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Microtubules (MTs), primarily composed of alpha and beta tubulin polymers, must often work in concert with microtubule-associated proteins (MAPs) in order to modulate their functional demands. In a mature brain neuron, one of the key MAPs that resides primarily in the axonal compartment is the tau protein. Tau, in the adult human brain, is a set of six protein isoforms, whose binding affinity to MTs can be modulated by phosphorylation. In addition to the role that phosphorylation of tau plays in the "normal" physiology of neurons, hyperphosphorylated tau is the primary component of the fibrillary pathology in Alzheimer's disease (AD). Although many protein kinases are known to phosphorylate tau in vitro, the in vivo players contributing to the hyperphosphorylation of tau remain elusive. The experiments in this study attempt to define which protein kinases and protein phosphatases reside in the associated network of microtubules, thereby being strategically positioned to influence the phosphorylation of tau. Microtubule fractions are utilized to determine which of the microtubule-associated kinases most readily impacts the phosphorylation of tau at "AD-like" sites. Results from this study indicate that PKA, CK1, GSK3beta, and cdk5 associate with microtubules. Among the MT-associated kinases, GSK3beta and cdk5 most readily contribute to the ATP-induced "AD-like" phosphorylation of tau., (Copyright 2000 Wiley-Liss, Inc.)

Abnormally phosphorylated tau protein is a major component of the cytoskeletal pathology of Alzheimer's disease (AD) found in the neurofibrillary tangle (NFT) and neuritic plaque (NP). Identification of the kinase responsible for this phosphorylation has been difficult. In the test tube, several proline-directed kinases, particularly mitogen-activated protein (MAP) and cdc2 kinase, phosphorylate tau on sites that appear to mimic the abnormally phosphorylated sites in AD. Important unanswered issues include: 1) whether this phosphorylation event occurs in the tightly regulated environment of a living cell; 2) whether this phosphorylation of tau affects its functional properties; and 3) what is the subcellular relationship of proline-directed kinases and tau. We show here that tau can be phosphorylated in cultured hippocampal neurons by the MAP kinase p44mpk, and phosphorylation of tau compromises its functional ability to assemble microtubules. We show further that MAP kinase copurifies with microtubule fractions where it is tyrosine phosphorylated and presumably active. These studies address and raise several important issues regarding the regulation of tau phosphorylation in normal and AD brain.

Immunohistochemical analysis was used to assess the distribution of the proline-directed kinase, cdc2, in Alzheimer's disease (AD) pathology. A robust signal was most prominent in the neurofibrillary tangle (NFT) of affected neurons that also contained abnormally phosphorylated tau protein. Biochemical analysis identified a pool of cdc2 in bovine brain microtubules that contain normal tau. These results strongly support the hypothesis that cdc2 is involved in the abnormal phosphorylation of tau in AD pathology and they raise important issues regarding regulation of tau phosphorylation in normal and diseased neurons.

Immunohistochemistry was used to assess the distribution of protein tyrosine kinases in Alzheimer's disease (AD) pathology. One of these, fyn, exhibited an interesting pattern of immunoreactivity. Low levels of immunoreactivity were apparent in neurons from both normal and AD brain. However a subset of neurons in AD brain exhibited intense fyn immunoreactivity. Double label immunohistochemistry revealed that fyn-positive neurons were also positive for abnormally phosphorylated microtubule-associated protein, tau. Proline-directed kinases, whose activity is regulated by phosphorylation on tyrosine, may be responsible for abnormal tau phosphorylation. These results identify fyn as a candidate kinase for regulating putative proline-directed kinases involved in abnormal tau phosphorylation.

In vitro assays were used to monitor and compare the kinetic behavior of bovine tubulin polymerization enhanced by tau proteins isolated from Alzheimer's disease (AD) and nondemented (ND) age-matched control brains. Tau from AD cases induced slower polymerization and a steady state turbidity value approximately 50% of that stimulated by tau from control cases. Tau from the most severe AD case was least effective at promoting polymerization. Dark-field light microscopy of the control samples revealed abundant microtubule formation and many microtubule bundles. Microtubule assembly was observed in AD samples as well, but bundling was not obvious. These results were confirmed by negative-stain electron microscopy. Morphological analysis showed that AD tau-induced microtubules were longer than control microtubules. Furthermore, our initial results suggest that the reduction of AD tau activity is correlated with neurofibrillary pathology in AD brains. Earlier reports indicated that AD tau is modified by phosphorylation (Grundke-Iqbal et al., 1986; Wood et al., 1986; Iqbal et al., 1989; Brion et al., 1991a,b; Lee et al., 1991). Our results support the hypothesis that tau modification compromises its function by altering its ability to nucleate and bundle microtubules.

Immunohistochemical techniques have been used to assess the distribution of phosphotyrosine-containing compartments in Alzheimer's disease (AD) pathology. Elevated levels of phosphotyrosine are apparent in the somatodendritic compartment of tangle-bearing neurons, in the neuritic plaque (NP) and in dystrophic neurites coursing through the neuropil. The only neuronal staining observed in non-AD tissue is in developing neurites. This suggests that some neuronal elements involved in AD pathology may be recapitulating a developmental profile or, alternately, that elevated phosphotyrosine levels may reflect a role for tyrosine kinase/phosphatase systems in the degeneration process directly. Cells in the neuritic plaque which strongly resemble microglia also contain elevated levels of phosphotyrosine compared to non-activated ramified microglia in the same tissue section. Thus, tyrosine phosphorylation systems may be involved in the response of microglia to degeneration in AD pathology. Implications of these results are discussed.

Immunohistochemistry of formalin-fixed human Alzheimer's disease (AD) tissue using an anti-tau antibody (Tau-1) reveals staining of neurofibrillary tangles (NFTs) and neuritic plaques (NPs), whereas normal axonal staining is less apparent. In this study, we used a combined biochemical and histochemical approach to assess effects of formalin on immunoreactivity of AD tau. Nitrocellulose blots were treated with fixative to mimic conditions used with tissue sections, a method that might be generally useful for assessing antigen sensitivity to different fixatives. A progressive decrease in Tau-1 immunoreactivity of the tau bands on a Western blot was observed with increasing times of formalin fixation. Phosphatase-digested blots demonstrated an increase in Tau-1 immunoreactivity compared to control blots. These results mimic the phosphatase-sensitive Tau-1 immunohistochemical staining of formalin-fixed AD tissue slices previously reported. Fixation of AD tissue with periodate-lysine-paraformaldehyde (PLP) preserves axonal tau antigenicity. Phosphatase digestion of PLP-fixed AD tissue enhances Tau-1 immunoreactivity of NFTs and NPs but does not alter axonal staining. These results indicate that axonal form(s) of tau are more sensitive to formalin fixation than pathology-associated tau. In addition, a modification of AD tau in pathological structures may protect it from the effects of formalin with regard to Tau-1 antigenicity.

Cytochemical and biochemical techniques have been used to assess the relationship of epitopes on the microtubule-associated protein, tau, to the cytoskeletal pathology of Alzheimer's disease. The main probes were Tau-1 and Alz-50, two monoclonal antibodies which recognize tau and a potentially related 68 kDa protein. Sequential treatment of tissue slices with combinations of the antibodies showed that each blocked the binding of the other to neurofibrillary tangles and neuritic plaques but not to normal axons. Western blot analysis of tau proteins isolated from Alzheimer's disease brains did not reveal such blocking patterns. The issue of steric hindrance affecting antibody binding in tissue sections was addressed by using Alz-50 in combination with Tau-2, another monoclonal antibody recognizing tau on blots and in Alzheimer's disease pathology. Neither antibody blocked the binding of the other to neurofibrillary tangles and neuritic plaques. These data suggest that the Alz-50 and Tau-1 epitopes are selectively organized in the tangles and plaques to be in close proximity which supports the hypothesis that in Alzheimer's disease pathology, tau is modified.

The relationship of the neurofibrillary tangle, found in Alzheimer disease and aged brains, to normal or abnormal cytoskeletal proteins remains elusive. Although immunohistochemical studies have yielded disparate results, most antigenic determinants localized to neurofibrillary tangles are cytoskeletal constituents normally present in neuronal perikarya or dendrites. We report light and electron microscopic immunolabeling of neurofibrillary tangles by a monoclonal antibody to the microtubule-associated protein tau (tau). Dephosphorylation of tissue slices not only increased the number of tau-positive tangles but also produced marked positive immunoreactivity of neuritic plaques. The localization of tau, an axonal protein, to neurofibrillary tangles in the perikaryon in particular suggests that abnormal synthesis, modification, or aggregation of tau may induce aberrant cytoskeletal--cell organelle interactions, subsequent interference with axonal flow, and resultant tangle formation.