DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models.

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. Here, we show that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders.

Antisense oligonucleotides restore excitability, GABA signalling and sodium current density in a Dravet syndrome model.

Dravet syndrome is an intractable developmental and epileptic encephalopathy caused by de novo variants in SCN1A resulting in haploinsufficiency of the voltage-gated sodium channel Nav1.1. We showed previously that administration of the antisense oligonucleotide STK-001, also called ASO-22, generated using targeted augmentation of nuclear gene output technology to prevent inclusion of the nonsense-mediated decay, or poison, exon 20N in human SCN1A, increased productive Scn1a transcript and Nav1.1 expression and reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy in a mouse model of Dravet syndrome. Here, we investigated the mechanism of action of ASO-84, a surrogate for ASO-22 that also targets splicing of SCN1A exon 20N, in Scn1a+/- Dravet syndrome mouse brain. Scn1a +/- Dravet syndrome and wild-type mice received a single intracerebroventricular injection of antisense oligonucleotide or vehicle at postnatal Day 2. We examined the electrophysiological properties of cortical pyramidal neurons and parvalbumin-positive fast-spiking interneurons in brain slices at postnatal Days 21-25 and measured sodium currents in parvalbumin-positive interneurons acutely dissociated from postnatal Day 21-25 brain slices. We show that, in untreated Dravet syndrome mice, intrinsic cortical pyramidal neuron excitability was unchanged while cortical parvalbumin-positive interneurons showed biphasic excitability with initial hyperexcitability followed by hypoexcitability and depolarization block. Dravet syndrome parvalbumin-positive interneuron sodium current density was decreased compared to wild-type. GABAergic signalling to cortical pyramidal neurons was reduced in Dravet syndrome mice, suggesting decreased GABA release from interneurons. ASO-84 treatment restored action potential firing, sodium current density and GABAergic signalling in Dravet syndrome parvalbumin-positive interneurons. Our work suggests that interneuron excitability is selectively affected by ASO-84. This new work provides critical insights into the mechanism of action of this antisense oligonucleotide and supports the potential of antisense oligonucleotide-mediated upregulation of Nav1.1 as a successful strategy to treat Dravet syndrome.

Therapeutic Potential of Targeting Regulated Intramembrane Proteolysis Mechanisms of Voltage-Gated Ion Channel Subunits and Cell Adhesion Molecules.

Several integral membrane proteins undergo regulated intramembrane proteolysis (RIP), a tightly controlled process through which cells transmit information across and between intracellular compartments. RIP generates biologically active peptides by a series of proteolytic cleavage events carried out by two primary groups of enzymes: sheddases and intramembrane-cleaving proteases (iCLiPs). Following RIP, fragments of both pore-forming and non-pore-forming ion channel subunits, as well as immunoglobulin super family (IgSF) members, have been shown to translocate to the nucleus to function in transcriptional regulation. As an example, the voltage-gated sodium channel ß1 subunit, which is also an IgSF-cell adhesion molecule (CAM), is a substrate for RIP. ß1 RIP results in generation of a soluble intracellular domain, which can regulate gene expression in the nucleus. In this review, we discuss the proposed RIP mechanisms of voltage-gated sodium, potassium, and calcium channel subunits as well as the roles of their generated proteolytic products in the nucleus. We also discuss other RIP substrates that are cleaved by similar sheddases and iCLiPs, such as IgSF macromolecules, including CAMs, whose proteolytically generated fragments function in the nucleus. Importantly, dysfunctional RIP mechanisms are linked to human disease. Thus, we will also review how understanding RIP events and subsequent signaling processes involving ion channel subunits and IgSF proteins may lead to the discovery of novel therapeutic targets. SIGNIFICANCE STATEMENT: Several ion channel subunits and immunoglobulin superfamily molecules have been identified as substrates of regulated intramembrane proteolysis (RIP). This signal transduction mechanism, which generates polypeptide fragments that translocate to the nucleus, is an important regulator of gene transcription. RIP may impact diseases of excitability, including epilepsy, cardiac arrhythmia, and sudden death syndromes. A thorough understanding of the role of RIP in gene regulation is critical as it may reveal novel therapeutic strategies for the treatment of previously intractable diseases.

XOB: A novel phenylalkylamine antagonist of 5-HT2A receptors and voltage-gated sodium channels.

Bipolar disorder impacts millions of patients in the United States but the mechanistic understanding of its pathophysiology and therapeutics is incomplete. Atypical antipsychotic serotonin2A (5-HT2A) receptor antagonists, such as quetiapine and olanzapine, and mood-stabilizing voltage-gated sodium channel (VGSC) blockers, such as lamotrigine, carbamazepine, and valproate, show therapeutic synergy and are often prescribed in combination for the treatment of bipolar disorder. Combination therapy is a complex task for clinicians and patients, often resulting in unexpected difficulties with dosing, drug tolerances, and decreased patient compliance. Thus, an unmet need for bipolar disorder treatment is to develop a therapeutic agent that targets both 5-HT2A receptors and VGSCs. Towards this goal, we developed a novel small molecule that simultaneously antagonizes 5-HT2A receptors and blocks sodium current. The new compound, N-(4-bromo-2,5-dimethoxyphenethyl)-6-(4-phenylbutoxy)hexan-1-amine (XOB) antagonizes 5-HT-stimulated, Gq-mediated, calcium flux at 5-HT2A receptors at low micromolar concentrations while displaying negligible affinity and activity at 5-HT1A, 5-HT2B, and 5-HT2C receptors. At similar concentrations, XOB administration inhibits sodium current in heterologous cells and results in reduced action potential (AP) firing and VGSC-related AP properties in mouse prefrontal cortex layer V pyramidal neurons. Thus, XOB represents a new, proof-of-principle tool that can be used for future preclinical investigations and therapeutic development. This polypharmacology approach of developing a single molecule to act upon two targets, which are currently independently targeted by combination therapies, may lead to safer alternatives for the treatment of psychiatric disorders that are increasingly being found to benefit from the simultaneous targeting of multiple receptors. Significance Statement We synthesized a novel small molecule (XOB) that simultaneously antagonizes two key therapeutic targets of bipolar disorder, 5-HT2A receptors and voltage-gated sodium channels (VGSCs), in heterologous cells, and inhibits the intrinsic excitability of mouse prefrontal cortex layer V pyramidal neurons in brain slices. XOB represents a valuable new proof-of-principle tool for future preclinical investigations and provides a novel molecular approach to the pharmacological treatment of complex neuropsychiatric disease, which often requires a combination of therapeutics for sufficient patient benefit.

Subcellular dynamics and functional activity of the cleaved intracellular domain of the Na+ channel ß1 subunit.

The voltage-gated Na+ channel ß1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits and participates in cell adhesion. ß1 is cleaved by α/ß and γ-secretases, releasing an extracellular domain and intracellular domain (ICD), respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on ß1 function in breast cancer cells. Using a series of GFP-tagged ß1 constructs, we show that ß1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal ß1-GFP levels occurred following γ-secretase inhibition, implicating endosomes and/or the preceding plasma membrane as important sites for secretase processing. Using live-cell imaging, we also report ß1ICD-GFP accumulation in the nucleus. Furthermore, ß1-GFP and ß1ICD-GFP both increased Na+ current, whereas ß1STOP-GFP, which lacks the ICD, did not, thus highlighting that the ß1-ICD is necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is tetrodotoxin (TTX)-resistant (carried by Nav1.5), the Na+ current increased by ß1-GFP or ß1ICD-GFP was TTX-sensitive. Finally, we found ß1-GFP increased mRNA levels of the TTX-sensitive α subunits SCN1A/Nav1.1 and SCN9A/Nav1.7. Taken together, this work suggests that the ß1-ICD is a critical regulator of α subunit function in cancer cells. Our data further highlight that γ-secretase may play a key role in regulating ß1 function in breast cancer.

Precision medicine for genetic epilepsy on the horizon: Recent advances, present challenges, and suggestions for continued progress.

The genetic basis of many epilepsies is increasingly understood, giving rise to the possibility of precision treatments tailored to specific genetic etiologies. Despite this, current medical therapy for most epilepsies remains imprecise, aimed primarily at empirical seizure reduction rather than targeting specific disease processes. Intellectual and technological leaps in diagnosis over the past 10 years have not yet translated to routine changes in clinical practice. However, the epilepsy community is poised to make impressive gains in precision therapy, with continued innovation in gene discovery, diagnostic ability, and bioinformatics; increased access to genetic testing and counseling; fuller understanding of natural histories; agility and rigor in preclinical research, including strategic use of emerging model systems; and engagement of an evolving group of stakeholders (including patient advocates, governmental resources, and clinicians and scientists in academia and industry). In each of these areas, we highlight notable examples of recent progress, new or persistent challenges, and future directions. The future of precision medicine for genetic epilepsy looks bright if key opportunities on the horizon can be pursued with strategic and coordinated effort.

Sodium channel ß1 subunits are post-translationally modified by tyrosine phosphorylation, S-palmitoylation, and regulated intramembrane proteolysis.

Voltage-gated sodium channel (VGSC) ß1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC α subunits and participate in cell-cell and cell-matrix adhesion, all with important implications for intracellular signal transduction, cell migration, and differentiation. Human loss-of-function variants in SCN1B, the gene encoding the VGSC ß1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that ß1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that ß1 subunits undergo regulated intramembrane proteolysis via the activity of ß-secretase 1 and γ-secretase, resulting in the generation of a soluble intracellular domain, ß1-ICD, which modulates transcription. Here, we report that ß1 subunits are phosphorylated by FYN kinase. Moreover, we show that ß1 subunits are S-palmitoylated. Substitution of a single residue in ß1, Cys-162, to alanine prevented palmitoylation, reduced the level of ß1 polypeptides at the plasma membrane, and reduced the extent of ß1-regulated intramembrane proteolysis, suggesting that the plasma membrane is the site of ß1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor, Dyngo-4a, re-stored the plasma membrane association of ß1-p.C162A to WT levels. Despite these observations, palmitoylation-null ß1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration of S-palmitoylation of a VGSC ß subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.

Gastrointestinal Symptoms and Channelopathy-Associated Epilepsy.

OBJECTIVE: To determine the prevalence of and identify factors associated with gastrointestinal (GI) symptoms among children with channelopathy-associated developmental and epileptic encephalopathy (DEE). STUDY DESIGN: Parents of 168 children with DEEs linked to SCN1A (n = 59), KCNB1 (n = 31), or KCNQ2 (n = 78) completed online CLIRINX surveys about their children's GI symptoms. Our analysis examined the prevalence, frequency, and severity of GI symptoms, as well as DEE type, functional mobility, feeding difficulties, ketogenic diet, antiseizure medication, autism spectrum disorder (ASD), and seizures. Statistical analyses included the χ2 test, Wilcoxon rank-sum analysis, and multiple logistic regression. RESULTS: GI symptoms were reported in 92 of 168 patients (55%), among whom 63 of 86 (73%) reported daily or weekly symptoms, 29 of 92 (32%) had frequent or serious discomfort, and 13 of 91 (14%) had frequent or serious appetite disturbances as a result. The prevalence of GI symptoms varied across DEE cohorts with 44% of SCN1A-DEE patients, 35% of KCNB1-DEE patients, and 71% of KCNQ2-DEE patients reporting GI symptoms in the previous month. After adjustment for DEE type, current use of ketogenic diet (6% reported), and gastrostomy tube (13% reported) were both associated with GI symptoms in a statistically, but not clinically, significant manner (P < .05). Patient age, functional mobility, feeding difficulties, ASD, and seizures were not clearly associated with GI symptoms. Overall, no individual antiseizure medication was significantly associated with GI symptoms across all DEE cohorts. CONCLUSIONS: GI symptoms are common and frequently severe in patients with DEE.

Variant-specific changes in persistent or resurgent sodium current in SCN8A-related epilepsy patient-derived neurons.

Missense variants in the SCN8A voltage-gated sodium channel gene are linked to early-infantile epileptic encephalopathy type 13, also known as SCN8A-related epilepsy. These patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy. The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current properties. To investigate these mechanisms using a patient-specific model, we generated induced pluripotent stem cells from three patients with missense variants in SCN8A: p.R1872>L (Patient 1); p.V1592>L (Patient 2); and p.N1759>S (Patient 3). Using small molecule differentiation into excitatory neurons, induced pluripotent stem cell-derived neurons from all three patients displayed altered sodium currents. Patients 1 and 2 had elevated persistent current, while Patient 3 had increased resurgent current compared to controls. Neurons from all three patients displayed shorter axon initial segment lengths compared to controls. Further analyses focused on one of the patients with increased persistent sodium current (Patient 1) and the patient with increased resurgent current (Patient 3). Excitatory cortical neurons from both patients had prolonged action potential repolarization. Using doxycycline-inducible expression of the neuronal transcription factors neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons, we investigated network activity and response to pharmacotherapies. Both small molecule differentiated and induced patient neurons displayed similar abnormalities in action potential repolarization. Patient induced neurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for SCN8A-related epilepsy patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent sodium currents, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the action potential firing threshold of patient-derived neurons to more depolarized potentials. Two of the patients in this study were prescribed riluzole off-label. Patient 1 had a 50% reduction in seizure frequency. Patient 3 experienced an immediate and dramatic seizure reduction with months of seizure freedom. An additional patient with a SCN8A variant in domain IV of Nav1.6 (p.V1757>I) had a dramatic reduction in seizure frequency for several months after starting riluzole treatment, but then seizures recurred. Our results indicate that patient-specific neurons are useful for modelling SCN8A-related epilepsy and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific neuronal disease modelling offers a useful platform for discovering precision epilepsy therapies.

Neuronal hyperexcitability in a mouse model of SCN8A epileptic encephalopathy.

Patients with early infantile epileptic encephalopathy (EIEE) experience severe seizures and cognitive impairment and are at increased risk for sudden unexpected death in epilepsy (SUDEP). EIEE13 [Online Mendelian Inheritance in Man (OMIM) # 614558] is caused by de novo missense mutations in the voltage-gated sodium channel gene SCN8A Here, we investigated the neuronal phenotype of a mouse model expressing the gain-of-function SCN8A patient mutation, p.Asn1768Asp (Nav1.6-N1768D). Our results revealed regional and neuronal subtype specificity in the effects of the N1768D mutation. Acutely dissociated hippocampal neurons from Scn8aN1768D/+ mice showed increases in persistent sodium current (INa) density in CA1 pyramidal but not bipolar neurons. In CA3, INa,P was increased in both bipolar and pyramidal neurons. Measurement of action potential (AP) firing in Scn8aN1768D/+ pyramidal neurons in brain slices revealed early afterdepolarization (EAD)-like AP waveforms in CA1 but not in CA3 hippocampal or layer II/III neocortical neurons. The maximum spike frequency evoked by depolarizing current injections in Scn8aN1768D/+ CA1, but not CA3 or neocortical, pyramidal cells was significantly reduced compared with WT. Spontaneous firing was observed in subsets of neurons in CA1 and CA3, but not in the neocortex. The EAD-like waveforms of Scn8aN1768D/+ CA1 hippocampal neurons were blocked by tetrodotoxin, riluzole, and SN-6, implicating elevated persistent INa and reverse mode Na/Ca exchange in the mechanism of hyperexcitability. Our results demonstrate that Scn8a plays a vital role in neuronal excitability and provide insight into the mechanism and future treatment of epileptogenesis in EIEE13.

Cardiac arrhythmia in a mouse model of sodium channel SCN8A epileptic encephalopathy.

Patients with early infantile epileptic encephalopathy (EIEE) are at increased risk for sudden unexpected death in epilepsy (SUDEP). De novo mutations of the sodium channel gene SCN8A, encoding the sodium channel Nav1.6, result in EIEE13 (OMIM 614558), which has a 10% risk of SUDEP. Here, we investigated the cardiac phenotype of a mouse model expressing the gain of function EIEE13 patient mutation p.Asn1768Asp in Scn8a (Nav1.6-N1768D). We tested Scn8aN1768D/+ mice for alterations in cardiac excitability. We observed prolongation of the early stages of action potential (AP) repolarization in mutant myocytes vs. controls. Scn8aN1768D/+ myocytes were hyperexcitable, with a lowered threshold for AP firing, increased incidence of delayed afterdepolarizations, increased calcium transient duration, increased incidence of diastolic calcium release, and ectopic contractility. Calcium transient duration and diastolic calcium release in the mutant myocytes were tetrodotoxin-sensitive. A selective inhibitor of reverse mode Na/Ca exchange blocked the increased incidence of diastolic calcium release in mutant cells. Scn8aN1768D/+ mice exhibited bradycardia compared with controls. This difference in heart rate dissipated after administration of norepinephrine, and there were no differences in heart rate in denervated ex vivo hearts, implicating parasympathetic hyperexcitability in the Scn8aN1768D/+ animals. When challenged with norepinephrine and caffeine to simulate a catecholaminergic surge, Scn8aN1768D/+ mice showed ventricular arrhythmias. Two of three mutant mice under continuous ECG telemetry recording experienced death, with severe bradycardia preceding asystole. Thus, in addition to central neuron hyperexcitability, Scn8aN1768D/+ mice have cardiac myoycte and parasympathetic neuron hyperexcitability. Simultaneous dysfunction in these systems may contribute to SUDEP associated with mutations of Scn8a.

Sodium channel ß subunits: emerging targets in channelopathies.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and ß subunits. Although ß subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. ß subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding ß subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC ß subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target.

Voltage-Gated Sodium Channel ß Subunits and Their Related Diseases.

Voltage-gated sodium channels are protein complexes comprised of one pore forming α subunit and two, non-pore forming, ß subunits. The voltage-gated sodium channel ß subunits were originally identified to function as auxiliary subunits, which modulate the gating, kinetics, and localization of the ion channel pore. Since that time, the five ß subunits have been shown to play crucial roles as multifunctional signaling molecules involved in cell adhesion, cell migration, neuronal pathfinding, fasciculation, and neurite outgrowth. Here, we provide an overview of the evidence implicating the ß subunits in their conducting and non-conducting roles. Mutations in the ß subunit genes (SCN1B-SCN4B) have been linked to a variety of diseases. These include cancer, epilepsy, cardiac arrhythmias, sudden infant death syndrome/sudden unexpected death in epilepsy, neuropathic pain, and multiple neurodegenerative disorders. ß subunits thus provide novel therapeutic targets for future drug discovery.

Drugging the undruggable: gabapentin, pregabalin and the calcium channel α2δ subunit.

In the post-genomic era, the idea of using the sequence of a protein to determine its potential role as a drug target has gained currency. The goal of this approach to drug discovery is to use the sequence of a protein that is known to bind a specific ligand or drug, along with the known structure of the ligand binding site, to predict other similar proteins that are also "druggable". Gabapentin (Neurontin) and pregabalin (Lyrica) are drugs currently in the clinic that were developed based on the hypothesis that generating non-hydrolyzable analogs of GABA would lead to the development of antiepileptic agents. While these compounds are indeed good anticonvulsants, their activity is surprisingly not due to activity in the GABAergic system. By purifying the protein to which gabapentin bound, and determining its identity as the α2δ1 subunit of voltage gated calcium channels, it was possible to make progress in developing new compounds with similar activities to gabapentin, including pregabalin. The recognition of the α2δ1 subunit as the receptor for these drugs also meant that related proteins, such as α2δ3, may be interesting targets for novel pain therapeutics.

ß1-C121W Is Down But Not Out: Epilepsy-Associated Scn1b-C121W Results in a Deleterious Gain-of-Function.

UNLABELLED: Voltage-gated sodium channel (VGSC) ß subunits signal through multiple pathways on multiple time scales. In addition to modulating sodium and potassium currents, ß subunits play nonconducting roles as cell adhesion molecules, which allow them to function in cell-cell communication, neuronal migration, neurite outgrowth, neuronal pathfinding, and axonal fasciculation. Mutations in SCN1B, encoding VGSC ß1 and ß1B, are associated with epilepsy. Autosomal-dominant SCN1B-C121W, the first epilepsy-associated VGSC mutation identified, results in genetic epilepsy with febrile seizures plus (GEFS+). This mutation has been shown to disrupt both the sodium-current-modulatory and cell-adhesive functions of ß1 subunits expressed in heterologous systems. The goal of this study was to compare mice heterozygous for Scn1b-C121W (Scn1b(+/W)) with mice heterozygous for the Scn1b-null allele (Scn1b(+/-)) to determine whether the C121W mutation results in loss-of-function in vivo We found that Scn1b(+/W) mice were more susceptible than Scn1b(+/-) and Scn1b(+/+) mice to hyperthermia-induced convulsions, a model of pediatric febrile seizures. ß1-C121W subunits are expressed at the neuronal cell surface in vivo However, despite this, ß1-C121W polypeptides are incompletely glycosylated and do not associate with VGSC α subunits in the brain. ß1-C121W subcellular localization is restricted to neuronal cell bodies and is not detected at axon initial segments in the cortex or cerebellum or at optic nerve nodes of Ranvier of Scn1b(W/W) mice. These data, together with our previous results showing that ß1-C121W cannot participate in trans-homophilic cell adhesion, lead to the hypothesis that SCN1B-C121W confers a deleterious gain-of-function in human GEFS+ patients. SIGNIFICANCE STATEMENT: The mechanisms underlying genetic epilepsy syndromes are poorly understood. Closing this gap in knowledge is essential to the development of new medicines to treat epilepsy. We have used mouse models to understand the mechanism of a mutation in the sodium channel gene SCN1B linked to genetic epilepsy with febrile seizures plus. We report that sodium channel ß1 subunit proteins encoded by this mutant gene are expressed at the surface of neuronal cell bodies; however, they do not associate with the ion channel complex nor are they transported to areas of the axon that are critical for proper neuronal firing. We conclude that this disease-causing mutation is not simply a loss-of-function, but instead results in a deleterious gain-of-function in the brain.

Abnormal neuronal patterning occurs during early postnatal brain development of Scn1b-null mice and precedes hyperexcitability.

Voltage-gated Na(+) channel (VGSC) ß1 subunits, encoded by SCN1B, are multifunctional channel modulators and cell adhesion molecules (CAMs). Mutations in SCN1B are associated with the genetic epilepsy with febrile seizures plus (GEFS+) spectrum disorders in humans, and Scn1b-null mice display severe spontaneous seizures and ataxia from postnatal day (P)10. The goal of this study was to determine changes in neuronal pathfinding during early postnatal brain development of Scn1b-null mice to test the hypothesis that these CAM-mediated roles of Scn1b may contribute to the development of hyperexcitability. c-Fos, a protein induced in response to seizure activity, was up-regulated in the Scn1b-null brain at P16 but not at P5. Consistent with this, epileptiform activity was observed in hippocampal and cortical slices prepared from the P16 but not from the P5-P7 Scn1b-null brain. On the basis of these results, we investigated neuronal pathfinding at P5. We observed disrupted fasciculation of parallel fibers in the P5 null cerebellum. Further, P5 null mice showed reduced neuron density in the dentate gyrus granule cell layer, increased proliferation of granule cell precursors in the hilus, and defective axonal extension and misorientation of somata and processes of inhibitory neurons in the dentate gyrus and CA1. Thus, Scn1b is critical for neuronal proliferation, migration, and pathfinding during the critical postnatal period of brain development. We propose that defective neuronal proliferation, migration, and pathfinding in response to Scn1b deletion may contribute to the development of hyperexcitability.

More than a pore: ion channel signaling complexes.

Voltage- and ligand-gated ion channels form the molecular basis of cellular excitability. With >400 members and accounting for ∼1.5% of the human genome, ion channels are some of the most well studied of all proteins in heterologous expression systems. Yet, ion channels often exhibit unexpected properties in vivo because of their interaction with a variety of signaling/scaffolding proteins. Such interactions can influence the function and localization of ion channels, as well as their coupling to intracellular second messengers and pathways, thus increasing the signaling potential of these ion channels in neurons. Moreover, functions have been ascribed to ion channels that are largely independent of their ion-conducting roles. Molecular and functional dissection of the ion channel proteome/interactome has yielded new insights into the composition of ion channel complexes and how their dysregulation leads to human disease.

Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism.

OBJECTIVE: Neuronal channelopathies cause brain disorders, including epilepsy, migraine, and ataxia. Despite the development of mouse models, pathophysiological mechanisms for these disorders remain uncertain. One particularly devastating channelopathy is Dravet syndrome (DS), a severe childhood epilepsy typically caused by de novo dominant mutations in the SCN1A gene encoding the voltage-gated sodium channel Na(v) 1.1. Heterologous expression of mutant channels suggests loss of function, raising the quandary of how loss of sodium channels underlying action potentials produces hyperexcitability. Mouse model studies suggest that decreased Na(v) 1.1 function in interneurons causes disinhibition. We aim to determine how mutant SCN1A affects human neurons using the induced pluripotent stem cell (iPSC) method to generate patient-specific neurons. METHODS: Here we derive forebrain-like pyramidal- and bipolar-shaped neurons from 2 DS subjects and 3 human controls by iPSC reprogramming of fibroblasts. DS and control iPSC-derived neurons are compared using whole-cell patch clamp recordings. Sodium current density and intrinsic neuronal excitability are examined. RESULTS: Neural progenitors from DS and human control iPSCs display a forebrain identity and differentiate into bipolar- and pyramidal-shaped neurons. DS patient-derived neurons show increased sodium currents in both bipolar- and pyramidal-shaped neurons. Consistent with increased sodium currents, both types of patient-derived neurons show spontaneous bursting and other evidence of hyperexcitability. Sodium channel transcripts are not elevated, consistent with a post-translational mechanism. INTERPRETATION: These data demonstrate that epilepsy patient-specific iPSC-derived neurons are useful for modeling epileptic-like hyperactivity. Our findings reveal a previously unrecognized cell-autonomous epilepsy mechanism potentially underlying DS, and offer a platform for screening new antiepileptic therapies.

The role of non-pore-forming ß subunits in physiology and pathophysiology of voltage-gated sodium channels.

Voltage-gated sodium channel ß1 and ß2 subunits were discovered as auxiliary proteins that co-purify with pore-forming α subunits in brain. The other family members, ß1B, ß3, and ß4, were identified by homology and shown to modulate sodium current in heterologous systems. Work over the past 2 decades, however, has provided strong evidence that these proteins are not simply ancillary ion channel subunits, but are multifunctional signaling proteins in their own right, playing both conducting (channel modulatory) and nonconducting roles in cell signaling. Here, we discuss evidence that sodium channel ß subunits not only regulate sodium channel function and localization but also modulate voltage-gated potassium channels. In their nonconducting roles, VGSC ß subunits function as immunoglobulin superfamily cell adhesion molecules that modulate brain development by influencing cell proliferation and migration, axon outgrowth, axonal fasciculation, and neuronal pathfinding. Mutations in genes encoding ß subunits are linked to paroxysmal diseases including epilepsy, cardiac arrhythmia, and sudden infant death syndrome. Finally, ß subunits may be targets for the future development of novel therapeutics.

The sodium channel accessory subunit Navß1 regulates neuronal excitability through modulation of repolarizing voltage-gated K⁺ channels.

The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (I(A)) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting, and cell-surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na⁺ channel accessory subunit Navß1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navß1 decreases I(A) densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b-null cortical pyramidal neurons lacking Navß1. Biochemical and voltage-clamp experiments further demonstrated that Navß1 interacts with and increases the stability of the heterologously expressed Kv4.2 protein, resulting in greater total and cell-surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Together, the results presented here identify Navß1 as a component of native neuronal Kv4.2-encoded I(A) channel complexes and a novel regulator of I(A) channel densities and neuronal excitability.