Project 1

肌萎缩侧索硬化症(ALS)是一种进行性、致命性神经退行性疾病，其特征是 运动神经元选择性死亡。虽然最常见的肌萎缩侧索硬化症是零星的，没有已知的原因， 由基因突变引起的病例子集是家族性的，其中由基因突变引起的病例 蛋白铜锌超氧化物歧化酶(SODl)是目前研究最广泛的ALS模型。这个 SODI连锁家族性遗传病的一个显著特征是脊髓中富含SOD1的纤维内含物的形成 在人类患者和这种疾病的动物模型中发现了ALS。在动物模型中，包涵体之前有 高分子量低聚体SOD1的形成，甚至在疾病发作之前就出现了 症状，表明SOD1的寡聚和聚集是 疾病病因学。了解多聚体S0D1如何在运动神经元死亡中起作用是首要的 计划项目的目标。在这个项目中，我们将讨论SODl多聚化的生物物理方面。 具体地说，目标包括(1)检查在体外产生或分离的多聚体SOD1的结构 从人和动物组织来源，(2)将已定义的标记SOD1的多聚体制剂应用于 培养运动神经元以研究它们是否有毒以及如何毒性(与项目2合作)，(3)检查 S0D1多聚化成纤维的机制了解结构因素如何破坏SOD1的稳定性 有助于这一过程，以及，(4)阐明家族性ALS引起的突变在调节 这些进程的速率。我们的研究将广泛使用我们在前一个奖项中开发的一种分析方法。 在温和的生理条件下，SOD1转变为可溶性、低聚物和淀粉样纤维的时间 相关条件。我们还将广泛使用各种高度敏感的生物物理方法来 研究各种结构性质，如折叠，金属含量和二硫化状态，可溶和 从动物组织中分离出来的不溶于水的SOD1。

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by the selective death of motor neurons. While the most common form of ALS is sporadic and has no known cause, a subset of cases caused by genetic mutations are familial, of which those caused by mutations in the protein copper-zinc superoxide dismutase (SODl) represent the most extensively studied model of ALS. The formation of SODl-rich fibrillar inclusions in the spinal cord is a prominent feature of SODI-linked familial ALS in human patients and animal models of this disease. In animal models, the inclusions are preceded by the formation of high-molecular-weight oligomeric forms of SODl that appear even before the onset of symptoms, suggesting that oligomerization and aggregation of SODl is an essential component of the disease etiology. Understanding how multimeric S0D1 contributes to motor neuron death is the overarching goal of the Program Project. In this project, we will address the biophysical aspects of SODl multimerization. Specifically, the goals include (1) examining the structure of multimeric SODl generated in vitro or isolated from human and animal tissue sources, (2) applying defined multimeric preparations of tagged SODl to cultured motor neurons to study if and how they are toxic (in collaboration with project 2), (3) examining the mechanism of S0D1 multimerization into fibrils to understand how structural factors that destabilize SODl contribute to this process and, (4) elucidating the role of familial ALS-causing mutations in modulating the rate of these processes. Our studies will make extensive use of an assay we developed in the prior award period for converting SODl into soluble, oligomeric species and amyloid fibrils under mild, physiologically relevant conditions. We will also make extensive use of a variety of highly sensitive biophysical methods to study a variety of structural properties such as folding,.metal content, and disulfide status of soluble and insoluble forms of SODl isolated from animal tissues.