PHOSPHATASE INHIBITION AND TAU PHOSPHORYLATION

这项建议的长期目标是了解其机制

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.