Neuronal hyperexcitability and seizures in a Scn2a deficient mouse model

项目总结/摘要 人类遗传学研究表明，SCN 2A的功能获得性变体与癫痫有关， 而SCN 2A的功能丧失变体与自闭症谱系障碍（ASD）相关。然而，在这方面， 大约三分之一的ASD儿童携带SCN 2A功能丧失或无义变体（统称为 SCN 2A缺陷）发展顽固性癫痫发作，与ASD相关的主要合并症。SCN 2A缺乏症 导致癫痫发作的原因尚不清楚为了解决这个问题，我们产生了Scn 2a缺陷小鼠， 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的 WT小鼠的Scn 2a表达水平，其模拟严重的Scn 2a缺乏。我们初步的膜片钳 记录显示具有严重Scn 2a缺陷的神经元显示神经元过度兴奋。这一发现 出乎意料，但可以为理解SCN 2A中令人困惑的癫痫合并症打开大门 与自闭症相关的自闭症然而，关于Scn 2a潜在的可能机制存在关键差距 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后果， 以及尿素相关表型的可逆性。填补这些空白将极大地增进理解 ASD中癫痫合并症的潜在机制;并为开发有效的 治疗干预。为此，在这里，我们建议测试一个总体假设，即一个实质性的 Scn 2a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，以及可以通过靶向遗传逆转的癫痫易感性升高， 干预措施。我们将测试我们的假设在细胞，电路和体内水平。在目标1中，我们将评估 Scn 2a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 Scn 2a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）SCN 2A缺乏是ASD的主要单基因形式之一， 约30%的受影响ASD患者患有癫痫发作合并症; ii）发现严重的SCN 2A缺乏 导致过度兴奋是潜在的范式转变，对它的研究可以揭示关键的见解， 关于与ASD相关的癫痫合并症;和iii）待评估的靶向遗传干预具有 明确的翻译相关性。我们的研究具有以下创新：i）使用新的Scn 2a缺陷小鼠 其揭示了神经元过度兴奋性意外发现; ii）实现遗传拯救的创新方法;以及 iii）使用尖端技术，包括高密度Neuropixels记录。申请人是一个早期 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。该小组非常适合全面完成拟议的工作 在项目时间框架内，并产生有影响力的成果，以推动该领域的发展。

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.