Functional Genetics of the Neuronal Sodium Channel Gene SCN8A

描述(申请人提供)：编码Nav1.6的电压门控钠通道SCN8A在中枢和外周神经系统的神经元中表达，并集中在兰维尔轴突起始段和结节。由于其广泛的表达，SCN8A的突变会影响大脑和周围神经功能的许多方面。这项建议的重点是在患者中鉴定人类SCN8A和相关钠通道的新突变，并使用体外和体内表达系统进行功能分析，以评估这些突变的致病性。我们已经建立了一条管道，用于通过整个外显子组测序项目确定神经和精神障碍患者的钠通道突变的功能特征。在上一次筹资期间，发现并确定了七种新的人类突变。癫痫脑病、自闭症和SUDEP患者中的一种新的SCN8A突变导致持续电流增加，导致神经元过度兴奋。我们将建立这种疾病的小鼠模型，以阐明其对神经和心脏功能的影响。SCN8A基因的单倍性不足会导致智力残疾和焦虑样行为。我们将我们的Scn8a小鼠与SERT-Cre和VIAAT-Cre小鼠杂交，以探讨5-羟色胺能神经元和抑制性中间神经元在异常行为中的作用。我们将通过Bira生物素连接酶转基因在体内对Avi标记的Nav1.6转基因进行生物素化，以无偏见地寻找与Nav1.6相互作用的蛋白质。我们将确定与MAP1B的相互作用是否介导了Nav1.6到Ranvier节点的囊泡运输。这些研究将有助于理解Nav1.6基因突变的基本功能和临床后果，Nav1.6基因突变是神经元活动的主要决定因素。

DESCRIPTION (provided by applicant): The voltage-gated sodium channel SCN8A encoding Nav1.6 is expressed in neurons throughout the central and peripheral nervous system, and is concentrated at axonal initial segments and nodes of Ranvier. Because of its widespread expression, mutations of SCN8A affect many aspects of brain and peripheral nerve function. This proposal is focused on identifying new mutations of human SCN8A and related sodium channels in patients, and carrying out functional analysis using in vitro and in vivo expression systems to evaluate the pathogenicity of the mutations. We have established a pipeline for functional characterization of sodium channel mutations identified by whole exome sequencing projects in patients with neurological and psychiatric disorders. Seven novel human mutations were identified and characterized during the previous funding period. A de novo SCN8A mutation in a patient with epileptic encephalopathy, features of autism, and SUDEP causes elevated persistent current leading to neuronal hyperexcitability. We will generate a mouse model of this disorder, to elucidate the effects on neuronal and cardiac function. Haploinsufficiency of SCN8A in results in intellectual disability and anxiety-like behavior. We wil cross our floxed Scn8a mouse with SERT-CRE and VIAAT-CRE mice to probe the contributions of serotonergic neurons and inhibitory interneurons to the abnormal behaviors. We will carry out in vivo biotinylation of an Avi-Tagged Nav1.6 transgene by the BirA biotin ligase transgene in an unbiased search for proteins that interact with Nav1.6. We will determine whether interaction with MAP1B mediates vesicular transport of Nav1.6 to the nodes of Ranvier. These studies will contribute to understanding basic function and clinical consequences of mutations in Nav1.6, a major determinant of neuronal activity.