Altered neurological and neurobehavioral phenotypes in a mouse model of the recurrent KCNB1-p.R306C voltage-sensor variant.

Pathogenic variants in KCNB1 are associated with a neurodevelopmental disorder spectrum that includes global developmental delays, cognitive impairment, abnormal electroencephalogram (EEG) patterns, and epilepsy with variable age of onset and severity. Additionally, there are prominent behavioral disturbances, including hyperactivity, aggression, and features of autism spectrum disorder. The most frequently identified recurrent variant is KCNB1-p.R306C, a missense variant located within the S4 voltage-sensing transmembrane domain. Individuals with the R306C variant exhibit mild to severe developmental delays, behavioral disorders, and a diverse spectrum of seizures. Previous in vitro characterization of R306C described altered sensitivity and cooperativity of the voltage sensor and impaired capacity for repetitive firing of neurons. Existing Kcnb1 mouse models include dominant negative missense variants, as well as knockout and frameshifts alleles. While all models recapitulate key features of KCNB1 encephalopathy, mice with dominant negative alleles were more severely affected. In contrast to existing loss-of-function and dominant-negative variants, KCNB1-p.R306C does not affect channel expression, but rather affects voltage-sensing. Thus, modeling R306C in mice provides a novel opportunity to explore impacts of a voltage-sensing mutation in Kcnb1. Using CRISPR/Cas9 genome editing, we generated the Kcnb1R306C mouse model and characterized the molecular and phenotypic effects. Consistent with the in vitro studies, neurons from Kcnb1R306C mice showed altered excitability. Heterozygous and homozygous R306C mice exhibited hyperactivity, altered susceptibility to chemoconvulsant-induced seizures, and frequent, long runs of slow spike wave discharges on EEG, reminiscent of the slow spike and wave activity characteristic of Lennox Gastaut syndrome. This novel model of channel dysfunction in Kcnb1 provides an additional, valuable tool to study KCNB1 encephalopathies. Furthermore, this allelic series of Kcnb1 mouse models will provide a unique platform to evaluate targeted therapies.

Strain-dependent effects on neurobehavioral and seizure phenotypes in Scn2aK1422E mice.

Pathogenic variants in SCN2A are associated with a range of neurodevelopmental disorders (NDD). Despite being largely monogenic, SCN2A-related NDD show considerable phenotypic variation and complex genotype-phenotype correlations. Genetic modifiers can contribute to variability in disease phenotypes associated with rare driver mutations. Accordingly, different genetic backgrounds across inbred rodent strains have been shown to influence disease-related phenotypes, including those associated with SCN2A-related NDD. Recently, we developed a mouse model of the variant SCN2A-p.K1422E that was maintained as an isogenic line on the C57BL/6J (B6) strain. Our initial characterization of NDD phenotypes in heterozygous Scn2aK1422E mice revealed alterations in anxiety-related behavior and seizure susceptibility. To determine if background strain affects phenotype severity in the Scn2aK1422E mouse model, phenotypes of mice on B6 and [DBA/2J×B6]F1 hybrid (F1D2) strains were compared. Convergent evidence from neurobehavioral assays demonstrated lower anxiety-like behavior in Scn2aK1422E mice compared to wild-type and further suggested that this effect is more pronounced on the B6 background compared to the F1D2 background. Although there were no strain-dependent differences in occurrence of rare spontaneous seizures, response to the chemoconvulsant kainic acid revealed differences in seizure generalization and lethality risk, with variation based on strain and sex. Continued examination of strain-dependent effects in the Scn2aK1422E mouse model could reveal genetic backgrounds with unique susceptibility profiles that would be relevant for future studies on specific traits and enable the identification of highly penetrant phenotypes and modifier genes that could provide clues about the primary pathogenic mechanism of the K1422E variant.

Evaluating the interplay between estrous cyclicity and induced seizure susceptibility in Scn2aK1422E mice.

Pathogenic variants in SCN2A are associated with a range of neurodevelopmental disorders (NDD). SCN2A-related NDD show wide phenotypic heterogeneity, suggesting that modifying factors must be considered in order to properly elucidate the mechanisms of pathogenic variants. Recently, we characterized neurological phenotypes in a mouse model of the variant SCN2A-p.K1422E. We demonstrated that heterozygous Scn2aK1422E female mice showed a distinct, reproducible distribution of flurothyl-induced seizure thresholds. Women with epilepsy often show a cyclical pattern of altered seizure susceptibility during specific phases of the menstrual cycle which can be attributed to fluctuations in hormones and corresponding changes in neurosteroid levels. Rodent models have been used extensively to examine the relationship between the estrous (menstrual) cycle, steroid hormones, and seizure susceptibility. However, the effects of the estrous cycle on seizure susceptibility have not been evaluated in the context of an epilepsy-associated genetic variant. To determine whether the estrous cycle affects susceptibility to flurothyl-induced seizures in Scn2aK1422E female mice, estrous cycle monitoring was performed in mice that had undergone ovariectomy (OVX), sham surgery, or no treatment prior to seizure induction. Removing the influence of circulating sex hormones via OVX did not affect the non-unimodal distribution of flurothyl seizure thresholds observed in Scn2aK1422E females. Additionally, flurothyl seizure thresholds were not associated with estrous cycle stage in mice that underwent sham surgery or were untreated. These data suggest that variation in Scn2aK1422E flurothyl seizure threshold is not significantly influenced by the estrous cycle and, by extension, fluctuations in ovarian hormones. Interestingly, untreated Scn2aK1422E females showed evidence of disrupted estrous cyclicity, an effect not previously described in a genetic epilepsy model. This unexpected result highlights the importance of considering sex specific effects and the estrous cycle in support of more inclusive biomedical research.

Altered neurological and neurobehavioral phenotypes in a mouse model of the recurrent KCNB1-p.R306C voltage-sensor variant.

Pathogenic variants in KCNB1 are associated with a neurodevelopmental disorder spectrum that includes global developmental delays, cognitive impairment, abnormal electroencephalogram (EEG) patterns, and epilepsy with variable age of onset and severity. Additionally, there are prominent behavioral disturbances, including hyperactivity, aggression, and features of autism spectrum disorder. The most frequently identified recurrent variant is KCNB1-p.R306C, a missense variant located within the S4 voltage-sensing transmembrane domain. Individuals with the R306C variant exhibit mild to severe developmental delays, behavioral disorders, and a diverse spectrum of seizures. Previous in vitro characterization of R306C described loss of voltage sensitivity and cooperativity of the sensor and inhibition of repetitive firing. Existing Kcnb1 mouse models include dominant negative missense variants, as well as knockout and frameshifts alleles. While all models recapitulate key features of KCNB1 encephalopathy, mice with dominant negative alleles were more severely affected. In contrast to existing loss-of-function and dominant-negative variants, KCNB1-p.R306C does not affect channel expression, but rather affects voltage-sensing. Thus, modeling R306C in mice provides a novel opportunity to explore impacts of a voltage-sensing mutation in Kcnb1. Using CRISPR/Cas9 genome editing, we generated the Kcnb1R306C mouse model and characterized the molecular and phenotypic effects. Heterozygous and homozygous R306C mice exhibited pronounced hyperactivity, altered susceptibility to flurothyl and kainic acid induced-seizures, and frequent, long runs of spike wave discharges on EEG. This novel model of channel dysfunction in Kcnb1 provides an additional, valuable tool to study KCNB1 encephalopathies. Furthermore, this allelic series of Kcnb1 mouse models will provide a unique platform to evaluate targeted therapies.

Evaluating the interplay between estrous cyclicity and flurothyl-induced seizure susceptibility in Scn2a K1422E mice.

Recently, we demonstrated that Scn2a K1422E female mice showed a distinct distribution of flurothyl-induced seizure thresholds. To evaluate whether the estrous cycle contributes to this effect, estrous cycle monitoring was performed in mice that had undergone ovariectomy, sham surgery, or no treatment prior to seizure induction. Ovariectomy did not affect the non-unimodal distribution of flurothyl seizure thresholds observed in Scn2a K1422E females. Additionally, seizure thresholds were not associated with estrous cycle stage in mice that underwent sham surgery or in non-surgerized (intact) mice. Interestingly, intact Scn2a K1422E females showed evidence of disrupted estrous cyclicity, an effect not previously described in a genetic epilepsy model.

Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice.

Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to developmental and epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to intellectual disability and/or autism spectrum disorder (ASD) with or without co-morbid seizures. One unique case less easily classified using this framework is the de novo missense variant SCN2A-p.K1422E, associated with infant-onset developmental delay, infantile spasms and features of ASD. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model incorporating variant channels that predicted reductions in peak action potential (AP) speed. We generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Cultured cortical neurons from heterozygous Scn2aK1422E/+ mice exhibited lower current density with a TTX-resistant component and reversal potential consistent with mixed ion permeation. Recordings from Scn2aK1442E/+ cortical slices demonstrated impaired AP initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the AP, suggesting complex effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal electroencephalogram abnormalities, altered induced seizure thresholds, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from other Scn2a models, consistent with complex effects of K1422E on NaV1.2 channel function.