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

KCNQ3 功能获得障碍的细胞和颞叶解剖

基本信息

批准号：
10591921
负责人：
TRISTAN T SANDS
金额：
$ 16.45万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-15 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10591921
关键词：
Action Potentials Alleles Arginine Axon Biophysics Cells Cerebral cortex Clinical Cre driver DNA cassette Data Development Disease Dissection Electroencephalography Electrophysiology (science)Enterobacteria phage P1 Cre recombinase Epilepsy Excision Fire - disasters Functional disorder Genes Genetic Genetic Predisposition to Disease Genetic Recombination Goals Grant Human In Vitro Intellectual functioning disability Internal Ribosome Entry Site Investigation Knock-in Knock-in Mouse Knowledge Lead LoxP-flanked allele Mediating Mission Modeling Mus Mutant Strains Mice Mutation Neurodevelopmental Disability Neurons Pathogenicity Pathologic Patient Care Patients Phenotype Physiology Population Potassium Potassium Channel Public Health Recurrence Reporter Reporting Research Seizures Site Sleep Subgroup Symptoms System Tamoxifen Testing Thalamic structure Therapeutic Translating United States National Institutes of Health Variant Voltage-Gated Potassium Channel Work autism spectrum disorder autistic beta Actin biophysical properties cell type critical period design disability disease phenotype excitatory neuron experimental study gain of function gain of function mutation improved inhibitory neuron innovation mouse model mutant neuronal excitability novel prevent programs recombinase targeted treatment tool translational approach

项目摘要

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.

抽象的智力障碍和自闭症是由反复出现的错义变异引起的（高达千分之一）改变 KCNQ3 基因中的单个精氨酸残基 (R230) 的 R230 变异患者具有非语言能力。智力障碍、自闭症症状，并出现近乎连续的多灶性尖峰脑电图 (EEG) 编码一个电压门控钾通道，该通道在阈下电位并集中在轴突初始段，在那里它反对去极化电流我们最近报道了 R230 变体赋予功能获得（GoF））体外通道的生物物理特性，但这些改变如何导致疾病表型尚不清楚。此外，遗传病因的鉴定尚未转化为靶向治疗。 KCNQ3 GoF 疾病小鼠模型，具有强大的电临床表型（频繁的尖峰波放电和降低电惊厥阈值）与人类状况相关。开发该模型的创新且多功能的条件版本，使我们能够控制 GoF 突变等位基因的表达允许对 KCNQ3 GoF 进行细胞和时间解剖该小鼠包含用于 Cre 重组酶介导的激活的 floxed 终止盒。 GoF 等位基因（带有 IRES-eGFP 报告基因）两侧为 frt 位点，以允许 Flpo 重组酶介导使用此工具按空间和时间顺序打开然后关闭 GoF 突变体通道。控制，我们将开始解剖（目标 1. 细胞解剖）哪些神经元群负责电临床表型以及（目标 2. 颞解剖）是否存在有限的时间窗口我们将通过两个测试实验来开始这些研究。在小鼠设计的不同方面，我们将选择性激活 1) 抑制性突变等位基因。使用 Gad2-IRES-Cre 敲入驱动线的神经元，或 2) 使用 Emx-IRES-Cre 敲入驱动线的兴奋性神经元在驱动线中，询问 GoF 等位基因在任一广泛神经亚群中的表达是否足以复制在目标 2 中，我们将使用在 GoF 突变小鼠中观察到的稳健的电临床表型。诱导型 Flpo 驱动线 R26FlpoER，用于检查 SWD 表型的可逆性。仅代表对这种条件模型所能提供的基因询问类型的介绍性尝试。这项基因工作是调查 KCNQ3 GoF 疾病的更大计划的一部分，其中包括广泛的研究对受影响的电路和神经元进行配对的基础生理学的电生理学研究。工具将增进我们的理解并为转化治疗方法提供信息。