The objective of this Fogarty International Collaborative Research Award proposal is to elucidate the mechanism of neurofibrillary degeneration in Alzheimer disease (AD) by (i) identifying the protein phosphatase activity(s) which can inhibit the binding of the AD abnormally phosphorylated tau (AD P-tau) to normal tau and restore the biological activity of the abnormal tau, and (ii) determining whether like tau, the high molecular weight microtubule associated proteins (HMW-MAPs) are also involved in Alzheimer neurofibrillary degeneration.
The Specific Aims of this proposal are (1) to study the effect of dephosphorylation of the AD abnormally phosphorylated tau (AD P-tau) on: a) its binding to normal tau and b) on its ability to disrupt the in vitro assembly of microtubules. The abnormal tau isolated from AD brains will be dephosphorylated with phosphoseryl/phosphothreonyl protein phosphatases PP-1, PP-2A and PP-2B. The binding of untreated and in vitro dephosphorylated AD P-tau to normal tau will be studied by dot overlay and sedimentation assays. The disruption of microtubule assembly will be studied by turbidimetric assays at 350 nm and the presence of microtubules confirmed by negative stain electron microscopy. The dephosphorylation of the abnormal phosphorylation sites of tau will be identified by reactivity of phosphorylation-dependent antibodies on Western blots; and (2) to study the binding of AD P-tau to high molecular weight (HMW) MAPs, MAP1 and MAP2 and inhibition of the microtubule assembly promoting activity. The binding of AD P-tau to bovine brain MAP1 and MAP2 will be examined by dot overlay assay. The effect of AD P-tau on the HMW MAPs stimulated microtubule assembly will be examined by turbidimetric assays at 350 nm and the presence of microtubules confirmed by negative stain electron microscopy. The binding of AD P-tau to HMW MAPs in AD brain will be studied by examining the co-sedimentation of HMW MAPs and AD P-tau. The levels of AD P-tau and HMW MAPs will be determined by radioimmunoslot blot assays. These studies will help elucidate (i) how the abnormal phosphorylation of MAPs might be causing the microtubule breakdown in the AD affected neurons, (ii) why the HMW MAPs do not compensate the effect of abnormal phosphorylation of tau, (iii) which are the critical phosphorylated sites in AD P-tau that lead to its biological inactivity and inhibitory properties, and (iv) the nature of the phosphatase activity that is able to reverse it. 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.
