to our understanding of disease mechanisms as well as our ability to develop new therapeutic strategies.
Commentary
Voltage-gated sodium channels (VGSCs) are critical for proper neuronal signaling. VGSC α subunits, including SCN1A, SCN2A, and SCN8A form heterotrimeric complexes with VGSC β subunits. VGSC α subunits form pores and allow conduction of sodium currents based on neuronal membrane potential. VGSC β subunits are multifunctional proteins that modulate VGSC α subunit activity as well as voltage-gated potassium channel activity. 1 The β subunits also function as cell adhesion molecules, play a role in neurodevelopment, 1 and are substrates for proteolytic cleavage by BACE1 and γ-secretase, which can regulate expression of VGSC α subunits. 2
SCN1B, encoding the VGSC β1 subunit, was first identified in generalized epilepsy with febrile seizures plus (GEFS+). 3 SCN1B variants have also been found in developmental and epileptic encephalopathies (DEEs), including Dravet syndrome and SCN1B encephalopathy (DEE52). 4 SCN1B variants associated with GEFS+, such as the p.C121W variant, are typically monoallelic. In contrast, biallelic variants are typical in SCN1B DEE.
SCN1B DEE variants are primarily missense. The p.R125C variant prevents normal trafficking of β1 to the cell surface. 5 Thus, homozygous p.R125C variants result in a functional null phenotype. Scn1b−/− null mice recapitulate spontaneous seizures and exhibit defects in neuronal development. 6 However, no functional experiments have been performed on the remaining SCN1B missense variants identified in DEE, so it is unclear whether SCN1B DEE is primarily the result of loss-of-function (LOF) or other mechanisms.
In the current study, Chen et al 7 identified the biallelic variant p.R89C in 2 siblings and 1 unrelated individual. One sibling was diagnosed with Dravet syndrome while the other had milder epilepsy. The unrelated individual had clinical features more severe than Dravet syndrome. To investigate the pathogenic effects of p.R89C, the team used CRISPR to create a mouse model of SCN1B epilepsy carrying the p.R89C variant.
Scn1bC89/C89 homozygous mutant mice exhibit spontaneous seizures and premature death. The p.R89C variant does not alter β1 protein expression nor prevent proteolytic cleavage. In contrast to p.R125C, p.R89C does not prevent β1 localization to the plasma membrane. Thus, p.R89C likely does not cause seizures by the same underlying mechanisms as p.R125C.
Co-expression of Nav1.1, Nav1.6, or Nav1.5 α subunits with WT b1 significantly increased transient sodium current density, as expected. The p.R89C b1 mutant increased Nav1.6 sodium current density to a similar degree as WTβ1. Intriguingly, p.R89C did not alter Nav1.1 or Nav1.5 sodium current density, indicating that p.R89C b1 subunits differentially affect VGSC α subunits. This experiment demonstrates that p.R89C does not behave exactly like the null allele.
After proteolytic cleavage, β1 enters the nucleus and can alter expression of VGSC genes. In Scn1b−/− mice, Scn1a mRNA levels were reduced by 30% in whole brain and by 50% in somatosensory cortex. 8 In contrast, Scn1bC89/C89 mice have elevated Scn1a mRNA levels in brainstem, while Scn1a mRNA levels remain unchanged in other areas. Scn2a, Scn3a, and Scn5a mRNAs were increased in somatosensory cortex, and levels of Scn1b mRNA itself were upregulated in somatosensory cortex and cerebellum. Again, p.R89C behaved differently than the null allele.
The underlying pathogenic mechanisms of SCN1B epilepsy remain unclear. Missense variants identified in GEFS+ (p.C121W, heterozygous) and in SCN1B DEE (p.R125C, homozygous) exhibit molecular phenotypes consistent with LOF. The Scn1b−/− null mouse confirmed that biallelic loss of Scn1b results in spontaneous seizures. However, Scn1b+/− heterozygous mice do not have spontaneous seizures, and parents heterozygous for variants found in biallelic DEE do not have epilepsy. Likewise, an Scn1bC121W mouse model only exhibited a severe phenotype when the p.C121W variant was homozygous, resulting in no production of β1 protein. 9 These findings indicate that simple LOF likely does not fully explain how Scn1b variants generate the GEFS+ or the complete SCN1B DEE phenotype.
It is clear that the p.R89C variant is not a functional null allele as it is present on cellular membranes and can modulate activity of Nav1.6. Curiously, it cannot modulate Nav1.1. The mechanistic explanation for this difference is unclear, since VGSC α subunits are highly similar in sequence and structure. Modeling based on the protein structures may help determine whether p.R98C b1 subunits are predicted to interact differently with Nav1.1 versus Nav1.6. It could also be useful to examine β1 protein distribution in different cell types in the Scn1bC89/C89 brain. For example, WT β1 is localized at axon initial segments (AIS) of excitatory and inhibitory neurons in mouse brain. 9 It would be interesting to determine whether the p.R89C is also present at AIS of excitatory neurons (with Nav1.6) as well as inhibitory neurons (with Nav1.1).
To quantify effects of p.R89C β1 subunits on neuronal firing, it will be necessary to perform electrophysiology in brain slices from Scn1bC89/C89 mice. Scn1b−/− mice exhibited hypoexcitability of inhibitory neurons and hyperexcitability of some populations of excitatory neurons. 10 These firing properties are partially explained by the reduction of Scn1a levels in Scn1b−/− brain. In contrast, levels of Scn1a and other α subunits are elevated in Scn1bC89/C89 brain, suggesting that inhibitory neuronal firing may not be reduced as strongly in this mouse model.
The finding that p.R89C is not complete LOF together with the differential regulation of Scn1a mRNA compared to the Scn1b−/− mice, raises questions about treatments for Scn1b DEE. Phenytoin was efficacious in an individual with a monogenic allele of p.R89H in SCN1B epilepsy, but carbamazepine was less effective in Scn1b−/− mice, suggesting that more study is needed to determine whether sodium channel blockers are helpful for SCN1B epilepsy.11,12 Fenfluramine reduced seizure frequency in some cases of Scn1a-linked Dravet syndrome and in some cases of SCN1B DEE, suggesting that convergent pathogenic mechanisms like reduced Scn1a expression may be involved. 4 However, differences highlighted by the current and previous mouse models suggest that individuals with SCN1B epilepsy may respond differently to pharmacological management based on their pathogenic variant. Additionally, ASO-based therapies that elevate Scn1a mRNA could be effective in some cases of SCN1B epilepsy but not others. It would be interesting to see whether these ASOs are therapeutic in either the Scn1b−/− or Scn1bC89/C89 mice.
Finally, the varying severity of disease in individuals with the p.R89C allele suggests that genetic modifiers may influence SCN1B epilepsy. This mouse model will be useful for mapping modifiers like those found for other sodium channel-related epilepsies. Overall, this study demonstrates the importance of modeling multiple missense variants in animal models. It is difficult to assess the effects of missense variants without assaying neurodevelopment and seizure pathology in the context of brain networks. It also allows for comprehensive assessment of gene expression and protein localization throughout the brain, which helps pinpoint targets for therapeutic intervention. This study reveals that the spectrum and the genotype-phenotype relationships in SCN1B epilepsy are more complex than SCN1B LOF alone and provides a new model to investigate the dynamic role of VGSCs in the brain.
