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

基本信息

批准号：
8803314
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
项目类别：
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/8803314
关键词：
Acetylation Acylation Adherence Affect Age Alzheimer&apos s Disease Amyloid Amyotrophic Lateral Sclerosis Animal Model Antibodies Antioxidants Appearance Asses Ataxia Autophagocytosis Biological Models Brain Caenorhabditis elegans Cell Respiration Cells Central Nervous System Part Chemical Agents Chemicals Cleaved cell Clinical Codon Nucleotides Complex Craniocerebral Trauma Defect Dementia Detergents Diagnostic Disease Drug Metabolic Detoxication Elderly Equilibrium Exposure to 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 Neurodegenerative Disorders Neurofibrillary Tangles Neurons Neuropathy Neurotoxins Parkinson Disease Parkinson&apos s Dementia Pathway interactions Patients Peptide Fragments Peptides Pharmaceutical Preparations Phosphorylation Play Post-Translational Protein Processing Procedures Process Property Proteins Psyche structure RNA Interference Recycling Relative (related person)Reporting Research Proposals Risk Role Sampling Scanning Severities Signal Pathway Site Specificity Stable Isotope Labeling Structural Protein Structure Superoxide Dismutase System Temperature Testing Therapeutic 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 human FRAP1 protein improved in vivo mind control multicatalytic endopeptidase complex neuropathology neurotoxic neurotoxicity oxidation oxidative damage polyglutamine prevent protein TDP-43 protein aggregate protein aggregation protein complex protein misfolding repaired repository synuclein tau Proteins ubiquilin

项目摘要

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损害中枢神经系统特定部分的有毒蛋白聚集体的形成。临床诊断受到影响的地区的指导，但是越来越明显的是，阿尔茨海默痴呆症的基本过程与导致帕金森氏症和亨廷顿疾病的痴呆症和肌萎缩性侧面硬化症非常相似。尽管所有细胞都必须维持蛋白质平衡，但由于氧化代谢率高，神经元特别有风险。特定的遗传病变（或可能是化学或病毒暴露，在零星的情况下）可能会根据促进和补偿因子的平衡来确定神经病部位是“最弱的联系”。有利于聚集的因素包括易受攻击的蛋白质的局部丰度和结构不稳定性（由天然折叠结构，裸露的反应式组确定，如果存在），氧化损伤，糖基化和蛋白质反应性代谢产物或产生交叉连接或加入的环境有毒物质。 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蛋白质。自噬可能能够清除对这些神经病理学的大型不溶性骨料的诊断，但是这仍然是有争议的，无论这些神经病理学是否确实是最神经毒性复合物，或者是由较小的，较小的，较小的可溶骨料形成的较小的可溶骨料，这些杂物是更有效的神经毒素。可以通过对可溶性和不溶性聚集体进行仔细的比较分析来解决这个问题，这是由在神经病中观察到的几种不同的引发剂蛋白“播种”的，从而定义了蛋白质和秀丽杆菌的翻译后修饰和人类神经病理学样本。当前的提案具有三个相互交织和互补的目的：（1.）定义在阿尔茨海默氏菌的秀丽隐杆线虫模型中汇总的蛋白质，该蛋白质由神经元特异性的tau和ass transes启动或播种。具有年龄依赖性或诱导骨料的线虫的蛋白质复合物将用抗Tau或Ass的抗体将其拉下，然后分离为洗涤剂 - 不溶性和可溶性分数。每种馏分的蛋白质将通过凝胶电泳解决，肽将通过胰蛋白酶裂解并通过LC-MS2分析，以定义和量化蛋白质及其合成后修饰。最敏感，最可靠的定量使用产品-ION扫描来寻求预测肽的诊断片段。同时，我们还将进行更一般但更敏感的“无偏扫描”，以识别和量化更丰富的蛋白质，即使不可预见。结果将通过不受限制的搜索进行交叉检查和扩展，以量化结合后修饰，包括磷酸化，乙酰化，氧化，酰基化，糖基化等，从而暗示了与其涉及的机制年龄依赖性的发生。双标签程序将允许鲁棒的相对定量，以及在体内汇总而不是在处理过程中汇总的蛋白质的验证。（2.）在人类样品中寻求类似的聚集体。将研究来自AD受试者的受影响和对照脑部位点的去识别样品，以及来自正常对照的可比位点样品，其骨料用抗体和TAU的抗体进行免疫沉淀。复合物将被分级和分离，以与AIM 1相分开，并且经过LC-MS2分析的聚集体如上所述，寻求蛋白质标记或修饰（包括任何的修饰）。（3.）为了评估哪些涉及的蛋白质和修饰在神经毒性中起功能的作用，将通过靶向保护基因的RNA干扰来使它变得更糟，或者通过治疗药物或RNAi对促促侵蚀性基因的释放。可溶性或不溶性聚集体中的蛋白质或修饰，与神经毒性一致，成为功能性候选者