Analysis and Therapy of Age-Dependent Proteostasis Failure in Neurodegeneration

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的蛋白质聚集体，损害中枢神经系统的特定部分。临床诊断一直以受影响的区域为指导，但越来越明显的是，阿尔茨海默病的基本过程与导致帕金森氏症、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，但神经元由于氧化代谢的高速率而处于特别危险的境地。特定的遗传损伤(或在零星病例中可能接触到化学物质或病毒)可能会基于促进聚集和对抗因素的平衡，将神经病变的位置确定为“最弱的一环”。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(由天然折叠结构、暴露的反应基团和三联体阵列长度决定)、氧化损伤、糖基化和产生交联物或加合物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(超氧化物歧化酶、谷胱甘肽转移酶、过氧化氢酶等)，解毒/清除系统，热休克蛋白/伴侣蛋白的水平，以重新折叠瞬时错误折叠的蛋白质(防止它们通过暴露的疏水残基附着到聚集体上)，有利于错误折叠的共价分子损伤的蛋白质修复，以及泛素化蛋白质的蛋白酶体降解。自噬可能能够清除诊断这些神经病理的大的不可溶聚集体，但这些是否真的是最具神经毒性的复合体，或者是毒性较低的隔离储存库，由更小的、可溶的聚集体形成，这是更有效的神经毒素，仍存在争议。这个问题可以通过对可溶聚集体和不可溶聚集体进行仔细的比较分析来解决，这些聚集体由在神经疾病中观察到的几种不同的启动蛋白“播种”，定义了线虫和人类神经病理样本中的蛋白质和翻译后修饰。目前的建议有三个相互交织和相辅相成的目标：(1)确定在线虫阿尔茨海默病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。线虫具有年龄依赖性或诱导聚集体的蛋白质复合体将被抗tau或Ass抗体拉下，然后分离成洗涤剂不可溶和可溶部分。每个组分的蛋白质将通过凝胶电泳法进行分解，多肽将被胰酶切割，并通过LC-MS2进行分析，以确定和定量蛋白质及其合成后的修饰。最灵敏和可靠的定量方法是使用产物离子扫描从预测的多肽中寻找诊断片段；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。结果将通过无限制的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、糖基化等，从而揭示其涉及的机制。 与年龄相关的发生。双标记程序将允许强大的相对定量，并验证在体内聚集的蛋白质，而不是在处理过程中。(2)在人类样本中寻找类似的聚集体。来自AD受试者的受影响和对照大脑部位的未识别样本将与来自正常对照组的可比部位样本一起进行研究，它们的聚集体将与Ass和tau抗体进行免疫沉淀。对于目标1，复合体将被分离和分离，并且聚集体经过如上所述的LC-MS2分析，寻找蛋白质标记或修饰，包括任何修饰，都与线虫模型有关。(3)为了评估哪些相关蛋白和修饰在神经毒性中发挥功能作用，针对保护性基因的RNA干扰将使其变得更糟，治疗药物或针对促聚集基因的RNAi将使其缓解。蛋白质或可溶或不可溶聚集体中的修饰，与神经毒性相一致，成为功能候选。

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