Mutational Analysis of Motoneuron Specific NMDA-R in ALS

ALS 中运动神经元特异性 NMDA-R 的突变分析

• 批准号: 6671954
• 负责人: YASUNORI HAYASHI
• 金额: $ 19.36万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6671954
• 关键词: NMDA receptors; amyotrophic lateral sclerosis; calcium flux; clinical research; family genetics; gene dosage; gene mutation; genetic mapping; genetic polymorphism; genetic susceptibility; genetically modified animals; genotype; human genetic material tag; human subject; laboratory mouse; motor neurons; neuromuscular disorder; patient oriented research; phenotype; receptor expression

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motoneurons. It affects 500,000 individuals worldwide annually and remains untreatable. To develop an effective treatment for ALS, a precise understanding of its pathogenesis is indispensable. One approach toward this goal involves identifying the gene(s) responsible for familial forms of ALS. Such an approach has uncovered mutations in the gene encoding Cu/Zn cytosolic superoxide dismutase (SOD1); these mutations only account for 20%-25% of familial cases. Despite intense efforts, the pathogenesis of the remainder of familial cases and all of sporadic cases are practically unknown. We have recently identified a new NMDA type glutamate receptor, NR3B. Interestingly, it is expressed almost exclusively in the motoneurons known to be affected in ALS and negatively regulates the function of NMDA receptor to limit Ca2+ influx. Because it is well documented that an overactivation of NMDA receptor causes the cell death associated with various neurological disorders, the identification of NR3B lead us to propose the "NR3B hypothesis" of the pathogenesis of ALS. This hypothesis proposes that defects in NR3B function may be neurotoxic in motoneurons through loss of the negative regulation of motoneuronal NMDA-mediated Ca2+ currents. Loss of NR3B function may increase the NMDA receptor current and Ca2+ influx in these cells, thereby predisposing motoneurons to excitotoxicity. This mechanism may cause ALS by itself or may predispose carrier to ALS when it is combined other genetic or epigenetic factors. In this proposal, we will test this NR3B hypothesis by genetic analysis of the NR3B gene in humans and modifications of NR3B gene dose in ALS mice. We will first analyzing DNA from ALS patients for mutations in the NR3B gene. We will first screen ALS families without a known genetic defect for genetic linkage to the NR3B gene (Aim 1). We will then perform mutational analysis of the full NR3B gene in linked cases and in a series of individuals with both familial and sporadic ALS, as well as controls (Aim 2). Thereafter (Aim 3) we will test the hypothesis that polymorphisms in the NR3B gene are associated with an increased risk of developing ALS or with some aspect of the clinical course of ALS (e.g. onset age, life span after onset, disease distribution). In related studies in mice, we will determine whether variations in the dose of the NR3B gene alter the phenotype of transgenic SOD1-G93A mice (Aim 4). In our view, this is a significant translational and explorative investigation that promises to relate a newly discovered, novel motoneuronal glutamate receptor subunit, NR3B, with a fatal human motor neuron disease; the findings have potential implications for the development of new approaches to therapy in ALS.

描述（由申请人提供）： 肌萎缩侧索硬化症（ALS）是一种神经退行性疾病，其特征是运动神经元选择性丧失。它每年影响全球 500,000 人，并且仍然无法治愈。为了开发 ALS 的有效治疗方法，准确了解其发病机制是必不可少的。实现这一目标的一种方法是确定导致家族性 ALS 的基因。这种方法发现了编码铜/锌胞质超氧化物歧化酶（SOD1）的基因发生突变；这些突变仅占家族病例的20%-25%。尽管付出了巨大的努力，其余家族性病例和所有散发病例的发病机制实际上仍不清楚。我们最近发现了一种新的 NMDA 型谷氨酸受体 NR3B。有趣的是，它几乎只在已知受 ALS 影响的运动神经元中表达，并负向调节 NMDA 受体的功能以限制 Ca2+ 流入。由于有充分证据表明，NMDA 受体过度激活会导致与各种神经系统疾病相关的细胞死亡，因此 NR3B 的鉴定使我们提出 ALS 发病机制的“NR3B 假说”。该假设提出，NR3B 功能缺陷可能通过运动神经元 NMDA 介导的 Ca2+ 电流负调节的丧失而对运动神经元产生神经毒性。 NR3B 功能的丧失可能会增加这些细胞中的 NMDA 受体电流和 Ca2+ 流入，从而使运动神经元容易出现兴奋性毒性。这种机制本身可能会导致 ALS，或者当与其他遗传或表观遗传因素结合时，可能会使携带者易患 ALS。 在本提案中，我们将通过对人类 NR3B 基因的遗传分析和 ALS 小鼠中 NR3B 基因剂量的修改来检验这一 NR3B 假设。我们将首先分析 ALS 患者的 DNA 中 NR3B 基因的突变。我们将首先筛选没有已知遗传缺陷的 ALS 家族与 NR3B 基因的遗传连锁（目标 1）。然后，我们将对相关病例、一系列患有家族性和散发性 ALS 的个体以及对照个体中的完整 NR3B 基因进行突变分析（目标 2）。此后（目标 3），我们将检验以下假设：NR3B 基因的多态性与发生 ALS 的风险增加或与 ALS 临床病程的某些方面（例如发病年龄、发病后寿命、疾病分布）相关。在小鼠的相关研究中，我们将确定NR3B基因剂量的变化是否会改变转基因SOD1-G93A小鼠的表型（目标4）。 我们认为，这是一项重要的转化和探索性研究，有望将新发现的新型运动神经元谷氨酸受体亚基 NR3B 与致命的人类运动神经元疾病联系起来；这些发现对 ALS 治疗新方法的开发具有潜在意义。