Luis F. Lopez-Santiago

A Functional Null Mutation of SCN1B in a Patient with Dravet Syndrome

Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Nav1.1 α subunits. Sodium channels are modulated by β1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Nav1.1 α subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of β1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b−/− versus Scn1b+/+ mice. Scn1b−/− CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a+/− model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b−/− mice seize spontaneously, the seizure susceptibility of Scn1b+/− mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.

Dravet syndrome patient‐derived neurons suggest a novel epilepsy mechanism

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 Nav1.1. 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 Nav1.1 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.

Sodium Channel β2 Subunits Regulate Tetrodotoxin-Sensitive Sodium Channels in Small Dorsal Root Ganglion Neurons and Modulate the Response to Pain

Voltage-gated sodium channel (Nav1) β2 subunits modulate channel gating, assembly, and cell-surface expression in CNS neurons in vitro and in vivo. β2 expression increases in sensory neurons after nerve injury, and development of mechanical allodynia in the spared nerve injury model is attenuated in β2-null mice. Thus, we hypothesized that β2 modulates electrical excitability in dorsal root ganglion (DRG) neurons in vivo. We compared sodium currents (INa) in small DRG neurons from β2+/+ and β2−/− mice to determine the effects of β2 on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Nav1 in vivo. Small-fast DRG neurons acutely isolated from β2−/− mice showed significant decreases in TTX-S INa compared with β2+/+ neurons. This decrease included a 51% reduction in maximal sodium conductance with no detectable changes in the voltage dependence of activation or inactivation. TTX-S, but not TTX-R, INa activation and inactivation kinetics in these cells were slower in β2−/− mice compared with controls. The selective regulation of TTX-S INa was supported by reductions in transcript and protein levels of TTX-S Nav1s, particularly Nav1.7. Low-threshold mechanical sensitivity was preserved in β2−/− mice, but they were more sensitive to noxious thermal stimuli than wild type whereas their response during the late phase of the formalin test was attenuated. Our results suggest that β2 modulates TTX-S Nav1 mRNA and protein expression resulting in increased TTX-S INa and increases the rates of TTX-S Nav1 activation and inactivation in small-fast DRG neurons in vivo. TTX-R INa were not significantly modulated by β2.

Voltage-Gated Na+ Channel β1B: A Secreted Cell Adhesion Molecule Involved in Human Epilepsy

Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, β1 and β1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of β1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to β1B. We show that wild-type β1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of β1B with voltage-gated Na+ channels Nav1.1 or Nav1.3 is not detectable by immunoprecipitation and β1B does not affect Nav1.3 cell surface expression as measured by [3H]saxitoxin binding. However, β1B coexpression results in subtle alteration of Nav1.3 currents in transfected cells, suggesting that β1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of β1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that β1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.

Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel α and β2 Subunits

Background: Voltage-gated Na+ channels are composed of α and β subunits. Results: We identified the cysteine residue in β2 responsible for disulfide linkage to α. Conclusion: α and β2 associate through a single disulfide bridge to achieve proper subcellular targeting in neurons. Significance: Understanding how Na+ channel complexes are formed in neurons is crucial for understanding the development of excitability. Voltage-gated Na+ channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked β subunit (β1 or β3), and one disulfide-linked β subunit (β2 or β4). The final step in Na+ channel biosynthesis in central neurons is concomitant α-β2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding β2) null mice have reduced Na+ channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant β2 cDNA constructs to investigate the cysteine residue(s) responsible for α-β2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and β2 subunits. Loss of α-β2 covalent complex formation disrupts the targeting of β2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal β2 subcellular localization in vivo. WT β2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant β2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-β2 covalent association via a single, extracellular disulfide bond is required for β2 targeting to specialized neuronal subcellular domains and for β2 association with the neuronal cytoskeleton within those domains.

Na+ channel Scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo.

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na+ current (INa) mediated by voltage-gated Na+ channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S INa inactivation, reduced persistent TTX-R INa, a prolonged rate of recovery of TTX-R INa from inactivation, and reduced cell surface expression of Nav1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K+ current (Ito) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both INa and IK.

The voltage-gated Na+ channel β3 subunit does not mediate trans homophilic cell adhesion or associate with the cell adhesion molecule contactin

Voltage-gated Na(+) channel (VGSC) beta1 and beta2 subunits are multifunctional, serving as both channel modulators and cell adhesion molecules (CAMs). The purpose of this study was to determine whether VGSC beta3 subunits function as CAMs. The beta3 extracellular domain is highly homologous to beta1, suggesting that beta3 may also be a functional CAM. We investigated the trans homophilic cell adhesive properties of beta3, its association with the beta1-interacting CAM contactin, as well as its ability to interact with the cytoskeletal protein ankyrin. Our results demonstrate that, unlike beta1, beta3 does not participate in trans homophilic cell-cell adhesion or associate with contactin. Further, beta3 does not associate with ankyrin(G) in a heterologous system. Previous studies have shown that beta3 interacts with the CAM neurofascin-186 but not with VGSC beta1. Taken together, these findings suggest that, although beta1 and beta3 exhibit similar channel modulatory properties in heterologous systems, these subunits differ with regard to their homophilic and heterophilic CAM binding profiles.

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

Significance Patients with epileptic encephalopathy have a high risk of sudden unexpected death in epilepsy (SUDEP), an event described as arrhythmia of brain and heart. We investigated the cardiac phenotype of a model of an epileptic encephalopathy caused by mutation of sodium channel SCN8A. We observed that mutant heart cells were hyperexcitable, exhibiting abnormal contraction and action potential wave forms. Mutant mice also had reduced heart rates compared with controls. This difference in heart rate was not observed in isolated hearts, implicating changes in cardiac regulation by the parasympathetic nervous system. When challenged with norepinephrine and caffeine, mutant mice had ventricular arrhythmias. These cardiac and parasympathetic abnormalities are predicted to contribute to the mechanism of SUDEP in patients with SCN8A mutations. 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.

Neuronal hyperexcitability in a mouse model of SCN8A epileptic encephalopathy

Significance Patients with early infantile epileptic encephalopathy experience severe seizures and cognitive impairment and are at increased risk for sudden unexpected death in epilepsy (SUDEP). Here, we investigated the neuronal phenotype of a mouse model of early infantile epileptic encephalopathy (EIEE) 13 caused by a mutation in the sodium channel gene SCN8A. We found that excitatory and inhibitory neurons from mutant mice had increased persistent sodium current density. Measurement of action potential firing in brain slices from mutant mice revealed hyperexcitability with spontaneous firing in a subset of neurons. These changes in neurons are predicted to contribute to the observed seizure phenotype in whole animals. Our results provide insights into the disease mechanism and future treatment of patients with EIEE13. 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.

Scn2b Deletion in Mice Results in Ventricular and Atrial Arrhythmias

Background—Mutations in SCN2B, encoding voltage-gated sodium channel &bgr;2-subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this, we propose that &bgr;2-subunits play critical roles in the establishment or maintenance of normal cardiac electric activity in vivo. Methods and Results—To understand the pathophysiological roles of &bgr;2 in the heart, we investigated the cardiac phenotype of Scn2b null mice. We observed reduced sodium and potassium current densities in ventricular myocytes, as well as conduction slowing in the right ventricular outflow tract region. Functional reentry, resulting from the interplay between slowed conduction, prolonged repolarization, and increased incidence of premature ventricular complexes, was found to underlie the mechanism of spontaneous polymorphic ventricular tachycardia. Scn5a transcript levels were similar in Scn2b null and wild-type ventricles, as were levels of Nav1.5 protein, suggesting that similar to the previous work in neurons, the major function of &bgr;2-subunits in the ventricle is to chaperone voltage-gated sodium channel &agr;-subunits to the plasma membrane. Interestingly, Scn2b deletion resulted in region-specific effects in the heart. Scn2b null atria had normal levels of sodium current density compared with wild type. Scn2b null hearts were more susceptible to atrial fibrillation, had increased levels of fibrosis, and higher repolarization dispersion than wild-type littermates. Conclusions—Genetic deletion of Scn2b in mice results in ventricular and atrial arrhythmias, consistent with reported SCN2B mutations in human patients.