Propofol directly increases tau phosphorylation

In Alzheimer's disease (AD) and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A) activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of neurofibrillary pathology.     

In Alzheimer's disease, heme oxygenase is coincident with Alz50, an epitope of tau induced by 4-hydroxy-2-nonenal modification

In this study, we compared the neuronal induction of the antioxidant heme oxygenase-1 (HO-1) in Alzheimer's disease with abnormalities in tau marked by antibodies recognizing either phosphorylation (AT8) or conformational change (Alz50). The epitope recognized by Alz50 shows a complete overlap with HO-1-containing neurons, but AT8 recognized these neurons as well as neurons not displaying HO-1. These findings suggest that tau phosphorylation precedes the HO-1 response and that HO-1 is coincident with the Alz50 epitope. This led us to consider whether oxidative damage plays a role in forming the Alz50 epitope. We found that 4-hydroxy-2-nonenal (HNE), a highly reactive product of lipid peroxidation, reacts with normal tau and induces the Alz50 epitope in tau. It is important that the ability of HNE to create the Alz50 epitope not only is dependent on lysine residues of tau but also requires tau phosphorylation because neither methylated, recombinant, nor dephosphorylated tau reacts with HNE to create the Alz50 epitope. Supporting the in vivo relevance of this observation, endogenous paired helical filament-tau isolated from subjects with Alzheimer's disease was immunoreactive with an antibody to a stable HNE-lysine adduct, as were all vulnerable neurons in subjects with Alzheimer's disease but not in control individuals. Together, these findings support the involvement of oxidative damage early in neurofibrillary tangle formation in Alzheimer's disease and also suggest that HNE modification contributes to the generation of the tau conformation defining the Alz50 epitope. These findings provide evidence that an interplay between phosphorylation of tau and neuronal oxidative stress-induced pathology is important in the formation of neurofibrillary tangles.     

Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells.

Tau protein, a neuronal microtubule-associated protein, is phosphorylated in situ and hyperphosphorylated when aggregated into the paired helical filaments of Alzheimer's disease. To study the phosphorylation of tau protein in vivo, we have stably transfected htau40, the largest human tau isoform, into Chinese hamster ovary cells. The distribution and phosphorylation of tau was monitored by gel shift, autoradiography, immunofluorescence, and immunoblotting, using the antibodies Tau-1, AT8, AT180, and PHF-1, which are sensitive to the phosphorylation of Ser202, Thr205, Thr231, Ser235, Ser396, and Ser404 and are used in the diagnosis of Alzheimer tau. In interphase cells, tau becomes phosphorylated to some extent, partly at these sites; most of the tau is associated with microtubules. In mitosis, the above Ser/Thr-Pro sites become almost completely phosphorylated, causing a pronounced shift in M(r) and an antibody reactivity similar to that of Alzheimer tau. Moreover, a substantial fraction of tau is found in the cytoplasm detached from microtubules. Autoradiographs of metabolically labeled Chinese hamster ovary cells in interphase and mitosis confirmed that tau protein is more highly phosphorylated during mitosis. The understanding of tau phosphorylation under physiological conditions might help elucidate possible mechanisms for the hyperphosphorylation in Alzheimer's disease.     

Interleukin-6 induces Alzheimer-type phosphorylation of tau protein by deregulating the cdk5/p35 pathway

Inflammation is a process that has been actively related with the onset of several neurodegenerative disorders including Alzheimer disease (AD). However, the precise implications of inflammatory response for neurodegeneration have not been elucidated. A current hypothesis considers that extracellular insults to neurons could trigger the production of inflammatory cytokines by astrocytes and microglia. These cytokines, namely, interleukin (IL)-1beta, TNFalpha, and IL-6, could affect the normal behavior of neuronal cells. In the present study, we describe the effect of the administration at physiologic doses of one of these cytokines, IL-6, to hippocampal neurons, on the protein kinase pathways as well as on the tau phosphorylation patterns. IL-6-treated neurons exhibited an increase in the amount of anomalously hyperphosphorylated tau protein in epitopes dependent on proline-directed protein kinases (PDPKs). On the basis of our data, the observed increase of tau epitopes of Alzheimer type is explained by an increase of intraneuronal levels of p35 activator and in the activity of the protein kinase cdk5 in response to this cytokine. Further confirmation of cdk5 involvement in this process was based on the findings that inhibition of the kinase activity with butyrolactone-I prevents the appearance of tau of Alzheimer type in IL-6-treated neurons. Additional studies suggest that an increase of cdk5 activity could be mediated by a known signaling cascade described for IL-6 function, namely, the MAPK-p38 signaling pathway. Stimulation of the IL-6 pathway appears to increase the tau epitopes of Alzheimer type, as demonstrated in studies with specific inhibitors. These results support the findings of a pathologic role for IL-6 in the neuroinflammatory response as related with the pathogenesis of neuronal degeneration.     

Prostate-derived Sterile 20-like Kinases (PSKs/TAOKs) Phosphorylate Tau Protein and Are Activated in Tangle-bearing Neurons in Alzheimer Disease

In Alzheimer disease (AD), the microtubule-associated protein tau is highly phosphorylated and aggregates into characteristic neurofibrillary tangles. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) 1 and 2, members of the sterile 20 family of kinases, have been shown to regulate microtubule stability and organization. Here we show that tau is a good substrate for PSK1 and PSK2 phosphorylation with mass spectrometric analysis of phosphorylated tau revealing more than 40 tau residues as targets of these kinases. Notably, phosphorylated residues include motifs located within the microtubule-binding repeat domain on tau (Ser-262, Ser-324, and Ser-356), sites that are known to regulate tau-microtubule interactions. PSK catalytic activity is enhanced in the entorhinal cortex and hippocampus, areas of the brain that are most susceptible to Alzheimer pathology, in comparison with the cerebellum, which is relatively spared. Activated PSK is associated with neurofibrillary tangles, dystrophic neurites surrounding neuritic plaques, neuropil threads, and granulovacuolar degeneration bodies in AD brain. By contrast, activated PSKs and phosphorylated tau are rarely detectible in immunostained control human brain. Our results demonstrate that tau is a substrate for PSK and suggest that this family of kinases could contribute to the development of AD pathology and dementia.     

CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival

The microtubule-binding protein tau has been implicated in the neurofibrillary pathology of Alzheimer's disease. Within affected cells, ubiquitinated and hyperphosphorylated tau assembles into massive filamentous polymers. Eventually these tangle-bearing neurons die. The formation of neurofibrillary tangles closely parallels the progression and anatomic distribution of neuronal loss in Alzheimer's disease, suggesting that these lesions play a role in the disease pathogenesis. Mutations in the human tau gene cause autosomal dominant neurodegenerative disorders. These and other neurodegenerative conditions are also characterized by extensive neurofibrillary pathology. The mechanisms underlying tau-mediated neurotoxicity remain unclear; however, phosphorylated tau is a strong candidate for a toxic molecule, particularly those isoforms phosphorylated by the kinases glycogen synthase kinase 3beta and Cdk5. Here we show that Alzheimer tau binds to Hsc70, and its phosphorylation is a recognition requirement for the addition of ubiquitin (Ub) by the E3 Ub ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) and the E2 conjugating enzyme UbcH5B. Other E3 Ub ligases including parkin and Cbl failed to ubiquitinate phosphorylated tau. CHIP could rescue phosphorylated tau-induced cell death, and therefore the CHIP-Hsc70 complex may provide a new therapeutic target for the tauopathies.     

Role of glycosylation in hyperphosphorylation of tau in Alzheimer's disease

In Alzheimer's disease (AD) brain, microtubule-associated protein tau is abnormally modified by hyperphosphorylation and glycosylation, and is aggregated as neurofibrillary tangles of paired helical filaments. To investigate the role of tau glycosylation in neurofibrillary pathology, we isolated various pools of tau protein from AD brain which represent different stages of tau pathology. We found that the non-hyperphosphorylated tau from AD brain but not normal brain tau was glycosylated. Monosaccharide composition analyses and specific lectin blots suggested that the tau in AD brain was glycosylated mainly through N-linkage. In vitro phosphorylation indicated that the glycosylated tau was a better substrate for cAMP-dependent protein kinase than the deglycosylated tau. These results suggest that the glycosylation of tau is an early abnormality that can facilitate the subsequent abnormal hyperphosphorylation of tau in AD brain.     

Cholesterol-dependent modulation of tau phosphorylation in cultured neurons

One of the hallmarks of Alzheimer's disease (AD) is the abnormal state of tau. It is both highly phosphorylated and aggregated into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). However, the mechanism underlying the hyperphosphorylation of tau in NFTs and neuronal degeneration in AD remains to be elucidated. The fact that hyperphosphorylation of tau in NFTs are also found in the patients with Niemann-Pick disease, type C (NPC), which is a cholesterol storage disease associated with defective intracellular trafficking of exogenous cholesterol, implies that perturbation of cholesterol metabolism may be involved in tau phosphorylation and neurodegeneration. Here, we report that cholesterol deficiency induced by inhibition of cholesterol biosynthesis in cultured neurons results in hyperphosphorylation of tau, accompanied by axonal degeneration associated with microtubule depolymerization. These changes were prevented by concurrent treatment with beta-migrating very low-density lipoprotein (beta-VLDL) or cholesterol. We propose that intracellular cholesterol plays an essential role in the modulation of tau phosphorylation and the maintenance of microtubule stability.     

Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain. Implications for neurofibrillary degeneration in Alzheimer's disease

Hyperphosphorylated tau, which is the major protein of the neurofibrillary tangles in Alzheimer's disease brain, is most probably the result of an imbalance of tau kinase and phosphatase activities in the affected neurons. By using metabolically competent rat brain slices as a model, we found that selective inhibition of protein phosphatase 2A by okadaic acid induced an Alzheimer-like hyperphosphorylation and accumulation of tau. The hyperphosphorylated tau had a reduced ability to bind to microtubules and to promote microtubule assembly in vitro. Immunocytochemical staining revealed hyperphosphorylated tau accumulation in pyramidal neurons in cornu ammonis and in neocortical neurons. The topography of these changes recalls the distribution of neurofibrillary tangles in Alzheimer's disease brain. Selective inhibition of protein phosphatase 2B with cyclosporin A did not have any significant effect on tau phosphorylation, accumulation, or function. These studies suggest that protein phosphatase 2A participates in regulation of tau phosphorylation, processing, and function in vivo. A down-regulation of protein phosphatase 2A activity can lead to Alzheimer-like abnormal hyperphosphorylation of tau.     

Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease

The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation.     

Stabilization of the cyclin-dependent kinase 5 activator,p35, by paclitaxel decreases beta-amyloid toxicity in cortical neurons

One hallmark of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid-beta (Abeta) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Abeta toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Abeta peptides, inhibits Abeta-induced activation of cyclin-dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Abeta-induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Abeta-induced activation of the cytosolic cdk5-p25 complex, but not the membrane-associated cdk5-p35 complex. MT-stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood-brain barrier, we assessed the potential of taxanes to attenuate Abeta toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood-brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Abeta-induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT-stabilizing agents may provide a therapeutic approach to decrease Abeta toxicity and neurofibrillary pathology in AD and other tauopathies.     

Pre-aggregated A??1?C42 peptide increases tau aggregation and hyperphosphorylation after short-term application

Neuritic amyloid plaques and neurofibrillary tangles, consisting of hyperphosphorylated tau protein, are the hallmarks of Alzheimer disease. It is not clear so far, how both structures are functionally and physiologically connected. We have investigated the role of Aβ1-42 on hyperphosphorylation and aggregation of tau in SY5Y cells by transfection and overexpression with two tau constructs, a shortened wildtype tau (2N4R) and a point mutation tau (P301L), found in fronto-temporal dementia. It was found that the tau protein becomes hyperphosphorylated and forms large aggregates inside cells, visualized by immunofluorescence, after short incubation of 90 min with preaggregated Aβ1-42. In Addition, Aβ1-42 caused a decrease of tau solubility in both tau constructs in this relatively short time period. Taken together, these experiments suggest that pathological preaggregated Aβ1-42 in physiological concentrations quickly induces hyperphosphorylation and pathological structural changes of tau protein and thereby directly linking the 'amyloid hypothesis' to tau pathology, observed in Alzheimer disease.     

Starvation induces tau hyperphosphorylation in mouse brain: implications for Alzheimer's disease

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease brains, and tau hyperphosphorylation is thought to be a critical event in the pathogenesis of this disease. The objective of this study was to reproduce tau hyperphosphorylation in an animal model by inducing hypoglycemia. Food deprivation of mice for 1 to 3 days progressively enhanced tau hyperphosphorylation in the hippocampus, to a lesser extent in the cerebral cortex, but the effect was least in the cerebellum, in correspondence with the regional selectivity of tauopathy in Alzheimer's disease. This hyperphosphorylation was reversible by refeeding for 1 day. We discuss possible mechanisms of this phenomenon, and propose the starved mouse as a simple model to study in vivo tau phosphorylation and dephosphorylation which are altered in Alzheimer's disease.     

Alzheimer neurofibrillary degeneration: significance, etiopathogenesis, therapeutics and prevention

Alzheimer disease (AD) is multi-factorial and heterogeneous. Independent of the aetiology, this disease is characterized clinically by chronic and progressive dementia and histopathologically by neurofibrillary degeneration of abnormally hyperphosphorylated tau seen as intraneuronal neurofibrillary tangles, neuropil threads and dystrophic neurites, and by neuritic (senile) plaques of beta-amyloid. The neurofibrillary degeneration is apparently required for the clinical expression of AD, and in related tauopathies it leads to dementia in the absence of amyloid plaques. While normal tau promotes assembly and stabilizes microtubules, the abnormally hyperphosphorylated tau sequesters normal tau, MAP1 and MAP2, and disrupts microtubules. The abnormal hyperphosphorylation of tau also promotes its self-assembly into tangles of paired helical and or straight filaments. Tau is phosphorylated by a number of protein kinases. Glycogen synthase kinase-3 (GSK-3) and cyclin dependent protein kinase 5 (cdk5) are among the kinases most implicated in the abnormal hyperphosphorylation of tau. Among the phosphatases which regulate the phosphorylation of tau, protein phosphatase-2A (PP-2A), the activity of which is down-regulated in AD brain, is by far the major enzyme. The inhibition of abnormal hyperphosphorylation of tau is one of the most promising therapeutic targets for the development of disease modifying drugs. A great advantage of inhibiting neurofibrillary degeneration is that it can be monitored by evaluating the levels of total tau and tau phosphorylated at various known abnormally hyperphosphorylated sites in the cerebrospinal fluid of patients, obtained by lumbar puncture. There are at least five subgroups of AD, each is probably caused by a different etiopathogenic mechanism. The AD subgroup identification of patients can help increase the success of clinical trials and the development of specific and potent disease modifying drugs.     

Somatodendritic localization and hyperphosphorylation of tau protein in transgenic mice expressing the longest human brain tau isoform.

Microtubule-associated protein tau is the major constituent of the paired helical filament, the main fibrous component of the neurofibrillary lesions of Alzheimer's disease. Tau is an axonal phosphoprotein in normal adult brain. In Alzheimer's disease brain tau is hyperphosphorylated and is found not only in axons, but also in cell bodies and dendrites of affected nerve cells. We report the production and analysis of transgenic mice that express the longest human brain tau isoform under the control of the human Thy-1 promoter. As in Alzheimer's disease, transgenic human tau protein was present in nerve cell bodies, axons and dendrites; moreover, it was phosphorylated at sites that are hyperphosphorylated in paired helical filaments. We conclude that transgenic human tau protein showed pre-tangle changes similar to those that precede the full neurofibrillary pathology in Alzheimer's disease.     

Proteasome–caspase–cathepsin sequence leading to tau pathology induced by prostaglandin J2 in neuronal cells

Neurofibrillary tangles (NFT) are a hallmark of Alzheimer's disease. The major neurofibrillary tangle component is tau that is truncated at Asp421 (Δtau), hyperphosphorylated and aggregates into insoluble paired helical filaments. Alzheimer's disease brains also exhibit signs of inflammation manifested by activated astrocytes and microglia, which produce cytotoxic agents among them prostaglandins. We show that prostaglandin (PG) J2, an endogenous product of inflammation, induces caspase-mediated cleavage of tau, generating Δtau, an aggregation prone form known to seed tau aggregation prior to neurofibrillary tangle formation. The initial event observed upon PGJ2-treatment of human neuroblastoma SK-N-SH cells was the build-up of ubiquitinated (Ub) proteins indicating an early disruption of the ubiquitin-proteasome pathway. Apoptosis kicked in later, manifested by caspase activation and caspase-mediated cleavage of tau at Asp421 and poly (ADP-ribose) polymerase. Furthermore, cathepsin inhibition stabilized Δtau suggesting its lysosomal clearance. Upon PGJ2-treatment tau accumulated in a large perinuclear aggregate. In rat E18 cortical neuronal cultures PGJ2-treatment also generated Δtau detected in dystrophic neurites. Levels of Δtau were diminished by caspase 3 knockdown using siRNA. PGD2, the precursor of PGJ2, produced some Δtau. PGE2 generated none. Our data suggest a potential sequence of events triggered by the neurotoxic product of inflammation PGJ2 leading to tau pathology. The accumulation of Ub proteins is an early response. If cells fail to overcome the toxic effects induced by PGJ2, including accumulation of Ub proteins, apoptosis kicks in triggering caspase activation and tau cleavage, the clearance of which by cathepsins could be compromised culminating in tau pathology. Our studies are the first to provide a mechanistic link between inflammation and tau pathology.     

Nitric oxide induces tau hyperphosphorylation via glycogen synthase kinase-3beta activation

Nitric oxide is associated with neurofibrillary tangle, which is composed mainly of hyperphosphorylated tau in the brain of Alzheimer's disease (AD). However, the role of nitric oxide in tau hyperphosphorylation is unclear. Here we show that nitric oxide produced by sodium nitroprusside (SNP), a recognized donor of nitric oxide, induces tau hyperphosphorylation at Ser396/404 and Ser262 in HEK293/tau441 cells with a simultaneous activation of glycogen synthase kinase-3beta (GSK-3beta). Pretreatment of the cells with 10 mM lithium chloride (LiCl), an inhibitor of GSK-3, 1 h before SNP administration inhibits GSK-3beta activation and prevents tau from hyperphosphorylation. This is the first direct evidence demonstrating that nitric oxide induces AD-like tau hyperphosphorylation in vitro, and GSK-3beta activation is partially responsible for the nitric oxide-induced tau hyperphosphorylation. It is suggested that nitric oxide may be an upstream element of tau abnormal hyperphosphorylation in AD.     

Epitope mapping of mAbs AT8 and Tau5 directed against hyperphosphorylated regions of the human tau protein

Post-mortem diagnosis of Alzheimer's disease relies on high numbers of senile plaques and neurofibrillary tangles (NFTs) stained in distinct brain areas. NFTs mostly consist of hyperphosphorylated versions of the microtubule attached tau protein (PHF-tau) with more than 30 serine and threonine phosphorylation sites identified so far. Characterization of hyperphosphorylated tau regions and the hope to develop robust assays for early AD diagnosis relies mostly on phosphorylation-dependent monoclonal antibodies (mAbs) recognizing only disease-specific phosphorylation patterns. Here, we report that anti-PHF-tau mAb AT8 recognizes an epitope doubly phosphorylated at serine 202 and threonine 205, which was not influenced by a third phosphate group at serine 199. But mAb AT8 was cross-reactive to two doubly phosphorylated motifs containing either serines 199 and 202 or serines 205 and 208 of the human tau sequence. The epitope of anti-tau mAb Tau5 was mapped to the human tau sequence 218-225, which is not phosphorylated in vivo.     

New Phosphorylation Sites Identified in Hyperphosphorylated Tau (Paired Helical Filament-Tau) from Alzheimer's Disease Brain Using Nanoelectrospray Mass Spectrometry

Abstract: Paired helical filaments (PHFs) are the structural constituents of neurofibrillary tangles in Alzheimer's disease and are composed of hyperphosphorylated forms of the microtubule-associated protein tau (PHF-tau). Pathological hyperphosphorylation of tau is believed to be an important contributor to the destabilisation of microtubules and their subsequent disappearance from tangle-bearing neurons in Alzheimer's disease, making elucidation of the mechanisms that regulate tau phosphorylation an important research goal. Thus, it is essential to identify, preferably by direct sequencing, all of the sites in PHF-tau that are phosphorylated, a task that is incomplete because of the difficulty to date of purifying insoluble PHF-tau to homogeneity and in sufficient quantities for structural analysis. Here we describe the solubilisation of PHF-tau followed by its purification by Mono Q chromatography and reversed-phase HPLC. Phosphopeptides from proteolytically digested PHF-tau were sequenced by nanoelectrospray mass spectrometry. We identified 22 phosphorylation sites in PHF-tau, including five sites not previously identified. The combination of our new data with previous reports shows that PHF-tau can be phosphorylated on at least 25 different sites.     

p38 kinase is activated in the Alzheimer's disease brain

The p38 mitogen-activated protein kinase is a stress-activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. In the Alzheimer's disease (AD) brain, increased levels of phosphorylated (active) p38 were detected relative to age-matched normal brain. Intense phospho-p38 immunoreactivity was associated with neuritic plaques, neuropil threads, and neurofibrillary tangle-bearing neurons. The antibody against phosphorylated p38 recognized many of the same structures as an antibody against aberrantly phosphorylated, paired helical filament (PHF) tau, although PHF-positive tau did not cross-react with the phospho-p38 antibody. These findings suggest a neuroinflammatory mechanism in the AD brain, in which aberrant protein phosphorylation affects signal transduction elements, including the p38 kinase cascade, as well as cytoskeletal components.