NR2B Mediated Neurotoxicity in Murine Huntington Disease Model

描述（由申请人提供）： 项目概要/摘要目标：我们的目标是描述亨廷顿病（HD）神经退行性变的近似机制，HD是一种不常见的成人发病的常染色体神经退行性疾病。HD是由CAG重复扩增引起的，翻译为多聚谷氨酰胺（polyQ），在亨廷顿基因座中占主导地位。几种不同的神经元死亡的近似机制被假设为引起HD中的神经变性。我们专注于批判性地评估，在体内，兴奋性神经元损伤和线粒体功能障碍的假设机制。我们也在讨论神经退行性变的细胞自主与非细胞自主原因的重要问题。在我们之前的资助期间，我们描述了一个优秀的HD小鼠遗传模型，并提供了强有力的体内证据，兴奋性神经元损伤作为HD神经变性的近因。研究计划：我们有三个主要的实验。我们将一个经过充分验证的敲入小鼠遗传模型的HD与3个其他线携带突变，使我们能够测试兴奋性神经元损伤，细胞自主与细胞非自主的神经变性的原因，和线粒体功能障碍的假设。每一个十字架都体现了基于每一个假设的特定预测，允许对每一个假设进行证伪或验证。在第一次杂交中，我们将HD样小鼠与携带NMDA受体NR 2B亚基前脑缺失的小鼠交配。在第二个实验中，我们将用携带NMDA受体NR 2B亚基纹状体特异性缺失的小鼠培育HD样小鼠。在第三个实验中，我们将培育具有线粒体缺陷突变体的HD样小鼠。在所有实验中，将使用行为和病理学的前瞻性评价来评估这些交叉的结果。方法：所有实验均使用同一组方法。HD样和其他小鼠品系相互杂交以产生体现来自每个假设的预测的双基因突变体。用长达2岁的一系列标准行为测试前瞻性地评估所得小鼠和对照动物。病理变化进行了评估与体视学，免疫组织化学和受体结合方法。所有的方法都在我们的实验室中得到了很好的建立。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人群的常见问题。神经变性的机制知之甚少，改善治疗需要了解神经变性的机制。更好地了解HD可能会导致更常见的神经退行性疾病的神经退行性变的机制的理解。我们正在研究两个近似的机制，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。 公共卫生相关性： 项目叙述：我们的目标是了解亨廷顿病（HD）中神经细胞死亡的机制。虽然HD背后的遗传缺陷已经知道了大约15年，但神经细胞死亡背后的细胞机制尚不清楚。已经提出了一些机制，并有良好的实验支持。我们将严格测试两个建议的机制，兴奋性神经元损伤和线粒体功能障碍，在体内。我们还将测试HD中的神经变性是否仅由受影响神经元内的毒性基因产物效应引起，或者是否需要神经回路异常。我们的方法是将经过充分验证的HD小鼠遗传模型与预测参与HD的途径中携带突变的其他突变株系杂交。预测这些突变对HD样表型的影响，可以验证或证伪HD神经变性的这些假设。这项工作将有助于确定适当的治疗靶点，用于HD和相关疾病的神经保护治疗。

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. PUBLIC HEALTH RELEVANCE: Project Narrative: Our goal is to understand the mechanisms of nerve cell death in Huntington disease (HD). While the genetic defect underlying HD has been known for approximately 15 years, the cellular mechanisms underlying nerve cell death are unclear. A number of mechanisms have been suggested and have good experimental support. We will critically test two suggested mechanisms, excitotoxic neuronal injury and mitochondrial dysfunction, in vivo. We will test also whether neurodegeneration in HD results solely from toxic gene product effects within affected neurons or if neural circuit abnormalities are required. Our approach is to cross-breed well validated murine genetic models of HD with other mutant lines carrying mutations in pathways predicted to be involved in HD. Predictions about the effects of these mutations on the HD-like phenotype allows verification or falsification of these hypotheses of neurodegeneration in HD. This work will help identify appropriate therapeutic targets for neuroprotective treatment of HD and related diseases.