HUMAN BRAIN PROTEOLYSIS IN AGING & ALZHEIMER'S DISEASE

衰老过程中的人脑蛋白水解

• 批准号: 3119841
• 负责人: RALPH A. NIXON
• 金额: $ 19.52万
• 依托单位: MCLEAN HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3119841
• 关键词: Alzheimer's disease; aging; chromatography; cytoskeleton; electrofocusing; gel electrophoresis; histochemistry /cytochemistry; human tissue; immunochemistry; isozymes; microfilaments; neurochemistry; neurofibrillary tangles; peptidases; postmortem; proteins; proteolysis; radioassay; scintillation counter

We propose to use new methods to study, for the first time, the proteolysis of neurofilament and other cytoskeletal proteins in postmortem human brain during normal aging and in Alzheimer's diseas (AD). In addition to increasing our understanding of this complex process in normal and aging brain, these experiments will provide information pertinent to the basic mechanism underlying the formation of neurofibrillary lessions and the degeneration of neurons in AD and other human neurodegenerative disorders. We have obtained evidence supporting our hypothesis that proteolysis is defective in AD brain due to abnormal proteolytic enzyme systems and/or to abnormally modified protein substrates that are resistant to degradation. Having developed methods to purify the four major proteinases from human brain (Ca++-activated neural proteinase(s) [CANP]; cathepsins D and B; and Ca++- independent neutral proteinases), we will seek age- and AD-related abnormalities in these enzyme systems by characterizing in detail the structural and enzymatic properties (including denaturation rates) of each purified enzyme and its isoenzymes from matched speciments of postmortem prefrontal isocortex from normal adults, aged individuals (Greater than 80 years) and AD patients. Interactions between various CANP forms and a specific regulatory factor purified from brain will also be investigated. Distribution of the activity and the content of each human brain proteinase will be measured by radioassay and radioimmunoassay respectively, in 15-20 selected regions of control, aged, and AD brains and correlated with the severity of neurofibrillary pathology quantitated morphometrically and biochemically. Based on our findings that paired helical filaments (PHF) in AD brain are resistent to digestion by brain proteinases, we will seek differences between normal and AD brain in the structure of specific cytoskeletal proteins by first studying the kinetics of degradation by each purified proteinase. Using monoclonal and polyclonal antibodies to individual NFPs and novel 2-D immunblot approaches, we will then compare in control and AD cases the patterns of NFP-immunreactive proteolytic products present in unincubated tissue or generated by either purified brain proteinases in vitro or by corresponding neuronal proteinases in situ within intact brain microslices. Finally, the susceptibility of PHF and recently observed PHF-immunoreactive (? precursor) variant forms to purified proteinases will be further investigated by immunoblot analyses using anti-PHF antibodies.

我们建议首次使用新方法来研究蛋白水解作用 死后人脑中神经丝和其他细胞骨架蛋白的研究 正常衰老期间和阿尔茨海默病 (AD) 中。 此外 增加我们对正常和衰老过程中这一复杂过程的理解 大脑，这些实验将提供与基本知识相关的信息 神经原纤维损伤形成的机制及 AD 和其他人类神经退行性疾病中的神经元变性。 我们已经获得证据支持我们的假设，即蛋白水解作用是 由于蛋白水解酶系统异常和/或 异常修饰的蛋白质底物具有抗降解能力。 开发出从人体中纯化四种主要蛋白酶的方法 脑（Ca++ 激活的神经蛋白酶 [CANP]；组织蛋白酶 D 和 B；以及 Ca++-独立的中性蛋白酶），我们将寻找与年龄和 AD 相关的 通过详细表征这些酶系统的异常 每个的结构和酶学特性（包括变性率） 从匹配的死后样本中纯化的酶及其同工酶 正常成年人、老年人（大于 80 年）和 AD 患者。 各种CANP形式和a之间的相互作用 还将研究从大脑中纯化的特定调节因子。 人脑各蛋白酶活性及含量分布 将分别在15-20年通过放射测定法和放射免疫测定法进行测定 控制、老年和 AD 大脑的选定区域，并与 神经原纤维病理学的严重程度通过形态测量进行定量 生物化学上。 根据我们的发现，成对螺旋丝 (PHF) AD 大脑对脑蛋白酶的消化具有抵抗力，我们将寻求 正常大脑和 AD 大脑在特定结构上的差异 首先研究细胞骨架蛋白的降解动力学 纯化的蛋白酶。 使用单克隆和多克隆抗体 个体 NFP 和新颖的 2-D 免疫印迹方法，然后我们将在 对照和 AD 病例 NFP 免疫反应性蛋白水解产物的模式 存在于未孵化的组织中或由纯化的大脑产生 体外蛋白酶或原位相应的神经元蛋白酶 在完整的大脑微切片内。 最后，PHF 的敏感性和 最近观察到的 PHF 免疫反应性（？前体）变异形式 纯化的蛋白酶将通过免疫印迹分析进一步研究 使用抗 PHF 抗体。