Role of Sodium Channel SCN1B in Inherited Epilepsy

钠通道 SCN1B 在遗传性癫痫中的作用

• 批准号: 9147046
• 负责人: Lori L. Isom
• 金额: $ 53.22万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9147046
• 关键词: Anions; Axon; Biopsy; Brain; Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Cell Line; Cell-Cell Adhesion; Cells; Cerebrum; Cessation of life; Childhood; Comorbidity; Complex; Data; Defect; Development; Developmental Delay Disorders; Encephalopathies; Epilepsy; Extracellular Matrix; Family; GABA-A Receptor; Generations; Genes; Genetic Models; Goals; Health; Human; Immunofluorescence Immunologic; Immunoglobulins; Impaired cognition; In Vitro; Inherited; Intellectual functioning disability; Ion Channel; Lead; Light; Link; Mediating; Mus; Muscle fasciculation; Mutation; Neurites; Neurons; Null Lymphocytes; Organoids; Parents; Patients; Phenotype; Phosphotransferases; Play; RNA Splicing; Rare Diseases; Regulation; Role; SCN1A protein; SCN8A gene; Seizures; Signal Transduction; Skin; Slice; Sodium Channel; Staining method; Stains; Syndrome; Testing; Time; Transcriptional Regulation; Tyrosine Phosphorylation; Variant; Voltage-Gated Potassium Channel; Wild Type Mouse; Work; cell type; critical period; design; early onset; high risk; in vivo; induced pluripotent stem cell; loss of function; migration; mortality; mutant; neuronal excitability; neuronal patterning; proband; research study; voltage

 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.

 描述（由申请人提供）：由于高度的神经发育损害，包括发育迟缓、认知下降和智力残疾，早发性儿科癫痫性脑病（EE）如Dravet综合征（DS）对家庭具有毁灭性影响。最令人担忧的是癫痫发作严重和突然意外死亡的高风险（SUDEP）。电压门控Na+通道（VGSC）α和β亚基基因的突变与DS相关。虽然大多数DS病例与SCN 1A单倍不足有关，但SCN 1B纯合突变也与DS（或DS样EE）有关。本工作的目的是了解人SCN 1B相关DS/EE的超兴奋机制。假设神经元中Scn 1b信号传导机制涉及细胞类型和亚细胞结构域特异性的Na+电流（INa）和K+电流（IK）变化以及细胞粘附介导的对神经元寻路和离子通道和转运蛋白转录调节的影响。有人提出，人类SCN 1B-DS突变导致神经元细胞间通讯和离子电流的缺陷，这与在Scn 1b-/-小鼠中观察到的缺陷相似。三个具体的目的是为了测试这一假设：1。确定人SCN 1B连锁DS突变是否导致体内功能丧失。假设SCN 1B-DS突变导致功能无效表型，但尚未在体内进行测试。一个具有遗传性隐性SCN 1B-DS突变的家庭捐赠了用于诱导多能干细胞（iPSC）生成的皮肤活检。将该突变以及先前鉴定的SCN 1B-R125 C人类突变引入小鼠Scn 1b基因座，以检测与Scn 1b-/-小鼠相比纯合子代的兴奋性、神经元寻路、INa、IK和GABA能信号传导的变化。同时，将产生SCN 1B连接的人DS iPSC神经元和脑类器官，并测试类似的缺陷。基因编辑将用于制备等基因对照，并产生纯合的无效患者源性神经元，以直接比较人类神经元的相同等基因iPSC系中的SCN 1B-DS突变体和无效细胞。2.确定Scn 1b-/-脑皮质切片中INa或IK的局部变化。SCN 1B-DS中癫痫发作的原因可能不是如先前提出的神经元寻路中断，而是INa或IK中神经元亚型特异性变化。在此，将使用有核斑块、从AIS拉出的斑块和免疫荧光染色的组合来确定-/-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Scn 1b介导的神经元寻路中断是否在DS的过度兴奋中起作用。scn 1b-/-小鼠在癫痫发作前有神经元寻路缺陷。有人提出，这些缺陷可能导致癫痫发作的发展。一个可诱导的，泛神经Cre线将被用来删除Scn 1b过去的小鼠大脑发育的关键时期，以确定癫痫发作和早期死亡是否发生在Scn 1b-/-小鼠。在这里，INa，IK，神经元模式，和GABA能信号的变化将进行性发育时间点的Scn 1b删除后进行研究。β1介导的神经突生长需要反式嗜同性β1-β1细胞粘附，导致β1与ankG在体外的细胞内缔合。在第二组实验中，将突变引入小鼠Scn 1b基因座，中断β1-ankG结合或β1酪氨酸磷酸化，以研究β1-CAM信号级联的破坏是否导致体内癫痫发作。尽管SCN 1B-连锁的DS/EE是一种罕见的疾病，但这项工作很重要，因为它将提供有关大脑发育缺陷和离子电流调节如何协同导致过度兴奋的新信息。