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Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies. 5th edition. New York: Oxford University Press; 2024. doi: 10.1093/med/9780197549469.003.0044
The genes SCN1A, SCN2A, and SCN8A encode the voltage-gated sodium channel (VGSC) α-subunits Nav1.1, Nav1.2, and Nav1.6, respectively. Dravet syndrome is eat the severe end of the disease spectrum caused by SCN1A variants, and Nav1.1 haploinsufficiency in parvalbumin-positive inhibitory neurons has been proposed as a primary cause for the disease. Nav1.2 is predominantly expressed in excitatory neurons in neocortex and hippocampus. SCN2A is the gene with the largest number of de novo variants in patients with autism spectrum disorder and/or intellectual disability, and those are mainly truncations. SCN2A variants also appear in patients with epileptic encephalopathies, but those are mostly missense. Variants of SCN8A have been reported in a number of patients with variable epilepsies. Gain-of-function missense variants are the most common in SCN8A developmental and epileptic encephalopathy associated with motor manifestations such as ataxia and choreoathetosis in the most severe cases. SCN1B encodes the non-pore-forming VGSCβ1 and β1B subunits, which are multifunctional channel regulatory molecules expressed in excitatory and inhibitory neurons. SCN1B variants are reported in patients with epilepsies of multiple etiologies. In vitro and animal model studies have provided critical insights into pathological mechanisms of the diseases caused by genetic variation in these genes.
Voltage-gated sodium channels (VGSCs) are indispensable for generation and propagation of action potentials in electrically excitable tissues, and consist of one pore-forming α-subunit and two non-pore-forming β-subunits that regulate channel kinetics and subcellular trafficking and also function as cell adhesion molecules (Catterall, 2020, 2012a). The human genome contains nine α-subunit (SCN1A~SCN11A; Table 44–1) and four β-subunit (SCN1B~SCN4B) genes. Expression sites of α-subunit genes in the body occasionally overlap but are largely distinct from each other, and therefore their variants lead to different disease phenotypes according to their expression levels and locations (Table 44–1). For example, SCN4A is mainly expressed in skeletal muscle and its variants cause skeletal muscle diseases such as myotonia and paralysis (Ptacek et al., 1991; McClatchey et al., 1992). SCN5A is mainly expressed in heart and its variants cause heart diseases such as Long-QT syndrome (Wang et al., 1995). SCN9A, SCN10A, and SCN11A are mainly expressed in the peripheral nervous system, and their variants have been found in patients with pain disorders (Cox et al., 2006; Faber et al., 2012; Leipold et al., 2013; Zhang et al., 2013). Among genes encoding α-subunits, SCN1A, SCN2A, SCN3A, and SCN8A are the major genes expressed in the central nervous system (CNS). Among these, SCN3A is mainly expressed embryonically (Brysch et al., 1991), and variants have been reported in patients with epilepsies associated with cortical malformations (Holland et al., 2008; Vanoye et al., 2014; Zaman et al., 2018), but SCN3A is not discussed in this review. SCN1A, SCN2A, and SCN8A are expressed postnatally, and a number of variants in these genes have been described in patients with epilepsy, intellectual disability (ID), autism spectrum disorder (ASD), and schizophrenia (SCZ). SCN1B, encoding the VGSC β1/β1B subunits, is widely expressed in the central and peripheral nervous systems, heart, and skeletal muscle. SCN1B mRNA is widely expressed throughout human and mouse brain, although distributions of β1 protein and its splice variant, β1B, may show heterogeneity. Multiple variants in SCN1B have been reported in patients with epilepsy and cardiac arrhythmia. In this review we focus on these four genes (SCN1A, SCN2A, SCN8A, and SCN1B), but see also other relevant reviews (Catterall, 2012b; O’Malley and Isom, 2015; Yamakawa, 2016; Musto et al., 2020; Meisler et al., 2021).
Voltage-Gated Sodium Channel α- and β1-Subunits and Diseases.
SCN1A variants appear in a wide spectrum of epilepsies. Inherited missense SCN1A variants are found in 10%–20% of patients with generalized epilepsy with febrile seizures plus (GEFS+) characterized by autosomal-dominant inheritance, febrile seizures that persist beyond the age of 6 years, and variable afebrile seizures such as generalized tonic-clonic, myoclonic, and absence seizures (Escayg et al., 2000, 2001; Sugawara et al., 2001a; Wallace et al., 2001; Annesi et al., 2003; Spampanato et al., 2004a; 2004b; Nagao et al., 2005; Osaka et al., 2007). SCN1A de novo variants are found in more than 80% of patients with Dravet syndrome characterized by a sporadic intractable epileptic encephalopathy characterized by early onset (6 months to 1 year after birth) epileptic seizures (which often appear first as febrile but later become afebrile), ID, autistic features, ataxia, and increased risk of sudden unexpected death in epilepsy (SUDEP; Claes et al., 2001, 2003; Sugawara et al., 2002; Ohmori et al., 2002; Fujiwara et al., 2003; Gennaro et al., 2003; Nabbout et al., 2003; Wallace 2013; Fukuma et al., 2004; Kimura et al., 2005; Harkin 2007; Depienne 2009; Nakayama 2010). Of these de novo variants of SCN1A, two-thirds result in protein truncation and one-third in missense substitutions. Microdeletion variants affecting the SCN1A 5′ promoter region in a small cohort of Dravet syndrome patients were also reported (Nakayama et al., 2010).
There are some familial Dravet syndrome cases where identical variants were found in multiple family members who manifest either as Dravet syndrome or milder epilepsies (Fujiwara et al., 2003; Nabbout et al., 2003; Kimura et al., 2005), and mosaicism was found in their parents (Morimoto et al., 2006; Gennaro et al., 2006). By using the highly sensitive digital PCR technique, Yang and colleagues (2017) reported mosaicism of SCN1A variants in 10 of the 56 paternal sperm samples of Dravet syndrome families with mutant allelic fractions (MAFs) ranging from 0.03% to 39.04%. These observations indicate that parents who had an affected child may have higher risks of Dravet syndrome in successive children than in general population, and they emphasize the clinical importance of genetic counseling and considerations of preimplantation or prenatal diagnoses.
SCN1A variants are also found in patients with other neurological and psychiatric disorders, including hemiplegic migraine (HM), panic disorder, and autism spectrum disorder (ASD), including Kanner’s and Asperger’s types (Weiss et al., 2003; Dichgans et al., 2005; Osaka et al., 2007; O’Roak et al., 2011, 2012; Hoischen et al., 2014; Deciphering Developmental Disorders Study, 2015).
Overall (with the exclusion of HM), inherited missense variants of SCN1A are found in patients with mild epilepsies such as GEFS+, and de novo loss-of-function variants are found in severe epilepsies such as Dravet syndrome. The former may cause milder and the latter may cause more severe impairments of sodium current in neocortical and hippocampal inhibitory neurons leading to milder and more severe disinhibition of excitatory neurons, and this may well explain the severity of epilepsies in the patients. Note that this situation is distinct or even opposite to that of SCN2A and SCN8A.
A number of in vitro patch-clamp analyses have been reported for Nav1.1 variants found in patients with epilepsy and/or neurodevelopmental disorders (Alekov et al., 2000, 2001; Spampanato et al., 2001, 2003; Lossin et al., 2002, 2003; Sugawara et al., 2003; Rhodes et al., 2004, 2005). For GEFS+ variants, several studies suggested increased activities of the mutant channels (Lossin et al., 2002; Spampanato et al., 2003), while other studies showed decreased or complete absence of sodium current (Alekov et al., 2001; Spampanato et al., 2001; Lossin et al., 2003). Meanwhile, a computer simulation experiment proposed increased neuronal firing with GEFS+ variants (Spampanato et al., 2004a). Studies of SCN1A variants found in patients with Dravet syndrome revealed that not only nonsense variants but also missense variants showed drastically attenuated sodium currents (Sugawara et al., 2003; Lossin et al., 2003; Barela et al., 2006), while some Dravet syndrome missense variants have been reported to have noninactivating channel activities with abnormal kinetics similar to those found in GEFS+ (Rhodes et al., 2004). Although ultimate functional effects for these variants still remain elusive, it has been proposed that these would be generally loss of function, in which milder GEFS+ phenotypes are explained by intermediate or partial loss of function and Dravet syndrome variants would result in complete loss of function or complete elimination of channel protein leading to haploinsufficiency of Nav1.1 (Yamakawa, 2005).
In contrast to the SCN1A variants found in patients with epilepsy and/or neurodevelopmental disorders, most of those found in hemiplegic migraine show gain-of-function (Dhifallah et al., 2018).
In many brain regions, Nav1.1 and Nav1.2 show mutually exclusive distribution (Yamagata et al., 2017, 2021), but Nav1.6 is often co-expressed with Nav1.1 (Van Wart et al., 2007; Duflocq et al., 2008; Lorincz and Nusser, 2008) or Nav1.2 (Hu et al., 2009; Lorincz and Nusser, 2010; Liao et al., 2010a; Tian et al., 2014; Ogiwara et al., 2018; Fig. 44–1).
Distributions of Nav1.1, Nav1.2 and Nav1.6 in brain. Nav1.1 and Nav1.2 are expressed mutually exclusively while Nav1.6 is co-expressed with Nav1.1 or Nav1.2 in most brain regions and neuron subtypes. CCa, CCb: cortico-cortical projection neuron subpopulations. (more...)
Although some previous studies reported somatodendritic distributions of Nav1.1 in neurons, including hippocampal excitatory neurons (Westenbroek et al., 1989; 1992; Gong et al., 1999; Yu et al., 2006), other studies revealed and confirmed that in hippocampus and neocortex the Nav1.1 protein is densely expressed in parvalbumin-positive (PV+) inhibitory neurons such as fast-spiking basket cells (Ogiwara et al., 2007; Van Wart 2007; Lorincz and Nusser 2008; 2010; Verret et al., 2012; Ogiwara, 2013; Dutton, 2013; Tian et al., 2014; Li et al., 2014; Yamagata et al., 2017) and somatostatin-positive (SST+) inhibitory neurons at lesser amount (Tai et al., 2014; Li et al., 2014; Yamagata et al., 2017) as well as Purkinje and basket cells in cerebellum (Van Wart et al., 2006; Ogiwara et al., 2007; Kalume et al., 2007) and thalamic reticular nucleus neurons (Kalume et al., 2015; Yamagata et al., 2020) at their axons (initial segments, node of Ranvier or intermediate trunks, and terminals) and occasionally somata (especially in hippocampus). Some of vasoactive intestinal peptide-positive (VIP+) inhibitory neurons are also reported to express Nav1.1 (Goff and Goldberg, 2019). Using single-cell RNA sequencing transcriptomic data from humans and mice, Du et al. (2020) reported that, while SCN1A/2A/3A/8A mRNAs are expressed in all neurons, they exhibit differential expression strength. SCN1A is predominantly expressed in inhibitory neurons, while SCN2/3/8A are mainly expressed in excitatory neurons, with SCN2/3A expression starting prenatally, followed by SCN1/8A neonatally.
In addition to inhibitory neurons, Nav1.1 is also expressed in a distinct subpopulation of neocortical but absent in hippocampal excitatory neurons (Ogiwara et al., 2013; Yamagata et al., 2017). A recent study of Scn1a- promoter-driven GFP transgenic mouse confirmed above observations including mutually exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons, and it further revealed that among neocortical excitatory projection neurons Nav1.1 is expressed in pyramidal tract (PT) and a subpopulation of cortico-cortical (CC) neurons but in neither intratelencephalic corticostriatal (iCS) nor cortico-thalamic (CT) neurons, and these Nav1.1-negative neurons (iCS, CT, and a subpopulation of CC) are instead Nav1.2-positive (Yamagata et al., 2023). The Nav1.1 expression in PT neurons proposed a pathological neural circuit for SUDEP of Dravet syndrome (Yamagata et al., 2023). Thalamocortical relay neurons, which are excitatory, also express Nav1.1 at their axon arborizations (Ogiwara et al., 2013). GPe (globus pallidus externus), GPi (globus pallidus internus), SNr (substantia nigra pars reticulata), and STN (subthalamic nucleus) are also positive for Nav1.1 (Yamagata et al., 2017, 2023), but their detailed natures (excitatory or inhibitory, projection- or interneurons, etc.) remain to be investigated.
In both excitatory and inhibitory neurons, Nav1.1 first appears at axon initial segments, and in later developmental stages the expression sites moved to distal axons, and instead Nav1.6 appears at axon initial segments (AISs) and continues to remain there (Ogiwara et al., 2013; Fig. 44–2). The Nav1.1 immunosignals reduced at callosal axons and remained as columnar structures at neocortical layer IV, where axons of thalamocortical excitatory projection neurons arborize, in adult Scn1afl/fl, Emx1-Cre mice also indicated Nav1.1 expressions at intermediate and distal axons (Ogiwara et al., 2013). Nav1.1 and Nav1.6 show polarized distribution at proximal and distal parts of AISs, respectively, including retinal ganglion cells (Van Wart et al., 2007; Lorincz and Nusser 2008), spinal motor neurons (Duflocq et al., 2008), and PV+ inhibitory neurons (Lorincz and Nusser 2008; Li et al., 2014). This segregated expression for Nav1.1 and Nav1.6 at the AIS is also the case for Nav1.2 and Nav1.6.
Developmental changes of subcellular localization for Nav1.1, Nav1.2 and Nav1.6 in mouse neurons. Nav1.1 or Nav1.2 firstly appears at proximal sites of axon initial segments (AISs) and nodes of Ranvier if myelinated, and then Nav1.6 appears there at (more...)
To understand ultimate functional consequences of SCN1A variants in patients’ brains, it is imperative to develop animal models harboring the gene defects. Yu and colleagues (2006) generated a mouse with SCN1A haplodeficiency (Scn1a+/–) in that the last exon has been eliminated. Another model (Scn1aRX/+), a knock-in mouse harboring an Scn1a nonsense variant (R1407X) originally found in three independent Dravet syndrome patients, has also been reported (Ogiwara et al., 2007). The Scn1aRX/+ mouse expressed half amount of normal Nav1.1 protein but no truncated protein, which may have been eliminated by NMD (nonsense-mediated mRNA decay) mechanism. Scn1a+/– and Scn1aRX/+ mice showed similar phenotypes, including spontaneous seizures, ataxia, and sporadic deaths after postnatal day 21. A patch-clamp analyses of Scn1a+/– hippocampal cultured neurons (Yu et al., 2006) or Scn1aRX/+ neocortex slices (Ogiwara et al., 2007) showed sodium current reduction specifically in inhibitory neurons but not in excitatory neurons. Epileptic phenotypes of these mice are highly temperature-sensitive, well reproducing the febrile seizures in Dravet syndrome patients (Oakley et al., 2009; Cao et al., 2012), and the dominant Nav1.1 expression in GABAergic neurons has been proposed as the basis for it (Yamakawa 2016). These observations indicate that, rather than a toxicity of truncated proteins, haploinsufficiency of Nav1.1 (becoming half amount of normal Nav1.1 protein) is truly the basis for the pathology of Dravet syndrome. A mouse model with deletion of exon 1 exhibits no observable phenotype on strain 129 and spontaneous seizures with SUDEP after crossing with strain C57BL/6J (Miller et al., 2014) due to low expression of Gabra2 in strain C57BL/6J (Mulligan et al., 2019).
In addition to epileptic seizures and SUDEP, Dravet syndrome patients also show autistic features and cognitive impairments. Both SCN1A+/– (Han et al., 2012) and Scn1aRX/+ (Ito et al., 2013) mice had impairments of spatial memory and sociability. Multiple studies of conditional haplo-elimination of Nav1.1 in GABAergic inhibitory neurons in mice reproduced epileptic seizures, sudden death, autistic-like features, and cognitive impairments (Cheah et al., 2012; Dutton et al., 2013; Ogiwara et al., 2013; Tai et al., 2014; Tatsukawa et al., 2018) in that Nav1.1 haploinsufficiency in PV+ inhibitory neurons play a major role while that of SST+ inhibitory neurons is rather minor (Tatsukawa et al., 2018). These results suggest that Nav1.1 haploinsufficiency in PV+ inhibitory neurons is the primary cause for the disease phenotypes of Dravet syndrome, including not only epileptic seizures and SUDEP but also autistic features and cognitive impairments.
Mice with conditional Nav1.1 elimination in dorsal telencephalic (including neocortex, hippocampus, amygdala, olfactory bulb, etc.) excitatory neurons were viable and did not show epileptic seizures or any noticeable behavioral abnormalities (Ogiwara et al., 2013; Dutton et al., 2013). Mice with conditional heterozygous elimination of Nav1.1 in global inhibitory neurons showed much severer epileptic seizures and sudden death than that of SCN1A+/– mouse, and additional haplo-elimination of Nav1.1 in neocortical excitatory neurons largely improved their seizure and sudden death phenotypes (Ogiwara et al., 2013). These findings indicate protective or ameliorating effect of Nav1.1 haploinsufficiency in neocortical excitatory neurons, which are PT neurons (Yamagata et al., 2023), for the risk of epileptic seizures or SUDEP in Dravet syndrome.
Nav1.6-deficient mouse models Scn8a(med) and Scn8a(med-jo) have been reported to be more seizure resistant than wild-type littermates, and furthermore Scn1a(+/–); Scn8a(med-jo/+) double heterozygous mice displayed seizure thresholds against a convulsant that were comparable to wild-type littermates (Martin et al., 2007). These results indicated that Nav1.6 haploinsufficiency is also ameliorating for Dravet syndrome.
Kalume and colleagues (2013) reported that sudden death in Scn1a+/– mouse occurred immediately after generalized tonic-clonic seizures and ictal bradycardia (slower heart rate), that the cardiac and sudden death phenotypes were reproduced in mice with forebrain GABAergic neuron-specific, but not cardiac neuron-specific heterozygous elimination of Scn1a, and that the ictal bradycardia and sudden death were suppressed by atropine, a competitive antagonist for muscarinic acetylcholine receptors, and therefore counteracts against parasympathetic nervous system. N-methyl scopolamine, a muscarinic receptor antagonist that does not cross the blood–brain barrier, also eliminated the bradycardia and peripheral blockade of muscarinic receptors was therefore sufficient to reduce sudden death in the mice. These results indicated that epileptic seizures cause parasympathetic hyperactivity, which then cause ictal bradycardia and finally result in seizure-associated sudden death in Scn1a+/– mice.
Yamagata and colleagues (2023) recently reported that Nav1.1 is expressed in neocortical layer V pyramidal tract projection neurons (PT). Together with the above-mentioned parasympathetic hyperactivity (Kalume et al., 2013) and the ameliorating effect of Nav1.1 haploinsufficiency in neocortical excitatory neurons (Ogiwara et al., 2013) observed in the sudden death of Scn1a+/– mice, the pathological neural circuit was assumed to be as follows, “Nav1.1 haploinsufficiency in neocortical PV+ inhibitory neurons disinhibits and activates PT as well as subsequent parasympathetic neurons (vagus nerve), and consequently suppresses heart activity and results in cardiac arrest” (Fig. 44–3). Nav1.1 haploinsufficiency in cardiac muscle, although Nav1.5 is a major VGSC there, may also provide substrates for arrhythmias independent of innervation (Auerbach et al., 2013).
Pathological neural circuit for SUDEP in Scn1a+/- mouse. Nav1.1 haploinsufficiency in neocortical parvalbumin-positive inhibitory neurons (PV-IN) fails to suppress and therefore activates neocortical layer V pyramidal tract projection neurons (L5-PT) (more...)
Several studies of iPS cells (induced pluripotent stem cells) from Dravet syndrome patients harboring SCN1A loss-of-function variants were reported (Liu et al., 2013; Jiao et al., 2013; Higurashi et al., 2013). Two groups reported hyperexcitability of excitatory neurons derived from iPS cells of Dravet syndrome patients harboring SCN1A truncation variants and suggested it as the basis for epileptic seizures of the patients (Liu et al., 2013; Jiao et al., 2013). However, this proposal is rather contradictory to the previous observation that excitatory neuron-specific Nav1.1 haploinsufficiency is ameliorating for epilepsy and sudden death of Scn1a+/– mouse (Ogiwara et al., 2013). In contrast, the third group reported a significant impairment in action potential generation in inhibitory neurons derived from iPS cells of a Dravet syndrome patient (Higurashi et al., 2013), which is consistent with the previous results of mouse models indicating that Nav1.1 haploinsufficiency in PV+ inhibitory neurons is the primary cause for the disease (Yu et al., 2006; Ogiwara et al., 2007, 2013; Cheah et al., 2012; Dutton et al., 2013). A recent study of gene therapy, in which inhibitory neuron-specific upregulation of Nav1.1 is ameliorating for epilepsy and sudden death of SCN1A+/– mouse (Yamagata et al., 2020), further supports that Nav1.1 haploinsufficiency in GABAergic inhibitory neurons is the primary basis for the pathology of Dravet syndrome. A study reported unaltered interneuron firing in SCN1A+/– mouse using in vivo recording (De Stasi et al., 2016) may require further investigation to understand the whole story. A report of patient iPS cell-derived cardiac myocytes suggested an additional effect of Nav1.1 haploinsufficiency in heart for SUDEP in Dravet patients (Frasier et al., 2018). See also a relevant review for iPS and precision therapy for epilepsy (Du and Parent, 2015).
In a chemical mutagenesis screen, Zebrafish Nav1.1 (scn1Lab) mutants were identified as models for Dravet syndrome (Baraban et al., 2013). The mutants showed spontaneous abnormal electrographic activity, hyperactivity, and convulsive behaviors that were effectively attenuated by ketogenic diet, diazepam, valproate, potassium bromide, and stiripentol. They also reported that clemizole, a histamine blocker, was effective for seizures of the mutants. They further found that clemizole binds to serotonin receptors and a clinically approved serotonin receptor agonist (lorcaserin) was effective for Dravet syndrome patients (Griffin et al., 2017).
Based on the CRISPR-ON technique in which nuclease-deficient Cas9 was fused to transcription activators to accelerate the transcription of Scn1a, two groups (Colasante et al., 2020; Yamagata et al., 2020) reported amelioration of disease symptoms in SCN1A+/– mice, including epilepsy, sudden death, and anxiety. Especially, Yamagata and colleagues (2020) reported that GABAergic inhibitory neuron-specific upregulation of Nav1.1 is ameliorating for epilepsy and sudden death of SCN1A+/– mouse, which supported the notion that Nav1.1 haploinsufficiency in GABAergic inhibitory neurons is the primary cause for Dravet syndrome. In these studies, Colasante and colleagues (2020) reported that one gRNA in downstream Scn1a promoter was solely potent to activate the transcription, but contrarily to this Yamagata and colleagues(2020) reported that multiple gRNAs in upstream rather than downstream promoter are synergistically potent. Further studies are required to solve these contradictions and to make this CRISPR-ON technique clinically applicable for the treatment of Dravet syndrome.
The first precision therapy for SCN1A-linked Dravet syndrome to reach clinical trials was STK-001, an antisense oligonucleotide (ASO) introduced by Stoke Therapeutics (Han et al., 2020; see Chapter 75, this volume, for more information). STK-001 was developed using Targeted Augmentation of Nuclear Gene Output (TANGO) technology (Lim et al., 2020; Scharner & Aznarez, 2021), which targets a naturally occurring, nonproductive alternative splicing event, or poison exon, in SCN1A to specifically reduce levels of nonproductive mRNA and increase levels of productive mRNA and Nav1.1 VGSC protein. This approach, which prevents expression of exon 20N in SCN1A (Carvill et al., 2018), upregulates expression of the wild-type allele to compensate for the mutant allele in the context of SCN1A haploinsufficiency. Preclinical work showed that intracerebroventricular (ICV) administration of STK-001 to wild-type C57BL/6J mouse brain in vivo increased the expression of productive, full-length Scn1a mRNA and Nav1.1 protein (Carvill et al., 2018; Mistry et al., 2014). A single ICV dose of STK-001 at postnatal day 2 (P2) in the Scn1aTmKea (F1:129S-Scn1a+/– x C57BL/6J) mouse model of Dravet syndrome (Mistry et al., 2014), in which exon 1 of Scn1a is deleted, increased productive Scn1a mRNA and Nav1.1 protein expression. Importantly, this single-dose treatment also prevented SUDEP in 97% of Dravet syndrome mice up to 90 days following the single injected dose. Electroencephalogram (EEG) recording of Dravet syndrome mice injected with STK-001 at P2 showed a reduction in seizure frequency with a prolonged latency to first seizure. Intrathecal lumbar bolus administration of STK-001 was subsequently evaluated at two different dosages in nonhuman primates for safety, brain biodistribution, target engagement, and pharmacodynamics (Liau et al., 2019). Taken together, this preclinical work led to the MONARCH Phase 1/2a clinical trial for STK-001, an open-label study of children and adolescents ages 2 to 18 who have an established diagnosis of Dravet syndrome linked to a pathogenic variant in SCN1A (Laux et al., 2020). Importantly, the clinical utility of STK-001 is limited to Dravet syndrome patients with SCN1A variants that result in Nav1.1 haploinsufficiency, for example, truncating, nonsense, or frame shift variants, in which the mutant allele undergoes nonsense-mediated decay, as well as partial or whole gene SCN1A deletions (Marini et al., 2011). In contrast, this therapy is contraindicated for Dravet syndrome patients with missense SCN1A variants that result in the generation of Nav1.1 polypeptides, which may have maladaptive gain-of-function or dominant negative effects (Berecki et al., 2019; Sadleir et al., 2017), as TANGO-mediated increases in protein expression would likely increase disease severity. Nevertheless, introduction of this ASO was a major advance in precision therapeutics for Dravet syndrome patients.
Another VGSC α-subunit gene SCN2A encoding the α2-subunit Nav1.2 (Table 44–1) also shows variants in patients with a wide spectrum of epilepsies and neurodevelopmental disorders.
Among SCN2A missense variants, de novo forms are found in severe epilepsies, including early-infantile epileptic encephalopathy (EIEE) or alternatively named Ohtahara syndrome, West syndrome, and Lennox-Gastaut syndrome (Kamiya et al., 2004; Ogiwara et al., 2009; Wong et al., 2014; Touma et al., 2013; Nakamura et al., 2013; Epi4K Consortium et al., 2013; Dhamija et al., 2013; Carvill et al., 2013; Martin et al., 2014; Hackenberg et al., 2014; Shi et al., 2009; Fukasawa et al., 2014; Wolff et al., 2017), while inherited forms appear in milder epilepsies such as atypical GEFS+ (Sugawara et al., 2001b) and benign familial neonatal-infantile seizures (BFNIS; Heron et al., 2002; Berkovic et al., 2004; Striano et al., 2006; Herlenius et al., 2007; Wolff et al., 2017) or those with intermediate severity (Liao et al., 2010b).
SCN2A truncation or loss-of-function variants appear in patients with ASD or ID associated with milder or severe but later-onset epilepsies compared to Ohtahara syndrome. Association of autism and SCN2A has been first suggested by Weiss and colleagues (2003) in that a missense variant in autism patient was in the calmodulin binding site and was found to reduce binding affinity for calcium-bound calmodulin. Subsequently a de novo SCN2A nonsense variant, R102X, was found in a patient with intractable epilepsy, severe ID, and autism (Kamiya et al., 2004). Thereafter, multiple whole-exome sequencing studies using hundreds or thousands of autism patients’ genomes revealed multiple de novo loss-of-function variants of SCN2A, now ranked as one of the top genes for neurodevelopmental disorders, including sporadic ASD and ID occasionally even without epilepsy (Sanders et al., 2012; Rauch et al., 2012; Jiang et al., 2013; Iossifov et al., 2014; De Rubeis et al., 2014; Fitzgerald et al., 2014; Tavassoli et al., 2014; Hoischen et al., 2014; Deciphering Developmental Disorders Study, 2015; Johnson et al., 2016; Wolff et al., 2017; Satterstrom et al., 2020). However, it should be noted that SCN2A truncation mutations also appear in patients with severe epilepsies including West syndrome and Lennox-Gastaut syndrome (Kamiya et al., 2004; Wolff et al., 2017). SCN2A truncation or loss-of-function variants also appear in patients with SCZ (Fromer et al., 2014; Carroll et al., 2016; Suddaby et al., 2019).
Contrarily to SCN1A in which missense variants cause milder epilepsies and truncation variants cause severer ones, as mentioned above de novo SCN2A missense variants cause severe (though inherited ones cause mild) epilepsies while SCN2A truncation variants cause ASD, ID, or SCZ associated with milder, later-onset epilepsies or even without epilepsy. Actually in patch-clamp analyses of cultured cells, the de novo SCN2A missense variants appearing in Ohtahara syndrome showed gain-of- accelerated function effects (Wolff et al., 2017) while not only SCN2A truncation but also missense variants appearing in ASD, ID, or SCZ associated with milder, later-onset, or no epilepsies showed impairments in channel function (Ben-Shalom et al., 2017). These observations are consistent to the dominant expression of Nav1.2 in excitatory neurons and also to the observation that sodium channel blocker is ameliorating to Ohtahara syndrome patients with SCN2A missense mutations but aggravating to ASD or ID patients with SCN2A truncation mutations (Wolff et al., 2017). Functional effects of Nav1.2 missense variants found in BFNIS (Scalmani et al., 2006; Misra et al., 2008; Liao et al., 2010a; Lauxman et al., 2013) still remain ambiguous.
Although at early infantile stage (P0.5) in whole brain a major half of Nav1.2 (~60%) is expressed in inhibitory neurons (Ogiwara et al., 2018), at 6 weeks of age or later in neocortex and hippocampus a major amount of Nav1.2 (~95%) is expressed in excitatory neurons including a major population of neocortical and all of hippocampal ones and densely localized at their axons (initial segments, node of Ranvier or intermediate trunks, and presynapses; Hu et al., 2009; Lorincz and Nusser 2010; Tian et al., 2014; Yamagata et al., 2017; Ogiwara et al., 2018). As mentioned, a recent study revealed that among neocortical excitatory neurons, intratelencephalic cortico-striatal (iCS), cortico- thalamic (CT), and a subpopulation of cortico-cortical (CC) projection neurons are Nav1.2-positive (Yamagata et al., 2023; Fig. 44–1).
A minor amount of Nav1.2 is expressed in CGE-derived inhibitory neurons such as VIP+ inhibitory neurons but not in MGE-derived neurons such as PV+ inhibitory neurons (Li et al., 2014; Yamagata et al., 2017). Although a major population of SST+ inhibitory neurons is Nav1.1-positive, one study reported that a minor population (10%) of SST+ inhibitory neurons are Nav1.2-positive/Nv1.1-negative (Li et al., 2014). In striatum, Nav1.2 is densely expressed at unmyelinated axonal fibers of medial spiny neurons (MSN), a major population (~95%) of striatal neurons which are GABAergic (Miyazaki et al., 2014; Fig. 44–1). It is also of note that some other unmyelinated axonal fibers such as hippocampal mossy fibers and cerebellar parallel fibers are also densely Nav1.2-positive (Boiko et al., 2001; Yamagata et al., 2017; Ogiwara et al., 2018; Fig. 44–2). Dopaminergic neurons such as substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) have been reported to be positive for Nav1.2 but negative for Nav1.1 and Nav1.6 (Yang et al., 2019).
Nav1.2 and Nav1.6 are often co-expressed in same neurons, where Nav1.2 appears first at axon initial segments (AISs) and nodes of Ranvier. In later developmental stages, the Nav1.2 expression extends to distal axons and becomes less intense at AISs, while Nav1.6 remains high at AISs and nodes of Ranvier (Boiko et al., 2001, 2003; Liao et al., 2010a; Miyazaki et al., 2014; Ogiwara et al., 2018; Fig. 44–2). Nav1.2 and Nav1.6 are concentrated at the proximal and distal portions of the AIS, respectively, which has been proposed to be responsible for the backpropagation of action potentials (Hu et al., 2009; Lorincz and Nusser 2010; Martínez-Hernández et al., 2013; Tian et al., 2014). This situation between Nav1.2 and Nav1.6 is similar to that between Nav1.1 and Nav1.6.
Presynaptic but not postsynaptic expression of Nav1.2 has been reported in hippocampal CA1 stratum radiatum (Lorincz and Nusser, 2010) and cerebellar granule cells (; Fig. 44–2). Contrarily, in matured neocortical pyramidal cells, recent electrophysiological studies suggested dense Nav1.2 expression in dendrites but not axons (Spratt et al., 2019, 2021; Fig. 44–2), and further immunohistochemical verifications are awaited. The gradual increase of diffused immunosignals in mice at adult stages is also observed for Nav1.1 (Ogiwara et al., 2013), but not for Nav1.6 (Ogiwara et al., 2018, Supplementary Fig. 10) suggesting much less expressions of Nav1.6 at distal axons and dendrites (Fig. 44–2). Although Nav1.6 expression at dendrites has been reported previously, the amount is actually much lower (a factor of 35 to 80) than that at AIS (Lorincz and Nusser, 2010).
Scn2a homozygous knockout mouse (Scn2a–/–) was reported to be morphologically and organogenically indistinguishable from their wild-type littermates but died perinatally with severe hypoxia and massive neuronal apoptosis in the brainstem (Planells-Cases et al., 2000; Ogiwara et al., 2018). Scn2a heterozygous knockout mouse (Scn2a+/–) showed mild epilepsy including absence-like abnormal EEG (spike-and-wave discharges) associated with behavioral arrest (Ogiwara et al., 2018), which may mimic the mild epileptic phenotypes of ASD or ID patients harboring SCN2A truncation variants. Scn2a heterozygous knock-in mouse (Scn2a+/RX) harboring a nonsense variant R102X (RX), which was originally found as de novo in a patient with sporadic intractable epilepsy, severe ID, and autism (Kamiya et al., 2004), also showed epileptic phenotypes mostly similar to that of Scn2a+/– mouse (Ogiwara et al., 2018). Truncated Nav1.2 protein was undetectable in the Scn2a+/RX mouse presumably because of a degradation by nonsense-mediated mRNA decay, and it therefore suggested that Nav1.2 haploinsufficiency is the primary basis for the epileptic phenotypes of Scn2a+/– and Scn2a+/RX mice as well as those of patients with SCN2A truncation/loss-of-function variants. However, the epileptic phenotypes of Scn2a+/RX mouse are surely similar to but slightly more severe than that of Scn2a+/– mouse (Ogiwara et al., 2018). Similarly, the epileptic phenotypes of the patient with R102X mutation (Kamiya et al., 2004) are surely milder than those of Ohtahara syndrome but still more severe than those with SCN2A truncation/loss-of-function variants who showed ASD or ID. A toxic effect of Nav1.2- R102X truncated protein has previously been suggested (Kamiya et al., 2004), and therefore undetectable in Western blots, but still a residual amount of truncated protein may possibly affect and have resulted in the rather more severe phenotypes of the patient and the mouse with R102X mutation (Kamiya et al., 2004; Ogiwara et al., 2018).
The above-mentioned absence-like epileptic phenotypes of Scn2a+/– mouse were reproduced or even more accelerated (~ 9 times) in mice with dorsal telencephalic (e.g., neocortical and hippocampal) excitatory but not global inhibitory neuron-specific heterozygous elimination of Scn2a (Ogiwara et al., 2018). Together with another result (Miyamoto et al., 2019; see next section), this result suggests that epileptic phenotypes in Scn2a+/– mouse as well as those in patients with SCN2A truncation/loss-of- function variants are primarily caused by Nav1.2 haploinsufficiency in excitatory neurons.
It is of note that, although Nav1.2 and Nav1.6 are both well expressed in neocortical excitatory neurons, mice with homozygous elimination of Nav1.2 in dorsal telencephalic excitatory neurons are perinatally-lethal (Ogiwara et al., 2018), while those of Nav1.6 did not display obvious behavioral abnormalities or reduced lifespan (Makinson et al., 2017). Mice with Nav1.6 global haploinsufficiency also cause absence epilepsy (Papale et al., 2009); however, absence seizures were not reproduced in mice with dorsal telencephalic excitatory neuron- specific elimination of Nav1.6 but in those with thalamic reticular nucleus neuron-specific ones (Makinson et al., 2017). These observations indicate largely distinct roles of Nav1.2 and Nav1.6 in neocortical excitatory neurons in regard to epilepsy and related lethality.
Variants of SCN2A as well as STXBP1 which encodes a synaptic protein Munc18-1 are both observed in Ohtahara syndrome (Saitsu et al., 2008; Ogiwara et al., 2009; Nakamura et al., 2013), and therefore a common pathological mechanism was suspected. Actually, impaired cortico-striatal excitatory neurotransmission has been shown as a common cause for epilepsy in both Scn2a+/– and Stxbp1+/– mice (Miyamoto et al., 2019), by using a number of experimental data, including the following: (1) Absence seizures occurred in both Scn2a+/– and Stxbp1+/– mice, and reproduced in mice with neocortical excitatory neuron-specific as well as in those with cortico-striatal projection neuron-specific conditional elimination of Stxbp1 or Scn2a, but not in mice with elimination in inhibitory neurons or cortico-thalamic projection neurons. (2) A “NeuRet” retrograde virus system also confirmed that cortico-striatal projection neuron-specific deletion of Stxbp1 reproduced absence seizures in mice. (3) Pharmacological suppression of striatal fast-spiking inhibitory neurons (FSIs) by NASPM, a selective blocker of AMPA receptors containing GluA4, triggers not only absence seizures but also dyskinesia, myoclonia, and tonic-clonic convulsive seizures in wild-type mice in a dose-dependent manner. Conversely, pharmacogenetic activation of striatal FSIs using the DREADD system rescued the epileptic phenotype of Stxbp1- deficient mice. (4) Direct recordings of neuronal activity from striatal FSIs and medium spiny neurons in vivo revealed that striatal FSI neuronal activity selectively and sharply declined at the onset of absence seizures in Stxbp1-deficient mice. Altogether, these results show that impaired cortico-striatal FSIs is a common pathological neural circuit responsible for the epileptic phenotypes including absence seizures in both Scn2a+–- and Stxbp1+/– mice. A recent finding of Nav1.2 expression in iCS neurons and Nav1.1 in PT neurons (Yamagata et al., 2023; Fig. 44–1), together with the observations that striatal direct and indirect-pathway medium spiny neurons (dMSNs and iMSNs) are predominantly targeted by IT and PT neurons, respectively (Lei et al., 2004; Deng et al., 2015), further refined this pathological circuit (Fig. 44–4), in which the hyperactivation of iMSNs is guaranteed by the intact excitatory input from Nav1.1-positive PT neurons and the decrease of Nav1.2+ iCS excitatory input onto dMSN may decrease its suppression onto substantia nigra pars reticulata/internal globus pallidus (SNr/GPi) in Scn2a+/– mouse, and both of these would contribute to the oversuppression onto thalamus. The much more severe epileptic phenotype (~9× more frequent absence-like seizures) of Scn2afl/+/Emx1-Cre dorsal telencephalic excitatory neuron-specific Nav1.2 haplodeficient mouse than that of Scn2aRX/+, which is equivalent to Scn2a+/– mouse (Ogiwara et al., 2018), suggests that full amount Nav1.2 remained in MSNs in Scn2afl/+/Emx1-Cre mouse is aggravating and further supports that the ultimate consequence in inhibitory input from iMSN onto GPe is increase and that the indirect pathway is still dominant in this pathological circuit. The circuit may provide answers to the multiple enigmatic questions: (1) Why and how does a VGSC haplodeficiency in excitatory neurons cause epilepsy? (2) Why has impaired excitatory neurotransmission been commonly observed in multiple animal models of absence epilepsy (Caddick et al., 1999)? (3) What is the true neural circuit for absence epilepsy? and (4) Why do neurodevelopmental disorders such as ASD often associate with epilepsy?
Pathological neural circuit for epilepsy in Scn2a+/- mouse. Impaired cortico-striatal fast-spiking inhibitory neurons causes epileptic phenotypes including absence seizures in Scn2a+/- mouse. See section 3-4-2 for detailed explanation. PT: pyramidal (more...)
In adult mice with conditional severe or full Scn2a deficiencies, recent studies reported cell-autonomous “paradoxical hyperexcitability” in prefrontal pyramidal cells (Spratt et al., 2021) or striatal medial spiny neurons (MSNs; Zhang et al., 2021), and proposed as plausible bases for epilepsies in patients with SCN2A loss-of-function mutations. However, the former study (Spratt et al., 2021) is rather conflicting with the above-mentioned observations of impaired cortico-striatal excitatory neurotransmission in Scn2a+/– mice (Miyamoto et al., 2019), as well as a recent report of reduced neuronal excitability in forebrain pyramidal neurons and decreased excitatory synaptic inputs to pyramidal neurons in medial prefrontal cortex and amygdala in adult mice with Scn2a heterozygous loss-of-function mutation (Wang et al., 2021). The latter (Zhang et al., 2021) is also inconsistent with the previous observation that the frequencies of SWDs (spike and wave discharges) as epileptic phenotype of Scn2a(+/–) mice were nine times more accelerated in mice with heterozygous conditional elimination of Scn2a only in neocortical excitatory neurons (Miyamoto et al., 2019), suggesting that haplodeficiency of Nav1.2 in MSN (Fig. 44–4) is rather ameliorating for the epileptic phenotype. Further studies are required to figure out actual implications of these “paradoxical hyperexcitability” of neurons for the pathological phenotypes in mice and patients with Nav1.2 deficiency.
Similar to the patients with ID and ASD harboring SCN2A truncation variants, Scn2a+/– mouse showed impairments in spatial memory and hippocampal replay (Middleton et al., 2018), impaired social memory, accelerated fear and impaired fear extinction, anxiety, and hyperactivity, which was reproduced by heterozygous inactivation of Scn2a in dorsal-telencephalic excitatory neurons but not in inhibitory neurons (Tatsukawa et al., 2019). Together with the observations that pharmacological augmentations of excitatory neurotransmission ameliorated the hyperactivity of Scn2a+/– (Tatsukawa et al., 2019) as well as the aggression of Stxbp1+/– mice (Miyamoto et al., 2017), these results suggest that impaired excitatory neurotransmission commonly plays a critical role in the pathology of neurodevelopmental disorders. ASD genes have been shown to be preferentially expressed in late mid-fetal prefrontal cortex and have concentrated expression in layer 5/6 cortical projection neurons (Willsey et al., 2013). Striatal abnormalities have been commonly reported in multiple animal models of ASD (Fuccillo 2016). Both SCN2A and TBR1 genes are ranked as the top genes for ASD (Sanders et al., 2015). The co-expression of Nav1.2 and TBR1 in iCS neurons (Yamagata et al., 2023; Fig. 44–1) would be of interest.
A transgenic mouse expressing Nav1.2 with a gain-of-function variant showed seizures and behavioral abnormalities (Kearney et al., 2001; Li et al., 2021), which may mimic the severe epileptic phenotypes of Ohtahara syndrome. However, although variants of SCN2A and STXBP1 are commonly observed in Ohtahara syndrome, the SCN2A variants are gain-of-accelerated function missense while those of STXBP1 are truncations or loss-of-function missense (Nakamura et al., 2013). One of the remaining questions is therefore why the opposite effects of SCN2A and Stxbp1 variants can cause a common disease phenotype, Ohtahara syndrome. Further studies are still warranted to be pursued.
SCN8A encodes the voltage-gated sodium channel Nav1.6, one of the most abundant sodium channels in the mammalian CNS. The clinical spectrum associated with SCN8A disorders has been growing with the increase in diagnostic exome sequencing at the time of seizure onset in newborns and infants. A wide range of clinical severity results from gain-of-function and loss-of-function mutations of SCN8A. The SCN8A disorders overlap with those for SCN1A and SCN2A, and they include seizures, ID, behavioral disorders, and movement disorders. Approximately 400 pathogenic variants were identified by the end of 2020. Channel function has been characterized for approximately 50 patient variants, mouse models are available for 4 patient variants, as well as several null alleles, and 1 zebrafish knockout line has been described.
Several hundred mutations of SCN8A mutations have been discovered in individuals with developmental and epileptic encephalopathy (DEE), the most severe disorder associated with this gene (Meisler et al 2016; Gardella et al., 2018; Larsen et al., 2015; Schreiber et al., 2020). This disorder is the result of de novo missense mutations that occur early in development. In a few cases, the pathogenic variant is inherited from a mosaic parent. The average age of seizure onset in SCN8A DEE is 4 months, but seizures can begin within the first few days after birth or even prenatally. In addition to drug-resistant seizures, patients exhibit developmental delay and impaired cognition and movement, and approximately half of affected individuals do not achieve intentional mobility. Detailed descriptions of the clinical course are available in the four papers cited above. Recurrent mutations account for approximately one-third of cases.
There are several examples of SCN8A mutations in individuals with cognitive impairment in the absence of seizures. This condition is designated OMIM 614306 and appears to be associated with either loss-of-function (SCN8A+/–) or partial loss-of-function variants. Impaired cognition and attention-deficit/hyperactivity disorder (ADHD) were first described in four heterozygous family members carrying a truncation mutation in the pore loop of domain IV (Trudeau et al., 2006). Wagnon et al. (2017) described two unrelated children with impaired intellectual development, IQs of 73 and 56, and developmental delay but without seizures. The children carried novel missense variants that altered the conserved residues p.Gly964Arg in transmembrane segment D2S6 and p.Glu1218Lys in transmembrane segment D3S1. Both variants failed to generate channel activity in transfected cells. Another recent report described patients with biallelic mutations. In these families, the heterozygous parents carrying loss-of-function alleles demonstrated cognitive impairment (Wengert et al., 2019). ID in the absence of seizures was also described for the partial loss-of-function variants p.Arg1620Leu and p.Gly964Arg (Liu et al., 2019). Finally, a 10-year-old boy with IQ of 59 and language delay, but no seizures, carried the de novo missense mutation L1379P in the pore loop of domain 3, a frequent site of loss-of-function mutations (E. K. Bijlsma, personal communication).
As the major channel localized at nodes of Ranvier in myelinated axons, Nav1.6 is required for propagation of action potentials to the neuromuscular junction. Ataxia and other impairments are common as comorbidities in SCN8A DEE. In rare cases, an SCN8A variant is associated with impaired movement in the absence of seizures. For example, the variant p.Pro1719Arg in the pore loop of domain IV was identified in five family members with autosomal dominant upper-limb isolated myoclonus; these individuals exhibited no seizures or cognitive impairment (Wagnon et al., 2018). Functional analysis of the mutant channel revealed greatly reduced channel activity with normal gating properties. In another study of 17 children with neuromuscular disease, an individual with DEE and biallelic de novo mutations of SCN8A was identified: p.Ala1622Asp in DIVS4 and p.Val1874Leu in the cytoplasmic C-terminus (Hoei-Hansen et al., 2021). The p.Ala1622Asp variant introduces a negative charge adjacent to a key positively charged arginine in a voltage-sensing transmembrane segment and is likely to be deleterious. Interestingly, peripheral nerve function was normal in this patient, and the motor defect was attributed to central origin. SCN8A variants have also been identified in patients with paroxysmal dyskinesia and seizures (Gardella et al., 2016; Tian et al., 2018).
Autism-like behaviors occur as comorbidities in many patients with SCN8A DEE, but few if any variants of SCN8A have been reported in individuals with a primary diagnosis of autism.
Functional studies of patient variants of Scn8a often find a combination of loss-of-function and gain-of-function effects. The complexity of these features makes it difficult to classify variants as simply gain-of-function or loss-of-function, or to compare their relative severity. Nonetheless, some relationships between genotype and phenotype can be recognized.
In patients with DEE, missense mutations of Nav1.6 are the rule. Variants occur throughout the four domains of the channel, predominantly in transmembrane segments, the inactivation gate, and the cytoplasmic N-terminus and C- terminus. These variants exhibit a variety of gain-of-function characteristics that lead to elevated firing in neuronal expression systems. The most common gain-of-function features are shown in Figure 44–5: hyperpolarizing shift in voltage-dependence of channel activation, delayed channel inactivation, and elevated persistent current. Another common effect is elevated resurgent current, which is important in generation of repetitive firing (Pan and Cummins, 2020). Each of these changes results in elevated firing when studied in cultured neurons. In vivo mouse models of patient mutations N1768D (elevated persistent current) and R1872W (delayed inactivation) result in spontaneous seizures and premature lethality (Wagnon et al., 2015a; Bunton-Stashyshn et al., 2019).
Effects of gain-of-function mutations of SCN8A in patients with epileptic encephalopathy. (A) The Thr767Ile substitution in transmembrane segment S1 of domain II causes a hyperpolarizing shift in the voltage dependence of activation, resulting in premature (more...)
Consistent with a gain-of-function mechanism, reduced expression of Scn8a was therapeutic in both mouse models. Mice carrying the patient mutation R1872W (Bunton-Stashyshn et al., 2019; Wagnon et al., 2015b) were treated with a gapmer ASO that led to mRNA degradation (Lenk, 2020). The abundance of the Scn8a mRNA was reduced to approximately 50% of wild-type level, resulting in increased survival from 2 weeks to 9 weeks in mutants receiving ASO (Lenk, 2020). Reduction of SCN8A expression in the CNS of patients is thus a potential therapeutic intervention.
In population databases such as gnomAD, there is a large deficiency of protein truncation mutations of SCN8A, indicating that loss-of-function alleles are disfavored by natural selection. In gnomAD, the probability that haplosufficiency is not tolerated (pLI) is 1.0 for SCN8A, with 80 loss-of-function variants predicted and only 5 observed in gnomAD. The few loss-of-function variants that have been identified are described below. It is possible that the SCN8A+/– genotype causes prenatal lethality in human, although this is not the case in the mouse (Burgess et al., 1995). It is more likely that the patient cohorts with heterozygous null mutations have not yet been identified.
A number of heterozygous loss-of-function variants (SCN8A+/–) were described in epilepsy patients (Johannesen et al., 2022). Unlike the de novo gain-of-function mutants, the loss-of- function variants are often inherited within families and are less severe. Absence seizures are the most common seizure type, observed in 64% of individuals with loss-of-function mutations. ID is most consistently associated with loss-of-function of SCN8A; the ID may occur in isolation or together with absence seizures (Wengert et al., 2019). Michael Hammer and colleagues demonstrated that gain-of-function and loss-of-function variants are clearly distinguished by time of onset and clinical phenotypes (Hack et al., 2023).
It is useful to make a distinction between partial loss-of-function alleles and complete loss-of-function alleles, since their clinical consequences can be quite different. For example, one partial loss-of-function variant resulted in neuromuscular disease without seizures or ID (Wagnon et al., 2017).
Nav1.6 expression is widespread in neurons. Transcripts of Scn8a have been detected in both inhibitory and excitatory neurons by single-cell RNAseq (Du et al., 2020) and are detected throughout the brain by in situ hybridization (Allen Brain Atlas). Recordings from mutant mice lacking Scn8a demonstrate that firing is reduced in many types of neurons, including both excitatory and inhibitory neurons (O’Brien and Meisler, 2013). The important role of Nav1.6 in motor neurons is evidenced by the sprouting of nerve terminals at the neuromuscular junction in sciatic nerve of mice lacking Scn8a.
Nav1.6 is concentrated at two subcellular sites, the axon initial segment (AIS) and the nodes of Ranvier of myelinated neurons. Localization to the AIS depends on interaction with several proteins, including ankyrin G, which binds a conserved element in intracellular loop 2 (Jenkins and Bennett, 2001); Map1b, which binds in the cytoplasmic N-terminal domain (O’Brien et al., 2012b; Solé et al., 2019); and ARF class II trafficking factors (Hosoi et al., 2019). SCN8A variants that disrupt the binding sites for these proteins are predicted to be pathogenic. Mutation of the ARF class II trafficking factors in the mouse reduces the abundance of Nav1.6 at the AIS of cerebellar Purkinje cells; these mice are considered a model of Parkinson disease (Hosoi et al., 2019). Variants that disrupt Nav1.6 phosphorylation at sites in intracellular loop 1 may also be pathogenic (Zybura et al., 2020).
There is an important difference in alternative splicing of Scn8a between neurons and other cell types (Plummer et al., 1997). Neuron-specific transcripts include the alternative exon 18A with an open reading frame; transcripts containing exon 18A encode the full-length channel protein. In non-neuronal cells, including glia, there is inclusion of the alternative exon 18N that introduces an in-frame stop codon. In current terminology, exon 18N is a “poison exon.” Transcripts containing exon 18N encode a protein that is truncated in domain 3. Transcripts containing exon 18N are susceptible to nonsense-mediated decay (O’Brien et al., 2012a). In purified astrocytes and oligodendrocytes from P1 hippocampus, we detected Scn8a transcripts containing exon 18N and transcripts that skip exon 18 completely, but no transcripts containing exon 18A (O’Brien et al., 2012a). These data indicate that functional Nav1.6 may not be produced by astrocytes or oligodendrocytes, a point of some uncertainty.
The developmental switch from exon 18N (neonatal) to exon 18A (adult) occurs during late fetal and early postnatal life in mouse and human. The time course has been investigated in human and mouse by qRT-PCR (Plummer et al., 1998; O’Brien et al., 2012a) and more recently by high-throughput sequencing of RNA from human prefrontal cortex and mouse brain (Liang et al., 2021).
Three mouse models with knock-in of human DEE mutations in the mouse Scn8a gene have been described. The p.Asn1768Asp variant represents a human gain-of-function mutation with a high level of persistent current (Veeramah et al., 2012). The knock-in mouse has onset of spontaneous seizures at 2 to 4 months of age, and death within a month after seizure onset (Wagnon et al., 2015). Excess spontaneous firing of hippocampal neurons may contribute to seizure onset in these mice, which also exhibit cardiac arrythmia (Frasier et al., 2016) and cortical hyperexcitability (Ottolini et al., 2017).
The conditional mouse model of the recurrent DEE mutation p.Arg1872Trp with delayed channel inactivation exhibits a more severe phenotype than p.Asn1768Asp, consistent with the more severe clinical course in patients (Bunton-Stashyshn et al., 2019). Global expression of this variant results in seizure onset at 2 weeks of age, followed by death, usually within a few minutes of the initial seizure. Activation of the p.Arg1872Trp mutant in the adult mouse using a tamoxifen-dependent CRE also generates spontaneous seizures and death within several weeks. Thus, susceptibility to SCN8A DEE persists into adulthood and does not require expression during early development.
To assess the level of mutant channel required to induce seizures, adult mice carrying the conditional R1872W variant and the tamoxifen-inducible CAG-CRE-ER were treated with a 70-fold range of tamoxifen (Yu et al., 2021). Seizures were consistently generated after activation of the R1872W variant in more than 16% of neurons, a high threshold for generation of seizures. Mice with expression of R1872W in <10% of neurons were susceptibility to enhanced seizure induction by kainate or acoustic stimulation.
These two gain-of-function mouse models were used to evaluate the effect of reducing Scn8a expression with a gapmer antisense oligonucleotide. The Scn8a ASO binds the 3′ UTR and leads to mRNA degradation, resulting in approximately 50% reduction in transcript abundance (Lenk et al., 2020). Survival of the p.Arg1872Trp mouse was extended from 2 weeks to 9 weeks by treatment with this ASO, providing proof of principle that targeting the Scn8a mRNA is an effective therapy for SCN8A DEE.
A recently described mouse model expresses the gain-of-function pathogenic variant p.Arg1620Leu that was identified in a patient with autism, ID, and behavioral seizures (Wong et al., 2021). The heterozygous mice reproduced autism-related phenotypes, including hyperactivity and learning and social deficits, without spontaneous seizures, providing a new model for analysis of the behavioral features of SCN8A DEE.
The conditional mouse mutant p.Arg1872Trp (Bunton-Stashyshn et al., 2019) can be used to evaluate the effect of the gain-of-function mutation in different classes of neurons, using targeted CRE constructs. Restriction of expression to excitatory forebrain neurons using Emx1-CRE resulted in seizures and death, while expression in inhibitory neurons was ineffective. This work identified excitatory neurons as targets for therapeutic intervention. A CRE specific for somatostatin-expressing inhibitory neurons also generated seizures, which may be explained by depolarization block leading to reduced activity of these inhibitory neurons (Wengert et al., 2021).
Analysis of pathogenic mutations on different mouse strain genetic backgrounds can identify “modifier genes” that modulate the severity of the monogenic disorder (Meisler et al., 2021). Strain C57BL/6J carries a splice site mutation in the Gabra2 gene that reduces expression to 25% of the level in other strains (Mulligan et al., 2019). The resulting deficit in the α2 subunit of the GABAA receptor accelerates the onset of seizures in mice with the p.Arg1872Trp mutation of Scn8a (Yu et al., 2020). Correction of the splice site mutation increased the survival of mice expressing the Scn8aR1872W allele in forebrain excitatory neurons from 27 days to 66 days (2.4×).
Several spontaneous loss-of-function mutations of mouse Scn8a were identified by their recessive lethal movement disorders (O’Brien and Meisler, 2013). In the Scn8amed-tg mouse, the random insertion of an alpha-fetoprotein transgene into the Scn8a gene resulted in a 20 kb deletion and a complete loss of function (Kohrman et al., 1995; Burgess et al., 1995). Scn8amed-tg/med-tg homozygotes exhibit failure of the neuromuscular junction beginning at 2 weeks of age, with hind limb hypotonia leading to lethality at 3 weeks of age. Scn8amed-tg/+ heterozygotes exhibit absence seizures (Papale et al., 2009) and elevated anxiety (McKinney et al., 2008) but do not have convulsive seizures or neuromuscular impairment. Loss of function of mouse Scn8a reduces neuronal activity, repetitive firing, and resurgent current in many types of neurons (O’Brien and Meisler, 2013; Raman et al., 1997). Three ENU-induced missense mutations in the pore loop of domain 3 have also been characterized (Buchner et al., 2004).
A floxed allele of mouse Scn8a has been used to test the functional consequences of knockout in specific types of neurons. Knockout of Scn8a in cerebellar Purkinje cells and granule cells result in impaired function on the rotarod test of motor coordination (Levin et al., 2006) and impaired learning (Woodruff-Pak et al., 2006). Inactivation of Scn8a in thalamic reticular nucleus neurons resulted in spike wave discharges and absence epilepsy of thalamic origin (Makinson et al., 2017).
An interesting splice site mutation in the Scn8amedJ mouse reduces the expression level of the wild-type channel to 5% of normal in strain C57BL/6J and 10% of normal in strain C3H (Kearney et al., 2001; Buchner et al., 2003). The major phenotype of Scn8amedJ/medJ homozygotes is dystonia with early onset and significant progression resulting in severe impairment by 1 year of age (Sprunger et al., 1999). It will be interesting to determine whether a recently identified SCN8A mutation in dystonia patients also exhibits partial loss of function (Sarmiento and Mencacci, 2021).
To gain insight into the mechanism of sudden death in SCN8A encephalopathy, Wenker et al carried out cardiorespiratory monitoring of knock-in mice with the patient mutations N1768D and R1872W (Wenker et al., 2021). In both models, a tonic phase seizure was followed by initiation of long-lasting apnea and eventual cessation of cardiac function. A continuous tonic diaphragm contraction during the tonic phase of the seizure may contribute to apnea by preventing exhalation. Recording of a nonfatal seizure in a human patient with the Leu257Val variant also demonstrated an extended tonic phase accompanied by apnea, which was suggested to be typical of SCN8A-associated sudden death. In a 14-month-old patient, in response to monthly seizures with apnea, brachycardia, and ictal asystole indicative of risk of SUDEP, a cardiac pacemaker was implanted; this patient carried the de novo mutation p.Met1645Thr in the linker between S4 and S5 in domain IV (Negishi et al., 2021).
Neuronal cultures derived from patient fibroblasts permit studies of pathogenesis in which the contributions of all variants in the patient genome are included. In studies of iPSC-derived neurons from three individuals with Dravet syndrome, elevated sodium currents were observed, suggesting that there might be compensation for the reduction of SCN1A by upregulation of other channels such as SCN8A (Liu et al., 2013). Recent studies of patient-derived neurons with three distinct SCN8A mutations detected elevated persistent current or elevated resurgent current due to the mutant channels (Tidball et al., 2020). Riluzole suppressed spontaneous firing in the iPSC-derived neurons and also resulted in improvement in the patients, demonstrating the promising role of iPSCs in development of precision therapies.
The zebrafish genome contains two copies of an ortholog of SCN8A whose expression is limited to the nervous system (Zakon, 2012). Evolutionarily conserved features of the zebrafish and human SCN8A genes include a noncoding sequence element in a 5′ noncoding exon (Drews, 2007) and the presence of alternatively spliced exons 18A and 18N (Plummer et al., 1997). Knockout of zebrafish Nav1.6 results in abnormal development of motor neurons (Pineda et al., 2006). In a fish model of Dravet syndrome, inhibition of Nav1.6 compensated for haploinsufficiency of Nav1.1 (Weuring et al., 2020), in agreement with observations in the mouse (Lenk et al., 2020). A zebrafish model of Scn8a epilepsy has not been described.
SCN1B, encoding the VGSC non-pore-forming transmembrane β1 subunit and the secreted splice variant, β1B (L.L. Isom et al., 1992; Kazen-Gillespie et al., 2000), was one of the first genes to be linked to epilepsy through discovery in 1998 of the inherited, monoallelic variant SCN1B-p.C121W, linked to genetic epilepsy with febrile seizures plus type 1 (GEFS+; Wallace et al., 1998). Monoallelic SCN1B variants were later also linked to cardiac arrhythmia, including Brugada syndrome, Long-QT syndrome, and atrial fibrillation (O’Malley & Isom, 2015). Genetic evaluation of additional epilepsy patients showed that inherited, biallelic variants in SCN1B are linked to DEE52 (OMIM), diagnosed either as Dravet syndrome or the more severe early infantile developmental and epileptic encephalopathy (EI-DEE). Patients with homozygous SCN1B variants are rare. The first patient, from consanguineous parents, was reported in 2009 by Patino et al. (Patino et al., 2009). Since that time, additional patients have been reported in the literature (Aeby et al., 2019; Darras et al., 2019; Ogiwara et al., 2012; Patino et al., 2009; Ramadan et al., 2017; Scala et al., 2021), including reports of multiple affected siblings in several unrelated consanguineous families. Other patients have been reported by online parent support groups (e.g., SCN1B Diagnosis, https://www.facebook.com/groups/557303204788099), including patients from nonconsanguineous parents.
While the majority of SCN1B DEE patients have been diagnosed with Dravet syndrome, their symptoms are generally more severe than the typical Dravet syndrome course. The first reported SCN1B DEE patient, with the variant SCN1B-p.R125C (Patino et al., 2009), presented with an abnormal EEG at 3 months of age (Fig. 44–6). While the neonatal period was not described, tetrapyramidal syndrome with global hypotonia was noted at 13 months. The second reported patient, with the variant SCN1B-p.I106F (Ogiwara et al., 2012), was more similar to Dravet syndrome in terms of the timing of seizure onset, but developmental decline occurred earlier than typical Dravet syndrome. Ramadan et al. reported five other cases of SCN1B-linked DEE from three consanguineous families (variants SCN1B-p.Y119D and SCN1B-c.449-2A>G; Ramadan et al., 2017). Here, seizure onset and developmental decline began at 1 to 2 months of age. The phenotype of one of the three families included microcephaly, dysmorphic features, and brain malformations. In the case reported by Aeby et al. (variant SCN1B-p.R85C; Aeby et al., 2019), development was abnormal at birth. Seizures were recorded at 3 months of age, and the disease course included profound motor and cognitive delay. This group suggested that the term “EI-DEE” was more appropriate for patients with homozygous SCN1B variants because their neurological prognosis may be caused by preexisting brain dysfunction that leads to severe epilepsy. Similar to Dravet syndrome, patients with homozygous SCN1B variants are often refractory to antiseizure medications, although for one patient, fenfluramine was effective in reducing seizure severity, suggesting possible therapeutic benefit for others (Aeby et al., 2019).
Variants in SCN1B are linked to epilepsy, cardiac disease, and sudden cardiac death. A. SCN1B encodes two splice forms: a transmembrane β1 subunit (left) and a secreted β1B subunit (right). Both subunits contain a common N-terminus and (more...)
VGSC β1/β1B subunits are developmentally regulated immunoglobulin superfamily cell adhesion molecules (Ig-CAMs) and ion channel modulators that play critical roles in the regulation of excitability (O’Malley & Isom, 2015). VGSCs were purified as heterotrimeric complexes of α and β subunits from mammalian brain (Hartshorne & Catterall, 1981). This work showed that a central α subunit forms the ion-conducting pore and is associated with two different non-pore-forming β subunits, one associated through noncovalent interactions (β1 or β3) and one associated through disulfide bonds (β2 or β4; Catterall, 2012; Messner & Catterall, 1985). Originally characterized as “auxiliary,” β1 subunits are now known to be dynamic, multifunctional molecules that engage in diverse and essential roles in multiple tissues (Bouza & Isom, 2018; Brackenbury & Isom, 2011; O’Malley & Isom, 2015). The ability of β1/β1B subunits to associate with multiple ion channel types and participate in both conducting and nonconducting roles may explain the severity and complexity of SCN1B-linked channelopathies.
The canonical role of β1 is to function in concert with VGSC α subunits to promote channel trafficking to the plasma membrane and to modulate channel biophysical properties. β1 modulates α subunit function in heterologous cells, with effects on peak sodium current (INa) density, voltage dependence of activation and inactivation, rate of inactivation, entry into slow inactivation, and persistent and resurgent INa (Calhoun & Isom, 2014). In general, co-expression of β1 in heterologous cells leads to increased α subunit cell surface expression and increased INa (Calhoun & Isom, 2014; L.L. Isom et al., 1992, 1995). β1-mediated shifts in voltage-dependent properties are cell-type and VGSC α subunit specific (Calhoun & Isom, 2014). In addition to VGSCs, β1 interacts with and functionally modulates some potassium channels (Bouza et al., 2021; Deschenes et al., 2008; Deschenes & Tomaselli, 2002; Marionneau et al., 2012; Nguyen et al., 2012) and calcium currents are reduced in Scn1b null mice (Bouza et al., 2021). This association of β1 with sodium, potassium, and calcium channels may provide a means for channel cross-talk in neurons and other excitable cells in vivo.
In addition to their roles in ion conduction, VGSC β1 subunits function as IgCAMs (Isom & Catterall, 1996; O’Malley & Isom, 2015). The breadth of β1 function hinges on the extracellular Ig domain, which is a key structural motif common to all members of this family of proteins, including the other VGSC β subunits (Brackenbury & Isom, 2011; Isom & Catterall, 1996; O’Malley & Isom, 2015). IgCAM-mediated cell-cell and cell-matrix adhesive functions are critical to brain development, including the processes of neurite outgrowth, axon pathfinding, fasciculation, and cell migration (Brackenbury & Isom, 2011; O’Malley & Isom, 2015). β1 subunits participate in trans homophilic cell-cell adhesion in cell culture (Malhotra et al., 2000) and drive cerebellar granule neuron process outgrowth through this mechanism (Davis et al., 2004). Integrity of the Ig loop is also critical for β1-mediated INa modulation in vivo (Kruger et al., 2016), making this domain multifunctional. Importantly, the majority of SCN1B-linked disease variants are located in the Ig domain, suggesting that cell-cell adhesion is clinically relevant (O’Malley & Isom, 2015; Fig. 44–6).
VGSC β1 subunits are substrates for sequential regulated intramembrane proteolysis (RIP) by the β-site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) and γ-secretase, resulting in shedding of the extracellular domain (ECD), which functions as a soluble ligand for cell adhesion (Davis et al., 2004; Patino et al., 2011), and generation of a soluble intracellular domain (ICD) that is translocated to the nucleus (Bouza et al., 2021; Wong et al., 2005). An unbiased RNA-seq approach identified a subset of genes, including those encoding ion channels, that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b null tissue which, by definition, lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo (Bouza et al., 2021). Thus, the β1-ICD may participate in transcriptional regulation in vivo and the absence of β1 RIP and downstream signaling may contribute to disease mechanisms in patients with loss-of-function SCN1B variants.
SCN1B is expressed as two developmentally regulated splice variants, β1 and β1B, which are both expressed in brain and heart (Kazen-Gillespie et al., 2000; Patino et al., 2011). β1B is generated by read-through from exon 3 to intron 3, which contains an open reading frame, termination codon, and polyadenylation site (Fig. 44–6). The amino acid sequence encoded by the retained intron is species specific, which led to the original confusion over naming this subunit (rat: β1A vs. human: β1B). Exons 1, 2, and 3 of SCN1B, encoding the N-terminal Ig domain, are common to both splice variants. While β1 subunits are type 1 transmembrane molecules, β1B is a soluble protein, similar to the β1 ECD that is generated by RIP (Bouza et al., 2021; Patino et al., 2011), which functions as a ligand for cell adhesion (Davis et al., 2004; Patino et al., 2011). In brain, β1B mRNA is expressed predominantly during embryonic development. In contrast, this subunit mRNA is expressed at all developmental time points in heart. β1B co-expression with VGSC α subunits in heterologous cells resulted in alteration of Nav1.3-generated INa, suggesting that β1B may modulate INa in developing brain (Patino et al., 2011). While SCN1B variants located in the Ig domain affect both β1 and β1B, some SCN1B variants, including SCN1B p.G257R, which is linked to epilepsy, are located in the retained intron region unique to β1B (Patino et al., 2011).
Heterologous expression studies of the SCN1B variants linked to epilepsy have shown, in general, loss of function. β1B-p.G257R and β1-p.R125C are retained inside the cell and unable to modulate INa (Patino et al., 2009, 2011). Cell surface biotinylation experiments with β1-p.R125C showed the absence of cell surface protein in cells cultured at 37, but appearance at the cell surface when cells were grown at a more permissive temperature of 27°C, suggesting a protein folding defect (Patino et al., 2009). In support of this hypothesis, co-expression of VGSC α and β1-p.R125C subunits in Xenopus oocytes, which are maintained at 18°C, resulted in INa modulation by the mutant β1 subunit, suggesting that therapeutic agents designed to normalize protein folding and promote subsequent cell surface expression may be effective in patients with this variant. In contract to β1-p.R125C, heterologous expression of β1-p.R85C showed robust cell surface expression in cells cultured at 37°C but the absence of modulation of INa density or voltage dependence, suggesting that this mutant subunit goes to the cell surface but does not functionally interact with VGSC α subunits (Aeby et al., 2019). The story with β1-p.C121W is even more complex. Early studies with this variant in Xenopus oocytes suggested loss of function (Wallace et al., 1998); however, in a subsequent study, injection of a 100-fold higher concentration of β1-p.C121W cRNA in oocytes resulted in INa modulation that was similar to wild-type (Meadows et al., 2002). Heterologous expression studies in mammalian cells showed that β1-p.C121W promotes the cell surface expression of VGSC α subunits and subtly, but incompletely modulates INa properties (Meadows et al., 2002). Expression studies in Drosophila S2 cells showed that, in spite of robust cell surface expression, β1-p.C121W does not participate in cell-cell adhesion (Meadows et al., 2002). To date, β1-p.C121W is the only SCN1B variant that has been studied for its cell adhesive properties. Because β1-mediated cell-cell and cell-matrix adhesion are proposed to be clinically relevant, this will be an important line of future investigation.
According to the Allen Brain Atlas, SCN1B mRNA is widely expressed throughout human and mouse brain, although more detailed studies using anti-β1 antibodies have shown heterogeneity in the intensity of staining between and within brain regions, possibly suggesting a mechanism for fine tuning of β1 modulation of excitability (Wimmer et al., 2015). Immunohistochemical analysis of β1 expression showed strong labeling in ankyrin-G-positive axon initial segments (AIS) of both excitatory and inhibitory neurons, as well as co-localization at the AIS with Nav1.1, Nav1.2, and Nav1.6 in many brain areas, including hippocampus, cortex, and subiculum (Kruger et al., 2016; Wimmer et al., 2015). The majority of hippocampal neurons express β1 at the AIS (Wimmer et al., 2015). In contrast, the intensity of anti-β1 staining across the somatosensory cortex is variable, suggesting possible layer-specific effects of β1. Scn1b null cerebellar granule neurons have reduced Nav1.6 staining at the AIS, with increased staining of Nav1.1 in its place, suggesting regulation of specific VGSC α subunit expression by β1 in this subcellular domain (Brackenbury et al., 2010). Anti-β1 staining is also observed at nodes of Ranvier in myelinated axons (Kruger et al., 2016; Wimmer et al., 2015). Scn1b null mice have fewer nodes of Ranvier in optic nerve, suggesting a mechanism for reduced saltatory conduction (Chen et al., 2004). Specific analysis of interneurons in the mouse cortex and hippocampus using fluorescent genetic markers showed anti-β1 staining at the AIS of essentially all parvalbumin positive (PV+) and somatostatin (SST)+ neurons, but only in a fraction of VIP+ neurons (Wimmer et al., 2015). In functional studies, PV+ interneuron specific Scn1b deletion using a Cre-lox strategy was sufficient to cause seizures and SUDEP in mice in vivo, confirming the functional expression of Scn1b in this neuronal population (Hull et al., 2020). Inducible Scn1b deletion using a Thy1 promoter driven Cre-lox strategy, which targets forebrain projection neurons, also resulted in seizures and SUDEP, confirming the functional expression of Scn1b in excitatory neurons (O’Malley et al., 2019). Thus, both excitatory and inhibitory neuronal populations contribute to Scn1b-linked DEE pathology. However, two different models of excitatory neuron-specific Scn1b deletion in mice, Emx1-Cre and Camk2a-Cre, showed no changes in life span or incidence of behavioral seizures, suggesting that further investigation of cell type–specific expression is required (Hull et al., 2020).
Only a single study, using a rat model, has described the protein localization of the Scn1b spice variant, β1B, previously called β1A (Kazen-Gillespie et al., 2000). This work, along with a subsequent mouse study of mRNA expression (Patino et al., 2011), showed that β1B mRNA is highly expressed in embryonic rodent and human brain but then is greatly diminished after birth. Staining of rat brain sections with a β1A-specific antibody showed expression in adult cerebellar Purkinje neurons as well as in subpopulations of cerebellar white matter neurons, neurons in the cerebellar dentate nucleus, cortical pyramidal neurons, and spinal cord neurons (Kazen-Gillespie et al., 2000).
Scn1b null mice phenocopy DEE52, with increased susceptibility to febrile seizures, spontaneous severe seizures beginning at ~P10, and death in 100% of mice by ~P21 (Chen et al., 2004, 2007; Kruger et al., 2016). Homozygous Scn1b-p.C121W mice, which model biallelic expression of a loss-of-function variant, have a similar phenotype (Wimmer et al., 2010) with differential glycosylation of the mutant β1 subunits in brain (Kruger et al., 2016). During the more than two decades since β1 subunits were identified, a growing body of research has shown the importance of these proteins not only in normal physiology but also in pathophysiology. In their roles as ion channel modulators, not only of VGSCs (O’Malley & Isom, 2015) but also potassium channels and calcium channels (Bouza et al., 2021; Deschenes et al., 2008; Deschenes & Tomaselli, 2002; Marionneau et al., 2012; Nguyen et al., 2012), as substrates for sequential cleavage by BACE1 and γ-secretase resulting in regulation of gene expression (Bouza et al., 2021; Kim et al., 2007; Wong et al., 2005), and as Ig-CAMs that regulate cell adhesion and neuronal patterning (Brackenbury & Isom, 2011), β1/β1B subunits are essential for the regulation of excitability. Reflecting this multifunctionality, Scn1b null mice have a complex and severe disease phenotype.
Scn1b null brains show altered excitability of excitatory and inhibitory neurons.
Scn1b null mice have impaired excitability and reduced INa density in PV+ cortical fast-spiking interneurons, suggesting disinhibition (Hull et al., 2020). Subpopulations of Scn1b null cortical pyramidal neurons show complex excitability phenotypes, consistent with the heterogeneity in anti-β1 antibody staining observed in this region (Wimmer et al., 2015). No genotypic differences in excitability or action potential (AP) properties were observed in cortical layer 5 pyramidal neurons. In contrast, subiculum and cortical layer 6 null neurons showed depolarized resting membrane potentials and increased input resistance. Decreased capacitance was recorded in layer 6. Increased firing, or hyperexcitability, of Scn1b null subicular and layer 6 pyramidal neurons was observed at low current injections with differential sensitivity to depolarization block, or hypoexcitability, at higher current injections. Transient and persistent INa density were reduced in null layer 6 pyramidal neurons compared to wild-type (Hull et al., 2020). Hippocampal slice recordings showed that Scn1b null CA3 pyramidal neurons fire evoked APs with higher peak voltage and greater amplitude than wild-type. However, in contrast to cortical neurons, there were no measurable differences in INa density observed in acutely dissociated CA3 hippocampal neurons (Patino et al., 2009) in spite of the robust anti-β1 staining observed in this region (Wimmer et al., 2015).
Scn1b null brains have deficits in neuronal pathfinding.
Multiple deficits in neuronal pathfinding are observed in Scn1b null brain, including defasciculation of cortiospinal tract axons, hilar ectopic dentate granule neurons, axonal pathfinding defects in the hippocampus, microorganization defects of PV+ interneurons in the hippocampus, migration deficits in cerebellar granule neurons, and aberrant patterning and defasciculation of cerebellar parallel fibers (Brackenbury et al., 2010, 2013). A number of these deficits were detected at P5, prior to seizure onset at ~P10, leading to the hypothesis that aberrant cell-adhesive interactions may result in hyperexcitability and contribute to epileptogenesis in the Scn1b null model of EI-DEE (Brackenbury et al., 2013). Interestingly, however, testing of this hypothesis using an inducible Cre-lox strategy under control of a Thy1 promoter showed that Scn1b deletion in adult mice, following normal brain development, resulted in seizures and death (O’Malley et al., 2019). Thus, while neuronal pathfinding defects may not lead to seizures in the Scn1b null model, they may contribute to other aspects of EI-DEE, including ID and behavioral disorders.
Scn1b null brains have altered VGSC α subunit expression.
Deletion of Scn1b in mice leads to alterations in the complement of α subunits expressed in specific brain regions, and these differences may contribute to EI-DEE. Scn1b null hippocampal CA3 pyramidal neurons have decreased levels of Nav1.1 and increased levels of Nav1.3, as assessed by immunofluorescence, although these measurements were made post seizure onset and could have been compensatory (Chen et al., 2004). In Scn1b null cerebellar granule neurons, AIS expression of Nav1.6 is reduced in favor of increased expression of Nav1.1, and conversely, expression of Nav1.6 is required for β1-mediated neurite outgrowth (Brackenbury et al., 2010). Taken together, and consistent with heterologous expression data (Calhoun & Isom, 2014), these studies suggest that β1 effects vary based on the α subunit being modulated as well as the specific cell type studied (Calhoun & Isom, 2014). Because the biophysical properties of each α subunit differ, this offers the potential to fine-tune excitability to specific cellular or subcellular requirements in a dynamic fashion via specific combinations of VGSC α and β1 subunits in vivo.
Maturation of GABAergic signaling.
Giant depolarizing potentials (GDPs), mediated by synchronous, network-driven excitatory action of GABA, are essential in early brain development (Ben-Ari et al., 2012). This excitatory GABAergic signaling, which is observed in normal mice during the first two postnatal weeks, is a consequence of a high intracellular Cl− in immature neurons that results in membrane depolarization in response to the opening of GABAA channels. Age-dependent expression of the Cl−-cation co-transporters Na+, K+-2Cl− co-transporter-1 (NKCC1) and K+-Cl− co-transporter-2 (KCC2) modulates neuronal intracellular Cl− regulation (Ben-Ari et al., 2012). NKCC1, which promotes Cl− influx, is expressed in immature neurons and becomes downregulated with brain development (Ben-Ari, 2017). In contrast, KCC2, which extrudes Cl−, is expressed at low levels at birth and is upregulated to adult levels by the end of the second postnatal week in rodent brain (Moore et al., 2017). As a result, activation of GABAA receptors results in Cl− efflux and membrane depolarization in early brain development and then, as development progresses, Cl− influx and hyperpolarization. GDPs in wild-type rodent brain disappear after the second postnatal week. In contrast, GDPs persist in pyramidal neurons of Scn1b null mice after P14 (Brackenbury et al., 2013) and the maturation time course of neuronal GABAergic signaling is delayed (Yuan et al., 2019). The reversal potential for GABAA-evoked currents in Scn1b null hippocampus and neocortex was found to be depolarized compared to wild-type at P16, suggesting developmental delay. Treatment with the NKCC1 antagonist bumetanide prolonged the lifespan of a subset of Scn1b null mice, suggesting that therapeutic strategies targeting GABAergic signaling polarity may be useful in reducing SUDEP risk in SCN1B-linked EI-DEE.
SUDEP is defined as Sudden, Unexpected, witnessed or unwitnessed, nontraumatic and nondrowning Death in patients with EPilepsy (Hirsch et al., 2011), excluding cases of documented status epilepticus. In the most widely used definition, death may occur with or without evidence of a seizure, and postmortem examination does not reveal a toxicological or anatomical cause of death. SUDEP is a leading cause of death in patients with epilepsy. SUDEP mechanisms are not understood, although there is evidence to implicate apnea, autonomic dysfunction, and cardiac arrhythmias (Bagnall et al., 2017; Massey et al., 2014; Schuele et al., 2007a; Schuele et al., 2007b; Shorvon & Tomson, 2011; Surges et al., 2009, 2010). The majority of SUDEP patients die during sleep and, by definition, autopsy findings are largely unremarkable. A myriad of genes has now been implicated in epilepsy (He et al., 2018; Myers et al., 2018). Careful dissection of the associated phenotypes has shown that SUDEP risk varies in a gene- specific manner (Bagnall et al., 2017; Thom et al., 2018). SCN1A, SCN8A, and SCN1B are high SUDEP risk genes critical for brain function (Catterall, 2012) with variants linked to DEE (Dravet, 2011; Meisler et al., 2016; O’Malley & Isom, 2015; Steel et al., 2017). DEE patients have the highest SUDEP risk, up to 20% (Cooper et al., 2016). No effective therapies exist for any of the genetic epilepsies, and no reliable biomarkers to predict SUDEP risk have been identified. SCN1A, SCN8A, and SCN1B are expressed in both the heart and brain of humans and mice (Frasier et al., 2016, 2018; Watanabe et al., 2008). Because of this dual localization and high SUDEP risk, cardiac arrhythmias have been proposed to contribute to the mechanism of SUDEP in channelopathy- linked DEEs. Mouse models of SCN1A-, SCN8A-, and SCN1B-linked DEE have altered cardiac myocyte (CM) INa density, calcium handling, and APs, as well as cardiac arrhythmias (Auerbach et al., 2013; Frasier et al., 2016; Lin et al., 2015; Lopez-Santiago et al., 2007).
The majority of SUDEP patients die during sleep (Bagnall et al., 2017). SUDEP is the most devastating consequence of epilepsy, yet little is understood about its causes and no biomarkers exist to identify at-risk patients. While SUDEP accounts for 7.5%–20% of all epilepsy deaths (Cooper et al., 2016; Schuele et al., 2007b; Shorvon & Tomson, 2011; Skluzacek et al., 2011), SUDEP risk in the genetic epilepsies varies with affected genes. Patients with ion channel gene variants have the highest SUDEP risk (Bagnall et al., 2016, 2017; Thom et al., 2018). Indirect evidence variably links SUDEP to seizure-induced apnea, pulmonary edema, dysregulation of cerebral circulation, autonomic dysfunction, and cardiac arrhythmias (Bagnall et al., 2017; Massey et al., 2014; Schuele et al., 2007a; Schuele et al., 2007b; Shorvon & Tomson, 2011; Surges et al., 2009, 2010). Arrhythmias may be primary or secondary to hormonal or metabolic changes, or autonomic discharges (Goldman et al., 2009; Schuele et al., 2007a; Schuele et al., 2007b; Surges et al., 2009, 2010). When SUDEP is compared to sudden cardiac death (SCD) secondary to long QT syndrome (LQTS), especially to LQT3 linked to variants in the VGSC gene SCN5A, there are parallels in the circumstances of death (Bagnall et al., 2017).
SCN1B variants are linked to human cardiac disease in addition to EI-DEE, including Brugada syndrome, Long-QT syndrome, and atrial fibrillation (Edokobi & Isom, 2018). Electrocardiograms in Scn1b null mice show bradycardia and QT prolongation (Lopez-Santiago et al., 2007). Scn1b null mouse ventricular cardiac myocytes have increased transient and persistent INa, AP prolongation, prolonged calcium transients, and increased incidence of delayed afterdepolarizations (Lin et al., 2015; Lopez-Santiago et al., 2007). Scn5a/Nav1.5 and Scn3a expression, as well as 3H-saxitoxin binding, which measures levels of tetrodotoxin- sensitive VGSC expression, are increased in Scn1b null heart. AP prolongation and aberrant calcium release in Scn1b null cardiac myocytes are also tetrodotoxin-sensitive. Scn1b null mice have disrupted cell-cell communication at the cardiac ventricular intercalated disk, suggesting that β1-mediated cell-cell adhesion may play a role in the regulation of cardiac excitability (Veeraraghavan et al., 2018). Finally, transcriptomic analysis of Scn1b null mouse cardiac ventricular tissue, in which the β1-ICD signaling pathway is deleted, showed that critical groups of genes, including those encoding VGSC α subunits and potassium channels, are upregulated, suggesting that the β1-ICD normally acts as a molecular brake on gene expression regulating excitability in the heart (Bouza et al., 2021). Taken together, these results suggest a neurocardiac mechanism of SUDEP in SCN1B-linked DEE.
In this review, we have described genetic variation in the VGSC genes SCN1A, SCN2A, SCN8A, and SCN1B that result in epilepsies and neurodevelopmental disorders. A substantial body of research has established the basic pathogenic mechanisms underlying many of these disorders. These studies reveal critical cellular and molecular targets for therapeutic approaches and demonstrate that targeting of specific classes of neurons will be required for the most effective interventions with avoidance of undesirable side effects. Dramatic advances in molecular biology, including new methods of gene editing, will facilitate specific targeting of therapeutic interventions, and future clinical trials are eagerly awaited.
A portion of Dr. Isom’s research was funded by a grant to the University of Michigan from Stoke Therapeutics.
This is an open access publication, available online and distributed under the terms of a Creative Commons Attribution-Non Commercial-No Derivatives 4.0 International licence (CC BY-NC-ND 4.0), a copy of which is available at https://creativecommons.org/licenses/by-nc-nd/4.0/. Subject to this license, all rights are reserved.
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