HUMAN BRAIN PROTEOLYSIS IN AGING & ALZHEIMER'S DISEASE

我们建议使用新的方法来研究，第一次， 神经丝和其他细胞骨架蛋白在死后人脑中的表达 在正常衰老和阿尔茨海默病（AD）中。 除了 增加我们对正常和衰老中这一复杂过程的理解 大脑，这些实验将提供有关的基本信息， 神经元损伤形成的机制和 AD和其他人类神经变性疾病中的神经元变性。 我们已经获得了支持我们假设的证据，即蛋白水解是 由于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。 建立了从人血清中分离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;和 Ca++非依赖性中性蛋白酶），我们将寻求年龄和AD相关的 通过详细描述这些酶系统的异常， 结构和酶性质（包括变性率） 从尸体配对标本中纯化的酶及其同工酶 正常成年人、老年人（80岁以上）的前额叶同皮层 年）和AD患者。 各种CANP表格和 还将研究从脑中纯化特异性调节因子。 人脑蛋白酶活性及含量的分布 分别用放射性分析法和放射免疫分析法测定， 选择的控制区域，老年人和AD大脑，并与 神经病理学的严重程度通过形态计量学定量， 生物化学 根据我们的发现，成对螺旋丝（PHF） 在AD中，脑抵抗脑蛋白酶消化，我们将寻求 正常和AD脑组织中特异性 细胞骨架蛋白的降解动力学，首先研究每个 纯化的蛋白酶。 使用单克隆和多克隆抗体， 单个NFP和新的2-D免疫印迹方法，然后我们将比较 对照组和AD组NFP免疫反应蛋白水解产物的模式 存在于未孵育的组织中或由纯化的大脑产生 蛋白酶或相应的神经元蛋白酶原位 在完整的大脑切片中。 最后，PHF的易感性和 最近观察到PHF免疫反应（？前体）变体形式， 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。

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.