Molecular Mechanisms of SOD1-linked ALS (P01)

SOD1 相关 ALS 的分子机制 (P01)

• 批准号: 8249456
• 负责人: JOAN Selverstone VALENTINE
• 金额: $ 101.23万
• 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/8249456
• 关键词: Address; Amyloid Fibrils; Amyotrophic Lateral Sclerosis; Animal Disease Models; Animal Model; Award; Biochemical; Biological Assay; Biological Models; Cell Culture Techniques; Cessation of life; Collaborations; Data; Disease; Disease Progression; Disease model; Disulfides; Etiology; Familial Amyotrophic Lateral Sclerosis; Gene Mutation; Goals; Homeostasis; Human; In Vitro; Investigation; Lead; Link; Metals; Methods; Modeling; Molecular; Molecular Weight; Motor Neurons; Mus; Mutation; Neurodegenerative Disorders; Neuroglia; Pathogenesis; Patients; Preparation; Principal Investigator; Process; Property; Proteins; Research Personnel; Role; Source; Spinal Cord; Stem cells; Structure; Study models; Symptoms; Testing; Tissues; Toxic effect; Vertebral column; Work; animal tissue; copper zinc superoxide dismutase; design; efficacy testing; human tissue; in vivo; inhibitor/antagonist; instrumentation; mutant; programs; public health relevance

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截然不同的专业知识和实验能力的研究人员，以阐明S0D1连接的家族性肌萎缩性侧面硬化症（FALS）发病机理的基础机制。众所周知，S0D1多聚体和较大的聚集体与疾病相关，但是有毒物种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，以进一步表征与突变体S0D1及其聚集相关的生化特性，以发现有关疾病起始和进展的线索，以利用这种理解以发展目标障碍物的靶向障碍者。 PPG合作将涵盖五名主要研究人员，其中有四个项目和一个技术核心。项目1（Joan Valentine博士）和4（David Eisenberg博士）将采用一种体外的结构和生物物理方法来研究S0D1多聚化的机制和多聚体的结构，并了解了共同聚集的多物质化和设计抑制剂的机制的目标。 Project 2（Martina Wiedau-Pazos博士）将使用干细胞衍生的运动神经元和GLIA和Project 3（David Borchelt博士）（David Borchelt博士）将使用小鼠，并使用细胞培养模型来探测多聚体的毒性，并表征突变体S0D1的变化，导致疾病。对后一个项目的特别强调将是与S0D1稳定性有关的体内金属载荷，而金属稳态与细胞毒性有关。核心A（Julian Whitelegge博士）将通过提供和维护必要的仪器和数据传递来充当这些调查的骨干。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组织将被用作验证发现并检验新假设的来源。 公共卫生相关性：一个关键的未解决的问题在理解肌萎缩性横向硬化症（ALS）和其他神经退行性疾病中是各种蛋白质蛋白质在引起疾病中的作用。该提案尤其是使用将一些最佳细胞培养和动物模型系统与先进的生物物理和生化方法相结合的方法解决了ALS的这个问题。 项目1 首席研究员：琼·瓦伦丁 标题：未提供。 描述（由申请人提供）：肌萎缩性侧面硬化症（ALS）是一种进行性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。尽管最常见的ALS形式是零星的，但没有已知原因，但由基因突变引起的一部分是家族性的，其中由蛋白质铜 - 锌超氧化物歧化酶（SODL）引起的病例代表了最广泛研究的ALS模型。脊髓中富含SODL的原纤维夹杂物的形成是人类患者和该疾病动物模型中SODI连接家族的重要特征。在动物模型中，夹杂物在症状发作之前就形成了高分子重量的SODL的高分子寡聚形式，这表明SODL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总体目标。在这个项目中，我们将解决SODL多聚化的生物物理方面。具体而言，目标包括（1）检查从人类和动物组织来源中产生或隔离的多聚体SODL的结构，（2）应用标记的SODL的定义多聚体制备对培养的运动神经元进行研究，以研究它们是否有毒以及与项目2中的合作（（3）检查构造的构造机制，以使命运构成如何进行命运，以使命运的效果构成如何进行命运，以使得命中率进行构成序列化的效果。在此过程中，以及（4）阐明家族性ALS突变在调节这些过程速率中的作用。我们的研究将广泛利用我们在上一项奖项期间开发的测定法，以将SODL转化为可溶性，低聚物种和淀粉样蛋白原纤维在轻度，生理上相关的条件下。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特性，例如折叠，金属含量以及从动物组织中分离出的SODL的可溶性和不溶性形式的二硫化状态。 公共卫生相关性：一个关键的未解决的问题在理解肌萎缩性横向硬化症（ALS）和其他神经退行性疾病中是各种蛋白质蛋白质在引起疾病中的作用。该项目特别是使用一些最佳的先进生物物理和生化方法来解决ALS的问题。