Molecular Mechanisms of SOD1-linked ALS (P01)

描述(由申请人提供)：该计划项目汇集了五名具有非常不同的专业知识和实验能力的研究人员，共同致力于阐明S0D1连锁的家族性肌萎缩侧索硬化症(FALS)发病的潜在机制。众所周知，S0D1多聚体和较大的聚集体与疾病有关，但其毒性种类和体内机制尚不清楚。这项建议的总体目标是广泛了解聚集在疾病中的作用，进一步表征与突变体S0D1及其聚集相关的生化特性，揭示疾病的发生和发展的线索，利用这一理解开发多聚体的靶向阻滞剂。PPG协作将包括五名主要调查人员、四个项目和一个技术核心。项目1(琼·瓦伦丁博士)和项目4(大卫·艾森伯格博士)将采用体外结构和生物物理方法研究S0D1多聚体的机制和多聚体的结构，目的是了解多聚体的机制(S)和设计聚集的抑制剂。项目2(Martina Wiedau-Pazos博士)将使用干细胞衍生的运动神经元和胶质细胞，项目3(David Borchelt博士)将使用小鼠和细胞培养模型来探索多聚体的毒性，并表征导致疾病的突变S0D1的变化。后一个项目的重点将是体内金属负载，因为它与S0D1的稳定性有关，以及金属动态平衡与细胞毒性有关。核心A(Julian Whitelege博士)将通过提供和维护必要的仪器和数据传输，作为这些调查的中坚力量。项目2和项目3的疾病模型将被用来测试项目1和项目4的抑制剂的有效性。最后，ALS组织将被用作验证发现和测试新假说的来源。 公共卫生相关性：在了解肌萎缩侧索硬化症(ALS)和其他神经退行性疾病方面，一个关键的悬而未决的问题是各种聚集形式的蛋白质在致病中的作用。这项建议特别针对肌萎缩侧索硬化症解决了这个问题，使用了一些最好的细胞培养和动物模型系统与先进的生物物理和生物化学方法相结合的方法。 项目1 主要调查者：琼·瓦伦丁 标题：未提供。 描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种进行性的、致命的神经退行性疾病，其特征是运动神经元的选择性死亡。虽然ALS最常见的形式是散发性的，原因尚不清楚，但由基因突变引起的一部分病例是家族性的，其中由蛋白铜锌超氧化物歧化酶(SOD1)突变引起的ALS是研究最广泛的ALS模型。在人类患者和动物模型的SODI连锁家族性ALS中，脊髓中富含SOD1的纤维包涵体的形成是一个显著的特征。在动物模型中，在包裹体之前，甚至在症状出现之前就已经出现了高相对分子质量的SOD1寡聚体形式，这表明SOD1的寡聚和聚集是疾病病因的重要组成部分。了解多聚体S0D1如何促进运动神经元死亡是该计划项目的首要目标。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)检查体外产生的或从人和动物组织来源分离的多聚体SOD1的结构；(2)将已定义的标记SOD1的多聚体制剂应用于培养的运动神经元，以研究它们是否以及如何有毒(与项目2合作)；(3)研究S0D1多聚体形成纤维的机制，以了解破坏SOD1稳定的结构因素如何参与这一过程；以及(4)阐明导致ALS的家族性突变在调节这些过程的速率中的作用。我们的研究将广泛使用我们在前一个获奖期开发的一种方法，在温和的生理相关条件下将SOD1转化为可溶的低聚物种和淀粉样纤维。我们还将广泛使用各种高度敏感的生物物理方法来研究从动物组织中分离出来的可溶和不溶SOD1的各种结构特性，如折叠、金属含量和二硫键状态。 公共卫生相关性：在了解肌萎缩侧索硬化症(ALS)和其他神经退行性疾病方面，一个关键的悬而未决的问题是各种聚集形式的蛋白质在致病中的作用。这个项目特别针对肌萎缩侧索硬化症解决了这个问题，使用了一些可用的最先进的生物物理和生化方法。

DESCRIPTION (provided by applicant): This Program Project brings together five researchers with very different expertise and experimental capabilities to work together on elucidating the underlying mechanisms of S0D1-linked familial amyotrophic lateral sclerosis (fALS) pathogenesis. It is well established that S0D1 multimers and larger aggregates are associated with disease but the toxic species and in vivo mechanism remain unknown. The overall goals of this proposal are to gain an extensive understanding of the role of aggregation in disease, to characterize further the biochemical properties associated with mutant S0D1 and its aggregation, to uncover clues about the initiation and progression of disease, to exploit this understanding to develop targeted blockers of multimerization. The PPG collaboration will encompass five primary investigators with four projects and a technical Core. Projects 1 (Dr. Joan Valentine) and 4 (Dr. David Eisenberg) will take an in vitro structural and biophysical approach to studying the mechanism of S0D1 multimerization and the structures of the multimers, with the goals of understanding the mechanism(s) of multimerization and designing inhibitors of aggregation. Project 2 (Dr. Martina Wiedau-Pazos) will use stem cell-derived motor neurons and glia and project 3 (Dr. David Borchelt) will use a mouse,and cell culture models to probe the toxicity of multimers and to characterize the changes in mutant S0D1 that lead toward disease. A particular emphasis on the latter project will be toward in vivo metal loading as it pertains to S0D1 stability, and metal homeostasis as it pertains toward cellular toxicity. Core A (Dr. Julian Whitelegge) will serve as the backbone of these investigations by providing and maintaining the necessary instrumentation and data delivery. Disease models from projects 2 and 3 will be used to test the efficacy of inhibitors from projects 1 and 4. Finally, ALS tissue will be used as a source to validate the findings and test new hypotheses. PUBLIC HEALTH RELEVANCE: A critical unsolved question in understanding amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases is the role of various aggregated forms of proteins in causing disease. This proposal addresses this question for ALS in particular using approaches that combine some of the best cell culture and animal model systems available with advanced biophysical and biochemical methods. PROJECT 1 Principal Investigator: Joan Valentine Title: Not provided. Description (provided by applicant): 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. Public Health Relevance: A critical unsolved question in understanding amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases is the role of various aggregated forms of proteins in causing disease. This project addresses this question for ALS in particular using some of the best advanced biophysical and biochemical methods available.