PHOSPHATASE INHIBITION AND TAU PHOSPHORYLATION

这项建议的长期目标是了解这一机制 阿尔茨海默病(AD)中神经原纤维变性的研究。具体的 这一试点项目的目的是研究抗精神病药物的作用， 氯丙嗪和三氟拉嗪在异常过度磷酸化中的作用 大鼠脑内tau和AD样神经原纤维蛋白病理的研究。这个 这项建议的具体目的是：(1)研究蛋白质的活性 慢性脑损伤后大鼠脑内磷酸酶(PP)、PP-1、PP-2A和PP-2B的变化 输注同为PP-2B的氯丙嗪和三氟拉嗪 抑制剂。使用阿尔茨海默氏液进行输液。 持续向侧脑室输送药物的微泵 大鼠脑内观察2~6个月。另一种PP-2B抑制剂环孢素A和 PP-2A/PP-1抑制剂、冈田酸和盏花素A也将被注入 单独和联合应用抗精神病药物来研究 通过抑制上述三种蛋白磷酸酶起作用。这个 大鼠脑提取液的磷酸酶活性将在体外进行测定 使用(32P)磷酸化酶(用于PP-1和PP2A)和[32P]磷酸化酶 蛋白激酶(PP-2B)为底物； 靶向1.福尔马林对处理和对照大鼠脑的影响 (10%)大鼠大脑的固定切片将为刚果红做准备 染色和免疫细胞化学。依赖磷酸化的tau 抗体Tau-1、AT8、PHF-1、12E8、M4、102C和R21657将用于 检查过度磷酸化的tau的积累。有没有神经原纤维 光学显微镜观察到的变化将被电子检查 显微镜下观察；(3)定量检测大鼠tau蛋白的磷酸化水平 来自特定目标的大脑#1.大鼠脑匀浆的蛋白质印迹 用[125I]标记的二抗研制并定量 将为此目的进行磷光成像仪。Tau抗体作为 将使用上述抗体作为主要抗体来检测 Tau在各自识别的特定位点(S)的磷酸化 抗体。 这些研究将有助于揭示在大鼠大脑中是否存在抑制 抗精神病药物和其他磷酸酶对蛋白质磷酸酶活性的影响 抑制剂诱导类阿尔茨海默病患者tau蛋白异常过度磷酸化 以及随之而来的神经原纤维变性。

The long term objective of this proposal is to understand the mechanism of neurofibrillary degeneration in Alzheimer disease (AD). The specific objective of this pilot project is to study the role of neuroleptics, chlorpromazine and trifluoperazine, in the abnormal hyperphosphorylation of tau and AD-like neurofibrillary protein pathology in rat brain. The specific aims of this proposal are: (1) study the activities of protein phosphatases (PP), PP-1 PP-2A, and PP-2B, in rat brains after chronic infusion with chlorpromazine and trifluoperazine which are also PP-2B inhibitors. Infusion will be carried out by using Alzet osomtic minipumps which will continually deliver drugs to lateral ventricle of rat brain for 2 to 6 months. Another PP-2B inhibitor cyclosporin A and PP-2A/PP-1 inhibitors, okadaic acid and calyculin A will also be infused individually and in combination with the neuroleptics to study the effect by inhibition of all the three protein phosphatases. The phosphatase activities of rat brain extracts will be assayed in vitro using (32P]phosphorylase (for PP-1 and PP2A) and [32P]phosphorylase kinase (for PP-2B) as substrates; (2) study the histopathological changes in treated and control rat brains from Specific Aim #1. Formalin (10%) fixed sections of the rat brain will be prepared for Congo red staining and immunocytochemistry. Phosphorylation-dependent tau antibodies Tau-1, AT8, PHF-1, 12E8, M4, 102c and R21657 will be used to examine the accumulation of hyperphosphorylated tau. Any neurofibrillary changes observed by light microscope will be examined by electron microscopy; (3) quantitate the phosphorylation level of tau in rat brains from Specific Aim #1. Western blots of rat brain homogenates developed with [125I]-labelled secondary antibody and quantitated by Phosphorimager will be carried out for this purpose. Tau antibodies as described above will be employed as primary antibodies to detect the phosphorylation of tau at the specific site(s) recognized by each antibody. These studies will help reveal whether in rat brain the inhibition of protein phosphatase activity by neuroleptics and other phosphatase inhibitors induces Alzheimer-like abnormal hyperphosphorylation of tau and consequent neurofibrillary degeneration.