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.

Merit的前期支持使我们能够开发新的免疫亲和分离方案， 非常纯的蛋白质聚集体的表征。我们从C开始。神经退行性疾病的elegans模型 疾病，然后应用相同的协议，以纯化Aβ42-和tau含有聚集体从受影响的 阿尔茨海默病患者的海马组织，寻找聚集蛋白，将其与年龄匹配的 对照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药物， 可能会延迟，预防，甚至逆转阿尔茨海默氏症和帕金森氏症的病理，通过以下目的： 目标1.我们通过我们的合作者苏·格里芬博士，进入了一个组织库， 阿尔茨海默氏病（尾海马）和帕金森氏病（黑质），我们将从 通过对Aβ42、tau和α-突触核蛋白免疫亲和性分离聚集体。我们建议将这些聚合物交联， 用胰蛋白酶彻底消化它们，并鉴定将揭示蛋白质-蛋白质的交联肽对 相互作用，介导了他们的聚集。我们将使用最先进的“点击化学”试剂来创造， 标记和恢复交联位点，以及Xlink Identifier软件来分析质谱数据。为了形象化， 整合并解释由此揭示的蛋白质相互作用，我们将创建蛋白质相互作用网络 并使用适用于在蛋白质聚集中占主导地位的非功能性相互作用的工具来分析它们。 目标2.来自Aim 1的蛋白质组学数据将定义聚集体内的关键蛋白质-蛋白质相互作用， tau、Aβ42或α-突触核蛋白，其在阿尔茨海默病或帕金森病脑样品中高度富集。这些蛋白质- 蛋白质相互作用将根据其预测倾向（通过ΔG估计）和稳定性（通过 分子动力学模拟）。然后，来自每个下拉的前10个蛋白质界面将作为靶点， 对开发用于破坏蛋白质-蛋白质相互作用的药物文库进行计算机筛选。这样的图书馆， 包括许多旨在阻断特定蛋白质对相互作用的“PPII”药物，但这些药物 经常发现超出其最初目标的应用。对于每个目标，我们将执行初始屏幕， PPII库药物在计算机上，从其中的顶级候选人将在分子动力学模拟重新测试。 目标3。计算机模拟预测的最有效破坏关键蛋白质相互作用的药物（Aim 2）将在 体内用于减少聚集体形成-首先在表达APPSwe的人类神经元细胞培养物中（形成 淀粉样蛋白聚集体）或分裂-GFP：：tau（在tau寡聚化后发荧光）。对于保护性药物， 无论是哪种测定，检测也将在C中进行。elegans聚集模型表达Aβ42、tau或α-突触核蛋白。 将评估4-6种顶级候选物的生物素化形式的活性，并将评估活性生物素化药物的活性。 用来拉下药物附着的蛋白质。这将首先在C中完成。elegans，使用内部 “全基因组”RNAi敲低文库，然后通过将shRNA构建体引入到 培养的神经细胞。然后将测试产生最大保护的靶点，以询问药物疗效是否 与RNAi敲除部分冗余，正如对介导药物保护作用的靶点所预期的那样。 通过发现专门用于减少聚集的新药，我们希望找到更有效的药物， 治疗和预防剂比目前可用的有限药物。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是共享的，至少其中一些是 药物也可证明对多种其它神经变性疾病包括孤儿病有效。