Molecular and circuit defects underlying different SCN2A mutations and ASDs

不同 SCN2A 突变和自闭症谱系障碍 (ASD) 背后的分子和电路缺陷

• 批准号: 10362623
• 负责人: Geoffrey S Pitt
• 金额: $ 64.34万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-07 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10362623
• 关键词: Action Potentials; Adult; Affect; Amygdaloid structure; Axon; Behavior; Behavioral; Biological Assay; Brain; C-terminal; CRISPR/Cas technology; Calmodulin; Cells; Characteristics; Clinical; Crystallization; Data; Defect; Detection; Development; Diagnosis; Disease model; Distal; Electrophysiology (science); Equilibrium; Fiber; Financial compensation; Functional disorder; Funding; Genes; Goals; Impairment; Interneurons; Lead; Link; Modeling; Molecular; Mus; Mutation; Neurons; Outcome; Output; Photometry; Point Mutation; Property; Protein Truncation; Reporting; Roentgen Rays; SCN2A protein; Social Behavior; Social Interaction; Sodium Channel; Structure; Synapses; Techniques; Testing; Therapeutic; Variant; Weaning; anxiety-like behavior; autism spectrum disorder; behavioral phenotyping; designer receptors exclusively activated by designer drugs; effective therapy; endophenotype; excitatory neuron; exome sequencing; genomic locus; in vivo; insight; loss of function; loss of function mutation; mouse model; mutant; neocortical; neural circuit; neuronal excitability; novel; restoration; social deficits; tool; voltage

ABSTRACT The recent wave of whole exome sequencing studies places SCN2A, which encodes the neuronal voltage- gated Na+ channel pore-forming α subunit NaV1.2, near top of the list of genetic loci linked to autism spectrum disorders (ASDs). On the one hand, that NaV1.2 is an essential Na+ channel responsible for initiating action potentials within excitatory neurons in the developing brain provides a rationale for the prominence of SCN2A. On the other, most SCN2A mutations associated with ASDs are loss-of-function and predicted to decrease neuronal excitability, an outcome that would lower the neocortical excitation/inhibition (E/I) balance and thus contrast with the generally accepted model that behavior defects in ASDs, such as social dysfunction, result from an increased E/I balance. This conundrum persists because of the absence of Scn2a mouse models that reveal ASD-associated endophenotypes, thus limiting our ability to dissect the cellular electrophysiological defects associated with Scn2a loss-of-function mutations and the consequent circuit level dysfunctions that lead to ASD-associated behaviors. Building on x-ray crystal structures of key regulatory components of NaV1.2 that we solved and analyzed during the previous funding period, we obtained specific insights into how ASD- associated mutations in NaV1.2 perturb channel function and alter E/I balance. Further, we generated two novel Scn2a mouse models by CRISPR/Cas9 to test the specific contribution of Scn2a mutations in vivo. Initial analyses of these models reveal abnormal Na+ channel function, decreased cortical neuron excitability, and dysfunctional behaviors consistent with ASDs, while simultaneously demonstrating informative differences between the two models. These models provide a unique set of tools that will allow us to trace abnormal channel function through altered neuronal electrical activity to the consequent circuit-level dysfunction and the resulting ASD endophenotypes. We propose to exploit these novel Scn2a mutant models for the following Aims: 1) We will obtain detailed information about their neuronal electrophysiological characteristics and synaptic properties, thereby defining how Scn2a mutations perturb neuronal function. 2) We will employ fiber photometry and chemogenetic tools (DREADDs) to test whether the Scn2a mutations decrease excitatory drive to the basolateral amygdala and thereby produce the social dysfunction and impaired danger detection observed in our Scn2a mouse models. 3) We will exploit our initial electrophysiological findings to test a potential therapeutic strategy in which we aim to counteract the reduced Na+ current associated with ASD-associated SCN2A loss-of-function mutations. Our overall goals are to define the range of cellular dysfunction that results from Scn2a mutations and trace those abnormalities through the circuit level to behavioral manifestations.

抽象的 最近一波全外显子组测序研究将编码神经元电压的 SCN2A 置于 门控 Na+ 通道成孔 α 亚基 NaV1.2，接近与自闭症谱系相关的遗传位点列表的顶部 障碍（ASD）。一方面，NaV1.2 是负责启动作用的重要 Na+ 通道 发育中大脑中兴奋性神经元的潜力为 SCN2A 的突出地位提供了理论基础。 另一方面，大多数与 ASD 相关的 SCN2A 突变都会导致功能丧失，并且预计会减少 神经元兴奋性，这种结果会降低新皮质兴奋/抑制（E/I）平衡，从而 与普遍接受的模型相反，该模型认为自闭症谱系障碍患者的行为缺陷（例如社交功能障碍）会导致 来自增加的 E/I 平衡。由于缺乏 Scn2a 小鼠模型，这个难题仍然存在 揭示 ASD 相关的内表型，从而限制了我们剖析细胞电生理学的能力 与 Scn2a 功能丧失突变相关的缺陷以及随之而来的电路水平功能障碍 导致 ASD 相关行为。以 NaV1.2 关键调控成分的 X 射线晶体结构为基础 我们在上一个资助期间解决和分析了这些问题，我们获得了关于 ASD 如何- NaV1.2 的相关突变会扰乱通道功能并改变 E/I 平衡。此外，我们生成了两个 CRISPR/Cas9 构建的新型 Scn2a 小鼠模型，用于测试体内 Scn2a 突变的具体贡献。最初的 对这些模型的分析揭示了 Na+ 通道功能异常、皮质神经元兴奋性降低以及 与自闭症谱系障碍一致的功能失调行为，同时表现出信息差异 两个模型之间。这些模型提供了一套独特的工具，使我们能够追踪异常情况 通过改变神经元电活动来调节通道功能，从而导致随后的电路水平功能障碍和 由此产生的 ASD 内表型。 我们建议利用这些新颖的 Scn2a 突变模型来实现以下目标：1）我们将获得详细的 有关其神经元电生理特征和突触特性的信息，从而定义 Scn2a 突变如何扰乱神经元功能。 2）我们将采用光纤光度测定和化学遗传学工具 （DREADD）测试 Scn2a 突变是否会降低基底外侧杏仁核的兴奋性驱动 从而产生在我们的 Scn2a 小鼠模型中观察到的社交功能障碍和危险检测受损。 3）我们将利用我们最初的电生理学发现来测试我们的目标的潜在治疗策略 抵消与 ASD 相关的 SCN2A 功能丧失突变相关的 Na+ 电流减少。我们的 总体目标是确定 Scn2a 突变导致的细胞功能障碍的范围并追踪这些突变 从电路层面到行为表现的异常。