Neuronal hyperexcitability and seizures in a Scn2a deficient mouse model

Scn2a 缺陷小鼠模型中的神经元过度兴奋和癫痫发作

• 批准号: 10281836
• 负责人: Yang Yang
• 金额: $ 38.75万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10281836
• 关键词: ASD patient; Action Potentials; Address; Adult; Affect; Brain; Brain region; Cells; Child; Corpus striatum structure; Data; Development; Disease; Down-Regulation; Electroencephalogram; Electrophysiology (science); Epilepsy; Foundations; Genetic; Genetic study; Human; Knock-out; Knockout Mice; Knowledge; Length; Light; Medial; Mission; Modeling; Molecular; Mus; Neuraxis; Neurons; Outcome; Pentylenetetrazole; Pharmacology; Phenotype; Physiology; Potassium Channel; Predisposition; Prefrontal Cortex; Research Personnel; Seizures; Signal Transduction; Slice; Sodium Channel; TNFSF5 gene; Technology; Testing; Therapeutic Intervention; United States National Institutes of Health; Variant; Work; autism spectrum disorder; comorbidity; density; efficacy evaluation; gain of function; gene therapy; hippocampal pyramidal neuron; in vivo; innovation; insight; loss of function; mouse model; neural network; neuronal excitability; novel; novel therapeutic intervention; novel therapeutics; patch clamp; restoration; therapeutically effective; transcriptome sequencing; voltage

Project Summary/Abstract Genetic studies in humans revealed that gain-of-function variants of SCN2A are associated with epilepsy, whereas loss-of-function variants of SCN2A are associated with autism spectrum disorder (ASD). However, about a third of ASD children carrying SCN2A loss-of-function or nonsense variants (collectively referred to as SCN2A deficiency) develop intractable seizures, major comorbidity associated with ASD. How SCN2A deficiency contributes to seizure is largely unknown. To address this problem, we generated a Scn2a-deficient mouse model by gene-trap knockout (gtKO) strategy. Homozygous gene-trap knockout mice have only a quarter of the Scn2a expression level of WT mice, which model severe Scn2a deficiency. Our preliminary patch-clamp recordings reveal that neurons with severe Scn2a deficiency displayed neuronal hyperexcitability. This finding is unexpected, but could open the door for the understanding of the puzzling seizure comorbidity in SCN2A deficiency-related ASD. However, critical gaps exist regarding the possible mechanisms underlying Scn2a deficiency-related neuronal hyperexcitability, its consequences on neural network and seizures susceptibility, as well as the reversibility of seizures-related phenotypes. Filling these gaps would greatly enhance understanding of the mechanisms underlying seizure comorbidity in ASD; and shed new light on the development of effective therapeutic interventions. To this end, here we propose to test an overarching hypothesis that a substantial reduction of Scn2a expression results in increased neuronal excitability related to K channel downregulation, hypersynchronization of in vivo firing, and elevated seizure susceptibility that can be reversed by targeted genetic interventions. We will test our hypothesis at the cellular, circuit, and in vivo levels. In Aim 1, we will assess ex vivo neuronal excitability of Scn2a-deficient mice. In Aim 2, we will determine in vivo neuronal firings and seizure susceptibility of the Scn2a-deficient mice. In Aim 3, we will test targeted genetic interventions. Our study is significant in the following ways: i) SCN2A deficiency is among the leading monogenetic forms of ASD and seizure comorbidity occurs in about 30% of affected ASD patients; ii) The finding that severe SCN2A deficiency resulting in hyperexcitability is potentially paradigm-shifting, and the study of which could reveal key insights regarding seizure comorbidity associated with ASD; and iii) Targeted genetic interventions to be evaluated have clear translational relevance. Our study has the following innovations: i) the use of novel Scn2a deficient mice that reveal unexpected finding of neuronal hyperexcitability; ii) innovative ways to achieve genetic rescue; and iii) the use of cutting-edge technologies including high-density Neuropixels recordings. The applicant is an early stage investigator (ESI), whose team has extensive expertise in sodium channel physiology, genetics, electrophysiology, and pharmacology. The team is well suited to carry out the proposed work to its full completion within the project timeframe, and generate impactful outcomes to advance the field.

项目概要/摘要 人类遗传学研究表明，SCN2A 的功能获得性变异与癫痫相关， 而 SCN2A 的功能丧失变异与自闭症谱系障碍 (ASD) 相关。然而， 大约三分之一的 ASD 儿童携带 SCN2A 功能丧失或无意义变异（统称为 SCN2A 缺陷）会导致顽固性癫痫发作，这是与 ASD 相关的主要合并症。 SCN2A缺乏如何发生 导致癫痫发作的因素在很大程度上尚不清楚。为了解决这个问题，我们生成了一个缺乏 Scn2a 的小鼠 通过基因陷阱敲除（gtKO）策略建立模型。纯合子基因陷阱敲除小鼠只有四分之一 WT 小鼠的 Scn2a 表达水平，该模型模拟了严重的 Scn2a 缺陷。我们初步的膜片钳 记录显示，严重缺乏 Scn2a 的神经元表现出神经元过度兴奋。这一发现是 出乎意料，但可能为理解 SCN2A 中令人困惑的癫痫合并症打开大门 缺乏相关的 ASD。然而，关于 Scn2a 的可能机制存在关键差距 与缺乏相关的神经元过度兴奋，其对神经网络和癫痫易感性的影响，如 以及癫痫相关表型的可逆性。填补这些空白将大大增强理解 自闭症谱系障碍 (ASD) 癫痫发作合并症的潜在机制；并为开发有效的 治疗干预。为此，我们在这里建议检验一个总体假设，即实质性 Scn2a 表达的减少导致与 K 通道下调相关的神经元兴奋性增加， 体内放电的超同步化和癫痫易感性升高，可以通过靶向遗传来逆转 干预措施。我们将在细胞、回路和体内水平上检验我们的假设。在目标 1 中，我们将评估 ex Scn2a 缺陷小鼠的体内神经元兴奋性。在目标 2 中，我们将确定体内神经元放电和癫痫发作 Scn2a 缺陷小鼠的易感性。在目标 3 中，我们将测试有针对性的基因干预措施。我们的研究是 i) SCN2A 缺陷是自闭症谱系障碍 (ASD) 的主要单基因形式之一， 大约 30% 的自闭症谱系障碍 (ASD) 患者会出现癫痫合并症； ii) 发现严重的 SCN2A 缺陷 导致过度兴奋可能会带来范式转变，对此的研究可以揭示关键见解 关于与 ASD 相关的癫痫合并症； iii) 待评估的有针对性的遗传干预措施 明确的翻译相关性。我们的研究有以下创新： i) 使用新型 Scn2a 缺陷小鼠 揭示了神经元过度兴奋的意外发现； ii) 实现基因拯救的创新方法；和 iii) 使用尖端技术，包括高密度 Neuropixels 记录。申请人是早期 阶段研究员（ESI），其团队在钠通道生理学、遗传学、 电生理学和药理学。该团队非常适合全面完成拟议的工作 在项目时间范围内，并产生有影响力的成果来推动该领域的发展。