Molecular and circuit defects underlying different SCN2A mutations and ASDs

摘要 最近的整个外显子组测序研究浪潮取代了SCN2A，它编码神经元电压- 门控钠通道造孔α亚单位NaV1.2，接近自闭症谱系遗传基因座的顶部 精神障碍(ASD)。一方面，NaV1.2是一个重要的钠通道，负责启动动作 发育中的大脑兴奋性神经元内的电位为SCN2A的突出提供了理论基础。 另一方面，大多数与自闭症相关的SCN2A突变是功能丧失的，预计会减少 神经元兴奋性，这一结果将降低新皮质兴奋/抑制(E/I)平衡，从而 与普遍接受的自闭症患者行为缺陷(如社交功能障碍)导致的模型形成对比 来自E/I余额的增加。这一难题之所以持续存在，是因为缺乏SCN2A小鼠模型 揭示ASD相关的内表型，从而限制了我们剖析细胞电生理的能力 与SCN2A功能丧失突变和随之而来的电路水平功能障碍相关的缺陷 导致与ASD相关的行为。以NaV1.2关键调节组件的X射线晶体结构为基础 我们在前一个资助期解决和分析的问题，我们获得了关于ASD如何- NaV1.2相关突变扰乱通道功能，改变E/I平衡。此外，我们生成了两个 通过CRISPR/Cas9建立新的SCN2A小鼠模型，以检测SCN2A突变在体内的特异性贡献。首字母 对这些模型的分析显示，钠通道功能异常，皮质神经元兴奋性降低，以及 功能障碍行为与自闭症一致，同时表现出信息性差异 在这两款车型之间。这些模型提供了一套独特的工具，使我们能够跟踪异常情况 通过神经元电活动改变的通道功能到随后的电路水平功能障碍和 由此产生的ASD内表型。 我们建议开发这些新的SCN2A突变模型，以实现以下目标：1)我们将获得详细的 关于它们的神经元电生理特性和突触特性的信息，从而定义 SCN2A突变是如何扰乱神经元功能的。2)我们将使用纤维光度学和化学发生工具 (DREADDS)来测试SCN2A突变是否减少了对杏仁基底外侧核的兴奋性驱动，以及 从而产生在我们的SCN2A小鼠模型中观察到的社会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发现来测试潜在的治疗策略，我们的目标是 以抵消与ASD相关的SCN2A功能丧失突变相关的钠电流减少。我们的 总体目标是定义由SCN2A突变导致的细胞功能障碍的范围，并追踪这些 异常通过电路层面表现为行为表现。

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.