Alpha-Tubulin Mutation and Motor Neuron Disease

项目摘要/摘要 肌萎缩侧索硬化症(ALS)或Lou Gehrig病，是一种由进行性疾病引起的致命疾病 运动神经元变性，每年影响约30,000美国人。目前，这些机制 肌萎缩侧索硬化症运动神经元死亡的原因尚不完全清楚，也没有有效的治疗方法来治愈或 减缓这种不可抗拒的疾病的发展。家族性ALS中新发现的TUBA4A突变 患者添加了一种新的基因，该基因与运动神经元的细胞骨架动力学有关。我们变得高度 有兴趣研究突变型-微管蛋白在肌萎缩侧索硬化症细胞骨架动力学和缺陷中的作用。冲向 为此，我们计划培育新型转基因小鼠，过度表达突变的人类野生型和 突变的-微管蛋白。我们的长期目标是了解突变型-微管蛋白对运动神经元的神经毒性 退化，并制定策略，以阻止其毒性。我们的近期目标是构建、开发一部小说 携带与在多个ALS家系中发现的突变相同的突变的动物模型。我们假设 突变的-微管蛋白在小鼠体内过表达将导致主要表型和 与肌萎缩侧索硬化症患者相似的病理。第一个目标是产生-微管蛋白转基因小鼠和 评估进行性运动障碍和类似肌萎缩侧索硬化症的表型。成年小鼠将被研究是否有迹象和 归因于功能性运动神经元变性的症状，将功能障碍的严重程度与 TUBA4A转基因拷贝数、蛋白表达水平和蛋白活性。 第二个目的是对hTUBA4AR320C和hTUBA4AWT转基因小鼠进行鉴定和比较 SOD1G93A和我们最近的hPFN1G118V转基因小鼠的神经肌肉病理。脑，脊椎 将研究脊髓和骨骼肌组织，以确定病理异常，包括损伤 神经元丢失、轴突损伤、氧化应激的产生和神经胶质细胞的激活。微管聚合 将进行α-微管蛋白活性分析，以确定R320C突变是否扰乱了 微管。 这项工作的影响可能是非常重要的，因为它在创造一种新的动物模型方面具有巨大的潜力 补充现有肌萎缩侧索硬化症研究的研究领域。这项研究将提供一种新的工具来确定 突变型-微管蛋白作为可能导致神经退行性变的基本机制的中心重要性 发现于肌萎缩侧索硬化症患者。我们期望这个小鼠模型也可以促进治疗的发展 肌萎缩侧索硬化症的策略。因此，预计结果将产生重大影响，因为我们的模型可能提供 发现预防或阻止肌萎缩侧索硬化症相关进展的基本机制和治疗的新靶点 神经退行性变。

PROJECT SUMMARY/ABSTRACT Amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease, is a fatal disease resulting from progressive degeneration of motor neurons and affects ~30,000 American each year. Currently, the mechanisms responsible for motor neuron death in ALS are not fully understood and there is no effective therapy to cure or slow the progression of this inexorable disease. The recent discovery of TUBA4A mutations in familial ALS patients added a new gene with implication in cytoskeleton dynamics in motor neurons. We became highly interested to investigate the role of mutant -tubulin in the cytoskeleton dynamics and defects in ALS. Toward that end, we have planned to generate novel transgenic mice overexpressing mutant human wildtype and mutant -tubulin. Our long-term goal is to understand the mutant -tubulin neurotoxicity in motor neuron degeneration and develop strategy to block its toxicity. Our immediate goal is to construct, develop a novel animal model that bears a mutation identical to that found in multiple families with ALS. We hypothesize that the overexpression of mutant -tubulin protein in mice will result in development of cardinal phenotypes and pathologies that resemble those in ALS patients. The first aim is to generate -tubulin transgenic mice and assess for progressive motor deficits and phenotypes resembling ALS. Adult mice will be studied for signs and symptoms that attributes to functional motor neuron degeneration, correlating the severity of the dysfunction to TUBA4A transgene copy number, protein expression level, and protein activity. The second aim is to characterize hTUBA4AR320C and hTUBA4AWT transgenic mice and compare neuromuscular pathologies with those of SOD1G93A and our recent hPFN1G118V transgenic mice. Brain, spinal cord and skeletal muscle tissues will be studied to determine pathological anomalies including damage to neuronal loss, axonal damage, production of oxidative stress, and glial activation. Microtubule polymerization and a-tubulin activity assays will be performed to determine if the R320C mutation disrupts the structure of the microtubule. The impact of this work could be highly significant for its potential in creating a new animal model for research areas complementary to existing ALS research. This study will provide a new tool to determine the central importance of mutant -tubulin as a basic mechanism that may contribute to the neurodegeneration found in ALS patients. We expect that this mouse model may also facilitate development of therapeutic strategies for ALS. Thus, the results are expected to have a significant impact because our model may provide new targets for discovery of basic mechanisms and therapies to prevent or stop progression of ALS-associated neurodegeneration.