Functional studies of Alzheimer's disease tau proteins

The neurofibrillary tangle (NFT) and neuritic plaque (NP) are the hallmark pathologies of Alzheimer's disease (AD). Abnormally phosphorylated tau protein is a major component of the cytoskeletal pathology found in the NFT and dystrophic neurites associated with the NP. Tau is predominantly an axonal microtubule-associated protein whose main function is to promote assembly and stabilization of microtubules. Since tau phosphorylation state is critical in this role, abnormal tau phosphorylation and its dislocation to the neuronal cell body in the NFT predict a fundamental role for tau in the cytoskeletal pathogenesis of AD. However, important unresolved issues include: (1) whether tau protein from AD brain is functionally competent to enhance microtubule assembly, (2) what are the kinases responsible for the abnormal phosphorylation of AD tau?; This dissertation examines the mechanisms by which tau protein becomes abnormal in AD pathology and its functional consequences. In vitro kinetic studies and microinjection of living cells in conjunction with immunocytochemistry were used to address these issues. The results establish that: (1) Fluorescently derivatized bovine tau protein enhances microtubule assembly and is associated with cytoplasmic microtubules when microinjected into Chinese Hamster Ovary (CHO) cells which do not contain endogenous tau, thus validating the use of labeled probe to study trafficking of both native tau and AD tau in real time using living cells; tau may also have functions other than microtubule association as fluorescent tau is also transported into the nucleolus and rapidly associates with centrosomes. (2) Soluble tau protein isolated from AD brains is functionally compromised in terms of its ability to induce microtubule assembly in vitro, and abnormal phosphorylation of aged non-demented human tau by proline-directed protein kinase mimics these effects. (3) p44{dollar}sp{lcub}rm mpk{rcub}{dollar} MAP kinase induces AD type alterations of tau in vitro and in primary hippocampal neurons; phosphotyrosine activated MAP kinase is associated with brain microtubules.; The functional studies performed here will enhance our understanding of the biological roles of tau protein and how abnormal tau modification affects its activity and, subsequently, the fate of cytoskeletal organelles with which modified tau interacts. The identification of proline-directed protein kinases as candidates for abnormally phosphorylating tau in living neurons will provide insight into mechanisms of cytoskeletal pathogenesis in AD.