Analysis and Therapy of Age-Dependent Proteostasis Failure in Neurodegeneration

神经变性中年龄依赖性蛋白质稳态失败的分析和治疗

• 批准号: 8971964
• 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 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8971964
• 关键词: Acetylation; Acylation; Adherence; Affect; Age; Alzheimer' s Disease; Amyloid; Amyotrophic Lateral Sclerosis; Animal Model; Antibodies; Antioxidants; Appearance; Asses; Ataxia; Autophagocytosis; Biological Models; Brain; Caenorhabditis elegans; Cell Respiration; Cells; Chemical Agents; Chemicals; Cleaved cell; Clinical; Codon Nucleotides; Complex; Craniocerebral Trauma; Defect; Dementia; Detergents; Diagnostic; Disease; Drug Metabolic Detoxication; Elderly; Equilibrium; Exposure to; FRAP1 gene; Failure; Familial disease; Generations; Genes; Genetic; Health; Human; Huntington Disease; Individual; Ions; Label; Lead; Learning; Length; Lesion; Link; MAPK8 gene; Maintenance; Mitochondria; Modeling; Modification; Molecular; Molecular Chaperones; Mus; Mutation; Nematoda; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Neuropathy; Neurotoxins; Parkinson Disease; Parkinson' s Dementia; Pathway interactions; Patients; Peptide Fragments; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phosphorylation; Play; Post-Translational Protein Processing; Procedures; Process; Property; Proteins; Psyche structure; RNA Interference; Recycling; Reporting; Research Proposals; Risk; Role; Sampling; Scanning; Severities; Signal Pathway; Site; Specificity; Stable Isotope Labeling; Structural Genes; Structure; Superoxide Dismutase; System; Temperature; Testing; Therapeutic; Thinking; Tissue Sample; Tissues; Toxic Environmental Substances; Toxic effect; Toxicant exposure; Transgenes; Transgenic Organisms; Trinucleotide Repeats; Triplet Multiple Birth; Trypsin; Ubiquitin; Validation; Veterans; Viral; age related; base; brain cell; catalase; clinical Diagnosis; clinically relevant; comparative; crosslink; design; gel electrophoresis; glycation; improved; in vivo; mind control; multicatalytic endopeptidase complex; neuropathology; neurotoxic; neurotoxicity; oxidation; oxidative damage; polyglutamine; prevent; protein TDP-43; protein aggregate; protein aggregation; protein biomarkers; protein complex; protein misfolding; repaired; repository; synuclein; tau Proteins; ubiquilin

DESCRIPTION (provided by applicant): Most major neurodegenerative diseases are characterized by the formation of toxic protein aggregates which compromise a specific part of the central nervous system. Clinical diagnosis has been guided by the regions affected, but it is becoming increasingly evident that the basic processes underlying Alzheimer dementia are remarkably similar to those leading to Parkinson's and Huntington's diseases, and Amyotrophic Lateral Sclerosis. Although all cells are obliged to maintain proteostatic equilibrium, neurons are at particular risk due to high rates of oxidative metabolism. Specific genetic lesions (or possibly chemical or viral exposure, in sporadic cases) may determine the site of neuropathy as the "weakest link" based on the balance of pro- aggregation and countervailing factors. Factors favoring aggregation include high local abundance of vulnerable proteins and structural instability (determined by the native folded structure, exposed reactive groups, and triplet-array length, if present), oxidative damage, glycation, and protein-reactive metabolites or environmental toxicants that produce cross-linking or adduction. Protective factors include antioxidant defenses (SODs, GSTs, catalases, etc.), detoxification/clearance systems, levels of HSP/chaperones to refold transiently misfolded proteins (preventing their adherence to aggregates via exposed hydrophobic residues), protein repair of covalent molecular damage that would favor misfolding, and proteasomal degradation of ubiquitinylated proteins. Autophagy may be able to clear the large insoluble aggregates diagnostic of these neuropathologies, but it remains controversial whether those are indeed the most neurotoxic complexes, or are instead less-toxic, sequestered repositories formed from smaller, soluble aggregates which are more potent neurotoxins. This issue could be resolved through a careful, comparative analysis of soluble vs. insoluble aggregates, "seeded" by several of the diverse initiator proteins observed in neuropathies, defining proteins and post-translational modifications in C. elegans and human neuropathology samples. The current proposal has three intertwined and complementary aims: (1.) To define proteins that aggregate in C. elegans models of Alzheimer's, initiated or seeded by neuron-specific tau and Ass transgenes. Protein complexes from nematodes with age-dependent or induced aggregates will be pulled down with antibody to tau or Ass, and then separated into detergent-insoluble and soluble fractions. Proteins from each fraction will be resolved by gel electrophoresis, and peptides will be cleaved by trypsin and analyzed by LC-MS2 to define and quantify the proteins and their post-synthetic modifications. The most sensitive and reliable quantitation uses product-ion scans to seek diagnostic fragments from predicted peptides; in parallel, we will also conduct more general but less sensitive "unbiased scans" to identify and quantify the more abundant proteins, even if unforeseen. Results will be cross-checked and extended by unrestrictive searches to quantify postsynthetic modifications including phosphorylation, acetylation, oxidation, acylation, glycation, etc., and thus implicate mechanisms involved in their age-dependent occurrence. Dual-label procedures will allow robust relative quantitation, and the validation of proteins that aggregated in vivo rather than during handling. (2.) To seek similar aggregates in human samples. De-identified samples from affected and control brain sites from AD subjects will be studied along with comparable-site samples from normal controls, and their aggregates immunoprecipitated with antibodies to Ass and tau. Complexes will be fractionated and separated as for Aim 1, and aggregates subjected to LC-MS2 analyses as above, seeking protein markers or modifications including any were implicated in the C. elegans models. (3.) To assess which implicated proteins and modifications play functional roles in neurotoxicity, it will be made worse by RNA interference targeting protective genes, or relieved by therapeutic drugs or RNAi against pro-aggregative genes. Proteins or modifications in either soluble or insoluble aggregates, which vary concordantly with neurotoxicity, become functional candidates

描述（由申请人提供）： 大多数主要的神经退行性疾病的特征是形成有毒蛋白质聚集体，损害中枢神经系统的特定部分。临床诊断以受影响的区域为指导，但越来越明显的是，阿尔茨海默氏痴呆的基本过程与导致帕金森病、亨廷顿病以及肌萎缩侧索硬化症的基本过程非常相似。尽管所有细胞都有义务维持蛋白质平衡，但神经元由于高氧化代谢率而面临特别的风险。特定的遗传病变（或可能是化学或病毒暴露，在散发病例中）可以根据促聚集因素和抵消因素的平衡将神经病变部位确定为“最薄弱的环节”。有利于聚集的因素包括脆弱蛋白质的高局部丰度和结构不稳定性（由天然折叠结构、暴露的反应基团和三联体阵列长度（如果存在）决定）、氧化损伤、糖化以及产生交联或加合的蛋白质反应性代谢物或环境毒物。保护因素包​​括抗氧化防御（SOD、GST、过氧化氢酶等）、解毒/清除系统、重新折叠短暂错误折叠蛋白质的 HSP/伴侣水平（防止它们通过暴露的疏水残基粘附到聚集体）、有利于错误折叠的共价分子损伤的蛋白质修复以及泛素化的蛋白酶体降解 蛋白质。自噬可能能够清除诊断这些神经病理学的大的不溶性聚集物，但这些是否确实是最具神经毒性的复合物，或者是由较小的可溶性聚集物形成的毒性较小的隔离库（更有效的神经毒素）仍然存在争议。这个问题可以通过对可溶性与不溶性聚集体进行仔细的比较分析来解决，这些聚集体是由神经病中观察到的几种不同的起始蛋白“播种”的，定义了秀丽隐杆线虫和人类神经病理学样本中的蛋白质和翻译后修饰。目前的提案具有三个相互交织且互补的目标：(1.) 定义在阿尔茨海默病的秀丽隐杆线虫模型中聚集的蛋白质，这些蛋白质由神经元特异性 tau 和 Ass 转基因启动或播种。来自线虫的蛋白质复合物具有年龄依赖性或诱导性聚集体，将用 tau 或 Ass 抗体将其拉下来，然后分离成洗涤剂不溶性和可溶性部分。每个级分中的蛋白质将通过凝胶电泳进行解析，肽将被胰蛋白酶切割并通过 LC-MS2 进行分析，以定义和量化蛋白质及其合成后修饰。最灵敏、最可靠的定量方法是使用产物离子扫描从预测的肽中寻找诊断片段；与此同时，我们还将进行更一般但不太敏感的“无偏扫描”，以识别和量化更丰富的蛋白质，即使是不可预见的。结果将通过无限制性搜索进行交叉检查和扩展，以量化合成后修饰，包括磷酸化、乙酰化、氧化、酰化、糖化等，从而暗示其涉及的机制。 年龄相关的发生。双标记程序将实现稳健的相对定量，并验证体内聚集的蛋白质，而不是在处理过程中聚集的蛋白质。 (2.) 在人类样本中寻找类似的聚集体。来自 AD 受试者受影响和对照大脑部位的去识别样本将与来自正常对照的类似部位样本一起进行研究，并用 Ass 和 tau 抗体对它们的聚集体进行免疫沉淀。复合物将按照目标 1 进行分级和分离，并对聚集体进行如上所述的 LC-MS2 分析，寻找蛋白质标记或修饰，包括线虫模型中涉及的任何标记或修饰。 (3.) 为了评估哪些相关蛋白质和修饰在神经毒性中发挥功能性作用，针对保护性基因的RNA干扰会使神经毒性变得更糟，或者通过针对促聚集基因的治疗药物或RNAi来缓解。可溶性或不溶性聚集体中的蛋白质或修饰，其变化与神经毒性一致，成为功能候选者