Phosphorylation regulates fibrillation of an aggregation core peptide in the second repeat of microtubule-binding domain of human tau

Hyperphosphorylation of the microtubule-associated protein tau is believed to play a crucial role in the neurofibrillary tangles formation in Alzheimer’s disease brain. In this study, fibril formation of peptides containing the critical sequences for tau aggregation VQIINK and a plausible serine phosphorylation site of tau at its C-terminal was investigated. All the peptides formed fibrils with the typical cross-β structural core. However, stability of the fibrils was highly sensitive to the pH conditions for the phosphorylated VQIINK peptide, suggesting a regulatory role of phosphorylation for the amyloid-formation of tau.     

The role of tau phosphorylation in transfected COS-1 cells

Tau cDNAs from each of the six human isoforms were transfected into COS- 1 cells and, in every case, more than one peptide was observed. The diversity of expressed isoforms was due to different levels of tau phosphorylation. Tau phosphorylation results in a decrease of the protein electrophoretic mobility. The major contribution to this mobility shift is due to the phosphorylation at the at the C-terminus of the molecule, as inferred from the expression of tau fragments. Phosphorylation takes place in some of the sites modified in neural cells and in the basis of AD patients. Copolymerization studies indicate that the level of phosphorylation, as well as the localization of the modified residues, may affect the binding of the protein to microtubules. These results indicate that phosphorylation regulates tau function inside the cell.     

Alzheimer-specific epitopes of tau represent lipid peroxidation-induced conformations

Several recent studies support a link between tau protein phosphorylation and adduction of tau by reactive carbonyls. Indeed, the phosphorylation-dependent adduction of tau by carbonyl products resulting from lipid peroxidation creates the neurofibrillary tangle-related antigen, Alz50. To determine whether epitopes of carbonyl-modified tau are major conformational changes associated with neurofibrillary tangle formation, we examined seven distinct antibodies raised against neurofibrillary tangles that recognize unique epitopes of tau in Alzheimer disease. Consistently, all seven antibodies recognize tau more strongly (4- to 34-fold) after treatment of normal tau with the reactive carbonyl, 4-hydroxy-2-nonenal (HNE), but only when tau is in the phosphorylated state. These findings not only support the idea that oxidative stress is involved in neurofibrillary tangle formation occurring in brains of Alzheimer disease patients, but also show, for the first time, that HNE modifications of tau promote and contribute to the generation of the major conformational properties defining neurofibrillary tangles.     

Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis

Tau in Alzheimer disease brain is highly phosphorylated and aggregated into paired helical filaments comprising characteristic neurofibrillary tangles. Here we have analyzed insoluble Tau (PHF-tau) extracted from Alzheimer brain by mass spectrometry and identified 11 novel phosphorylation sites, 10 of which were assigned unambiguously to specific amino acid residues. This brings the number of directly identified sites in PHF-tau to 39, with an additional six sites indicated by reactivity with phosphospecific antibodies to Tau. We also identified five new phosphorylation sites in soluble Tau from control adult human brain, bringing the total number of reported sites to nine. To assess which kinases might be responsible for Tau phosphorylation, we used mass spectrometry to determine which sites were phosphorylated in vitro by several kinases. Casein kinase 1delta and glycogen synthase kinase-3beta were each found to phosphorylate numerous sites, and each kinase phosphorylated at least 15 sites that are also phosphorylated in PHF-tau from Alzheimer brain. A combination of casein kinase 1delta and glycogen synthase kinase-3beta activities could account for over three-quarters of the serine/threonine phosphorylation sites identified in PHF-tau, indicating that casein kinase 1delta may have a role, together with glycogen synthase kinase-3beta, in the pathogenesis of Alzheimer disease.     

Abnormal tau species are produced during Alzheimer's disease neurodegenerating process

Tau proteins were detected in human brain using two polyclonal antibodies: anti-paired helical filaments and anti-human native tau proteins. Both antisera detected identically the normal set of tau proteins in control brains. Moreover they detected two abnormal tau variants of 64 and 69 kDa exclusively in brain areas showing neurofibrillary tangles and senile plaques. Tau 64 and 69 were abnormally phosphorylated as revealed by the decrease in their molecular mass observed after alkaline phosphatase treatment. Therefore, tau 64 and 69 are specific markers of the neurofibrillary degeneration of the Alzheimer type and might be useful tools for studying the first pathological events that lead to neuronal death.     

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.     

Pinning down phosphorylated tau and tauopathies

Neurofibrillary tangles (NFTs) are prominent neuronal lesions in a large subset of neurodegenerative diseases, including Alzheimer's disease (AD). NFTs are mainly composed of insoluble Tau that is hyperphosphorylated on many serine or threonine residues preceding proline (pSer/Thr-Pro). Tau hyperphosphorylation abolishes its biological function to bind microtubules and promotes microtubule assembly and precedes neurodegeneration. Not much is known about how tau is further regulated following phosphorylation. Notably, we have recently shown that phosphorylated Ser/Thr-Pro motifs exist in two distinct conformations. The conversion between two conformations in some proteins is catalyzed by the prolyl isomerase Pin1. Pin1 binds to tau phosphorylated specifically on the Thr231-Pro site and probably catalyzes cis/trans isomerization of pSer/Thr-Pro motif(s), thereby inducing conformational changes in tau. Such conformational changes can directly restore the ability of phosphorylated Tau to bind microtubules and promote microtubule assembly and/or facilitate tau dephosphorylation by its phosphatase PP2A, as PP2A activity is conformation-specific. Furthermore, Pin1 expression inversely correlates with the predicted neuronal vulnerability in normally aged brain and also with actual neurofibrillary degeneration in AD brain. Moreover, deletion of the gene encoding Pin1 in mice causes progressive age-dependent neuropathy characterized by motor and behavioral deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Distinct from all other mouse models where transgenic overexpression of specific proteins elicits tau-related pathologies, Pin1 is the first protein whose depletion causes age-dependent neurodegeneration and tau pathologies. Thus, Pin1 is pivotal in maintaining normal neuronal function and preventing age-dependent neurodegeneration. This could represent a promising interventive target to prevent neurodegenerative diseases.     

Turning down tau phosphorylation

Phosphorylation and glycosylation of the tau protein, which is implicated in neurodegenerative diseases, are intimately linked. In vivo pharmacological inhibition of tau deglycosylation may be a new way to suppress abnormal tau phosphorylation, known to be involved in the formation of neurofibrillary tangles in the brain.     

Developmental regulation of tau phosphorylation, tau kinases, and tau phosphatases

Tau is a neuronal microtubule-associated protein. Its hyperphosphorylation plays a critical role in Alzheimer disease (AD). Expression and phosphorylation of tau are regulated developmentally, but its dynamic regulation and the responsible kinases or phosphatases remain elusive. Here, we studied the developmental regulation of tau in rats during development from embryonic day 15 through the age of 24 months. We found that tau expression increased sharply during the embryonic stage and then became relatively stable, whereas tau phosphorylation was much higher in developing brain than in mature brain. However, the extent of tau phosphorylation at seven of the 14 sites studied was much less in developing brain than in AD brain. Tau phosphorylation during development matched the period of active neurite outgrowth in general. Tau phosphorylation at various sites had different topographic distributions. Several tau kinases appeared to regulate tau phosphorylation collectively at overlapping sites, and the decrease of overall tau phosphorylation in adult brain might be due to the higher levels of tau phosphatases in mature brain. These studies provide new insight into the developmental regulation of site-specific tau phosphorylation and identify the likely sites required for the abnormal hyperphosphorylation of tau in AD.     

Phosphorylation of FTDP-17 mutant tau by cyclin-dependent kinase 5 complexed with p35, p25, or p39

One of the major pathological hallmarks of Alzheimer disease is neurofibrillary tangles. Neurofibrillary tangles are bundles of paired helical filaments composed of hyperphosphorylated tau. Cyclin-dependent kinase 5 (Cdk5) is one of the tau protein kinases that increase paired helical filament epitopes in tau by phosphorylation. Recently, various mutations of tau have been identified in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we investigated the phosphorylation of FTDP-17 mutant tau proteins, K257T, P301L, P301S, and R406W, by Cdk5 complexed with p35, p25, or p39 in vitro and in cultured cells. The extent of phosphorylation by all Cdk5 species was slightly lower in mutant tau than in wild-type tau. Major phosphorylation sites, including Ser202, Ser235, and Ser404, were the same among the wild-type, K257T, P301L, and P301S tau proteins phosphorylated by any Cdk5. On the other hand, R406W tau was less phosphorylated at Ser404 than were the other variants. This was not due to the simple replacement of amino acid Arg406 with Trp close to the phosphorylation site, because Ser404 in a R406W peptide was equally phosphorylated in a wild-type peptide. The decreased phosphorylation of mutant tau by Cdk5s was canceled when tau protein bound to microtubules was phosphorylated. These results indicate that FTDP-17 mutations do not affect the phosphorylatability of tau by Cdk5 complexed with p35, p25, or p39 and may explain part of the discrepancy reported previously between in vivo and in vitro phosphorylation of FTDP-17 tau mutants.     

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.     

Reversible beta-pleated sheet formation of a phosphorylated synthetic tau peptide.

Serine416 of human tau protein is believed to be phosphorylated in Alzheimer neurofibrillary tangles. We synthesized a fragment of tau, consisting of amino acids 408-421 in both non-phosphorylated and serine416-phosphorylated forms. Circular dichroism in a trifluoroethanol-water mixture indicated a beta-turn----beta-pleated sheet conformational transition upon phosphorylation. The beta-structure formation is intermolecular and can be inhibited by addition of Ca2+ ions or a phosphorylated tripeptide, but not with its non-phosphorylated analog. The presence of the phosphorylated tau peptide did not facilitate the formation of beta-pleated sheets of a phosphorylated neurofilament fragment. Multivalent cations induced a conformational transition of this phosphorylated neurofilament peptide, but the effect was less specific than the transition induced in the tau fragment, and it could also be reversed with the competing phosphorylated tripeptide.     

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.     

Phosphorylation of tau at Ser214 mediates its interaction with 14-3-3 protein: implications for the mechanism of tau aggregation

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine –phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro . The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.     

Epitopes that span the tau molecule are shared with paired helical filaments

Tau protein has been shown to be an integral component of Alzheimer paired helical filaments (PHF). However, the extent to which tau is incorporated into PHF has not been clear because the antibodies used to label PHF generally do not have precisely defined epitopes. Here we define the antigenic sites for five monoclonal antibodies that react with tau and cross-react with SDS-extracted neurofibrillary tangles. The reactive sites were determined by screening a lambda gt11 sublibrary expressing small fragments of the tau sequence. The mapped epitopes were found to span almost the entire length of tau, suggesting that PHF contains tau in its entirety or nearly in its entirety. One antibody was found to cross-react with microtubule-associated protein 2, implying some degree of homology between the two proteins.     

Hyperphosphorylation of tau induces local polyproline II helix

Alzheimer's disease is characterized by two protein precipitates, extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs). The primary constituent of NFTs is a hyperphosphorylated form of the microtubule-binding protein tau. Hyperphosphorylation of tau on over 30 residues, primarily within proline-rich sequences, is associated with conformational changes whose nature is poorly defined. Peptides derived from the proline-rich region of tau (residues 174-242) were synthesized, and the conformations were analyzed for the nonphosphorylated and phosphorylated peptides. CD and NMR data indicate that phosphorylation of serine and threonine residues in proline-rich sequences induces a conformational change to a type II polyproline helix. The largest phosphorylation-dependent conformational changes observed by CD were for tau peptides incorporating residues 174-183 or residues 229-238. Phosphoserine and phosphothreonine residues exhibited ordered values of (3)J(alphaN) (3.1-6.2 Hz; mean = 4.7 Hz) compared to nonphosphorylated serine and threonine. Phosphorylation of a tau peptide consisting of tau residues 196-209 resulted in the disruption of a nascent alpha-helix. These results suggest that global reorganization of tau may occur upon hyperphosphorylation of proline-rich sequences in tau.     

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.     

Proline-directed phosphorylation of human Tau protein.

The primary sequence of the microtubule-associated protein tau contains multiple repeats of the sequence -X-Ser/Thr-Pro-X-, the consensus sequence for the proline-directed protein kinase (p34cdc2/p58cyclin A). When phosphorylated by proline-directed protein kinase in vitro, tau was found to incorporate up to 4.4 mol of phosphate/mol of protein. Isoelectric focusing of the tryptic phosphopeptides demonstrated the presence of five distinct peptides with pI values of approximately 6.9, 6.5, 5.6-5.9, 4.7, and 3.6. Mapping of the tryptic phosphopeptides by high performance liquid chromatography techniques demonstrated three distinct peaks. Data from gas phase sequencing, amino acid analysis, and phosphoamino acid analysis suggest that proline-directed protein kinase phosphorylates tau at four sites. Each site demonstrates the presence of a proline residue on the carboxyl-terminal side of the phosphorylated residue. Two phosphorylation sites are located adjacent to the three-repeat microtubule-binding domain that has been found to be required for the in vivo co-localization of tau protein to microtubules. Two other putative phosphorylation sites are located within the identified epitope of the monoclonal antibody Tau-1. Phosphorylation of these sites altered the immunoreactivity of tau to Tau-1 antibody. Since the neuronal microtubule-associated protein tau is multiply phosphorylated in Alzheimer's disease, and Tau-1 immunoreactivity is similarly reduced in neurofibrillary tangles and enhanced after dephosphorylation, phosphorylation at one or more of these sites may correlate with abnormally phosphorylated sites in tau protein in Alzheimer's disease.     

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.