NMDAR Mutations & Neurodevelopmental Disorder: from Mechanism to Targeted Therapy

摘要 N-甲基-D-天冬氨酸受体（NMDAR）是配体门控的离子通道，其介导缓慢的细胞内信号传导。 它是兴奋性突触后电流的组成部分，在正常的脑功能中发挥重要作用。遗传 编码GluN亚基的GRIN基因的变异与神经发育障碍有关， 包括癫痫性脑病、自闭症和智力残疾，这些疾病会带来毁灭性的精神和 对个人、家庭和社会造成的经济后果。在第一份关于疾病的报告之后- 在2010年引起GRIN变异，大量的人类GRIN基因变异（>300）已被确定 在患有各种神经问题的儿科患者中。我们在上一个供资周期的研究表明， 相似的表型（即癫痫发作）可能由功能获得性（GoF）和功能丧失性变体引起 (LoF)同一个基因。我们的工作还表明，不同的GRIN变体对 FDA批准的药物，并且在三个无关患者中对相同治疗有不同的反应 在“N of 1”试验中拥有相同的变体。拟议的实验将提供第一个详细的评估 的电路功能后，LoF和GoF NMDAR在不同的发展阶段，并解决如何这两个 对NMDAR的相反作用可能产生相似的表型。这些研究将确定一个关键窗口， 建立了与异常活动相关的电路连接，并将为 通过提出缓解功能变化的新治疗策略来实现个性化医疗。 具体目标1：新鉴定的疾病相关GRIN变体的功能评估和 GRIN 2A变异在一般人群中。我们将分析所有新发表的功能特性 和未发表的疾病相关GRIN变异体在受体的未充分研究的区域。我们将 确定健康个体中蛋白质功能和等位基因频率之间的关系，并评估 不耐受基因的变异可能是神经系统疾病的危险因素。 具体目标2：NMDAR活性的丧失如何促进网络过度兴奋并诱导癫痫 表型？我们将在体内评估携带两种LoF变体的敲入小鼠和GluN 2A敲除小鼠， 探索NMDAR功能的丧失是否减少了突触抑制并导致网络过度兴奋。 具体目标3：功能获得性GRIN变异体相关早发性癫痫的机制是什么 脑病？我们将使用三种GoF GRIN变体的体内敲入小鼠模型来确定 增强的NMDAR功能是否驱动癫痫发作和早发性癫痫性脑病。 具体目标4：GRIN/NMDAR通道病的最佳治疗方法是什么？我们将筛选约2，000个FDA- 批准的药物，因为它们能够纠正GRIN变体诱导的过度兴奋。我们将评估 FDA批准的药物在转基因小鼠上，以确定是否可以恢复兴奋/抑制失衡。

ABSTRACT N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels that mediate the slow component of excitatory post-synaptic currents and play important roles in normal brain function. Genetic variations in GRIN genes, which encode the GluN subunits, are linked to neurodevelopmental disorders, including epileptic encephalopathy, autism, and intellectual disability, which carry devastating mental and economic consequences for the individuals, their families, and society. Following the first report on disease- causing GRIN variants in 2010, a large number of human variants (>300) in GRIN genes have been identified in pediatric patients with various neurologic problems. Our studies in the previous funding cycle indicated that similar phenotypes (i.e. seizures) could result from both gain-of-function (GoF) and loss-of-function variants (LoF) in the same gene. Our work also revealed that different GRIN variants present differential sensitivity to FDA-approved drugs, and there are divergent responses to the same treatment among three unrelated patients hosting the same variant in “N of 1” trials. The proposed experiments will provide the first detailed evaluation of circuit function following LoF and GoF NMDAR at different developmental stages, and address how the two opposite effects on NMDARs might generate a similar phenotype. These studies will define a critical window in which circuit connections relevant for aberrant activity are established, and will advance opportunities for personalized medicine by suggesting new therapeutic strategies for mitigation of functional changes. Specific Aim 1: Functional assessment of newly identified disease-associated GRIN variants and evaluation of GRIN2A variants in the general population. We will analyze the functional properties of all newly published and unpublished disease-associated GRIN variants in the understudied regions of the receptors. We will determine the relationship between protein function and allelic frequency in healthy individuals, and evaluate the idea that variation of intolerant genes can act as risk factors for neurological disorders. Specific Aim 2: How does the loss of NMDAR activity promote network hyperexcitability and induce epileptic phenotypes? We will evaluate in vivo knockin mice hosting two LoF variants and GluN2A knockout mice to explore whether loss of NMDAR function reduces synaptic inhibition and leads to network hyperexcitability. Specific Aim 3: What is the mechanism of gain-of-function GRIN variant-associated early-onset epileptic encephalopathy? We will use in vivo knockin mouse models for three GoF GRIN variants to determine whether enhanced NMDAR function drives seizures and early-onset epileptic encephalopathy. Specific Aim 4: How can GRIN/NMDAR channelopathies best be treated? We will screen ~2,000 FDA- approved drugs for their ability to rectify GRIN variant-induced hyperexcitability. We will evaluate actions of FDA-approved drugs on transgenic mice to determine if the excitation/inhibition imbalance can be restored.