The objective of this Fogarty International Research Collaboration Award (FIRCA) proposal is to elucidate the mechanism of neurofibrillary degeneration in Alzheimer's disease (AD) by (I) studying the association of the AD abnormally hyperphosphorylated tau (AD P-tau) to various normal human tau isoforms, and (2) identifying the protein kinase(s) the phosphorylation of normal tau by which converts it to an AD-like state in sequestering normal microtubule associated proteins (MAP), MAP1 and MAP2 and depolymerizing microtubles.
For Aim #1, each of the six recombinant human tau isoforms will be purified by column chromatography. The AD P-tau will be isolated from unfixed AD autopsied brains obtained within six hours postmortem and stored at -75 degrees C. The association between the AD P-tau and various recombinant tau isoforms will be assayed both by dot overlay and by the sedimentation assays, followed by quantitation of normal and AD P-tau with a radioimmuno-dot-blot assay using phosphorylation-dependent tau antibody, Tau-1, as the primary antibody.
For Aim #2, we will isolate protein kinase C and CaM kinase-II from rat brain, glycogen synthase kinase-3, cyclin-dependent kinase-5 and casein kinase-I from bovine brain. The tau isoform found to associate the most to the AD P-tau will be in vitro phosphorylated by the above kinases and as well as protein kinase-A (PKA) and mitogen activated kinase (MAP kinase) obtained commercially, and the association of this in vitro phosphorylated tau with MAP1 and MAP2, and as a control with tau, will be examined as in Aim #1. Furthermore, the ability of the phosphorylated tau to depolymerize microtubules assembled by MAP1 and MAP2 will be investigated. The microtubule assembly and disassembly studies will be carried out by light scattering/turbidimeteric changes at 350 mm in microcuvettes, and by negative stain electron microscopy. These studies will help identify the role of different tau isoforms, the abnormai hyperphosphorylation of which convert them to a toxic molecule that can depolymerize the microtubule network, and identify the protein kinase(s) the phosphorylation of tau by which convert it into AD-like state and lead to neurofibrillary degeneration. This information will not only elucidate the mechanism of Alzheimer neurofibrillary degeneration but also provide a lead towards developing a rational therapeutical approach to the disease.
