HUMAN BRAIN PROTEOLYSIS IN AGING & ALZHEIMER'S DISEASE

我们首次提出用新的方法来研究蛋白质的分解。 神经丝和其他细胞骨架蛋白在死后人脑中的分布 在正常衰老和阿尔茨海默病(AD)期间。除了……之外 增加我们对正常和衰老这一复杂过程的理解 这些实验将提供与大脑相关的基本信息 神经纤维样变形成的机制及其机制 阿尔茨海默病和其他人类神经退行性疾病中的神经元变性。 我们已经获得了支持我们的假设的证据，即蛋白质分解是 AD脑内蛋白水解酶系统异常和/或 抗降解的异常修饰的蛋白质底物。 已经开发出从人类体内纯化四种主要蛋白酶的方法 脑(钙激活的神经蛋白水解酶S)；组织蛋白D和B；以及 不依赖CA的中性蛋白)，我们将寻找与年龄和AD相关的 这些酶系统的异常通过详细地表征 每种的结构和酶性质(包括变性速率) 从配对身体标本中提取纯化的酶及其同工酶 正常成人、老年人(>80岁)的前额叶等皮质 年)和AD患者。不同CANP形式之间的相互作用 从大脑中提纯的特定调节因子也将被研究。 人脑组织中各蛋白水解酶活性及含量的分布 将在15-20年内分别用放射测定法和放射免疫测定法进行测定 控制、衰老和阿尔茨海默病大脑的选定区域，并与 神经原纤维病变的严重程度通过形态计量学和 生化方面的。根据我们的发现，成对的螺旋丝(PHF) 在阿尔茨海默病的大脑中是抵抗被脑部蛋白酶消化的，我们将寻求 正常与阿尔茨海默病患者脑内特定结构的差异 首先研究细胞骨架蛋白的降解动力学 纯化的蛋白酶。使用单抗和多克隆抗体 单个NFP和新的2-D免疫印迹方法，然后我们将在 对照和阿尔茨海默病患者NFP免疫反应蛋白分解产物的模式 存在于未孵化的组织中或由纯化的大脑产生 体外蛋白水解酶或相应的神经细胞原位蛋白水解酶 在完整的脑微切片中。最后，PHF和PHF的敏感性 最近观察到的PHF免疫反应阳性(？前身)变体形式到 纯化的蛋白水解酶将通过免疫印迹分析进行进一步研究 使用抗PHF抗体。

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.