Molecular Mechanisms of SOD1-linked ALS (P01)

SOD1 相关 ALS 的分子机制 (P01)

• 批准号: 8110503
• 负责人: JOAN Selverstone VALENTINE
• 金额: $ 103.74万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-11 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8110503
• 关键词: Molecular; Mechanisms; SOD1; linked; ALS

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.

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S 0 D1相关家族性肌萎缩侧索硬化症（fALS）发病机制的潜在机制。已经确定S 0 D1多聚体和较大的聚集体与疾病相关，但毒性物质和体内机制仍然未知。该提案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S 0 D1及其聚集相关的生化特性，揭示疾病起始和进展的线索，利用这种理解开发多聚化的靶向阻断剂。PPG合作将包括五名主要研究人员，四个项目和一个技术核心。项目1（Joan Valentine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S 0 D1多聚化的机制和多聚体的结构，目的是了解多聚化的机制和设计聚集抑制剂。项目2（Martina Wiedau-Pazos博士）将使用干细胞衍生的运动神经元和神经胶质细胞，项目3（大卫Borchelt博士）将使用小鼠和细胞培养模型来探测多聚体的毒性并表征导致疾病的突变S 0 D 1的变化。后一个项目的特别重点将是体内金属负载，因为它涉及S 0 D1的稳定性，和金属稳态，因为它涉及细胞毒性。核心A（Julian Whitelegge博士）将作为这些调查的骨干，提供和维护必要的仪器和数据传输。项目2和3的疾病模型将用于测试项目1和4的抑制剂的功效。最后，ALS组织将被用作验证研究结果和测试新假设的来源。 公共卫生相关性：了解肌萎缩侧索硬化症（ALS）和其他神经退行性疾病的一个关键未解决的问题是各种聚集形式的蛋白质在引起疾病中的作用。该提案解决了ALS的这个问题，特别是使用将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 主要研究者：Joan Valentine 标题：未提供。 描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种进行性、致死性神经退行性疾病，其特征是运动神经元的选择性死亡。虽然ALS的最常见形式是散发性的并且没有已知的原因，但是由基因突变引起的病例的子集是家族性的，其中由蛋白质铜锌超氧化物歧化酶（SODl）中的突变引起的那些代表ALS的最广泛研究的模型。脊髓中富含SOD 1的纤维状内含物的形成是该疾病的人类患者和动物模型中的SOD 1相关家族性ALS的突出特征。在动物模型中，在包含物之前形成甚至在症状发作之前出现的SODl的高分子量寡聚体形式，表明SODl的寡聚化和聚集是疾病病因学的重要组成部分。了解多聚体S 0 D1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，我们将解决SOD 1多聚化的生物物理方面。具体地，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SOD 1的结构，（2）将标记的SOD 1的限定的多聚体制剂应用于培养的运动神经元以研究它们是否有毒以及如何有毒（与项目2合作），（3）检查SOD 1多聚化成原纤维的机制以理解使SOD 1不稳定的结构因子如何促成该过程，（4）阐明家族性ALS致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之前的授予期开发的用于在温和的生理学相关条件下将SODl转化为可溶性寡聚物种类和淀粉样蛋白原纤维的测定。我们还将广泛使用各种高度灵敏的生物物理方法来研究从动物组织分离的可溶和不可溶形式的SOD 1的各种结构特性，例如折叠、金属含量和二硫化物状态。 公共卫生相关性：了解肌萎缩侧索硬化症（ALS）和其他神经退行性疾病的一个关键未解决的问题是各种聚集形式的蛋白质在引起疾病中的作用。该项目解决了ALS的这个问题，特别是使用一些最先进的生物物理和生物化学方法。