Pathogenesis of epilepsy in a SCN8A human mutation mouse model

SCN 8A（编码钠（Na）通道亚型Nav1.6的基因）的从头突变是 已知引起早期婴儿癫痫性脑病13（EIEE 13）。迄今为止，超过150架SCN 8A 突变已被确认。患者经历各种癫痫发作类型和运动特征， 对轮椅的依赖智力残疾从轻微到严重不等，并逐渐恶化 癫痫发作癫痫猝死（Sudden unexpected death in epilepsy，SUDEP）发生在约10%的患者中。 如果癫痫不被控制，病情会显著加重。不幸的是，大多数患者都有药物 难治性癫痫或对抗癫痫药物（AED）的混合反应。 关于SCN 8A癫痫性脑病的发病机制或SCN 8A癫痫性脑病的治疗选择知之甚少。 患者在这项提议中，我们将使用一种高度新颖和创新的敲入小鼠模型，由 Meisler实验室，携带人类SCN 8A脑病突变p.Asn1768Asp（N1768 D）。突变 在一名6个月大的儿童中发现了难治性癫痫，智力残疾， 15岁时共济失调和SUDEP。小鼠模型表现出许多病理表型， 患者，包括自发性癫痫发作和猝死。在纯合突变小鼠（D/D）中，癫痫发作开始 并在24小时内进展至死亡。杂合子突变小鼠（D/+）癫痫发作较晚 从8周龄开始发病，并在1至2个月内进展至死亡。这个的可用性 小鼠模型提供了一个独特的机会，充分研究这种毁灭性的人类疾病的发病机制， 癫痫性脑病，并测试新的和选择性的治疗方法。 在这个提议中，我们将研究当Na通道生理学和膜 兴奋性开始出现在我们的癫痫脑病模型中，测试兴奋性和抑制性 已知与癫痫发作活动有关的大脑区域内的神经元。在目标1中，我们将确定 使用突变小鼠在癫痫发作之前和之后的特定时间点观察这些改变的发病机制。在 目的2，我们将使用病毒递送的、dox诱导的Nav1.6 shRNA沉默Nav1.6，靶向兴奋性 或抑制性神经元，并确定对Na通道活性、神经元兴奋性和癫痫发作活性的影响 在突变小鼠中。我们将确定我们是否可以通过在癫痫发作前的一个时间点进行靶向治疗来延迟癫痫发作。 癫痫发作，以及我们是否可以调节自发性癫痫发作小鼠的癫痫发作活动。在目标3中， 将测试我们的Nav1.6亚型选择性化合物（MV 1505）是否可以减少杂合子的癫痫发作活动。 （D/+）小鼠和延迟SUDEP。我们还将评估两种临床使用的抗惊厥药（苯妥英和 拉考沙胺）。这些研究将极大地影响我们目前对生理学的理解。 SCN 8A癫痫性脑病中Nav1.6活性增加的后果。他们还将提供 这是对Nav1.6选择性靶向治疗的重要见解。

De novo mutations of SCN8A, the gene that encodes for the sodium (Na) channel isoform Nav1.6, are known to cause early infantile epileptic encephalopathy 13 (EIEE13). To date, more than 150 SCN8A mutations have been identified. Patients experience a variety of seizure types and motor features that can lead to wheelchair dependence. Intellectual disability varies from mild to severe and becomes progressively worst with seizure onset. Sudden unexpected death in epilepsy (SUDEP) occurs in approximately 10% of patients and increases significantly if seizures are not controlled. Unfortunately, a majority of patients have drug refractory epilepsy or a mixed response to anti-epileptic drugs (AEDs). Very little is known about the pathogenesis of SCN8A epileptic encephalopathy or treatment options for patients. In this proposal we will use a highly novel and innovative knock-in mouse model, developed by the Meisler lab, carrying the human SCN8A encephalopathy mutation p.Asn1768Asp (N1768D). The mutation was identified in a child who presented with refractory epilepsy at the age of 6 months, intellectual disability, ataxia and SUDEP at 15 years of age. The mouse model exhibits many of the pathological phenotypes seen in patients, including spontaneous seizures and sudden death. In homozygous mutant mice (D/D), seizures begin at 3 weeks of age and progress to death within 24 hours. Heterozygous mutant mice (D/+) have later seizure onset starting at 8 weeks of age and progression to death within one to two months. The availability of this mouse model provides a unique opportunity to fully investigate the pathogenesis of this devastating human epileptic encephalopathy and to also test new and selective therapies. In this proposal we will investigate when alterations in Na channel physiology and membrane excitability begin to appear in our model of epileptic encephalopathy, testing both excitatory and inhibitory neurons within brain regions known to be involved in seizure activity. In Aim 1 we will determine the pathogenesis of these alterations at specific time points before and after seizure onset using mutant mice. In Aim 2, we will silence Nav1.6 using virally delivered, dox-inducible, Nav1.6 shRNA, targeting either excitatory or inhibitory neurons and determine the effects on Na channel activity, neuronal excitability and seizure activity in mutant mice. We will determine if we can delay the onset of seizures by targeting at a time point before the onset of seizures and also if we can modulate seizure activity in mice with spontaneous seizures. In Aim 3, we will test whether our Nav1.6 subtype selective compound (MV1505) can reduce seizure activity in heterozygous (D/+) mice and delay SUDEP. We will also evaluate two clinically used anticonvulsants (phenytoin and lacosamide). These studies will significantly impact our current understanding of the physiological consequences of increased Nav1.6 activity in SCN8A epileptic encephalopathy. They will also provide important insight into the selective targeting of Nav1.6 for therapy.