Gustavo A. Patino

Loss of Plakophilin-2 Expression Leads to Decreased Sodium Current and Slower Conduction Velocity in Cultured Cardiac Myocytes

Rationale: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (NaV1.5) to this region. Objective: To establish the association of PKP2 with sodium channels and its role on action potential propagation. Methods and Results: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with NaV1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. Conclusions: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed.

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.

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.

β1-C121W Is Down But Not Out: Epilepsy-Associated Scn1b-C121W Results in a Deleterious Gain-of-Function

Voltage-gated sodium channel (VGSC) β subunits signal through multiple pathways on multiple time scales. In addition to modulating sodium and potassium currents, β 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 β1 and β1B, 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 β1 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. β1-C121W subunits are expressed at the neuronal cell surface in vivo. However, despite this, β1-C121W polypeptides are incompletely glycosylated and do not associate with VGSC α subunits in the brain. β1-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 Scn1bW/W mice. These data, together with our previous results showing that β1-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 β1 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.

Of fish and men

Commentary Dravet syndrome (DS) is a severe pediatric epilepsy that presents with multiple seizure types commonly resistant to pharmacologic treatment, as well as intellectual disability, behavioral abnormalities, gait and motor dysfunction, and increased mortality (1). In most cases, the disease is caused by heterozygous de novo mutations or gene deletions of SCN1A, the gene encoding the pore-forming protein Nav1.1 of the voltage-gated sodium channel (VGSC) (2). DS is considered an epileptic encephalopathy, meaning that seizures contribute to the progressive worsening of the neurologic condition (1). Unfortunately, most available anticonvulsants are ineffective in controlling the seizures; some anticonvulsants, such as phenytoin, carbamazepine, and lamotrigine, are sodium channel blockers and may worsen seizure control. Among the medications that have shown some efficacy are valproate, clobazam, clonazepam, topiramate, levetiracetam, bromides, and stiripentol (3). The ketogenic diet is also useful and may reduce seizure frequency in up to 75% of patients (4). These treatments are neither fully effective nor curative, however, and thus a need exists for finding better therapies for DS. The search for such new compounds would be greatly aided by the availability of a DS model amenable to high-throughput screening (HTPS). Available models of DS include Scn1a knock-out mice (5), mice engineered with a human DS SCN1A mutation (6), and DS patient-induced pluripotent–stem cell (iPSC)-derived neurons (7, 8). While the murine models reproduce many of the behavioral and electrographic characteristics of the disease, breeding is often difficult and takes substantial time, seizure phenotypes vary considerably between different mouse genetic backgrounds, and screening techniques are not easily scalable. The DS iPSC model has the advantage of being patient derived and shows in vitro electrophysiological abnormalities, but at present no consensus exists on the best method to monitor responses to pharmacologic treatments in this model. The study by Baraban et al. aims to investigate the feasibility of using zebrafish as an animal model of DS to investigate disease mechanisms and the feasibility of HTPS of pharmacologic compounds. Given the short breeding times and the possibility of simultaneously monitoring dozens of fish, this is an intriguing idea. The authors take advantage of a previously described mutant fish line carrying a homozygous mutation in the gene Scn1Lab (9), which shares 77% identity with the human SCN1A gene. Because the zebrafish genome underwent a duplication during evolution, the fish have two genes homologous to SCN1A: Scn1Lab and Scn1Laa. Thus, a homozygous mutation in one of these genes would be equivalent to a heterozygous condition in the human. Through the use of quantitative PCR and in situ hybridization, the authors demonstrate that Scn1Lab is expressed during early CNS development. The mutation causes a reduction in the expression levels of this gene, while those of other VGSC genes are not affected. Fortuitously and for unknown reasons, the mutant fish exhibit hyperpigmentation, facilitating the recognition of mutants from their wild-type siblings. Microarray and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated mild changes in the expression level of 1,099 genes, including some associated with CNS disorders, and this database should provide a valuable resource for future studies. Drug Screening in Scn1a Zebrafish Mutant Identifies Clemizole as a Potential Dravet Syndrome Treatment.

Runaway Dendrites: Blame the Older Siblings

Commentary Temporal lobe epilepsy (TLE) is one of the most common intractable epilepsy syndromes encountered in the clinic. Hippocampal pathology in TLE patients includes loss of hilar cells and pyramidal neurons in CA1 and CA3, astroand microgliosis, and architectural abnormalities of the dentate granule cells (DGCs). The latter comprises three main structural alterations. First is an abnormal localization of the DGC soma, resulting in dispersion of the granule cell layer and hilar ectopic DGCs. Second, the axonal projections of DGCs (termed “mossy fibers”) remodel abnormally as well, resulting in mossy fiber sprouting into the dentate inner molecular layer, where DGCs normally receive synaptic inputs (1). Third, many DGCs exhibit abnormal dendritic trees with dendrites projecting into the hilus; these are known as hilar basal dendrites (HBDs) (2, 3). Multiple animal models of TLE exist. These models are typically created by local or systemic administration of chemicals, toxins, or electrical stimulation to induce status epilepticus (4). Although they differ in the degree to which they recapitulate the aforementioned neuropathologic abnormalities, the presence of mossy-fiber sprouting seems to be a constant finding (5). The presence of HBDs, however, has only been examined and identified in a subset of TLE models to date, including those involving intraperitoneal application of either kainic acid (KA) or pilocarpine (2, 3). Of interest, in both of these animal models, the induction of epilepsy markedly increases DGC neurogenesis (6). Furthermore, only DGCs maturing during, or especially those generated after, the epileptogenic insult exhibit persistent HBDs (7–9). The study by Murphy et al. aims to investigate whether HBDs might be present in the only known adult rodent model Abnormalities of Granule Cell Dendritic Structure Are a Prominent Feature of the Intrahippocampal Kainic Acid Model of Epilepsy Despite Reduced Postinjury Neurogenesis.