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.

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.

Fine mapping and candidate gene analysis of Dravet syndrome modifier loci on mouse chromosomes 7 and 8.

Dravet syndrome is a developmental and epileptic encephalopathy (DEE) characterized by intractable seizures, comorbidities related to developmental, cognitive, and motor delays, and a high mortality burden due to sudden unexpected death in epilepsy (SUDEP). Most Dravet syndrome cases are attributed to SCN1A haploinsufficiency, with genetic modifiers and environmental factors influencing disease severity. Mouse models with heterozygous deletion of Scn1a recapitulate key features of Dravet syndrome, including seizures and premature mortality; however, severity varies depending on genetic background. Here, we refined two Dravet survival modifier (Dsm) loci, Dsm2 on chromosome 7 and Dsm3 on chromosome 8, using interval-specific congenic (ISC) mapping. Dsm2 was complex and encompassed at least two separate loci, while Dsm3 was refined to a single locus. Candidate modifier genes within these refined loci were prioritized based on brain expression, strain-dependent differences, and biological relevance to seizures or epilepsy. High priority candidate genes for Dsm2 include Nav2, Ptpn5, Ldha, Dbx1, Prmt3 and Slc6a5, while Dsm3 has a single high priority candidate, Psd3. This study underscores the complex genetic architecture underlying Dravet syndrome and provides insights into potential modifier genes that could influence disease severity and serve as novel therapeutic targets.

Fine mapping and candidate gene analysis of a dravet syndrome modifier locus on mouse chromosome 11.

Pathogenic variants in SCN1A result in a spectrum of phenotypes ranging from mild febrile seizures to Dravet syndrome, a severe infant-onset epileptic encephalopathy. Individuals with Dravet syndrome have developmental delays, elevated risk for sudden unexpected death in epilepsy (SUDEP), and have multiple seizure types that are often refractory to treatment. Although most Dravet syndrome variants arise de novo, there are cases where an SCN1A variant was inherited from mildly affected parents, as well as some individuals with de novo loss-of-function or truncation mutations that presented with milder phenotypes. This suggests that disease severity is influenced by other factors that modify expressivity of the primary mutation, which likely includes genetic modifiers. Consistent with this, the Scn1a+/- mouse model of Dravet syndrome exhibits strain-dependent variable phenotype severity. Scn1a+/- mice on the 129S6/SvEvTac (129) strain have no overt phenotype and a normal lifespan, while [C57BL/6Jx129]F1.Scn1a+/- mice have severe epilepsy with high rates of premature death. Low resolution genetic mapping identified several Dravet syndrome modifier (Dsm) loci responsible for the strain-dependent difference in survival of Scn1a+/- mice. To confirm the Dsm5 locus and refine its position, we generated interval-specific congenic strains carrying 129-derived chromosome 11 alleles on the C57BL/6J strain and localized Dsm5 to a 5.9 Mb minimal region. We then performed candidate gene analysis in the modifier region. Consideration of brain-expressed genes with expression or coding sequence differences between strains along with gene function suggested numerous strong candidates, including several protein coding genes and two miRNAs that may regulate Scn1a transcript.

Epilepsy and neurobehavioral abnormalities in mice with a dominant-negative KCNB1 pathogenic variant.

Developmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay, and often have elevated risk for premature mortality. Numerous genes have been identified as a monogenic cause of DEE, including KCNB1. The voltage-gated potassium channel KV2.1, encoded by KCNB1, is primarily responsible for delayed rectifier potassium currents that are important regulators of excitability in electrically excitable cells, including neurons. In addition to its canonical role as a voltage-gated potassium conductance, KV2.1 also serves a highly conserved structural function organizing endoplasmic reticulum-plasma membrane junctions clustered in the soma and proximal dendrites of neurons. The de novo pathogenic variant KCNB1-p.G379R was identified in an infant with epileptic spasms, and atonic, focal and tonic-clonic seizures that were refractory to treatment with standard antiepileptic drugs. Previous work demonstrated deficits in potassium conductance, but did not assess non-conducting functions. To determine if the G379R variant affected KV2.1 clustering at endoplasmic reticulum-plasma membrane junctions, KV2.1-G379R was expressed in HEK293T cells. KV2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and co-expression of KV2.1-G379R with KV2.1-wild-type lowered induction of these structures relative to KV2.1-WT alone, consistent with a dominant negative effect. To model this variant in vivo, we introduced Kcnb1G379R into mice using CRISPR/Cas9 genome editing. We characterized neuronal expression, neurological and neurobehavioral phenotypes of Kcnb1G379R/+ (Kcnb1R/+) and Kcnb1G379R/G379R (Kcnb1R/R) mice. Immunohistochemistry studies on brains from Kcnb1+/+, Kcnb1R/+ and Kcnb1R/R mice revealed genotype-dependent differences in the expression levels of KV2.1 protein, as well as associated KV2.2 and AMIGO-1 proteins. Kcnb1R/+ and Kcnb1R/R mice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. Spontaneous seizures were observed in Kcnb1R/R mice, as well as seizures induced by exposure to novel environments and/or handling. Both Kcnb1R/+ and Kcnb1R/R mutants were more susceptible to proconvulsant-induced seizures. In addition, both Kcnb1R/+ and Kcnb1R/R mice exhibited abnormal interictal EEG activity, including isolated spike and slow waves. Overall, the Kcnb1G379R mice recapitulate many features observed in individuals with DEE due to pathogenic variants in KCNB1. This new mouse model of KCNB1-associated DEE will be valuable for improving the understanding of the underlying pathophysiology and will provide a valuable tool for the development of therapies to treat this pharmacoresistant DEE.

Gabra2 is a genetic modifier of Dravet syndrome in mice.

Pathogenic variants in epilepsy genes result in a spectrum of clinical severity. One source of phenotypic heterogeneity is modifier genes that affect expressivity of a primary pathogenic variant. Mouse epilepsy models also display varying degrees of clinical severity on different genetic backgrounds. Mice with heterozygous deletion of Scn1a (Scn1a+/-) model Dravet syndrome, a severe epilepsy most often caused by SCN1A haploinsufficiency. Scn1a+/- mice recapitulate features of Dravet syndrome, including spontaneous seizures, sudden death, and cognitive/behavioral deficits. Scn1a+/- mice maintained on the 129S6/SvEvTac (129) strain have normal lifespan and no spontaneous seizures. In contrast, admixture with C57BL/6J (B6) results in epilepsy and premature lethality. We previously mapped Dravet Survival Modifier loci (Dsm1-Dsm5) responsible for strain-dependent differences in survival. Gabra2, encoding the GABAA α2 subunit, was nominated as a candidate modifier at Dsm1. Direct measurement of GABAA receptors found lower abundance of α2-containing receptors in hippocampal synapses of B6 mice relative to 129. We also identified a B6-specific single nucleotide deletion within Gabra2 that lowers mRNA and protein by nearly 50%. Repair of this deletion reestablished normal levels of Gabra2 expression. In this study, we used B6 mice with a repaired Gabra2 allele to evaluate Gabra2 as a genetic modifier of severity in Scn1a+/- mice. Gabra2 repair restored transcript and protein expression, increased abundance of α2-containing GABAA receptors in hippocampal synapses, and rescued epilepsy phenotypes of Scn1a+/- mice. These findings validate Gabra2 as a genetic modifier of Dravet syndrome, and support enhancing function of α2-containing GABAA receptors as treatment strategy for Dravet syndrome.

Soticlestat, a novel cholesterol 24-hydroxylase inhibitor, reduces seizures and premature death in Dravet syndrome mice.

OBJECTIVE: Dravet syndrome is a severe developmental and epileptic encephalopathy (DEE) most often caused by de novo pathogenic variants in SCN1A. Individuals with Dravet syndrome rarely achieve seizure control and have significantly elevated risk for sudden unexplained death in epilepsy (SUDEP). Heterozygous deletion of Scn1a in mice (Scn1a+/- ) recapitulates several core phenotypes, including temperature-dependent and spontaneous seizures, SUDEP, and behavioral abnormalities. Furthermore, Scn1a+/- mice exhibit a similar clinical response to standard anticonvulsants. Cholesterol 24-hydroxlase (CH24H) is a brain-specific enzyme responsible for cholesterol catabolism. Recent research has indicated the therapeutic potential of CH24H inhibition for diseases associated with neural excitation, including seizures. METHODS: In this study, the novel compound soticlestat, a CH24H inhibitor, was administered to Scn1a+/- mice to investigate its ability to improve Dravet-like phenotypes in this preclinical model. RESULTS: Soticlestat treatment reduced seizure burden, protected against hyperthermia-induced seizures, and completely prevented SUDEP in Scn1a+/- mice. Video-electroencephalography (EEG) analysis confirmed the ability of soticlestat to reduce occurrence of electroclinical seizures. SIGNIFICANCE: This study demonstrates that soticlestat-mediated inhibition of CH24H provides therapeutic benefit for the treatment of Dravet syndrome in mice and has the potential for treatment of DEEs.

CaMKII modulates sodium current in neurons from epileptic Scn2a mutant mice.

Monogenic epilepsies with wide-ranging clinical severity have been associated with mutations in voltage-gated sodium channel genes. In the Scn2aQ54 mouse model of epilepsy, a focal epilepsy phenotype is caused by transgenic expression of an engineered NaV1.2 mutation displaying enhanced persistent sodium current. Seizure frequency and other phenotypic features in Scn2aQ54 mice depend on genetic background. We investigated the neurophysiological and molecular correlates of strain-dependent epilepsy severity in this model. Scn2aQ54 mice on the C57BL/6J background (B6.Q54) exhibit a mild disorder, whereas animals intercrossed with SJL/J mice (F1.Q54) have a severe phenotype. Whole-cell recording revealed that hippocampal pyramidal neurons from B6.Q54 and F1.Q54 animals exhibit spontaneous action potentials, but F1.Q54 neurons exhibited higher firing frequency and greater evoked activity compared with B6.Q54 neurons. These findings correlated with larger persistent sodium current and depolarized inactivation in neurons from F1.Q54 animals. Because calcium/calmodulin protein kinase II (CaMKII) is known to modify persistent current and channel inactivation in the heart, we investigated CaMKII as a plausible modulator of neuronal sodium channels. CaMKII activity in hippocampal protein lysates exhibited a strain-dependence in Scn2aQ54 mice with higher activity in F1.Q54 animals. Heterologously expressed NaV1.2 channels exposed to activated CaMKII had enhanced persistent current and depolarized channel inactivation resembling the properties of F1.Q54 neuronal sodium channels. By contrast, inhibition of CaMKII attenuated persistent current, evoked a hyperpolarized channel inactivation, and suppressed neuronal excitability. We conclude that CaMKII-mediated modulation of neuronal sodium current impacts neuronal excitability in Scn2aQ54 mice and may represent a therapeutic target for the treatment of epilepsy.

Potentiating α2 subunit containing perisomatic GABAA receptors protects against seizures in a mouse model of Dravet syndrome.

KEY POINTS: Dravet syndrome mice (Scn1a+/- ) demonstrate a marked strain dependence for the severity of seizures which is correlated with GABAA receptor α2 subunit expression. The α2 /α3 subunit selective positive allosteric modulator (PAM) AZD7325 potentiates inhibitory postsynaptic currents (IPSCs) specifically in perisomatic synapses. AZD7325 demonstrates stronger effects on IPSCs in the seizure resistant mouse strain, consistent with higher α2 subunit expression. AZD7325 demonstrates seizure protective effects in Scn1a+/- mice without apparent sedative effects in vivo. ABSTRACT: GABAA receptor potentiators are commonly used for the treatment of epilepsy, but it is not clear whether targeting distinct GABAA receptor subtypes will have disproportionate benefits over adverse effects. Here we demonstrate that the α2 /α3 selective positive allosteric modulator (PAM) AZD7325 preferentially potentiates hippocampal inhibitory responses at synapses proximal to the soma of CA1 neurons. The effect of AZD7325 on synaptic responses was more prominent in mice on the 129S6/SvEvTac background strain, which have been demonstrated to be seizure resistant in the model of Dravet syndrome (Scn1a+/- ), and in which the α2 GABAA receptor subunits are expressed at higher levels relative to in the seizure prone C57BL/6J background strain. Consistent with this, treatment of Scn1a+/- mice with AZD7325 elevated the temperature threshold for hyperthermia-induced seizures without apparent sedative effects. Our results in a model system indicate that selectively targeting α2 is a potential therapeutic option for Dravet syndrome.

Fine Mapping of a Dravet Syndrome Modifier Locus on Mouse Chromosome 5 and Candidate Gene Analysis by RNA-Seq.

A substantial number of mutations have been identified in voltage-gated sodium channel genes that result in various forms of human epilepsy. SCN1A mutations result in a spectrum of severity ranging from mild febrile seizures to Dravet syndrome, an infant-onset epileptic encephalopathy. Dravet syndrome patients experience multiple seizures types that are often refractory to treatment, developmental delays, and elevated risk for SUDEP. The same sodium channel mutation can produce epilepsy phenotypes of varying clinical severity. This suggests that other factors, including genetic, modify the primary mutation and change disease severity. Mouse models provide a useful tool in studying the genetic basis of epilepsy. The mouse strain background can alter phenotype severity, supporting a contribution of genetic modifiers in epilepsy. The Scn1a+/- mouse model has a strain-dependent epilepsy phenotype. Scn1a+/- mice on the 129S6/SvEvTac (129) strain have a normal phenotype and lifespan, while [129xC57BL/6J]F1-Scn1a+/- mice experience spontaneous seizures, hyperthermia-induced seizures and high rates of premature death. We hypothesize the phenotypic differences are due to strain-specific genetic modifiers that influence expressivity of the Scn1a+/- phenotype. Low resolution mapping of Scn1a+/- identified several Dravet syndrome modifier (Dsm) loci responsible for the strain-dependent difference in survival. One locus of interest, Dsm1 located on chromosome 5, was fine mapped to a 9 Mb region using interval specific congenics. RNA-Seq was then utilized to identify candidate modifier genes within this narrowed region. Three genes with significant total gene expression differences between 129S6/SvEvTac and [129xC57BL/6J]F1 were identified, including the GABAA receptor subunit, Gabra2. Further analysis of Gabra2 demonstrated allele-specific expression. Pharmological manipulation by clobazam, a common anticonvulsant with preferential affinity for the GABRA2 receptor, revealed dose-dependent protection against hyperthermia-induced seizures in Scn1a+/- mice. These findings support Gabra2 as a genetic modifier of the Scn1a+/- mouse model of Dravet syndrome.

The novel sodium channel modulator GS-458967 (GS967) is an effective treatment in a mouse model of SCN8A encephalopathy.

OBJECTIVE: De novo mutations of SCN8A, encoding the voltage-gated sodium channel NaV 1.6, have been associated with a severe infant onset epileptic encephalopathy. Individuals with SCN8A encephalopathy have a mean age of seizure onset of 4-5 months, with multiple seizure types that are often refractory to treatment with available drugs. Anecdotal reports suggest that high-dose phenytoin is effective for some patients, but there are associated adverse effects and potential for toxicity. Functional characterization of several SCN8A encephalopathy variants has shown that elevated persistent sodium current is one of several common biophysical defects. Therefore, specifically targeting elevated persistent current may be a useful therapeutic strategy in some cases. METHODS: The novel sodium channel modulator GS967 has greater preference for persistent as opposed to peak current and nearly 10-fold greater potency than phenytoin. We evaluated the therapeutic effect of GS967 in the Scn8aN1768D/+ mouse model carrying an SCN8A patient mutation that results in elevated persistent sodium current. We also performed patch clamp recordings to assess the effect of GS967 on peak and persistent sodium current and excitability in hippocampal neurons from Scn8aN1768D/+ mice. RESULTS: GS967 potently blocked persistent sodium current without affecting peak current, normalized action potential morphology, and attenuated excitability in neurons from heterozygous Scn8aN1768D/+ mice. Acute treatment with GS967 provided dose-dependent protection against maximal electroshock-induced seizures in Scn8aN1768D/+ and wild-type mice. Chronic treatment of Scn8aN1768D/+ mice with GS967 resulted in lower seizure burden and complete protection from seizure-associated lethality observed in untreated Scn8aN1768D/+ mice. Protection was achieved at a chronic dose that did not cause overt behavioral toxicity or sedation. SIGNIFICANCE: Persistent sodium current modulators like GS967 may be an effective precision targeting strategy for SCN8A encephalopathy and other functionally similar channelopathies when elevated persistent sodium current is the primary dysfunction.

Cacna1g is a genetic modifier of epilepsy in a mouse model of Dravet syndrome.

Dravet syndrome, an early onset epileptic encephalopathy, is most often caused by de novo mutation of the neuronal voltage-gated sodium channel gene SCN1A. Mouse models with deletion of Scn1a recapitulate Dravet syndrome phenotypes, including spontaneous generalized tonic-clonic seizures, susceptibility to seizures induced by elevated body temperature, and elevated risk of sudden unexpected death in epilepsy. Importantly, the epilepsy phenotype of Dravet mouse models is highly strain-dependent, suggesting a strong influence of genetic modifiers. We previously identified Cacna1g, encoding the Cav3.1 subunit of the T-type calcium channel family, as an epilepsy modifier in the Scn2aQ54 transgenic epilepsy mouse model. In this study, we asked whether transgenic alteration of Cacna1g expression modifies severity of the Scn1a+/- Dravet phenotype. Scn1a+/- mice with decreased Cacna1g expression showed partial amelioration of disease phenotypes with improved survival and reduced spontaneous seizure frequency. However, reduced Cacna1g expression did not alter susceptibility to hyperthermia-induced seizures. Transgenic elevation of Cacna1g expression had no effect on the Scn1a+/- epilepsy phenotype. These results provide support for Cacna1g as a genetic modifier in a mouse model of Dravet syndrome and suggest that Cav3.1 may be a potential molecular target for therapeutic intervention in patients.

Cacna1g is a genetic modifier of epilepsy caused by mutation of voltage-gated sodium channel Scn2a.

More than 1,200 mutations in neuronal voltage-gated sodium channel (VGSC) genes have been identified in patients with several epilepsy syndromes. A common feature of genetic epilepsies is variable expressivity among individuals with the same mutation. The Scn2a(Q54) transgenic mouse model has a mutation in Scn2a that results in spontaneous epilepsy. Scn2a(Q54) phenotype severity varies depending on the genetic strain background, making it a useful model for identifying and characterizing epilepsy modifier genes. Scn2a(Q54) mice on the [C57BL/6JxSJL/J]F1 background exhibit earlier seizure onset, elevated spontaneous seizure frequency, and decreased survival compared to Scn2a(Q54) mice congenic on the C57BL/6J strain. Genetic mapping and RNA-Seq analysis identified Cacna1g as a candidate modifier gene at the Moe1 locus, which influences Scn2a(Q54) phenotype severity. In this study, we evaluated the modifier potential of Cacna1g, encoding the Cav3.1 voltage-gated calcium channel, by testing whether transgenic alteration of Cacna1g expression modifies severity of the Scn2a(Q54) seizure phenotype. Scn2a(Q54) mice exhibited increased spontaneous seizure frequency with elevated Cacna1g expression and decreased seizure frequency with decreased Cacna1g expression. These results provide support for Cacna1g as an epilepsy modifier gene and suggest that modulation of Cav3.1 may be an effective therapeutic strategy.

Antiepileptic activity of preferential inhibitors of persistent sodium current.

OBJECTIVE: Evidence from basic neurophysiology and molecular genetics has implicated persistent sodium current conducted by voltage-gated sodium (NaV ) channels as a contributor to the pathogenesis of epilepsy. Many antiepileptic drugs target NaV channels and modulate neuronal excitability, mainly by a use-dependent block of transient sodium current, although suppression of persistent current may also contribute to the efficacy of these drugs. We hypothesized that a drug or compound capable of preferential inhibition of persistent sodium current would have antiepileptic activity. METHODS: We examined the antiepileptic activity of two selective persistent sodium current blockers ranolazine, a U.S. Food and Drug Administration (FDA)-approved drug for treatment of angina pectoris, and GS967, a novel compound with more potent effects on persistent current, in the epileptic Scn2a(Q54) mouse model. We also examined the effect of GS967 in the maximal electroshock model and evaluated effects of the compound on neuronal excitability, propensity for hilar neuron loss, development of mossy fiber sprouting, and survival of Scn2a(Q54) mice. RESULTS: We found that ranolazine was capable of reducing seizure frequency by approximately 50% in Scn2a(Q54) mice. The more potent persistent current blocker GS967 reduced seizure frequency by >90% in Scn2a(Q54) mice and protected against induced seizures in the maximal electroshock model. GS967 greatly attenuated abnormal spontaneous action potential firing in pyramidal neurons acutely isolated from Scn2a(Q54) mice. In addition to seizure suppression in vivo, GS967 treatment greatly improved the survival of Scn2a(Q54) mice, prevented hilar neuron loss, and suppressed the development of hippocampal mossy fiber sprouting. SIGNIFICANCE: Our findings indicate that the selective persistent sodium current blocker GS967 has potent antiepileptic activity and that this compound could inform development of new agents.

Fine Mapping and Candidate Gene Analysis of Dravet Syndrome Modifier Loci on Mouse Chromosomes 7 and 8.

Dravet syndrome is a developmental and epileptic encephalopathy (DEE) characterized by intractable seizures, comorbidities related to developmental, cognitive, and motor delays, and a high mortality burden due to sudden unexpected death in epilepsy (SUDEP). Most Dravet syndrome cases are attributed to SCN1A haploinsufficiency, with genetic modifiers and environmental factors influencing disease severity. Mouse models with heterozygous deletion of Scn1a recapitulate key features of Dravet syndrome, including seizures and premature mortality; however, severity varies depending on genetic background. Here, we refined two Dravet survival modifier (Dsm) loci, Dsm2 on chromosome 7 and Dsm3 on chromosome 8, using interval-specific congenic (ISC) mapping. Dsm2 was complex and encompassed at least two separate loci, while Dsm3 was refined to a single locus. Candidate modifier genes within these refined loci were prioritized based on brain expression, strain-dependent differences, and biological relevance to seizures or epilepsy. High priority candidate genes for Dsm2 include Nav2, Ptpn5, Ldha, Dbx1, Prmt3 and Slc6a5, while Dsm3 has a single high priority candidate, Psd3. This study underscores the complex genetic architecture underlying Dravet syndrome and provides insights into potential modifier genes that could influence disease severity and serve as novel therapeutic targets.

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.

Sex and Gene Influence Arrhythmia Susceptibility in Murine Models of Calmodulinopathy.

BACKGROUND: Pathogenic variants in genes encoding CaM (calmodulin) are associated with a life-threatening ventricular arrhythmia syndrome (calmodulinopathy). The in vivo consequences of CaM variants have not been studied extensively and there is incomplete understanding of the genotype-phenotype relationship for recurrent variants. We investigated effects of different factors on calmodulinopathy phenotypes using 2 mouse models with a recurrent pathogenic variant (N98S) in Calm1 or Calm2. METHODS: Genetically engineered mice with heterozygous N98S pathogenic variants in Calm1 or Calm2 were generated. Differences between the sexes and affected genes were assessed using multiple physiological assays at the cellular and whole animal levels. Statistical significance among groups was evaluated using 1-way ANOVA or the Kruskal-Wallis test when data were not normally distributed. RESULTS: Calm1N98S/+ (Calm1S/+) or Calm2N98S/+ (Calm2S/+) mice exhibited sinus bradycardia and were more susceptible to arrhythmias after exposure to epinephrine and caffeine. Male Calm1S/+ mice had the most severe arrhythmia phenotype with evidence of early embryonic lethality, greater susceptibility for arrhythmic events, frequent premature beats, corrected QT prolongation, and more heart rate variability after epinephrine and caffeine than females with the same genotype. Calm2 S/+ mice exhibited a less severe phenotype, with female Calm2 S/+ mice having the least severe arrhythmia susceptibility. Flecainide was not effective in preventing arrhythmias in heterozygous CaM-N98S mice. Intracellular Ca2+ transients observed in isolated ventricular cardiomyocytes from male heterozygous CaM-N98S mice had lower peak amplitudes and slower sarcoplasmic reticulum Ca2+ release following in vitro exposure to epinephrine and caffeine, which were not observed in cardiomyocytes from heterozygous female CaM-N98S mice. CONCLUSIONS: We report heterogeneity in arrhythmia susceptibility and cardiomyocyte Ca2+ dynamics among male and female mice heterozygous for a recurrent pathogenic variant in Calm1 or Calm2, illustrating a complex calmodulinopathy phenotype in vivo. Further investigation of sex and genetic differences may help identify the molecular basis for this heterogeneity.

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.

Heterozygous deletion of Gpr55 does not affect a hyperthermia-induced seizure, spontaneous seizures or survival in the Scn1a+/- mouse model of Dravet syndrome.

A purified preparation of cannabidiol (CBD), a cannabis constituent, has been approved for the treatment of intractable childhood epilepsies such as Dravet syndrome. Extensive pharmacological characterization of CBD shows activity at numerous molecular targets but its anticonvulsant mechanism(s) of action is yet to be delineated. Many suggest that the anticonvulsant action of CBD is the result of G protein-coupled receptor 55 (GPR55) inhibition. Here we assessed whether Gpr55 contributes to the strain-dependent seizure phenotypes of the Scn1a+/- mouse model of Dravet syndrome. The Scn1a+/- mice on a 129S6/SvEvTac (129) genetic background have no overt phenotype, while those on a [129 x C57BL/6J] F1 background exhibit a severe phenotype that includes hyperthermia-induced seizures, spontaneous seizures and reduced survival. We observed greater Gpr55 transcript expression in the cortex and hippocampus of mice on the seizure-susceptible F1 background compared to those on the seizure-resistant 129 genetic background, suggesting that Gpr55 might be a genetic modifier of Scn1a+/- mice. We examined the effect of heterozygous genetic deletion of Gpr55 and pharmacological inhibition of GPR55 on the seizure phenotypes of F1.Scn1a+/- mice. Heterozygous Gpr55 deletion and inhibition of GPR55 with CID2921524 did not affect the temperature threshold of a thermally-induced seizure in F1.Scn1a+/- mice. Neither was there an effect of heterozygous Gpr55 deletion observed on spontaneous seizure frequency or survival of F1.Scn1a+/- mice. Our results suggest that GPR55 antagonism may not be a suitable anticonvulsant target for Dravet syndrome drug development programs, although future research is needed to provide more definitive conclusions.

Rescue of neuropsychiatric phenotypes in a mouse model of 16p11.2 duplication syndrome by genetic correction of an epilepsy network hub.

Neuropsychiatric disorders (NPDs) are frequently co-morbid with epilepsy, but the biological basis of shared risk remains poorly understood. The 16p11.2 duplication is a copy number variant that confers risk for diverse NPDs including autism spectrum disorder, schizophrenia, intellectual disability and epilepsy. We used a mouse model of the 16p11.2 duplication (16p11.2dup/+) to uncover molecular and circuit properties associated with this broad phenotypic spectrum, and examined genes within the locus capable of phenotype reversal. Quantitative proteomics revealed alterations to synaptic networks and products of NPD risk genes. We identified an epilepsy-associated subnetwork that was dysregulated in 16p11.2dup/+ mice and altered in brain tissue from individuals with NPDs. Cortical circuits from 16p11.2dup/+ mice exhibited hypersynchronous activity and enhanced network glutamate release, which increased susceptibility to seizures. Using gene co-expression and interactome analysis, we show that PRRT2 is a major hub in the epilepsy subnetwork. Remarkably, correcting Prrt2 copy number rescued aberrant circuit properties, seizure susceptibility and social deficits in 16p11.2dup/+ mice. We show that proteomics and network biology can identify important disease hubs in multigenic disorders, and reveal mechanisms relevant to the complex symptomatology of 16p11.2 duplication carriers.