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Objective: Phenotypic heterogeneity presents challenges in providing clinical care to patients with pathogenic SCN8A variants, which underly a wide disease spectrum ranging from neurodevelopmental delays without seizures to a continuum of mild to severe developmental and epileptic encephalopathies (DEEs). An important unanswered question is whether there are clinically important subgroups within this wide spectrum. Using both supervised and unsupervised machine learning (ML) approaches, we previously found statistical support for two and three subgroups associated with loss- and gain- of- function vari-ants, respectively. Here, we test the hypothesis that the unsupervised subgroups (U1-U3) are distinguished by differential contributions of developmental and epileptic components., Methods: We predicted that patients in the U1 and U2 subgroups would differ in timing of developmental delay and seizure onset, with earlier and concurrent onset of both features for the U3 subgroup. Standard statistical procedures were used to test these predictions, as well as to investigate clinically relevant associations among all five subgroups., Results: Two-population proportion and Kruskal-Wallis tests supported the hypothesis of a reversed order of developmental delay and seizure onset for patients in U1 and U2, and nearly synchronous developmental delay/seizure onset for the U3 (termed DEE) subgroup. Association testing identified subgroup variation in treatment response, frequency of initial seizure type, and comorbidities, as well as different median ages of developmental delay onset for all five subgroups., Significance: Unsupervised ML approaches discern differential developmental and epileptic components among patients with SCN8A-related epilepsy. Patients in U1 (termed developmental encephalopathy) typically gain seizure control yet rarely experience improvements in development, whereas those in U2 (termed epileptic encephalopathy) have fewer if any developmental impairments despite difficulty in achieving seizure control. This understanding improves prognosis and clinical management and provides a framework to discover mechanisms underlying variability in clinical outcome of patients with SCN8A-related disorders., (© 2024 International League Against Epilepsy.)

Objective: Developmental and epileptic encephalopathies (DEEs) can result from dominant, gain of function variants of neuronal ion channels. More than 450 de novo missense variants of the sodium channel gene SCN8A have been identified in individuals with DEE., Methods: We studied a mouse model carrying the patient Scn8a variant p.Asn1768Asp. An AAV-PHP.eB virus carrying an allele-specific single guide RNA (sgRNA) was administered by intracerebroventricular injection. Cas9 was provided by an inherited transgene., Results: Allele-specific disruption of the reading frame of the pathogenic transcript generated out-of-frame indels in 1/4 to 1/3 of transcripts throughout the brain. This editing efficiency was sufficient to rescue lethality and seizures. Neuronal hyperexcitability was reduced in cells expressing the virus., Interpretation: The data demonstrate efficient allele-specific editing of a dominant missense variant and support the feasibility of allele-specific therapy for DEE epilepsy. ANN NEUROL 2024;96:958-969., (© 2024 The Author(s). Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

SCN8A developmental and epileptic encephalopathy (DEE) is a severe epilepsy syndrome resulting from mutations in the voltage-gated sodium channel Nav1.6, encoded by the gene SCN8A. Nav1.6 is expressed in excitatory and inhibitory neurons, yet previous studies primarily focus on how SCN8A mutations affect excitatory neurons, with limited studies on the importance of inhibitory interneurons. Parvalbumin (PV) interneurons are a prominent inhibitory interneuron subtype that expresses Nav1.6. To assess PV interneuron function within SCN8A DEE, we used 2 mouse models harboring patient-derived SCN8A gain-of-function variants, Scn8aD/+, where the SCN8A variant N1768D is expressed globally, and Scn8aW/+-PV, where the SCN8A variant R1872W is selectively expressed in PV interneurons. Expression of the R1872W SCN8A variant selectively in PV interneurons led to development of spontaneous seizures and seizure-induced death. Electrophysiology studies showed that Scn8aD/+ and Scn8aW/+-PV interneurons were susceptible to depolarization block and exhibited increased persistent sodium current. Evaluation of synaptic connections between PV interneurons and pyramidal cells showed synaptic transmission deficits in Scn8aD/+ and Scn8aW/+-PV interneurons. Together, our findings indicate that PV interneuron failure via depolarization block along with inhibitory synaptic impairment likely elicits an overall inhibitory reduction in SCN8A DEE, leading to unchecked excitation and ultimately resulting in seizures and seizure-induced death.

ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide., (© 2024 COLLAÇO et al.)

Background and Purpose: Inhibitors of voltage-gated sodium channels (Na V s) are important anti-epileptic drugs, but the contribution of specific channel isoforms is unknown since available inhibitors are non-selective. We aimed to create novel, isoform selective inhibitors of Na v channels as a means of informing the development of improved antiseizure drugs., Experimental Approach: We created a series of compounds with diverse selectivity profiles enabling block of Na V 1.6 alone or together with Na V 1.2. These novel Na V inhibitors were evaluated for their ability to inhibit electrically evoked seizures in mice with a heterozygous gain-of-function mutation (N1768D/+) in Scn8a (encoding Na V 1.6) and in wild-type mice., Key Results: Pharmacologic inhibition of Na V 1.6 in Scn8a N1768D/+ mice prevented seizures evoked by a 6-Hz shock. Inhibitors were also effective in a direct current maximal electroshock seizure assay in wild-type mice. Na V 1.6 inhibition correlated with efficacy in both models, even without inhibition of other CNS Na V isoforms., Conclusions and Implications: Our data suggest Na V 1.6 inhibition is a driver of efficacy for Na V inhibitor anti-seizure medicines. Sparing the Na V 1.1 channels of inhibitory interneurons did not compromise efficacy. Selective Na V 1.6 inhibitors may provide targeted therapies for human Scn8a developmental and epileptic encephalopathies and improved treatments for idiopathic epilepsies., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Blockade of Angiotensin type 1 receptor (AT1R) has potential therapeutic utility in the treatment of numerous detrimental consequences of epileptogenesis, including oxidative stress, neuroinflammation, and blood-brain barrier (BBB) dysfunction. We have recently shown that many of these pathological processes play a critical role in seizure onset and propagation in the Scn8a-N1768D mouse model. Here we investigate the efficacy and potential mechanism(s) of action of candesartan (CND), an FDA-approved angiotensin receptor blocker (ARB) indicated for hypertension, in improving outcomes in this model of pediatric epilepsy. We compared length of lifespan, seizure frequency, and BBB permeability in juvenile (D/D) and adult (D/+) mice treated with CND at times after seizure onset. We performed RNAseq on hippocampal tissue to quantify differences in genome-wide patterns of transcript abundance and inferred beneficial and detrimental effects of canonical pathways identified by enrichment methods in untreated and treated mice. Our results demonstrate that treatment with CND gives rise to increased survival, longer periods of seizure freedom, and diminished BBB permeability. CND treatment also partially reversed or 'normalized' disease-induced genome-wide gene expression profiles associated with inhibition of NF-κB, TNFα, IL-6, and TGF-β signaling in juvenile and adult mice. Pathway analyses reveal that efficacy of CND is due to its known dual mechanism of action as both an AT1R antagonist and a PPARγ agonist. The robust effectiveness of CND across ages, sexes and mouse strains is a positive indication for its translation to humans and its suitability of use for clinical trials in children with SCN8A epilepsy., (© 2024 The Author(s).)

Gain-of-function mutations in SCN8A cause developmental and epileptic encephalopathy (DEE), a disorder characterized by early-onset refractory seizures, deficits in motor and intellectual functions, and increased risk of sudden unexpected death in epilepsy. Altered activity of neurons in the corticohippocampal circuit has been reported in mouse models of DEE. We examined the effect of chronic seizures on gene expression in the hippocampus by single-nucleus RNA sequencing in mice expressing the patient mutation SCN8A-p.Asn1768Asp (N1768D). One hundred and eighty four differentially expressed genes were identified in dentate gyrus granule cells, many more than in other cell types. Electrophysiological recording from dentate gyrus granule cells demonstrated an elevated firing rate. Targeted reduction of Scn8a expression in the dentate gyrus by viral delivery of an shRNA resulted in doubling of median survival time from 4 months to 8 months, whereas delivery of shRNA to the CA1 and CA3 regions did not result in lengthened survival. These data indicate that granule cells of the dentate gyrus are a specific locus of pathology in SCN8A-DEE., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Patients affected by glioma frequently experience epileptic discharges; however, the causes of brain tumor-related epilepsy (BTRE) are still not completely understood. We investigated the mechanisms underlying BTRE by analyzing the effects of exosomes released by U87 glioma cells and by patient-derived glioma cells. Rat hippocampal neurons incubated for 24 hours with these exosomes exhibited increased spontaneous firing, while their resting membrane potential shifted positively by 10-15 mV. Voltage clamp recordings demonstrated that the activation of the Na+ current shifted toward more hyperpolarized voltages by 10-15 mV. To understand the factors inducing hyperexcitability, we focused on exosomal cytokines. Western blot and ELISAs showed that TNF-α was present inside glioma-derived exosomes. Remarkably, incubation with TNF-α fully mimicked the phenotype induced by exosomes, with neurons firing continuously, while their resting membrane potential shifted positively. Real-time PCR revealed that both exosomes and TNF-α induced overexpression of the voltage-gated Na+ channel Nav1.6, a low-threshold Na+ channel responsible for hyperexcitability. When neurons were preincubated with infliximab, a specific TNF-α inhibitor, the hyperexcitability induced by exosomes and TNF-α was drastically reduced. We propose that infliximab, an FDA-approved drug to treat rheumatoid arthritis, could ameliorate the conditions of glioma patients with BTRE.

Objective: We aimed to develop consensus for diagnosis/management of SCN8A-related disorders. Utilizing a modified Delphi process, a global cohort of experienced clinicians and caregivers provided input on diagnosis, phenotypes, treatment, and management of SCN8A-related disorders., Methods: A Core Panel (13 clinicians, one researcher, six caregivers), divided into three subgroups (diagnosis/phenotypes, treatment, comorbidities/prognosis), performed a literature review and developed questions for the modified Delphi process. Twenty-eight expert clinicians, one researcher, and 13 caregivers from 16 countries participated in the subsequent three survey rounds. We defined consensus as follows: strong consensus, ≥80% fully agree; moderate consensus, ≥80% fully/partially agree, <10% disagree; and modest consensus, 67%-79% fully/partially agree, <10% disagree., Results: Early diagnosis is important for long-term clinical outcomes in SCN8A-related disorders. There are five phenotypes: three with early seizure onset (severe developmental and epileptic encephalopathy [DEE], mild/moderate DEE, self-limited (familial) infantile epilepsy [SeL(F)IE]) and two with later/no seizure onset (neurodevelopmental delay with generalized epilepsy [NDDwGE], NDD without epilepsy [NDDwoE]). Caregivers represented six patients with severe DEE, five mild/moderate DEE, one NDDwGE, and one NDDwoE. Phenotypes vary by age at seizures/developmental delay onset, seizure type, electroencephalographic/magnetic resonance imaging findings, and first-line treatment. Gain of function (GOF) versus loss of function (LOF) is valuable for informing treatment. Sodium channel blockers are optimal first-line treatment for GOF, severe DEE, mild/moderate DEE, and SeL(F)IE; levetiracetam is relatively contraindicated in GOF patients. First-line treatment for NDDwGE is valproate, ethosuximide, or lamotrigine; sodium channel blockers are relatively contraindicated in LOF patients., Significance: This is the first-ever global consensus for the diagnosis and treatment of SCN8A-related disorders. This consensus will reduce knowledge gaps in disease recognition and inform preferred treatment across this heterogeneous disorder. Consensus of this type allows more clinicians to provide evidence-based care and empowers SCN8A families to advocate for their children., (© 2024 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

SCN8A variants cause a spectrum of epilepsy phenotypes ranging from self-limited infantile epilepsy (SeLIE) to developmental and epileptic encephalopathy. SeLIE is an infantile onset focal epilepsy, occurring in developmentally normal infants, which often resolves by 3 years. Our aim was to ascertain when epilepsy resolves in SCN8A-SeLIE. We identified unpublished individuals with SCN8A-SeLIE and performed detailed phenotyping. Literature was searched for published SCN8A-SeLIE cases. Nine unpublished individuals from four families were identified (age at study = 3.5-66 years). Six had their last seizure after 3 years (range = 4-21 years); although drug-responsive and despite multiple weaning attempts (1-5), five of six remain on antiseizure medications (carbamazepine, n = 3; lamotrigine, n = 2). We identified 29 published individuals with SCN8A-SeLIE who had data on seizure progression. Of the 22 individuals aged at least 10 years, reported here or in the literature, nine of 22 (41%) had seizure offset prior to 3 years, five of 22 (23%) had seizure offset between 3 and 10 years, and eight of 22 (36%) had seizures after 10 years. Our data highlight that more than half of individuals with SCN8A-SeLIE continue to have seizures into late childhood. In contrast to SeLIE due to other etiologies, many individuals have a more persistent, albeit drug-responsive, form of epilepsy., (© 2024 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Background: Voltage-gated sodium channels are involved in the initial depolarisation of neurones. As such, they play important roles in neurotransmission. Variants in the genes encoding these channels may lead to altered functionality and neurodevelopmental disorders. Pathogenic variants of SCN8A, which encodes the voltage-gated Na+ channel Nav1.6, have been associated with various encephalopathies characterised by developmental delay and epileptic seizures. Herein, we discuss the genotype-phenotype associations in a group of 17 novel Polish patients with SCN8A mutations, further expanding the molecular and phenotypic spectrum of SCN8A-related diseases., Methods: The participants were recruited from five clinical centres in Poland. Pathogenic and likely pathogenic SCN8A variants were identified using a next-generation sequencing (NGS) panel and exome sequencing, respectively. Magnetic resonance imaging (MRI) and electroencephalography (EEG) recordings were performed to obtain relevant clinical data on brain malformations and epileptic seizures., Results: Three phenotypes were observed in the study group: developmental and epileptic encephalopathy, early onset epileptic encephalopathy, and neurodevelopmental disorders without epilepsy. Patients in the first two phenotypic subgroups presented with epileptic seizures within the first few months of life. Their semiology evolved with age, comprising mostly tonic, clonic, and tonic-clonic seizures, with eyelid myoclonia, myoclonic seizures, and epileptic spasms. The most prevalent neurological feature was developmental delay. Alterations in muscle tone were more frequent than in previous reports., Conclusions: Seventeen patients with 11 novel mutations in SCN8A had alterations in muscular tone accompanied by typical features of SCN8A-related encephalopathies (i.e., developmental delay and a wide range of seizures)., Competing Interests: Declaration of competing interest The Authors declare no conflict of interests., (Copyright © 2024. Published by Elsevier Ltd.)

Objectives: We aimed to develop consensus on comorbidities (frequency, severity, and prognosis) and overall outcomes in epilepsy, development, and cognition for the five phenotypes of SCN8A-related disorders., Methods: A core panel consisting of 13 clinicians, 1 researcher, and 6 caregivers was formed and split into three workgroups. One group focused on comorbidities and prognosis. All groups performed a literature review and developed questions for use in a modified-Delphi process. Twenty-eight clinicians, one researcher, and 13 caregivers from 16 countries participated in three rounds of the modified-Delphi process. Consensus was defined as follows: strong consensus ≥80% fully agree; moderate consensus ≥80% fully or partially agree, <10% disagree; and modest consensus 67%-79% fully or partially agree, <10% disagree., Results: Consensus was reached on the presence of 14 comorbidities in patients with Severe Developmental and Epileptic Encephalopathy (Severe DEE) spanning non-seizure neurological disorders and other organ systems; impacts were mostly severe and unlikely to improve or resolve. Across Mild/Moderate Developmental and Epileptic Encephalopathy (Mild/Moderate DEE), Neurodevelopmental Delay with Generalized Epilepsy (NDDwGE), and NDD without Epilepsy (NDDwoE) phenotypes, cognitive and sleep-related comorbidities as well as fine and gross motor delays may be present but are less severe and more likely to improve compared to Severe DEE. There was no consensus on comorbidities in the SeL(F)IE phenotype but strong conesensus that seizures would largely resolve. Seizure freedom is rare in patients with Severe DEE but may occur in some with Mild/Moderate DEE and NDDwGE., Significance: Significant comorbidities are present in most phenotypes of SCN8A-related disorders but are most severe and pervasive in the Severe DEE phenotype. We hope that this work will improve recognition, early intervention, and long-term management for patients with these comorbidities and provide the basis for future evidence-based studies on optimal treatments of SCN8A-related disorders. Identifying the prognosis of patients with SCN8A-related disorders will also improve care and quality-of-life for patients and their caregivers., (© 2024 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel Na V 1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of Na V 1.6 G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant Na V 1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of Na V 1.6 G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in Na V 1.6 G1625R . Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

Background: Sudden unexpected death in epilepsy (SUDEP) is a fatal complication experienced by otherwise healthy epilepsy patients. Dravet syndrome (DS) is an inherited epileptic disorder resulting from loss of function of the voltage-gated sodium channel, Na V 1.1, and is associated with particularly high SUDEP risk. Evidence is mounting that Na V s abundant in the brain also occur in the heart, suggesting that the very molecular mechanisms underlying epilepsy could also precipitate cardiac arrhythmias and sudden death. Despite marked reduction of Na V 1.1 functional expression in DS, pathogenic late sodium current (I Na,L ) is paradoxically increased in DS hearts. However, the mechanisms by which DS directly impacts the heart to promote sudden death remain unclear., Objectives: In this study, the authors sought to provide evidence implicating remodeling of Na + - and Ca 2+ -handling machinery, including Na V 1.6 and Na + /Ca 2+ exchanger (NCX) within transverse (T)-tubules in DS-associated arrhythmias., Methods: The authors undertook scanning ion conductance microscopy (SICM)-guided patch clamp, super-resolution microscopy, confocal Ca 2+ imaging, and in vivo electrocardiography studies in Scn1a haploinsufficient murine model of DS., Results: DS promotes I Na,L in T-tubular nanodomains, but not in other subcellular regions. Consistent with increased Na V activity in these regions, super-resolution microscopy revealed increased Na V 1.6 density near Ca 2+ release channels, the ryanodine receptors (RyR2) and NCX in DS relative to WT hearts. The resulting I Na,L in these regions promoted aberrant Ca 2+ release, leading to ventricular arrhythmias in vivo. Cardiac-specific deletion of Na V 1.6 protects adult DS mice from increased T-tubular late Na V activity and the resulting arrhythmias, as well as sudden death., Conclusions: These data demonstrate that Na V 1.6 undergoes remodeling within T-tubules of adult DS hearts serving as a substrate for Ca 2+ -mediated cardiac arrhythmias and may be a druggable target for the prevention of SUDEP in adult DS subjects., Competing Interests: Funding Support and Author Disclosures This work was supported by National Institutes of Health grants R01NS121234 and R01HL155378 (to Dr Radwański), R01HL148736 (to Dr Veeraraghavan), R01HL156652 (to Dr Hund), K99HL157684 (to Dr Nassal), and T32HL149637-02 and L40NS129034 (to Dr King); National Science Foundation Graduate Research Fellowship, NSF Fellow ID: 2019259354 (to Dr Struckman); and American Heart Association postdoctoral fellowships 915300 (to Dr Tarasov) and 908824 (to Dr Moise). The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Distinguishing clinical subgroups for patients suffering with diseases characterized by a wide phenotypic spectrum is essential for developing precision therapies. Patients with gain-of-function (GOF) variants in the SCN8A gene exhibit substantial clinical heterogeneity, viewed historically as a linear spectrum ranging from mild to severe. To test for hidden clinical subgroups, we applied two machine-learning algorithms to analyze a dataset of patient features collected by the International SCN8A Patient Registry. We used two research methodologies: a supervised approach that incorporated feature severity cutoffs based on clinical conventions, and an unsupervised approach employing an entirely data-driven strategy. Both approaches found statistical support for three distinct subgroups and were validated by correlation analyses using external variables. However, distinguishing features of the three subgroups within each approach were not concordant, suggesting a more complex phenotypic landscape. The unsupervised approach yielded strong support for a model involving three partially ordered subgroups rather than a linear spectrum. Application of these machine-learning approaches may lead to improved prognosis and clinical management of individuals with SCN8A GOF variants and provide insights into the underlying mechanisms of the disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Pathogenic variants in SCN8A are associated with a broad phenotypic spectrum, including Self-Limiting Familial Infantile Epilepsy (SeLFIE), characterized by infancy-onset age-related seizures with normal development and cognition. Movement disorders, particularly paroxysmal kinesigenic dyskinesia typically arising after puberty, may represent another core symptom. We present the case of a 1-year-old girl with a familial disposition to self-limiting focal seizures from the maternal side and early-onset orofacial movement disorders associated with SCN8A-SeLFIE. Brain MRI was normal. Genetic testing revealed a maternally inherited SCN8A variant [c.4447G > A; p.(Glu1483Lys)]. After the introduction of valproic acid, she promptly achieved seizure control as well as complete remission of strabismus and a significant decrease in episodes of tongue deviation. Family history, genetic findings, and epilepsy phenotype are consistent with SCN8A-SeLFIE. Movement disorders are an important part of the SCN8A phenotypic spectrum, and this case highlights the novel early-onset orofacial movement disorders associated with this condition. The episodes of tongue deviation and protrusion suggest focal oromandibular (lingual) dystonia. Additionally, while infantile strabismus or esophoria is a common finding in healthy individuals, our case raises the possibility of an ictal origin of the strabismus. This study underscores the importance of recognizing and addressing movement disorders in SCN8A-SeLFIE patients, particularly the rare early-onset orofacial manifestations. It adds to the growing body of knowledge regarding the diverse clinical presentations of SCN8A-associated disorders and suggests potential avenues for clinical management and further research., (© 2024 The Authors. Epileptic Disorders published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Objective: De novo mutations of the voltage-gated sodium channel gene SCN8A cause developmental and epileptic encephalopathy (DEE). Most pathogenic variants result in gain-of-function changes in activity of the sodium channel Na v 1.6, poorly controlled seizures, and significant comorbidities. In previous work, an antisense oligonucleotide (ASO) reduced Scn8a transcripts and increased lifespan after neonatal administration to a mouse model. Here, we tested long-term ASO treatment initiated after seizure onset, as required for clinical application., Methods: ASO treatment was initiated after observation of a convulsive seizure and repeated at 4 to 6 week intervals for 1 year. We also tested the long-term efficacy of an AAV10-short hairpin RNA (shRNA) virus administered on P1., Results: Repeated treatment with the Scn8a ASO initiated after seizure onset provided long-term survival and reduced seizure frequency during a 12 month observation period. A single treatment with viral shRNA was also protective during 12 months of observation., Interpretation: Downregulation of Scn8a expression that is initiated after the onset of seizures is effective for long-term treatment in a model of SCN8A-DEE. Repeated ASO administration or a single dose of viral shRNA prevented seizures and extended survival through 12 months of observation. ANN NEUROL 2024;95:754-759., (© 2023 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Background: Epilepsy is a common neurological disease; however, few if any of the currently marketed antiseizure medications prevent or cure epilepsy. Discovery of pathological processes in the early stages of epileptogenesis has been challenging given the common use of preclinical models that induce seizures in physiologically normal animals. Moreover, despite known sex dimorphism in neurological diseases, females are rarely included in preclinical epilepsy models., Methods: We characterized sex differences in mice carrying a pathogenic knockin variant (p.N1768D) in the Scn8a gene that causes spontaneous tonic-clonic seizures (TCs) at ∼3 months of age and found that heterozygous females are more resilient than males in mortality and morbidity. To investigate the cellular mechanisms that underlie female resilience, we utilized blood-brain barrier (BBB) and hippocampal transcriptomic analyses in heterozygous mice before seizure onset (pre-TC) and in mice that experienced ∼20 TCs (post-TC)., Results: In the pre-TC latent phase, both sexes exhibited leaky BBB; however, patterns of gene expression were sexually dimorphic. Females exhibited enhanced oxidative phosphorylation and protein biogenesis, while males activated gliosis and CREB signaling. After seizure onset (chronic phase), females exhibited a metabolic switch to lipid metabolism, while males exhibited increased gliosis and BBB dysfunction and a strong activation of neuroinflammatory pathways., Conclusion: The results underscore the central role of oxidative stress and BBB permeability in the early stages of epileptogenesis, as well as sex dimorphism in response to increasing neuronal hyperexcitability. Our results also highlight the need to include both sexes in preclinical studies to effectively translate results of drug efficacy studies., (© 2024 The Author(s).)

Objective: To explore the clinical and genetic characteristics of five children with epilepsies due to variants of SCN8A gene., Methods: Clinical data of five children (four males and one female) admitted to Linyi People's Hospital due to hereditary epilepsies between August 2015 and August 2022 were collected. Whole exome sequencing was carried out for these children, and candidate variants were verified by Sanger sequencing., Results: All of the five children were found to harbor variants of the SCN8A gene. Case 1, who had benign familial infantile epilepsy, inherited a known pathogenic c.4840A>G variant from his father with similar symptoms. Cases 2 to 4 had presented with intermediate epilepsy. Among these, case 2 has harbored a de novo c.3967G>A variant which was rated as pathogenic (PS1+PS2+PM1+PM2&#95;Supporting+PP3) based on the guidelines from the American College of Medical Genetics and Genomics. Cases 3 and 4 were found to respectively harbor a de novo c.415A>T and a c.4697C>T variant, which were both rated as likely pathogenic (PS2+PM1+PM2&#95;Supporting+PP3). Case 5, who had early-onset infantile epileptic encephalopathy transformed into Lennox Gastaut-like syndrome, has harbored a de novo c.5615G>A variant, which was known to be pathogenic. The children had their age of onset ranging from 2 to 14 months, and all had focal seizures and generalized tonic clonic seizures. Four children (cases 1, 2, 3 and 5) had cluster seizures, four (cases 1 to 4) had become seizure-free after single or dual treatment and showed normal growth and development, whilst case 5 was drug-resistant and showed severe developmental retardation., Conclusion: The five children had new features such as cluster seizures, occasional benign seizures in adulthood, and intermediate epilepsy which are prone to relapse after discontinuation of medication, which may be attributed to the pathogenic variants of the SCN8A gene.

Quinidine has been used as an anticonvulsant to treat patients with KCNT1-related epilepsy by targeting gain-of-function KCNT1 pathogenic mutant variants. However, the detailed mechanism underlying quinidine's blockade against KCNT1 (Slack) remains elusive. Here, we report a functional and physical coupling of the voltage-gated sodium channel Na V 1.6 and Slack. Na V 1.6 binds to and highly sensitizes Slack to quinidine blockade. Homozygous knockout of Na V 1.6 reduces the sensitivity of native sodium-activated potassium currents to quinidine blockade. Na V 1.6-mediated sensitization requires the involvement of Na V 1.6's N- and C-termini binding to Slack's C-terminus and is enhanced by transient sodium influx through Na V 1.6. Moreover, disrupting the Slack-Na V 1.6 interaction by viral expression of Slack's C-terminus can protect against Slack G269S -induced seizures in mice. These insights about a Slack-Na V 1.6 complex challenge the traditional view of 'Slack as an isolated target' for anti-epileptic drug discovery efforts and can guide the development of innovative therapeutic strategies for KCNT1-related epilepsy., Competing Interests: TY, YW, YJ, HY, SX, HZ, QC, NL, XM, HS, CP, ZG, JD, GD, QS, YY, FY, ZH No competing interests declared, (© 2023, Yuan, Wang, Jin et al.)

Background: SCN8A-related disorders are a group of variable conditions caused by pathogenic variations in SCN8A. Online Mendelian Inheritance in Man (OMIM) terms them as developmental and epileptic encephalopathy 13, benign familial infantile seizures 5 or cognitive impairment with or without cerebellar ataxia., Methods: In this study, we describe clinical and genetic results on eight individuals from six families with SCN8A pathogenic variants identified via exome sequencing., Results: Clinical findings ranged from normal development with well-controlled epilepsy to significant developmental delay with treatment-resistant epilepsy. Three novel and three reported variants were observed in SCN8A. Electrophysiological analysis in transfected cells revealed a loss-of-function variant in Patient 4., Conclusions: This work expands the clinical and genotypic spectrum of SCN8A-related disorders and provides electrophysiological results on a novel loss-of-function SCN8A variant., (© 2024. The Author(s).)

Gastric cancer (GC) is a major contributor to cancer-related deaths and is characterized by high heterogeneity in epidemiology and histopathology worldwide. Increasing evidence indicates that circular RNAs (circRNAs) play multifaceted roles in cellular processes in human cancers. Here, we demonstrated that circFNTA high expression increases the proliferation, metastasis, and epithelial-mesenchymal transition process and tumorigenicity of GC cells. First, we found that circFNTA was upregulated in GC cells and tissues, and the high circFNTA levels were positively associated with the poor prognosis in GC patients. Using luciferase reporter and RNA-pull down assays, we elucidated that circFNTA sponged two microRNAs, miR-604 and miR-647. In addition, the proliferation and metastatic ability of GC cell reduction caused by silencing circFNTA was hindered by inhibitors of miR-604 and miR-647. Moreover, SCN8A was predicted by miRDB as a common target gene of miR-604 and miR-647, which was then verified by the luciferase reporter assay. Knockdown of circFNTA causes messenger RNA and protein levels in SCN8A to be downregulated in GC cells. However, this effect was overturned by cotransfection miR-604 and miR-647. Also, we identified that SCN8A was downregulated in GC tissues, which was positively correlated with circFNTA expression. In rescue experiments, the attenuated cell proliferation and metastatic ability caused by circFNTA knockdown was reversed by miR-604 and miR-647 inhibitors and SCN8A overexpression. Collectively, our findings suggest an oncogenic role of circFNTA in GC progression and elucidate that circFNTA exerts its function by modulating the miR-604/miR-647/SCN8A axis., (© 2023 The Authors. Journal of Biochemical and Molecular Toxicology published by Wiley Periodicals LLC.)

Background: Variants in SCN8A are associated with a spectrum of epilepsies and neurodevelopmental disorders. Ataxia as a predominant symptom of SCN8A variation has not been well studied. We set out to investigate disease mechanisms and genotype-phenotype correlations of SCN8A-related ataxia., Methods: We collected genetic and electro-clinical data of ten individuals from nine unrelated families carrying novel SCN8A variants associated with chronic progressive or episodic ataxia. Electrophysiological characterizations of these variants were performed in ND7/23 cells and cultured neurons., Findings: Variants associated with chronic progressive ataxia either decreased Na + current densities and shifted activation curves towards more depolarized potentials (p.Asn995Asp, p.Lys1498Glu and p.Trp1266Cys) or resulted in a premature stop codon (p.Trp937Ter). Three variants (p.Arg847Gln and biallelic p.Arg191Trp/p.Asp1525Tyr) were associated with episodic ataxia causing loss-of-function by decreasing Na + current densities or a hyperpolarizing shift of the inactivation curve. Two additional episodic ataxia-associated variants caused mixed gain- and loss-of function effects in ND7/23 cells and were further examined in primary murine hippocampal neuronal cultures. Neuronal firing in excitatory neurons was increased by p.Arg1629His, but decreased by p.Glu1201Lys. Neuronal firing in inhibitory neurons was decreased for both variants. No functional effect was observed for p.Arg1913Trp. In four individuals, treatment with sodium channel blockers exacerbated symptoms., Interpretation: We identified episodic or chronic ataxia as predominant phenotypes caused by variants in SCN8A. Genotype-phenotype correlations revealed a more pronounced loss-of-function effect for variants causing chronic ataxia. Sodium channel blockers should be avoided under these conditions., Funding: BMBF, DFG, the Italian Ministry of Health, University of Tuebingen., Competing Interests: Declaration of interests MK reports a doctoral grant from DAAD. JDOE is coordinator of the Chorea and Huntington Disease Group (ERN-RND). EB has received funding from the Dutch Epilepsy Foundation, on behalf of the Dravet syndrome Foundation Netherlands/Flanders, the JANIVO foundation and the K.F. Hein Foundation. LS declares grants from the German Research Foundation, German Ministry of Heath, European Commission, and Innovationsfond, and has received consulting fees from Vico Therapeutics and Novartis. RSM has received consulting and speaker fees from UCB and Orion and speaker fees from EISAI and Angelini Pharma. All other authors report no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is a common neurodegenerative disease in elderly people, which is characterized by motor disabilities in PD patients. Nav1.6 is the most abundant subtype of voltage-gated sodium channels (VGSCs) in the brain of adult mammals and rodents. Here we investigated the role of Nav1.6 in the external globus pallidus (GP) involved in the pathogenesis of motor deficits in unilateral 6-OHDA(6-hydroxydopamine)lesioned rats. The results show that Nav1.6 is dramatically increased in reactive astrocytes of the ipsilateral GP in the middle stage, but not different from the control rats in the later stage of the pathological process in 6-OHDA lesioned rats. Furthermore, the down-regulation of Nav1.6 expression in the ipsilateral GP can significantly improve motor deficits in 6-OHDA lesioned rats in the middle stage of the pathological process. The electrophysiological experiments show that the down-regulation of Nav1.6 expression in the ipsilateral GP significantly decreases the abnormal high synchronization between the ipsilateral M1 (the primary motor cortex) and GP in 6-OHDA lesioned rats. Ca 2+ imaging reveals that the down-regulation of Nav1.6 expression reduces the intracellular concentration of Ca 2+ ([Ca 2+ ]i) in primary cultured astrocytes. These findings suggest that the increased Nav1.6 expression of reactive astrocytes in the GP play an important role in the pathogenesis of motor dysfunction in the middle stage in 6-OHDA lesioned rats, which may participate in astrocyte-neuron communication by regulating [Ca 2+ ]i of astrocytes, thereby contributing to the formation of abnormal electrical signals of the basal ganglia (BG) in 6-OHDA lesioned rats., (© 2023 Wiley Periodicals LLC.)

Objective: Genetic variants in the SCN8A gene underlie a wide spectrum of neurodevelopmental phenotypes including several distinct seizure types and a host of comorbidities. One of the major challenges facing clinicians and researchers alike is to identify genotype-phenotype (G-P) correlations that may improve prognosis, guide treatment decisions, and lead to precision medicine approaches., Methods: We investigated G-P correlations among 270 participants harboring gain-of-function (GOF) variants enrolled in the International SCN8A Registry, a patient-driven online database. We performed correlation analyses stratifying the cohort by clinical phenotypes to identify diagnostic features that differ among patients with varying levels of clinical severity, and that differ among patients with distinct GOF variants., Results: Our analyses confirm positive correlations between age at seizure onset and developmental skills acquisition (developmental quotient), rate of seizure freedom, and percentage of cohort with developmental delays, and identify negative correlations with number of current and weaned antiseizure medications. This set of features is more detrimentally affected in individuals with a priori expectations of more severe clinical phenotypes. Our analyses also reveal a significant correlation between a severity index combining clinical features of individuals with a particular highly recurrent variant and an independent electrophysiological score assigned to each variant based on in vitro testing., Significance: This is one of the first studies to identify statistically significant G-P correlations for individual SCN8A variants with GOF properties. The results suggest that individual GOF variants (1) are predictive of clinical severity for individuals carrying those variants and (2) may underlie distinct clinical phenotypes of SCN8A disease, thus helping to explain the wide SCN8A-related epilepsy disease spectrum. These results also suggest that certain features present at initial diagnosis are predictive of clinical severity, and with more informed treatment plans, may serve to improve prognosis for patients with SCN8A GOF variants., (© 2023 International League Against Epilepsy.)

Variants in SCN8A are associated with several diseases, including developmental and epileptic encephalopathy, intermediate epilepsy or mild-to-moderate developmental and epileptic encephalopathy, self-limited familial infantile epilepsy, neurodevelopmental delays with generalized epilepsy, neurodevelopmental disorder without epilepsy, hypotonia, and movement disorders. Herein, we report an 8-year-old Moroccan boy with intermediate epilepsy of unknown origin, intellectual disability, autism spectrum disorder, and hyperactivity. The patient presented a normal 46, XY karyotype and a normal comparative genomic hybridization profile. Whole-exome sequencing was performed, and heterozygous variants were identified in KCNK4 and SCN8A . The SCN8A variant [c.4499C > T (p.Pro1500Leu)] was also detected in the healthy mother and was classified as a variant of uncertain clinical significance. This variant occurs in a highly conserved domain, which may affect the function of the encoded protein. More studies are needed to confirm the pathogenicity of this novel variant to establish the effective care, management, and genetic counselling of affected individuals.

Background and Purpose: Variants in SCN8A, the Na V 1.6 channel's coding gene, are characterized by a variety of symptoms, including intractable epileptic seizures, psychomotor delay, progressive cognitive decline, autistic features, ataxia or dystonia. Standard anticonvulsant treatment has a limited impact on the course of disease., Experimental Approach: We investigated the therapeutic potential of eslicarbazepine (S-licarbazepine; S-lic), an enhancer of slow inactivation of voltage gated sodium channels, on two variants with biophysical and neuronal gain-of-function (G1475R and M1760I) and one variant with biophysical gain-of-function but neuronal loss-of-function (A1622D) in neuroblastoma cells and in murine primary hippocampal neuron cultures. These three variants cover the broad spectrum of Na V 1.6-associated disease and are linked to representative phenotypes of mild to moderate epilepsy (G1475R), developmental and epileptic encephalopathy (M1760I) and intellectual disability without epilepsy (A1622D)., Key Results: Similar to known effects on Na V 1.6 wildtype channels, S-lic predominantly enhances slow inactivation on all tested variants, irrespective of their particular biophysical mechanisms. Beyond that, S-lic exhibits variant-specific effects including a partial reversal of pathologically slowed fast inactivation dynamics (A1622D and M1760I) and a trend to reduce enhanced persistent Na + current by A1622D variant channels. Furthermore, our data in primary transfected neurons reveal that not only variant-associated hyperexcitability (M1760I and G1475R) but also hypoexcitability (A1622D) can be modulated by S-lic., Conclusions and Implications: S-lic has not only substance-specific effects but also variant-specific effects. Personalized treatment regimens optimized to achieve such variant-specific pharmacological modulation may help to reduce adverse side effects and improve the overall therapeutic outcome of SCN8A-related disease., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

SINE-VNTR-Alu (SVA) retrotransposons arose and expanded in the genome of hominoid primates concurrent with the slowing of brain maturation. We report genes with intronic SVA transposons are enriched for neurodevelopmental disease and transcribed into long non-coding SVA-lncRNAs. Human-specific SVAs in microcephaly CDK5RAP2 and epilepsy SCN8A gene introns repress their expression via transcription factor ZNF91 to delay neuronal maturation. Deleting the SVA in CDK5RAP2 initiates multi-dimensional and in SCN8A selective sodium current neuronal maturation by upregulating these genes. SVA-lncRNA AK057321 forms RNA:DNA heteroduplexes with the genomic SVAs and upregulates these genes to initiate neuronal maturation. SVA-lncRNA AK057321 also promotes species-specific cortex and cerebellum-enriched expression upregulating human genes with intronic SVAs (e.g., HTT, CHAF1B and KCNJ6) but not mouse orthologs. The diversity of neuronal genes with intronic SVAs suggest this hominoid-specific SVA transposon-based gene regulatory mechanism may act at multiple steps to specialize and achieve neoteny of the human brain., (© 2023. The Author(s).)

Genetic variants in the SCN8A gene underlie a wide spectrum of neurodevelopmental phenotypes that range from severe epileptic encephalopathy to benign familial infantile epilepsy to neurodevelopmental delays with or without seizures. A host of additional comorbidities also contribute to the phenotypic spectrum. As a result of the recent identification of the genetic etiology and the length of time it often takes to diagnose patients, little data are available on the natural history of these conditions. The International SCN8A Patient Registry was developed in 2015 to fill gaps in understanding the spectrum of the disease and its natural history, as well as the lived experiences of individuals with SCN8A syndrome. Another goal of the registry is to collect longitudinal data from participants on a regular basis. In this article, we describe the construction and structure of the International SCN8A Patient Registry, present the type of information available, and highlight particular analyses that demonstrate how registry data can provide insights into the clinical management of SCN8A syndrome., (© 2023 National Cancer Registrars Association.)

Voltage-gated sodium channel Na v 1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Na v 1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Na v 1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Na v 1.6 in the presence of auxiliary subunits β1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the β1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Na v 1.6-specific blockers. Structural mapping of disease-related Na v 1.6 mutations provides insights into their pathogenic mechanism.

De novo missense mutations in SCN8A gene encoding voltage-gated sodium channel Na V 1.6 are linked to a severe form of early infantile epileptic encephalopathy named early infantile epileptic encephalopathy type13 (EIEE13). The majority of the patients with EIEE13 does not respond favorably to the antiepileptic drugs (AEDs) in clinic and has a significantly increased risk of death. Although more than 60 EIEE13-associated mutations have been discovered, only few mutations have been functionally analyzed. In this study we investigated the functional influences of mutations N1466T and N1466K, two EIEE13-associated mutations located in the inactivation gate, on sodium channel properties. Sodium currents were recorded from CHO cells expressing the mutant and wide-type (WT) channels using the whole-cell patch-clamp technique. We found that, in comparison with WT channels, both the mutant channels exhibited increased window currents, persistent currents (I NaP ) and ramp currents, suggesting that N1466T and N1466K were gain-of-function (GoF) mutations. Sodium channel inhibition is one common mechanism of currently available AEDs, in which topiramate (TPM) was effective in controlling seizures of patients carrying either of the two mutations. We found that TPM (100 µM) preferentially inhibited I NaP and ramp currents but did not affect transient currents (I NaT ) mediated by N1466T or N1466K. Among the other 6 sodium channel-inhibiting AEDs tested, phenytoin and carbamazepine displayed greater efficacy than TPM in suppressing both I NaP and ramp currents. Functional characterization of mutants N1466T and N1466K is beneficial for understanding the pathogenesis of EIEE13. The divergent effects of sodium channel-inhibiting AEDs on I NaP and ramp currents provide insight into the development of therapeutic strategies for the N1466T and N1466K-associated EIEE13., (© 2022. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

A 13-year-old girl with a voltage-gated sodium channel mutation (SCN8A)-associated intractable epilepsy presented for bilateral mastectomy for painful juvenile fibroadenomatosis. Sodium channel mutations are more frequently diagnosed with continued advances in genetic testing. Understanding the effects of sodium channel mutations is important to provide safe anesthetic care to these patients. In this article, we discuss what is known regarding the physiology of SCN8A channels and the anesthetic considerations when caring for patients with an SCN8A mutation., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 International Anesthesia Research Society.)

Cancer-induced bone pain (CIBP) occurs frequently among advanced cancer patients. Voltage-gated sodium channels (VGSCs) have been associated with chronic pain, but how VGSCs function in CIBP is poorly understood. Here, we aimed to investigate the specific role of VGSCs in the dorsal root ganglia (DRGs) in CIBP. A CIBP rat model was generated by the intratibial inoculation of MRMT-1 breast carcinoma cells. Transcriptome sequencing was conducted to assess the gene expression profiles. The expression levels of key genes and differentiated genes related to activated pathways were measured by Western blotting and qPCR. We implanted a catheter intrathecally for the administration of lentivirus and drugs. Then, the changes in the mechanical withdrawal threshold (MWT) were measured. We identified 149 differentially expressed mRNAs (DEmRNAs) in the DRGs of CIBP model rats. The expression of Na v 1.6, which was among these DEmRNAs, was significantly upregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEmRNAs showed that they were mainly enriched in the mitogen-activated protein kinase (MAPK) pathway. The decrease in MWT induced by bone cancer was attenuated by Na v 1.6 knockdown. Western blot analysis revealed that a p38 inhibitor decreased the expression of Na v 1.6 and attenuated pain behavior. Our study shows that the upregulation of Na v 1.6 expression by p38 MAPK in the DRGs of rats contributes to CIBP.

Epilepsy is a common neurological disorder characterized by recurrent unprovoked seizures. SCN1A encodes Na V 1.1, a neuronal voltage-gated Na + channel that is highly expressed throughout the central nervous system. Na V 1.1 is localized within the axon initial segment where it plays a critical role in the initiation and propagation of action potentials and neuronal firing, predominantly in γ-amino-butyric-acid (GABA)ergic neurons of the hippocampus. The objective of this study was to characterize a de novo missense variant of uncertain significance in the SCN1A gene of a proband presented with febrile status epilepticus characterized by generalized tonic clonic movements associated with ictal emesis and an abnormal breathing pattern. Screening a gene panel revealed a heterozygous missense variant of uncertain significance in the SCN1A gene, designated c.4379A>G, p.(Tyr1460Cys). The Na V 1.1 wild-type (WT) and mutant channel reproduced in vivo and were transfected in HEK 293 cells. Na + currents were recorded using the whole-cell configuration of the patch-clamp technique. This Na V 1.1 variant (Tyr1460Cys) failed to express functional Na + currents when expressed in HEK293 cells, most probably due to a pore defect of the channel given that the cell surface expression of the channel was normal. Currents generated after co-transfection with functional WT channels exhibited biophysical properties comparable to those of WT channels, which was mainly due to the functional WT channels at the cell surface. The Na V 1.1 variant failed to express functional Na + currents, most probably due to pore impairment and exhibited a well-established loss of function mechanism. The present study highlights the added-value of functional testing for understanding the pathophysiology and potential treatment decisions for patients with undiagnosed developmental epileptic encephalopathy., (© 2022. The Author(s).)

Voltage-gated sodium and potassium channels regulate the initiation and termination of neuronal action potentials. Gain-of-function mutations of sodium channel Scn8a and loss-of-function mutations of potassium channels Kcna1 and Kcnq2 increase neuronal activity and lead to seizure disorders. We tested the hypothesis that reducing the expression of Scn8a would compensate for loss-of-function mutations of Kcna1 or Kcnq2. Scn8a expression was reduced by the administration of an antisense oligonucleotide (ASO). This treatment lengthened the survival of the Kcn1a and Kcnq2 mutants, and reduced the seizure frequency in the Kcnq2 mutant mice. These observations suggest that reduction of SCN8A may be therapeutic for genetic epilepsies resulting from mutations in these potassium channel genes., (© 2022 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

We report detailed functional analyses and genotype-phenotype correlations in 392 individuals carrying disease-causing variants in SCN8A, encoding the voltage-gated Na+ channel Nav1.6, with the aim of describing clinical phenotypes related to functional effects. Six different clinical subgroups were identified: Group 1, benign familial infantile epilepsy (n = 15, normal cognition, treatable seizures); Group 2, intermediate epilepsy (n = 33, mild intellectual disability, partially pharmaco-responsive); Group 3, developmental and epileptic encephalopathy (n = 177, severe intellectual disability, majority pharmaco-resistant); Group 4, generalized epilepsy (n = 20, mild to moderate intellectual disability, frequently with absence seizures); Group 5, unclassifiable epilepsy (n = 127); and Group 6, neurodevelopmental disorder without epilepsy (n = 20, mild to moderate intellectual disability). Those in Groups 1-3 presented with focal or multifocal seizures (median age of onset: 4 months) and focal epileptiform discharges, whereas the onset of seizures in patients with generalized epilepsy was later (median: 42 months) with generalized epileptiform discharges. We performed functional studies expressing missense variants in ND7/23 neuroblastoma cells and primary neuronal cultures using recombinant tetrodotoxin-insensitive human Nav1.6 channels and whole-cell patch-clamping. Two variants causing developmental and epileptic encephalopathy showed a strong gain-of-function (hyperpolarizing shift of steady-state activation, strongly increased neuronal firing rate) and one variant causing benign familial infantile epilepsy or intermediate epilepsy showed a mild gain-of-function (defective fast inactivation, less increased firing). In contrast, all three variants causing generalized epilepsy induced a loss-of-function (reduced current amplitudes, depolarizing shift of steady-state activation, reduced neuronal firing). Functional effects were known for 170 individuals. All 136 individuals carrying a functionally tested gain-of-function variant had either focal (n = 97, Groups 1-3) or unclassifiable (n = 39) epilepsy, whereas 34 individuals with a loss-of-function variant had either generalized (n = 14), no (n = 11) or unclassifiable (n = 6) epilepsy; only three had developmental and epileptic encephalopathy. Computational modelling in the gain-of-function group revealed a significant correlation between the severity of the electrophysiological and clinical phenotypes. Gain-of-function variant carriers responded significantly better to sodium channel blockers than to other anti-seizure medications, and the same applied for all individuals in Groups 1-3. In conclusion, our data reveal clear genotype-phenotype correlations between age at seizure onset, type of epilepsy and gain- or loss-of-function effects of SCN8A variants. Generalized epilepsy with absence seizures is the main epilepsy phenotype of loss-of-function variant carriers and the extent of the electrophysiological dysfunction of the gain-of-function variants is a main determinant of the severity of the clinical phenotype in focal epilepsies. Our pharmacological data indicate that sodium channel blockers present a treatment option in SCN8A-related focal epilepsy with onset in the first year of life., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

SCN8A-developmental and epileptic encephalopathy is caused by pathogenic variants in the SCN8A gene encoding the Na v 1.6 sodium channel, and is characterized by intractable multivariate seizures and developmental regression. Fenfluramine is a repurposed drug with proven antiseizure efficacy in Dravet syndrome and Lennox-Gastaut syndrome. The effect of fenfluramine treatment was assessed in a retrospective series of three patients with intractable SCN8A epilepsy and severe neurodevelopmental comorbidity (n = 2 females; age 2.8-13 years; 8-16 prior failed antiseizure medications [ASM]; treatment duration: 0.75-4.2 years). In the 6 months prior to receiving fenfluramine, patients experienced multiple seizure types, including generalized tonic-clonic, focal and myoclonic seizures, and status epilepticus. Overall seizure reduction was 60%-90% in the last 3, 6, and 12 months of fenfluramine treatment. Clinically meaningful improvement was noted in ≥1 non-seizure comorbidity per patient after fenfluramine, as assessed by physician-ratings of ≥"Much Improved" on the Clinical Global Impression of Improvement scale. Improvements included ambulation in a previously non-ambulant patient and better attention, sleep, and language. One patient showed mild irritability which resolved; no other treatment-related adverse events were reported. There were no reports of valvular heart disease or pulmonary arterial hypertension. Fenfluramine may be a promising ASM for randomized clinical trials in SCN8A-related disorders., (© 2022 The Authors. Epilepsia Open published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

SCN8A is linked to early infantile epileptic encephalopathy type 13 (EIEE13). We generate a human induced pluripotent stem cell (iPSC) line from a child diagnosed with EIEE, caused by SCN8A variation. The iPSC line expresses high pluripotency markers, retains SCN8A variation and is able to differentiate into three germ layers in vitro. The iPSC lines will provide useful resources for studying the pathogenesis and drug screening of SCN8A-related epilepsy., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab . Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.

The impact of gene-related early infancy onset epilepsies in cognitive development can be potentially devastating. Here we report two cases of SCN8A-related epilepsy that highlight the neuropsychological heterogeneity seen with differing de-novo pathogenic variants. Case 1 is a 6-year-old right-handed girl who presented with SCN8A-developmental and epileptic encephalopathy (SCN8A-DEE) and a missense pathogenic variant (c.802A > C), not previously documented in the literature. Her history includes speech and motor delay, with focal motor seizures starting at 4-months. Early EEG showed bilateral centroparietal epileptiform discharges. She shows motor and language delays and prominent motor tics. Testing documented Intellectual Disability (ID) (Mild) with widespread neuropsychological deficits (i.e., academics, attention/executive functions, memory, visual-spatial skills, fine motor, language). Case 2 is an 8-year-old right-handed girl who presented with SCN8A-related epilepsy with c.5630A > G pathogenic variant with seizure onset at 5-months. Her initial EEG showed right occipital spikes. She shows low average intellect and average academics, but evaluation documented attention deficits, fine motor delays, and behavioral issues in addition to tics; she was diagnosed with Attention-Deficit/Hyperactivity Disorder, Oppositional Defiant Disorder, Obsessive Compulsive Disorder, and Tourette's. These cases expand limited knowledge regarding neuropsychological functioning of children with SCN8A-related epilepsy with unique de-novo pathogenic variants. While SCN8A-DEE is clearly associated with ID, other pathogenic variants may show better preserved intellect, despite other neuropsychological and behavioral concerns.

Objective: SCN8A epileptic encephalopathy is caused predominantly by de novo gain-of-function mutations in the voltage-gated sodium channel Na v 1.6. The disorder is characterized by early onset of seizures and developmental delay. Most patients with SCN8A epileptic encephalopathy are refractory to current anti-seizure medications. Previous studies determining the mechanisms of this disease have focused on neuronal dysfunction as Na v 1.6 is expressed by neurons and plays a critical role in controlling neuronal excitability. However, glial dysfunction has been implicated in epilepsy and alterations in glial physiology could contribute to the pathology of SCN8A encephalopathy. In the current study, we examined alterations in astrocyte and microglia physiology in the development of seizures in a mouse model of SCN8A epileptic encephalopathy., Methods: Using immunohistochemistry, we assessed microglia and astrocyte reactivity before and after the onset of spontaneous seizures. Expression of glutamine synthetase and Na v 1.6, and K ir 4.1 channel currents were assessed in astrocytes in wild-type (WT) mice and mice carrying the N1768D SCN8A mutation (D/+)., Results: Astrocytes in spontaneously seizing D/+ mice become reactive and increase expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. These same astrocytes exhibited reduced barium-sensitive K ir 4.1 currents compared to age-matched WT mice and decreased expression of glutamine synthetase. These alterations were only observed in spontaneously seizing mice and not before the onset of seizures. In contrast, microglial morphology remained unchanged before and after the onset of seizures., Significance: Astrocytes, but not microglia, become reactive only after the onset of spontaneous seizures in a mouse model of SCN8A encephalopathy. Reactive astrocytes have reduced K ir 4.1-mediated currents, which would impair their ability to buffer potassium. Reduced expression of glutamine synthetase would modulate the availability of neurotransmitters to excitatory and inhibitory neurons. These deficits in potassium and glutamate handling by astrocytes could exacerbate seizures in SCN8A epileptic encephalopathy. Targeting astrocytes may provide a new therapeutic approach to seizure suppression., (© 2021 The Authors. Epilepsia Open published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

This study was aimed to analyze the commonalities and distinctions of voltage-gated sodium channels, Nav1.2, Nav1.6, in neurodevelopmental disorders. An observational study was performed including two patients with neurodevelopmental disorders. The demographic, electroclinical, genetic, and neuropsychological characteristics were analyzed and compared with each other and then with the subjects carrying the same genetic variants reported in the literature. The clinical features of one of them argued for autism spectrum disorder and developmental delay, the other for intellectual disability, diagnoses confirmed by the neuropsychological assessment. The first patient was a carrier of SCN2A (p.R379H) variant while the second was carrier of SCN8A (p.E936K) variant, both involving the pore loop of the two channels. The results of this study suggest that the neurodevelopmental disorders without overt epilepsy of both patients can be the consequences of loss of function of Nav1.2/Nav1.6 channels. Notably, the SCN2A variant, with an earlier expression timing in brain development, resulted in a more severe phenotype as autism spectrum disorder and developmental delay, while the SCN8A variant, with a later expression timing, resulted in a less severe phenotype as intellectual disability., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Purpose: SCN8A developmental epileptic encephalopathy (SCN8A-DEE) is a rare and severe genetic epilepsy syndrome characterized by early-onset developmental delay, cognitive impairment, and intractable seizures. SCN8A gene variants are associated with a broad phenotypic spectrum and variable disease severity. A caregiver survey, solicited by the advocacy group The Cute Syndrome Foundation (TCSF), was conducted to gather information on the demographics/disease presentation, seizure history, and treatment of patients with SCN8A-related epilepsies., Methods: A 36-question online survey was developed to obtain de-identified data from caregivers of children with SCN8A-related epilepsy. The survey included questions on genetic diagnosis, disease manifestations/comorbidities, seizure severity/type, current/prior use of antiseizure medicines (ASMs), and best/worst treatments per caregiver perception., Results: In total, 116 survey responses (87 USA, 12 Canada, 12 UK, 5 Australia) were quantitatively analyzed. Generalized tonic/clonic was the most common seizure type at onset and time of survey; absence and partial/focal seizures were also common. Most patients (77%) were currently taking ≥2 ASMs; 50% had previously tried and stopped ≥4 ASMs. Sodium channel blockers (oxcarbazepine, phenytoin, lamotrigine) provided the best subjective seizure control and quality of life., Conclusion: The SCN8A-DEE patient population is heterogeneous in seizure characteristics and ASMs taken and is difficult to treat, with high seizure burden and multiple comorbidities. The high proportion of patients who previously tried and stopped ASMs indicates large unmet treatment need. Further collaboration between families, caregivers, patient advocates, clinicians, researchers, and industry can increase awareness and understanding of SCN8A-related epilepsies, improve clinical trial design, and potentially improve patient outcomes., (Copyright © 2022. Published by Elsevier Ltd.)

NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target Na V 1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A -related epilepsy syndromes ( SCN8A -RES), where gain-of-function (GoF) mutations lead to excess Na V 1.6 sodium current, or other indications where Na V 1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of Na V 1.6 (IC 50 0.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for Na V 1.1, 134 X for Na V 1.2, 276 X for Na V 1.7, and >583 Xfor Na V 1.3, Na V 1.4, and Na V 1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting Na V 1.6 currents, including resurgent and persistent Na V 1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where Na V 1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A- developmental epileptic encephalopathy ( SCN8A -DEE) and adult focal-onset seizures., Competing Interests: JJ, TF, KK, CD, SG, JA, GB, KB, EC, SC, RD, Gd, SD, CD, MF, WG, MG, AH, AH, QJ, SL, JL, SL, AL, VL, JM, RN, KN, NS, LS, SS, MT, MW, DW, SW, AW, MW, ZX, RY, CY, AZ, WZ, AC, RS, SP, RW, CC, JE is employed by Xenon Pharmaceuticals, Inc and may hold equity in Xenon Pharmaceuticals, Inc, PT, DB was previously employed by Xenon Pharmaceuticals, Inc and may hold equity in Xenon Pharmaceuticals, Inc, (© 2022, Johnson et al.)

Objective: This study investigates the effects of PRAX-562 on sodium current (I Na ), intrinsic neuronal excitability, and protection from evoked seizures to determine whether a preferential persistent I Na inhibitor would exhibit improved preclinical efficacy and tolerability compared to two standard voltage-gated sodium channel (Na V ) blockers., Methods: Inhibition of I Na  was characterized using patch clamp analysis. The effect on intrinsic excitability was measured using evoked action potentials recorded from hippocampal CA1 pyramidal neurons in mouse brain slices. Anticonvulsant activity was evaluated using the maximal electroshock seizure (MES) model, and tolerability was assessed by measuring spontaneous locomotor activity (sLMA)., Results: PRAX-562 potently and preferentially inhibited persistent I Na induced by ATX-II or the SCN8A mutation N1768D (half-maximal inhibitory concentration [IC 50 ] = 141 and 75 nmol·L -1 , respectively) relative to peak I Na tonic/resting block (60× preference). PRAX-562 also exhibited potent use-dependent block (31× preference to tonic block). This profile is considerably different from standard Na V blockers, including carbamazepine (CBZ; persistent I Na IC 50 = 77 500 nmol·L -1 , preference ratios of 30× [tonic block], less use-dependent block observed at various frequencies). In contrast to CBZ, PRAX-562 reduced neuronal intrinsic excitability with only a minor reduction in action potential amplitude. PRAX-562 (10 mg/kg po) completely prevented evoked seizures without affecting sLMA (MES unbound brain half-maximal efficacious concentration = 4.3 nmol·L -1 , sLMA half-maximal tolerated concentration = 69.7 nmol·L -1 , protective index [PI] = 16×). In contrast, CBZ and lamotrigine (LTG) had PIs of approximately 5.5×, with significant overlap between doses that were anticonvulsant and that reduced locomotor activity., Significance: PRAX-562 demonstrated robust preclinical anticonvulsant activity similar to CBZ but improved compared to LTG. PRAX-562 exhibited significantly improved preclinical tolerability compared with standard Na V blockers (CBZ and LTG), potentially due to the preference for persistent I Na . Preferential targeting of persistent I Na may represent a differentiated therapeutic option for diseases of hyperexcitability, where standard Na V blockers have demonstrated efficacy but poor tolerability., (© 2022 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)