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.

上一个值得支持的时期使我们能够制定新的免疫亲属隔离方案，并 非常纯的蛋白质聚集体的表征。我们从秀丽隐杆线虫的神经退行性模型开始 疾病，然后应用相同的方案以净化Aβ42-和含Tau的骨料 阿尔茨海默氏症患者的海马组织，寻求分化它们与年龄匹配的蛋白质 控件。现在，我们建议我们在这些模型中运用我们的经验，以开发新颖的治疗剂 可能会通过以下目的延迟，预防甚至逆转阿尔茨海默氏症和帕金森氏病理学： 目标1。我们可以通过我们的合作者Sue Griffin博士进入纸巾样本 阿尔茨海默氏病（尾海马）和帕金森氏病（黑质），我们将从中 通过免疫亲和力的Aβ42，tau和α-突触核蛋白的分离株。我们建议交叉链接这些聚集体， 用胰蛋白酶彻底消化它们，并识别将揭示蛋白质蛋白的交联肽对 介导其集团的互动。我们将使用最先进的“点击化学”试剂来创建 标记和恢复交联站点和Xlink标识符软件，以分析质谱数据。可视化， 整合并解释了这样揭示的蛋白质 - 蛋白质相互作用，我们将创建蛋白质交互网络 并使用适合于蛋白质聚集的非功能相互作用的工具分析它们。 AIM 2。来自AIM 1的蛋白质组学数据将定义包含骨料中的关键蛋白质 - 蛋白质相互作用 tau，Aβ42或α-突触核蛋白，高度富含阿尔茨海默氏症或帕金森脑样品。这些蛋白质 - 蛋白质相互作用将根据其预测承诺（通过ΔG估计）和稳定性进行排名（通过 分子动力模拟）。然后，来自每个下拉的前10个蛋白质界面将作为目标 在用于破坏蛋白质蛋白质相互作用的药物库的硅筛查中。这样的库，构建 在潜水员方面，包括许多旨在阻止特定蛋白质相互作用的“ PPII”药物 经常发现的应用程序超出其原始目标。对于每个目标，我们将执行 硅中的PPII-上生药物，将在该药物中重新测试，以分子动态模拟进行重新测试。 AIM 3。在硅中预测的药物最有效地破坏关键蛋白质相互作用（AIM 2）将在 减少骨料形成的体内 - 表达Appswe（形成）的人类神经元细胞培养 淀粉样蛋白聚集体）或split-gfp :: tau（在tau低聚时荧光）。对于受到保护的药物 分析也将在表达Aβ42，TAU或α-突触核蛋白的秀丽隐杆线虫聚集模型中进行测试。 将评估4-6个顶级候选者的生物素化版本的活动，活性生物素化药物将是 用于拉下药物附着的蛋白质。这将最初在秀丽隐杆线虫中进行，并使用内部 “全基因组” RNAi-KNOCKDOWN库，然后通过将shrna构建体引入 培养的神经元细胞。然后，将测试产生最大保护的目标，以询问药物效率是否 对于介导该药物的受保护作用的靶标的预期，RNAi敲低是部分冗余的。 通过发现专门设计用于减少聚集的新型药物，我们希望发现更有效 与目前可用的有限药物相比，治疗和预防剂。根据我们的观察 许多骨料组件在潜水员神经退行性疾病模型之间共享，至少其中一些 药物还可能证明对包括孤儿疾病在内的各种其他神经退行性疾病有效。