Role of Sodium Channel SCN1B in Inherited Epilepsy

 描述(由申请人提供)：早发性儿童癫痫脑病(EES)，如德拉韦综合征(DS)，由于高度的神经发育损害，包括发育迟缓、认知能力下降和智力残疾，对家庭来说是毁灭性的。最令人担忧的是癫痫的严重发作和猝死的高风险(SUDEP)。电压门控钠通道α和β亚单位基因突变与DS相关。虽然大多数DS病例与SCN1A单倍体功能不全有关，但SCN1B纯合子突变也与DS(或类似DS的EE)有关。本工作的目的是了解人SCN1B连锁的DS/EE的超兴奋性的机制。推测Scn1b在神经元中的信号转导机制涉及细胞类型和亚细胞区特异性钠电流(Ina)和钾电流(Ik)的改变，以及细胞黏附介导的神经元通路和离子通道和转运蛋白的转录调控。有人认为，人类SCN1B-DS突变导致神经元细胞间通讯和离子电流的缺陷，这与在Scn1b-/-小鼠中观察到的类似。我们设计了三个特定的目标来验证这一假说：1.确定人类SCN1B连锁DS突变是否会在体内导致功能丧失。SCN1B-DS突变被认为会导致功能性零表型，然而，这还没有在体内进行测试。一个具有遗传、隐性、SCN1B-DS突变的家庭捐赠了皮肤活检，用于诱导多能干细胞(IPSC)的生成。这个突变，以及之前发现的SCN1B-R125C人类突变，将被引入小鼠Scn1b基因座，以测试与Scn1b-/-小鼠相比，纯合子后代在兴奋性、神经元寻路、INA、IK和GABA能信号方面的变化。同时，将产生与SCN1B连接的人类DS iPSC神经元和脑器官，并测试是否存在类似的缺陷。基因编辑将被用来进行等基因对照，并产生纯合子零患者来源的神经元，以直接比较相同的、同基因的人类神经细胞系中SCN1B-DS突变和零细胞。2.确定Scn1b-/-大脑皮层脑片INA或Ik的定位变化。SCN1B-DS癫痫发作的原因可能不是先前提出的神经元寻路中断，而是INA或Ik的神经元亚型特异性改变。在这里，有核斑块、从AIS中拔出的斑块和免疫荧光染色的组合将被用来确定-/-与//皮质中INA和VGSC表达的差异变化。还将测试IK和电压门控K通道(VGKC)表达的变化。3.确定Scn1b介导的神经元寻路中断是否在DS的超兴奋性中起作用。Scn1b-/-小鼠在癫痫发作之前就存在神经元寻路缺陷。有人提出，这些缺陷可能导致癫痫的发展。一个可诱导的泛神经元Cre系将被用来在小鼠大脑发育的关键期之后删除Scn1b，以确定是否像Scn1b-/-小鼠那样发生癫痫发作和早期死亡。在这里，将研究在渐进性发育时间点Scn1b缺失后INA、IK、神经元模式和GABA能信号的变化。β-1介导的神经突起生长需要跨亲同的β-β-1细胞黏附，从而在体外导致β-1与ankG的细胞内结合。在第二组实验中，将在小鼠Scn1b基因座引入突变，从而中断β1-ankG关联或β1酪氨酸磷酸化，以询问β1-cam信号级联的中断是否会导致体内癫痫发作。尽管SCN1B连锁的DS/EE是一种罕见的疾病，但这项工作非常重要，因为它将提供关于大脑发育缺陷和离子电流调节如何协同导致超兴奋性的新信息。

 DESCRIPTION (provided by applicant): Early onset pediatric epileptic encephalopathies (EEs) such as Dravet Syndrome (DS) are devastating to families because of the high degree of neurodevelopmental compromise, including developmental delay, cognitive decline, and intellectual disability. Most concerning are the severe seizures and high risk of sudden unexpected death in epilepsy (SUDEP). Mutations in voltage-gated Na+ channel (VGSC) α and β subunit genes are linked to DS. While the majority of DS cases are linked to SCN1A haploinsufficiency, SCN1B homozygous mutations are also linked to DS (or a DS-like EE). The objective of this work is to understand the mechanism of hyperexcitability in human SCN1B-linked DS/EE. It is hypothesized that the mechanism of Scn1b signaling in neurons involves cell type and subcellular domain specific changes in Na+ current (INa) and K+ current (IK) as well as cell adhesion-mediated effects on neuronal pathfinding and transcriptional regulation of ion channels and transporters. It is proposed that human SCN1B-DS mutations result in defects in neuronal cell-cell communication and ionic currents that are similar to those observed in Scn1b-/- mice. Three Specific Aims are designed to test this hypothesis: 1. To determine whether human SCN1B-linked DS mutations result in loss-of-function in vivo. SCN1B-DS mutations are assumed to result in a functional null phenotype, however, this has not been tested in vivo. A family with an inherited, recessive, SCN1B-DS mutation has donated skin biopsies for induced pluripotent stem cell (iPSC) generation. This mutation, as well as the previously identified SCN1B-R125C human mutation, will be introduced into the mouse Scn1b locus to test homozygous progeny for changes in excitability, neuronal pathfinding, INa, IK, and GABAergic signaling in comparison with Scn1b-/- mice. In parallel, SCN1B-linked human DS iPSC neurons and cerebral organoids will be generated and tested for similar deficits. Gene editing will be used to make isogenic controls, and to generate homozygous null patient-derived neurons to directly compare SCN1B-DS mutant and null cells in the same, isogenic, iPSC line of human neurons. 2. To determine localized changes in INa or IK in Scn1b-/- brain cortical slices. It is possible that the cause of seizures in SCN1B-DS is not disrupted neuronal pathfinding, as previously proposed, but instead neuronal subtype specific changes in INa or IK. Here, a combination of nucleated patch, pulled patches from the AIS, and immunofluorescence staining will be used to determine differential changes in INa and VGSC expression in -/- vs. +/+ cortex. Changes in IK and voltage-gated K+ channel (VGKC) expression will also be tested. 3. To determine whether disruption of Scn1b-mediated neuronal pathfinding plays a role in hyperexcitability in DS. Scn1b-/- mice have neuronal pathfinding defects that precede seizure onset. It was proposed that these defects might lead to the development of seizures. An inducible, pan-neuronal Cre line will be used to delete Scn1b past the critical period of mouse brain development to determine whether seizures and early mortality occur as in Scn1b-/- mice. Here, changes in INa, IK, neuronal patterning, and GABAergic signaling will be investigated following Scn1b deletion at progressive developmental time points. β1-mediated neurite outgrowth requires trans homophilic β1-β1 cell adhesion leading to intracellular association of β1 with ankG in vitro. In a second set of experiments, mutations will be introduced to the mouse Scn1b locus that interrupt β1-ankG association or β1 tyrosine phosphorylation to ask whether disruption of the β1-CAM signaling cascade leads to seizures in vivo. Even though SCN1B- linked DS/EE is a rare disease, this work is important because it will provide new information regarding how deficits in brain development and regulation of ionic currents can synergize to result in hyperexcitability.