Phosphorylation, conformation, and aggregation of tau

Abstract

In Alzheimer's disease (AD), post-translationally modified forms of tau aggregate into filamentous structures (paired helical filaments or PHF) that contribute to the neurofibrillary degeneration seen in this disease. An Alz-50 immunoaffinity column was created that allows the purification of abnormal tau from Alzheimer brain homogenates. The eluent from this column was used to create more extensive panels of antibodies, including MC-1 and TG-3, and has allowed an initial characterization of an associated protein kinase activity referred to as the PHF kinase. Research goals included a characterization of Alz-50, MC-1, TG-3, and the PHF kinase. Alz-50 and MC-1 have now been shown to recognize a specific conformation of tau found in the disease state, involving an intramolecular association between the extreme N-terminus (amino acids 7--9) and the third microtubule-binding domain (amino acids 312--322). Characterization of TG-3 reactivity includes a demonstration of the disease-specificity of this antibody, which is shown to have dual requirements for reactivity including the p34-cdc2 induced phosphorylation of thr231 and a beta-turn motif between residues 231 and 235 of tau. Recombinant tau proteins expressing fronto-temporal dementia with Parkinsonism-17 mutations were created, analyzed, and found to exhibit structural alterations in comparison to the wild-type molecule. Further analysis of immunoaffinity purified material from AD brain demonstrates that conformationally altered tau molecules self-assemble in a concentration-dependent manner. Characterization of the PHF kinase includes the demonstration that cAMP-dependent protein kinase (PKA) phosphorylations of ser356 and ser409 are required for the sequential phosphorylations of thr361 and 412 on tau. The phosphorylation of ser409 by PKA is shown to induce conformational alterations on recombinant tau similar to those seen in the disease state. Sequential phosphorylation by PKA and the PHF kinase is shown to facilitate recombinant tau aggregation into existing PHF. The creation and characterization of monoclonal antibodies that react specifically with PKA phosphorylations of ser214 and ser409 on tau further demonstrate that these phosphorylations are limited to the disease-state. Additional data demonstrate that the PKA-AKAP 79 holoenzyme complex is tightly associated with neurofibrillary pathology. These data demonstrate that conformational alterations in tau, initiation of the mitotic cascade, and aberrant activation and/or localization of PKA participate in neurofibrillary degeneration in AD.