Cellular and Temporal Dissection of KCNQ3 Gain-of-Function Disorder

摘要 智力残疾和自闭症--在1000例中高达1例--是由反复出现的从头开始的错义变异引起的 这会改变KCNQ3基因中的一个精氨酸残基(R230)。携带R230变异的患者有非语言的 智力残疾、自闭症症状，并在以下方面发展为几乎连续的多灶性棘波 脑电(EEG)。KCNQ3编码电压门控钾通道，该通道在 亚阈值电位，集中在轴突起始段，在那里它反对去极化电流 驱动神经元产生动作电位。我们最近报道了R230变体提供了功能增益(GoF)) 在体外对该通道的生物物理特性进行了研究，但这些变化如何导致疾病表型尚不清楚。 此外，对遗传病因的识别尚未转化为有针对性的治疗。我们已经开发出一种 具有强健电临床表型(频繁棘波)的KCNQ3Gof病小鼠模型 放电和降低电惊厥阈值)与人体状况相关。我们现在提议 开发该模型的创新和通用的有条件版本，以使我们能够控制 Gof突变等位基因的表达从而允许KCNQ3Gof的细胞和时间解剖 无序。这种小鼠包括一个用于Cre重组酶介导的激活的带花线的停止盒。此外， Gof等位基因(带有IRES-EGFP报告基因)的两侧将有FRT位点，以允许Flpo重组酶介导 停用。使用此工具按顺序打开和关闭Gof突变通道的空间和时间 对照，我们将开始解剖(目标1.细胞解剖)哪些神经元群体负责 电临床表型和(目标2.颞叶解剖)是否存在有限的时间窗 阻止或逆转这些表型。我们将从两个实验开始这些调查，这些实验证明 鼠标设计的不同方面。在目标1中，我们将在1)抑制中选择性地激活突变等位基因 使用GAD2-IRES-Cre敲入驱动器系的神经元，或使用EMX-IRES-Cre敲打的兴奋性神经元 在驾驶员系中，询问Gof等位基因在任何一个广泛的神经元亚群中的表达是否足以繁殖 在结构性Gof突变小鼠中观察到的强健的电临床表型。在目标2中，我们将使用 可诱导的Flpo驱动系R26FlpoER，以检测SWD表型的可逆性。这些实验 只代表了对这种条件模型所能承受的遗传审讯的介绍性尝试。 这项基因工作是一个更大的项目的一部分，该项目调查KCNQ3 Gof障碍，包括广泛的 对受冲击的电路和神经元的潜在生理学的电生理学研究。将这些配对 工具将促进我们的理解，并为治疗提供翻译方法。

ABSTRACT Intellectual disability and autism are caused – in up to 1 in 1,000 cases – by recurrent de novo missense variants that alter a single arginine residue (R230) in the KCNQ3 gene. Patients with R230 variants have non-verbal intellectual disability, autistic symptoms, and develop near-continuous multifocal spikes on electroencephalography (EEG). KCNQ3 encodes a voltage-gated potassium channel that is active at subthreshold potentials and concentrated at the axon initial segment, where it opposes depolarizing currents that drive neurons to fire action potentials. We recently reported that R230 variants impart gain-of-function (GoF)) biophysical properties to the channel in vitro, but how these alterations lead to disease phenotypes is unknown. Moreover, identification of the genetic etiology has yet to translate into targeted therapy. We have developed a mouse model of the KCNQ3 GoF Disorder with a robust electroclinical phenotypes (frequent spike-wave discharges and lowered electroconvulsive threshold) with relevance to the human condition. We now propose the development of an innovative and versatile conditional version of this model to grant us control over expression of the GoF mutant allele thereby permitting cellular and temporal dissection of the KCNQ3 GoF Disorder. This mouse includes a floxed stop cassette for Cre recombinase mediated activation. In addition, the GoF allele, (with an IRES-eGFP reporter) will be flanked by frt sites to permit Flpo recombinase mediated deactivation. Using this tool to sequentially turn on and then off the GoF mutant channel with spatial and temporal control, we will begin to dissect out (Aim 1. Cellular dissection) which neuronal populations are responsible for the electroclinical phenotypes and (Aim 2. Temporal dissection) whether there are limited temporal windows for preventing or reversing these phenotypes. We will begin these investigations with a pair of experiments that test different aspects of the mouse design. In Aim 1, we will selectively activate the mutant allele in 1) inhibitory neurons using a Gad2-IRES-Cre knock-in driver line, or in 2) excitatory neurons using an Emx-IRES-Cre knock- in driver line, to ask if expression of the GoF allele in either broad neuronal subgroup is sufficient to reproduce the robust electroclinical phenotypes observed in the constitutive GoF mutant mouse. In Aim 2, we will use an inducible Flpo driver line, R26FlpoER, to examine the reversibility of the SWD phenotype. These experiments represent only the introductory forays into the kind of genetic interrogations this conditional model will afford. This genetic work is part of a larger program investigating the KCNQ3 GoF disorder, including extensive electrophysiological investigations of the underlying physiology of impacted circuits and neurons. Pairing these tools will advance our understanding and inform translational approaches to treatment.