RESUMO
Mutations in voltage-gated sodium channels expressed highly in the brain (SCN1A, SCN2A, SCN3A, and SCN8A) are responsible for an increasing number of epilepsy syndromes. In particular, mutations in the SCN3A gene, encoding the pore-forming Nav1.3 α subunit, have been identified in patients with focal epilepsy. Biophysical characterization of epilepsy-associated SCN3A variants suggests that both gain- and loss-of-function SCN3A mutations may lead to increased seizure susceptibility. In this report, we identified a novel SCN3A variant (L247P) by whole exome sequencing of a child with focal epilepsy, developmental delay, and autonomic nervous system dysfunction. Voltage clamp analysis showed no detectable sodium current in a heterologous expression system expressing the SCN3A-L247P variant. Furthermore, cell surface biotinylation demonstrated a reduction in the amount of SCN3A-L247P at the cell surface, suggesting the SCN3A-L247P variant is a trafficking-deficient mutant. To further explore the possible clinical consequences of reduced SCN3A activity, we investigated the effect of a hypomorphic Scn3a allele (Scn3aHyp) on seizure susceptibility and behavior using a gene trap mouse line. Heterozygous Scn3a mutant mice (Scn3a+/Hyp) did not exhibit spontaneous seizures nor were they susceptible to hyperthermia-induced seizures. However, they displayed increased susceptibility to electroconvulsive (6Hz) and chemiconvulsive (flurothyl and kainic acid) induced seizures. Scn3a+/Hyp mice also exhibited deficits in locomotor activity and motor learning. Taken together, these results provide evidence that loss-of-function of SCN3A caused by reduced protein expression or deficient trafficking to the plasma membrane may contribute to increased seizure susceptibility.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.3/deficiência , Canal de Sódio Disparado por Voltagem NAV1.3/genética , Convulsões/genética , Convulsões/metabolismo , Canais de Sódio/deficiência , Canais de Sódio/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Epilepsias Parciais/genética , Epilepsias Parciais/metabolismo , Feminino , Predisposição Genética para Doença , Variação Genética , Células HEK293 , Humanos , Lactente , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , RNA Mensageiro/metabolismoRESUMO
SCN1A mutations are the main cause of the epilepsy disorders Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Mutations that reduce the activity of the mouse Scn8a gene, in contrast, are found to confer seizure resistance and extend the lifespan of mouse models of DS and GEFS+. To investigate the mechanism by which reduced Scn8a expression confers seizure resistance, we induced interictal-like burst discharges in hippocampal slices of heterozygous Scn8a null mice (Scn8a(med/+)) with elevated extracellular potassium. Scn8a(med/+) mutants exhibited reduced epileptiform burst discharge activity after P20, indicating an age-dependent increased threshold for induction of epileptiform discharges. Scn8a deficiency also reduced the occurrence of burst discharges in a GEFS+ mouse model (Scn1a(R1648H/+)). There was no detectable change in the expression levels of Scn1a (Nav1.1) or Scn2a (Nav1.2) in the hippocampus of adult Scn8a(med/+) mutants. To determine whether the increased seizure resistance associated with reduced Scn8a expression was due to alterations that occurred during development, we examined the effect of deleting Scn8a in adult mice. Global Cre-mediated deletion of a heterozygous floxed Scn8a allele in adult mice was found to increase thresholds to chemically and electrically induced seizures. Finally, knockdown of Scn8a gene expression in the adult hippocampus via lentiviral Cre injection resulted in a reduction in the number of EEG-confirmed seizures following the administration of picrotoxin. Our results identify the hippocampus as an important structure in the mediation of Scn8a-dependent seizure protection and suggest that selective targeting of Scn8a activity might be efficacious in patients with epilepsy.
Assuntos
Hipocampo/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Convulsões/metabolismo , Convulsões/patologia , Fatores Etários , Animais , Animais Recém-Nascidos , Convulsivantes/toxicidade , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/patologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Potássio/metabolismo , Desempenho Psicomotor , Tempo de Reação/genética , Tempo de Reação/fisiologia , Convulsões/etiologia , Convulsões/genéticaRESUMO
We previously found that genetic mutants with reduced expression or activity of Scn8a are resistant to induced seizures and that co-segregation of a mutant Scn8a allele can increase survival and seizure resistance of Scn1a mutant mice. In contrast, Scn8a expression is increased in the hippocampus following status epilepticus and amygdala kindling. These findings point to Scn8a as a promising therapeutic target for epilepsy and raise the possibility that aberrant overexpression of Scn8a in limbic structures may contribute to some epilepsies, including temporal lobe epilepsy. Using a small-hairpin-interfering RNA directed against the Scn8a gene, we selectively reduced Scn8a expression in the hippocampus of the intrahippocampal kainic acid (KA) mouse model of mesial temporal lobe epilepsy. We found that Scn8a knockdown prevented the development of spontaneous seizures in 9/10 mice, ameliorated KA-induced hyperactivity, and reduced reactive gliosis. These results support the potential of selectively targeting Scn8a for the treatment of refractory epilepsy.
Assuntos
Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Convulsões/genética , Convulsões/metabolismo , Animais , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia do Lobo Temporal/diagnóstico , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Hipocampo/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , RNA Interferente Pequeno/genética , Convulsões/diagnósticoRESUMO
We report the profiling of the 5-methyl cytosine distribution within single genomic-sized DNA molecules at a gene-relevant resolution. This method linearizes and stretches DNA molecules by confinement to channels with a dimension of about 250×200 nm(2). The methylation state is detected using fluorescently labeled methyl-CpG binding domain proteins (MBD), with high signal contrast and low background. DNA barcodes consisting of methylated and non-methylated segments are generated, with both short and long concatemers demonstrating spatially resolved MBD binding. The resolution of the technique is better than 10 kbp, and single-molecule read-lengths exceeding 140 kbp have been achieved.