A monoclonal antibody to the microtubuleassociated protein X (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical

Just as neuronal activity is essential to normal brain function, microtubule-associated protein tau appears to be critical to normal neuronal activity in the mammalian brain, especially in the evolutionary most advanced species, the homo sapiens. While the loss of functional tau can be compensated by the other two neuronal microtubule-associated proteins, MAP1A/MAP1B and MAP2, it is the dysfunctional, i.e., the toxic tau, which forces an affected neuron in a long and losing battle resulting in a slow but progressive retrograde neurodegeneration. It is this pathology which is characteristic of Alzheimer disease (AD) and other tauopathies. To date, the most established and the most compelling cause of dysfunctional tau in AD and other tauopathies is the abnormal hyperphosphorylation of tau. The abnormal hyperphosphorylation not only results in the loss of tau function of promoting assembly and stabilizing microtubules but also in a gain of a toxic function whereby the pathological tau sequesters normal tau, MAP1A/MAP1B and MAP2, and causes inhibition and disruption of microtubules. This toxic gain of function of the pathological tau appears to be solely due to its abnormal hyperphosphorylation because dephosphorylation converts it functionally into a normal-like state. The affected neurons battle the toxic tau both by continually synthesizing new normal tau and as well as by packaging the abnormally hyperphosphorylated tau into inert polymers, i.e., neurofibrillary tangles of paired helical filaments, twisted ribbons and straight filaments. Slowly but progressively, the affected neurons undergo a retrograde degeneration. The hyperphosphorylation of tau results both from an imbalance between the activities of tau kinases and tau phosphatases and as well as changes in tau's conformation which affect its interaction with these enzymes. A decrease in the activity of protein phosphatase-2A (PP-2A) in AD brain and certain missense mutations seen in frontotemporal dementia promotes the abnormal hyperphosphorylation of tau. Inhibition of this tau abnormality is one of the most promising therapeutic approaches to AD and other tauopathies.

The microtubule-associated protein is a family of six isoforms that becomes abnormally hyperphosphorylated and accumulates in the form of paired helical filaments (PHF) in the brains of patients with Alzheimer's disease (AD) and patients with several other tauopathies. Here, we show that the abnormally hyperphosphorylated from AD brain cytosol (AD P-) self-aggregates into PHF-like structures on incubation at pH 6.9 under reducing conditions at 35°C during 90 min. In vitro dephosphorylation, but not deglycosylation, of AD P-inhibits its self-association into PHF. Furthermore, hyperphosphorylation induces self-assembly of each of the six isoforms into tangles of PHF and straight filaments, and the microtubule binding domains͞repeats region in the absence of the rest of the molecule can also self-assemble into PHF. Thus, it appears that self-assembles by association of the microtubule binding domains͞repeats and that the abnormal hyperphosphorylation promotes the self-assembly of into tangles of PHF and straight filaments by neutralizing the inhibitory basic charges of the flanking regions.

Tau is the major microtubule associated protein (MAP) of a mature neuron. The other two neuronal MAPs are MAP1 and MAP2. An established function of MAPs is their interaction with tubulin and promotion of its assembly into microtubules and stabilization of the microtubule network. The microtubule assembly promoting activity of tau, a phosphoprotein, is regulated by its degree of phosphorylation. Normal adult human brain tau contains 2–3 moles phosphate/mole of tau protein. Hyperphosphorylation of tau depresses this biological activity of tau. In Alzheimer disease (AD) brain tau is ∼three to four-fold more hyperphosphorylated than the normal adult brain tau and in this hyperphosphorylated state it is polymerized into paired helical filaments ([PHF) admixed with straight filaments (SF) forming neurofibrillary tangles. Tau is transiently hyperphosphorylated during development and during anesthesia and hypothermia but not to the same state as in AD brain. The abnormally hyperphosphorylated tau in AD brain is distinguished from transiently hyperphosphorylated tau by its ability (1) to sequester normal tau, MAP1 and MAP2 and disrupt microtubules, and (2) to self-assemble into PHF/SF. The cytosolic abnormally hyperphosphorylated tau, because of oligomerization, unlike normal tau, is sedimentable and on self-assembly into PHF/SF, loses its ability to sequester normal MAPs. Some of the tau in AD brain is truncated which also promotes its self-assembly. Tau mutations found in frontotemporal dementia apparently promote its abnormal hyperphosphorylation. Thus, the AD abnormally hyperphosphorylated tau (1) is distinguishable from both normal and transiently hyperphosphorylated taus, and (2) is inhibitory when in a cytosolic/oligomeric state but not when it is self-assembled into PHF/SF. Inhibition of abnormal hyperphosphorylation of tau offers a promising therapeutic target for AD and related tauopathies.

Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains of individuals with Alzheimer's disease (AD) and other tauopathies. Tau pathology is critical to pathogenesis and correlates to the severity of dementia. However, the mechanisms leading to abnormal hyperphosphorylation are unknown. Here, we demonstrate that human brain tau was modified by O-GlcNAcylation, a type of protein O-glycosylation by which the monosaccharide ␤-N-acetylglucosamine (GlcNAc) attaches to serine͞threonine residues via an O-linked glycosidic bond. O-GlcNAcylation regulated phosphorylation of tau in a site-specific manner both in vitro and in vivo. At most of the phosphorylation sites, O-GlcNAcylation negatively regulated tau phosphorylation. In an animal model of starved mice, low glucose uptake͞metabolism that mimicked those observed in AD brain produced a decrease in O-GlcNAcylation and consequent hyperphosphorylation of tau at the majority of the phosphorylation sites. The O-GlcNAcylation level in AD brain extracts was decreased as compared to that in controls. These results reveal a mechanism of regulation of tau phosphorylation and suggest that abnormal hyperphosphorylation of tau could result from decreased tau O-GlcNAcylation, which probably is induced by deficient brain glucose uptake͞metabolism in AD and other tauopathies.

Abnormal hyperphosphorylation of tau is believed to lead to neurofibrillary degeneration in Alzheimer's disease (AD) and other tauopathies. Recent studies have shown that protein phosphatases (PPs) PP1, PP2A, PP2B and PP5 dephosphorylate tau in vitro, but the exact role of each of these phosphatases in the regulation of site-specific phosphorylation of tau in the human brain was unknown. Hence, we investigated the contributions of these PPs to the regulation of tau phosphorylation quantitatively. We found that these four phosphatases all dephosphorylated tau at Ser199, Ser202, Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404 and Ser409, but with different efficiencies toward different sites. The K(m) values of tau dephosphorylation catalysed by PP1, PP2A and PP5 were 8-12 microm, similar to the intraneuronal tau concentration of human brain, whereas the K(m) of PP2B was fivefold higher. PP2A, PP1, PP5 and PP2B accounted for approximately 71%, approximately 11%, approximately 10% and approximately 7%, respectively, of the total tau phosphatase activity of human brain. The total phosphatase activity and the activities of PP2A and PP5 toward tau were significantly decreased, whereas that of PP2B was increased in AD brain. PP2A activity negatively correlated to the level of tau phosphorylation at the most phosphorylation sites in human brains. Our findings indicate that PP2A is the major tau phosphatase that regulates its phosphorylation at multiple sites in human brain. The abnormal hyperphosphorylation of tau is partially due to a downregulation of PP2A activity in AD brain.

Microtubule-associated protein tau becomes abnormally hyperphosphorylated in Alzheimer's disease (AD) and accumulates as tangles of paired helical filaments in neurons undergoing degeneration. We now show that in solution normal tau associates with the AD hyperphosphorylated tau (AD P-tau) in a nonsaturable fashion, forming large tangles of filaments 3.3 +/- 0.7 nm in diameter. These tangles, which are not detected in identically treated normal tau or AD P-tau alone, are made up of filaments several microns in length and are labeled with tau antibodies. Dephosphorylation with alkaline phosphatase abolishes the ability of AD P-tau to aggregate with normal tau and prevents tangle formation. AD P-tau disassembles microtubules assembled from normal tau and tubulin. These data provide insight into how the hyperphosphorylation of tau might lead to the formation of the neurofibrillary tangles and the degeneration of the affected neurons in AD.

The microtubule assembly-promoting activity of different pools of tau protein isolated from Alzheimer disease (AD) and control brains and the effect of dephosphorylation on this activity were studied. Tau isolated from a 2.5% perchloric extract of AD brain had almost the same activity as that obtained from control brain, and this activity did not change fitly on dephosphorylation. Abnormally phosphorylated tau (AD P-tau) isolated from brain homogenate of AD patients had little activity, and upon dephosphorylation with alkaline phospbatase, its activity increased to approximately the same level as the acid-soluble tau. Addition of AD P-tau to a mixture of normal tau and tubulin inhibited microtubule assembly. AD P-tau bound to normal tau but not to tubulin. These studies suggest that the abnormal phosphorylation of tau might be responsible for the breakdown of microtubules in affected neurons in AD not only because the altered protein has little microtubule-promoting activity but also because it interacts with normal tau, making the latter unavailable for promoting the assembly of tubulin into microtubules.In the brains of patients with Alzheimer disease (AD), the cytoskeleton is progressively disrupted and displaced by the appearance of bundles of paired helical filaments (PHF), which are composed mainly ofhyperphosphorylated forms of tau protein (1, 2). Unlike normal tau, which contains two or three phosphate groups, the soluble hyperphosphorylated tau from AD brain (AD P-tau) contains 5-9 mol of phosphate per mol of the protein (3). Levels of tau are severalfold higher in AD than in the age-matched control brains, and this increase is in the form of the abnormally phosphorylated tau (4). Neurons with neurofibrillary tangles of PHF lack microtubules, and microtubule assembly from brain cytosol in the absence of an added polycation, DEAE-dextran, is not observed (5). The reason for this disruption might therefore be some alteration in either tau or other microtubuleassociated proteins. In AD brain, tau can be isolated from different pools: (i) a cytosolic fraction, (ii) abnormally phosphorylated tau that is not polymerized into PHF and sediments at 200,000 x g, and (iii) as a component of PHF. To understand the role of the abnormal phosphorylation of tau in microtubule disruption in AD brain, we studied the ability of the normal cytosolic tau and the AD P-tau to bind to tubulin and to promote microtubule assembly and investigated the effect of alkaline phosphatase treatment of tau on microtubule assembly. The studies described in this paper suggest that the abnormal phosphorylation of tau is a likely cause of the breakdown of the microtubule system in AD because the AD P-tau does not bind to tubulin and inhibits the in vitro assembly ofnormal tau and tubulin into microtubules. The altered tau inhibits microtubule assembly, probably through its binding to normal tau, making the latter unavailable for interaction with tubulin.