NR2B Mediated Neurotoxicity in Murine Huntington Disease Model

描述(由申请人提供)： 项目摘要/摘要目的：我们的目标是描述亨廷顿病(HD)神经变性的最新机制，HD是一种罕见的成人起病的常染色体神经退行性疾病。HD是由一个扩展的CAG重复序列引起的，翻译为多聚谷氨酰胺(PolyQ)，在Huntingtin基因座上占主导地位。几种不同的神经元死亡机制被假设为导致HD的神经变性。我们的重点是在体内对兴奋性毒性神经元损伤和线粒体功能障碍的假想机制进行批判性评估。我们还在解决神经变性的细胞自主与非细胞自主原因这一重要问题。在我们之前的资助期间，我们描述了一种优秀的HD小鼠遗传模型，并在体内提供了强有力的证据，证明兴奋性神经元损伤是HD神经变性的直接原因。研究计划：我们有3个初步实验。我们将把一个经过良好验证的HD敲击小鼠遗传模型与其他3个携带突变的品系进行交叉，这些突变使我们能够测试兴奋性毒性神经元损伤、细胞自主与细胞非自主导致神经变性和线粒体功能障碍的假设。每个十字都包含一个基于每个假设的特定预测，从而允许对每个假设进行证伪或验证。在第一个杂交中，我们将用前脑携带NMDA受体NR2B亚单位缺失的小鼠培育类似HD的小鼠。在第二个实验中，我们将用携带NMDA受体NR2B亚单位纹状体特异性缺失的小鼠培育类似HD的小鼠。在第三个实验中，我们将培育具有线粒体缺陷突变的类HD小鼠。在所有实验中，行为和病理的前瞻性评估将被用来评估这些交叉试验的结果。方法：在所有实验中使用相同的方法。类HD和其他小鼠品系交叉产生双基因突变，体现了从每个假说中得出的预测。结果小鼠和对照动物通过一组标准的2岁以下的行为测试进行前瞻性评估。病理变化通过体视学、免疫组织化学和受体结合的方法进行评估。所有的方法都在我们的实验室里得到了很好的证实。临床相关性(如果是基础科学研究)：神经退行性疾病是老年人和美国退伍军人中常见的问题。神经退行性变的机制目前知之甚少，改进的治疗方法需要了解神经退行性变的机制。更好地了解HD可能有助于了解更常见的神经退行性疾病的神经退行性变的机制。我们正在研究两种最接近的机制，兴奋性毒性和线粒体功能障碍，被认为在几种神经退行性疾病中起主要作用。

DESCRIPTION (provided by applicant): Project Summary/Abstract Objectives: Our objective is to delineate proximate mechanisms of neurodegeneration in Huntington disease (HD), an uncommon, adult-onset, autosomal neurodegenerative disorder. HD is caused by an expanded CAG repeat, translated as polyglutamine (polyQ), dominant in the huntingtin locus. Several different proximate mechanisms of neuronal death are hypothesized to cause neurodegeneration in HD. We are focused on critically evaluating, in vivo, the hypothesized mechanisms of excitotoxic neuronal injury and mitochondrial dysfunction. We are also addressing the important issue of cell autonomous versus non-cell autonomous causes of neurodegeneration. In our prior funding period, we characterized an excellent murine genetic model of HD and provided strong in vivo evidence for excitotoxic neuronal injury as a proximate cause of neurodegeneration in HD. Research Plan: We have 3 primary experiments. We will cross a well validated knockin murine genetic model of HD with 3 other lines carrying mutations that allow us to test the hypotheses of excitotoxic neuronal injury, cell autonomous versus cell non-autonomous causes of neurodegeneration, and mitochondrial dysfunction. Each cross embodies a specific prediction based on each hypothesis, allowing falsification or verification of each hypothesis. In the first cross, we will breed HD-like mice with mice carrying a forebrain deletion of the NR2B subunit of the NMDA receptor. In the second experiment, we will breed HD-like mice with mice carrying a striatal specific deletion of the NR2B subunit of the NMDA receptor. In the third experiment, we will breed HD-like mice with a mitochondrial deficiency mutant. In all experiments, prospective evaluations of behavior and pathology will be used to assess outcomes of these crosses. Methods: The same set of methods are used across all experiments. HD-like and other mouse lines are intercrossed to generate bigenic mutants embodying predictions derived from each hypothesis. The resulting mice and control animals are evaluated prospectively with a standard battery of behavioral tests up to 2 years of age. Pathologic changes are assessed with stereology, immunohistochemistry, and receptor binding methods. All methods are well established in our laboratory. Clinical Relevance (if basic science study): Neurodegenerative disorders are common problems among the elderly and in the US Veteran population. The mechanisms of neurodegeneration are understood poorly and improved treatment requires understanding mechanisms of neurodegeneration. Better understanding of HD may lead to understanding of mechanisms of neurodegeneration in more common neurodegenerative disorders. We are investigating 2 proximate mechanisms, excitotoxicity and mitochondrial dysfunction, thought to play a major role in several neurodegenerative disorders.