Elevating Black fathers' experiences in the National Fatherhood Initiative: Strengths and areas for improvement of 24/7 Dad® curriculum and case management services.

The Healthy Marriage and Responsible Fatherhood grant program is one of the most robust mechanisms supporting fatherhood programming in the United States, with the gold standard of care including fatherhood curriculum (e.g., 24/7 Dad®) and case management services. Despite the fact that fatherhood in the United States is not a monolith and Black fathers make up over 40% of those served through Responsible Fatherhood (RF) program, little research explicitly centers the lived experiences of low-income Black fathers. The current study used focus groups with fathers (n = 23) and staff (n = 8) enrolled in a RF grantee program. Thematic analysis was used to determine programmatic mechanisms supporting these fathers' success and areas for improvement. Our findings highlight that the creation of a "safe space" by facilitators and the power of the group dynamic were essential elements for fathers' success overall. We also identified numerous programmatic mechanisms supporting Black fathers' success specific to fathering, healthy relationships, and economic stability. Finally, we highlighted important limitations that may impede the success of low-income, Black fathers related to the 24/7 Dad® curriculum, engagement efforts, and economic stability, and discuss related practice, policy, and research recommendations.

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 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.

A high seizure burden increases several prostaglandin species in the hippocampus of a Scn1a+/- mouse model of Dravet syndrome.

Dravet syndrome is an intractable epilepsy with a high seizure burden that is resistant to current anti-seizure medications. There is evidence that neuroinflammation plays a role in epilepsy and seizures, however few studies have specifically examined neuroinflammation in Dravet syndrome under conditions of a higher seizure burden. Here we used an established genetic mouse model of Dravet syndrome (Scn1a+/- mice), to examine whether a higher seizure burden impacts the number and morphology of microglia in the hippocampus. Moreover, we examined whether a high seizure burden influences classical inflammatory mediators in this brain region. Scn1a+/- mice with a high seizure burden induced by thermal priming displayed a localised reduction in microglial cell density in the granule cell layer and subgranular zone of the dentate gyrus, regions important to postnatal neurogenesis. However, microglial cell number and morphology remained unchanged in other hippocampal subfields. The high seizure burden in Scn1a+/- mice did not affect hippocampal mRNA expression of classical inflammatory mediators such as interleukin 1ß and tumour necrosis factor α, but increased cyclooxygenase 2 (COX-2) expression. We then quantified hippocampal levels of prostanoids that arise from COX-2 mediated metabolism of fatty acids and found that Scn1a+/- mice with a high seizure burden displayed increased hippocampal concentrations of numerous prostaglandins, notably PGF2α, PGE2, PGD2, and 6-K-PGF1A, compared to Scn1a+/- mice with a low seizure burden. In conclusion, a high seizure burden increased hippocampal concentrations of various prostaglandin mediators in a mouse model of Dravet syndrome. Future studies could interrogate the prostaglandin pathways to further better understand their role in the pathophysiology of Dravet syndrome.

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.

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.

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.

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.

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.

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.

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.

Cell-Selective Adeno-Associated Virus-Mediated SCN1A Gene Regulation Therapy Rescues Mortality and Seizure Phenotypes in a Dravet Syndrome Mouse Model and Is Well Tolerated in Nonhuman Primates.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy caused by monoallelic loss-of-function variants in the SCN1A gene. SCN1A encodes for the alpha subunit of the voltage-gated type I sodium channel (NaV1.1), the primary voltage-gated sodium channel responsible for generation of action potentials in GABAergic inhibitory interneurons. In these studies, we tested the efficacy of an adeno-associated virus serotype 9 (AAV9) SCN1A gene regulation therapy, AAV9-REGABA-eTFSCN1A, designed to target transgene expression to GABAergic inhibitory neurons and reduce off-target expression within excitatory cells, in the Scn1a+/- mouse model of DS. Biodistribution and preliminary safety were evaluated in nonhuman primates (NHPs). AAV9-REGABA-eTFSCN1A was engineered to upregulate SCN1A expression levels within GABAergic inhibitory interneurons to correct the underlying haploinsufficiency and circuit dysfunction. A single bilateral intracerebroventricular (ICV) injection of AAV9-REGABA-eTFSCN1A in Scn1a+/- postnatal day 1 mice led to increased SCN1A mRNA transcripts, specifically within GABAergic inhibitory interneurons, and NaV1.1 protein levels in the brain. This was associated with a significant decrease in the occurrence of spontaneous and hyperthermia-induced seizures, and prolonged survival for over a year. In NHPs, delivery of AAV9-REGABA-eTFSCN1A by unilateral ICV injection led to widespread vector biodistribution and transgene expression throughout the brain, including key structures involved in epilepsy and cognitive behaviors, such as hippocampus and cortex. AAV9-REGABA-eTFSCN1A was well tolerated, with no adverse events during administration, no detectable changes in clinical observations, no adverse findings in histopathology, and no dorsal root ganglion-related toxicity. Our results support the clinical development of AAV9-REGABA-eTFSCN1A (ETX101) as an effective and targeted disease-modifying approach to SCN1A+ DS.

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.

KNa1.1 gain-of-function preferentially dampens excitability of murine parvalbumin-positive interneurons.

KCNT1 encodes the sodium-activated potassium channel KNa1.1, expressed preferentially in the frontal cortex, hippocampus, cerebellum, and brainstem. Pathogenic missense variants in KCNT1 are associated with intractable epilepsy, namely epilepsy of infancy with migrating focal seizures (EIMFS), and sleep-related hypermotor epilepsy (SHE). In vitro studies of pathogenic KCNT1 variants support predominantly a gain-of-function molecular mechanism, but how these variants behave in a neuron or ultimately drive formation of an epileptogenic circuit is an important and timely question. Using CRISPR/Cas9 gene editing, we introduced a gain-of-function variant into the endogenous mouse Kcnt1 gene. Compared to wild-type (WT) littermates, heterozygous and homozygous knock-in mice displayed greater seizure susceptibility to the chemoconvulsants kainate and pentylenetetrazole (PTZ), but not to flurothyl. Using acute slice electrophysiology in heterozygous and homozygous Kcnt1 knock-in and WT littermates, we demonstrated that CA1 hippocampal pyramidal neurons exhibit greater amplitude of miniature inhibitory postsynaptic currents in mutant mice with no difference in frequency, suggesting greater inhibitory tone associated with the Kcnt1 mutation. To address alterations in GABAergic signaling, we bred Kcnt1 knock-in mice to a parvalbumin-tdTomato reporter line, and found that parvalbumin-expressing (PV+) interneurons failed to fire repetitively with large amplitude current injections and were more prone to depolarization block. These alterations in firing can be recapitulated by direct application of the KNa1.1 channel activator loxapine in WT but are occluded in knock-in littermates, supporting a direct channel gain-of-function mechanism. Taken together, these results suggest that KNa1.1 gain-of-function dampens interneuron excitability to a greater extent than it impacts pyramidal neuron excitability, driving seizure susceptibility in a mouse model of KCNT1-associated epilepsy.

The endocannabinoid system impacts seizures in a mouse model of Dravet syndrome.

Dravet syndrome is a catastrophic childhood epilepsy with multiple seizure types that are refractory to treatment. The endocannabinoid system regulates neuronal excitability so a deficit in endocannabinoid signaling could lead to hyperexcitability and seizures. Thus, we sought to determine whether a deficiency in the endocannabinoid system might contribute to seizure phenotypes in a mouse model of Dravet syndrome and whether enhancing endocannabinoid tone is anticonvulsant. Scn1a+/- mice model the clinical features of Dravet syndrome: hyperthermia-induced seizures, spontaneous seizures and reduced survival. We examined whether Scn1a+/- mice exhibit deficits in the endocannabinoid system by measuring brain cannabinoid receptor expression and endocannabinoid concentrations. Next, we determined whether pharmacologically enhanced endocannabinoid tone was anticonvulsant in Scn1a+/- mice. We used GAT229, a positive allosteric modulator of the cannabinoid (CB1) receptor, and ABX-1431, a compound that inhibits the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). The Scn1a+/- phenotype is strain-dependent with mice on a 129S6/SvEvTac (129) genetic background having no overt phenotype and those on an F1 (129S6/SvEvTac x C57BL/6J) background exhibiting a severe epilepsy phenotype. We observed lower brain cannabinoid CB1 receptor expression in the seizure-susceptible F1 compared to seizure-resistant 129 strain, suggesting an endocannabinoid deficiency might contribute to seizure susceptibility. GAT229 and ABX-1431 were anticonvulsant against hyperthermia-induced seizures. However, subchronic ABX1431 treatment increased spontaneous seizure frequency despite reducing seizure severity. Cnr1 is a putative genetic modifier of epilepsy in the Scn1a+/- mouse model of Dravet syndrome. Compounds that increase endocannabinoid tone could be developed as novel treatments 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.

Cannabigerolic acid, a major biosynthetic precursor molecule in cannabis, exhibits divergent effects on seizures in mouse models of epilepsy.

BACKGROUND AND PURPOSE: Cannabis has been used to treat epilepsy for millennia, with such use validated by regulatory approval of cannabidiol (CBD) for Dravet syndrome. Unregulated artisanal cannabis-based products used to treat children with intractable epilepsies often contain relatively low doses of CBD but are enriched in other phytocannabinoids. This raises the possibility that other cannabis constituents might have anticonvulsant properties. EXPERIMENTAL APPROACH: We used the Scn1a+/- mouse model of Dravet syndrome to investigate the cannabis plant for phytocannabinoids with anticonvulsant effects against hyperthermia-induced seizures. The most promising, cannabigerolic acid (CBGA), was further examined against spontaneous seizures and survival in Scn1a+/- mice and in electroshock seizure models. Pharmacological effects of CBGA were surveyed across multiple drug targets. KEY RESULTS: The initial screen identified three phytocannabinoids with novel anticonvulsant properties: CBGA, cannabidivarinic acid (CBDVA) and cannabigerovarinic acid (CBGVA). CBGA was most potent and potentiated the anticonvulsant effects of clobazam against hyperthermia-induced and spontaneous seizures, and was anticonvulsant in the MES threshold test. However, CBGA was proconvulsant in the 6-Hz threshold test and a high dose increased spontaneous seizure frequency in Scn1a+/- mice. CBGA was found to interact with numerous epilepsy-relevant targets including GPR55, TRPV1 channels and GABAA receptors. CONCLUSION AND IMPLICATIONS: These results suggest that CBGA, CBDVA and CBGVA may contribute to the effects of cannabis-based products in childhood epilepsy. Although these phytocannabinoids have anticonvulsant potential and could be lead compounds for drug development programmes, several liabilities would need to be overcome before CBD is superseded by another in this class.

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.