Analysis and therapy of age-dependent proteostasis failure in neurodegeneration

神经退行性变中年龄依赖性蛋白质稳态失败的分析和治疗

• 批准号: 10082413
• 负责人: Robert Joseph Shmookler Reis
• 金额: --
• 依托单位: CENTRAL ARKANSAS VETERANS HLTHCARE SYS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082413
• 关键词: Affect; Affinity; Age; Alzheimer' s Disease; Alzheimer' s disease patient; Amyloid; Amyloid beta-42; Amyotrophic Lateral Sclerosis; Binding; Biological Assay; Brain; Caenorhabditis elegans; Cell Culture Techniques; Cells; Chemical Agents; Chemistry; Chronic; Combined Modality Therapy; Complex; Computer Simulation; Computer software; Craniocerebral Trauma; Data; Dementia; Development; Diagnosis; Disease; Disease model; Docking; Drug Design; Drug Screening; Drug Targeting; Elderly; Environmental Risk Factor; Experimental Designs; Exposure to; Failure; Free Energy; Genetic; Heart Diseases; Hippocampus (Brain); Human; Huntington Disease; Hypertension; Impairment; Individual; Knowledge; Lead; Learning; Libraries; Link; Mass Spectrum Analysis; Mechanics; Mediating; Modeling; Molecular; Mutate; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neuropathy; Oxidation-Reduction; Oxidative Stress; Oxides; Parkinson Disease; Pathology; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Phosphorylated Peptide; Post-Translational Protein Processing; Process; Property; Protective Agents; Proteins; Proteomics; Protocols documentation; Psyche structure; RNA Interference; Rare Diseases; Reagent; Research Proposals; Resistance; Risk; Role; Sampling; Signal Pathway; Site; Substantia nigra structure; Testing; Therapeutic Agents; Tissue Banks; Tissue Sample; Tissues; Trypsin; Veterans; active duty; age related; alpha synuclein; base; brain cell; cohesion; crosslink; design; drug candidate; drug efficacy; effective therapy; experience; first-in-human; genome-wide; high risk; human old age (65+); human tissue; in silico; in vivo; in vivo evaluation; inhibitor/antagonist; insight; knock-down; novel; novel therapeutics; prevent; protective effect; protein aggregation; protein protein interaction; proteostasis; quantum; screening; simulation; small hairpin RNA; tau Proteins; therapeutically effective; tool

The previous period of Merit support allowed us to develop novel protocols for immuno-affinity isolation and characterization of very pure protein aggregates. We began with C. elegans models of neurodegenerative diseases, and then applied the same protocols to purify Aβ42- and tau-containing aggregates from affected hippocampal tissue of Alzheimer's patients, seeking aggregate proteins that differentiate them from age-matched controls. We now propose to apply our experience with these models to develop novel therapeutic agents that might delay, prevent, or even reverse Alzheimer's and Parkinson's pathology, through the following Aims: Aim 1. We have access through Dr. Sue Griffin, our collaborator, to a tissue bank of tissue samples from Alzheimer's Disease (caudal hippocampus) and Parkinson's Disease (substantia nigra), from which we will isolate aggregates by immuno-affinity for Aβ42, tau and α-synuclein. We propose to cross-link these aggregates, thoroughly digest them with trypsin, and identify cross-linked peptide pairs that will reveal the protein-protein interactions that mediated their conglomeration. We will use state-of-the-art “click chemistry” reagents to create, tag and recover cross-link sites, and Xlink Identifier software to analyze mass-spectrometry data. To visualize, integrate and interpret the protein-protein interactions thus revealed, we will create protein-interaction networks and analyze them with tools adapted to the nonfunctional interactions that predominate in protein aggregation. Aim 2. Proteomics data from Aim 1 will define critical protein-protein interactions within aggregates containing tau, Aβ42 or α-synuclein, which are highly enriched in Alzheimer or Parkinson brain samples. These protein- protein interactions will be ranked on the basis of their predicted propensity (by ΔG estimation) and stability (by molecular-dynamic simulations). The top 10 protein interfaces from each pulldown will then serve as targets for in silico screening of drug libraries developed to disrupt protein-protein interactions. Such libraries, constructed in diverse ways, include many `PPII' drugs designed to block specific protein-pair interactions, but which have frequently found applications beyond their original targets. For each target, we will perform initial screens of PPII-library drugs in silico, from which the top candidates will be retested in molecular-dynamic simulations. Aim 3. The drugs predicted in silico to most effectively disrupt key protein interactions (Aim 2) will be tested in vivo for reduction of aggregate formation―first in human neuronal-cell cultures expressing APPSwe (forming amyloid aggregates) or split-GFP::tau (fluorescing upon tau oligomerization). For drugs that are protective in either assay, tests will also be conducted in C. elegans aggregation models expressing Aβ42, tau, or α-synuclein. Biotinylated versions of 4–6 top candidates will be assessed for activity, and active biotinylated drugs will be used to pull down proteins to which the drug attaches. This will be done initially in C. elegans, using an in-house `genomewide' RNAi-knockdown library, and will then be cross-checked by introducing shRNA constructs into cultured neuronal cells. Targets producing maximal protection will then be tested to ask whether drug efficacy is partially redundant with RNAi knockdown, as expected for a target that mediates the drug's protective effects. By discovery of novel drugs designed specifically to reduce aggregation, we expect to find far more effective therapeutic and preventative agents than the limited drugs currently available. Based on our observations that many aggregate components are shared among diverse neurodegenerative-disease models, at least some of these drugs may also prove effective against a variety of other neurodegenerative diseases including orphan diseases.

上一期的Merit支持使我们能够开发新的免疫亲和分离和 非常纯净的蛋白质聚集体的特征。我们从线虫神经退行性变的模型开始 疾病，然后应用相同的方法从受影响的人中纯化含有β42和tau的聚集体 阿尔茨海默病患者的海马区组织，寻找将他们与年龄匹配的人区分开来的聚合蛋白 控制。我们现在建议应用我们在这些模型上的经验来开发新的治疗剂， 可能通过以下目的延缓、预防甚至逆转阿尔茨海默氏症和帕金森氏症的病理： 目标1.我们可以通过我们的合作者苏·格里芬博士访问组织库中的组织样本 阿尔茨海默氏症(尾侧海马体)和帕金森氏病(黑质)，我们将从 通过Aβ42、tau和α突触核蛋白的免疫亲和力分离聚集体。我们建议将这些聚合体进行交叉连接， 用胰酶彻底消化它们，并鉴定将揭示蛋白质-蛋白质的交联肽对 相互作用促成了它们的凝聚。我们将使用最先进的“点击化学”试剂来创造， 标记和恢复交叉链接站点，以及用于分析质谱学数据的Xlink识别器软件。想像一下， 整合和解释由此揭示的蛋白质-蛋白质相互作用，我们将创建蛋白质-相互作用网络 并用适应于蛋白质聚集中占主导地位的非功能相互作用的工具对它们进行分析。 目标2.来自目标1的蛋白质组学数据将定义包含以下内容的聚集体中的关键蛋白质-蛋白质相互作用 Tau、Aβ42或α-Synuclein，它们在阿尔茨海默氏症或帕金森病脑样本中高度浓缩。这些蛋白质- 蛋白质相互作用将根据它们的预测倾向(通过ΔG估计)和稳定性(通过 分子动力学模拟)。然后，每个下拉菜单中的前10个蛋白质界面将作为目标 在电子筛选中，开发了用来破坏蛋白质-蛋白质相互作用的药物文库。这样的库，构建 在不同的方面，包括许多旨在阻止特定蛋白质对相互作用的‘PPII’药物，但它们 经常发现应用程序超出其原始目标。对于每个目标，我们将执行以下初始筛选 PPII-硅胶药物库，其中排名靠前的候选药物将在分子动力学模拟中进行重新测试。 目标3：在电子计算机中预测最有效地破坏关键蛋白质相互作用的药物(目标2)将在 减少聚集形成的活体--首先在人神经细胞培养中表达APPsWE(形成 淀粉样蛋白聚集体)或裂解GFP：：tau(tau齐聚后发出荧光)。对于具有保护性的药物 无论是哪种检测方法，也将在表达Aβ42、tau或α-突触核蛋白的线虫聚集模型中进行检测。 将对4-6个顶级候选药物的生物素化版本进行活性评估，并将活性生物素化药物 用来拉下药物附着的蛋白质。这将首先在线虫中完成，使用内部的 全基因组RNAi敲除文库，然后通过将shRNA结构引入到 培养的神经细胞。然后将对产生最大保护的目标进行测试，以询问药物疗效 RNAi敲除部分是多余的，正如预期的那样，作为介导药物保护作用的靶点。 通过发现专为减少聚集而设计的新药，我们预计会发现更有效的药物 治疗和预防药物比目前可用的有限药物更多。根据我们的观察， 许多聚集成分在不同的神经退行性疾病模型中共享，至少其中一些是这样的 药物还可能被证明对包括孤儿在内的各种其他神经退行性疾病有效。