The State of Phosphorylation of Normal Adult Brain τ, Fetal τ, and τ from Alzheimer Paired Helical Filaments at Amino Acid Residue Ser262

Abstract: Antibody Ab262 was raised against a synthetic τ peptide (SKIGSTENLK, amino acids 258–267 of τ, termed Ser²⁶² peptide). The antibody was more reactive with Ser²⁶² peptide and unphosphorylated τ than a related phosphopeptide [SKIGS(P)TENLK, termed P-Ser²⁶² peptide] and τ phosphorylated by a partially purified kinase, glycogen synthase kinase (GSK) 3β. Ab262 reacted poorly with a peptide having the sequence DRVQSKIGSLD (amino acids 348–358). Treatment of P-Ser²⁶² peptide or GSK 3β phosphorylated τ with alkaline phosphatase increased Ab262 immunoreactivity, indicating that Ab262 is a reagent useful for studying τ phosphorylation at the Ser²⁶² residue. The Ab262 immunoreactivity was detected in τ from normal brains and Alzheimer paired helical filament (PHF-τ) and in PHFs. Alkaline phosphatase treatment had no effect on the Ab262 immunoreactivity of normal τ and PHF-τ but altered the Tau-1 and PHF-1 immunoreactivities. τ proteins from rat brains at 3 and 8 h postmortem exhibited 5 and 19%, respectively, more Ab262 immunoreactivity than τ from fresh tissues. In comparison, rat τ at 8 h postmortem was 40% more immunoreactive with Tau-1. The results suggest that Ser²⁶² is not a major phosphorylation site in vivo. Moreover, there is little or no difference between PHF-τ and normal τ in the extent of phosphorylation at Ser²⁶².     

Neurofilament Monoclonal Antibodies RT97 and 8D8 Recognize Different Modified Epitopes in Paired Helical Filament-τ in Alzheimer's Disease

Abstract: Neurofibrillary tangles in Alzheimer's disease have been previously found to be labeled by some neurofilament antibodies that also recognize τ proteins. We have studied the reactivity of two such monoclonal antibodies, RT97 and 8D8, and of an anti-ubiquitin serum with the abnormal paired helical filaments (PHF)-τ (A68) polypeptides known to be the main component of the PHFs constituting the neurofibrillary tangles. 8D8 recognized the three major PHF-τ polypeptides, but RT97 reacted only with the two larger PHF-τ species. PHF-τ polypeptides were labeled by 8D8 and RT97 much more strongly than normal human τ and this labeling was decreased after alkaline phosphatase treatment. Anti-ubiquitin and anti-phosphotyrosine antibodies did not label PHF-τ polypeptides. The immunoreactivity of proteolytic fragments of PHF-τ polypeptides was studied with RT97, 8D8, and a panel of τ antibodies. The epitope for 8D8 on PHF-τ was localized between amino acids 222 and 427 in the carboxyl half of τ. The RT97 epitope on PHF-τ was localized in the amino domain of τ, probably in the 29-amino-acid insertion (insert 1) found towards the amino terminus of some τ isoforms. These results show that the basis for the labeling of neurofibrillary tangles by antibodies 8D8 and RT97 to neurofilament is their ability to react with PHF-τ polypeptides by recognizing sites specifically modified on PHF-τ, including a site specific to some τ isoforms.     

Characterization of Two Distinct Monoclonal Antibodies to Paired Helical Filaments: Further Evidence for Fetal-Type Phosphorylation of the τ in Paired Helical Filaments

Abstract: Two monoclonal antibodies C5 and M4 raised against Sarkosyl-insoluble paired helical filaments (PHF) specifically labeled fetal τ, but hardly labeled normal adult τ. C5 immunoreactivity was eliminated by alkaline phosphatase treatment at 37°C, whereas M4 reactivity could be removed only by the treatment at 67°C. Epitope analysis showed that C5 and M4 recognition sites are in residues 386–406 and 198–250, respectively, according to the numbering of the longest human τ isoform. Thus, the phosphorylation sites are located in the amino- and carboxyl-terminal portions of the microtubule-binding region. These two well-characterized monoclonals should be valuable in the identification of a protein kinase(s) that converts normal τ into PHF-τ.     

Involvement of τ Protein Kinase I in Paired Helical Filament-Like Phosphorylation of the Juvenile τ in Rat Brain

Abstract: τ protein kinase I (TPKI) phosphorylates τ and forms paired helical filament epitopes in vitro. We studied temporal expression and histochemical distribution of τ phosphoserine epitopes at sites known to be phosphorylated by TPKI. Antibodies directed against phosphorylated Ser¹⁹⁹ (anti-PS 199) or phosphorylated Ser³⁹⁶ (C5 or anti-PS 396) were used. TPKI is abundantly expressed in the young rat brain and the highly phosphorylated juvenile form of τ occurs in the same period. The activity peak of TPKI coincided with the high level of phosphorylation of Ser¹⁹⁹ and Ser³⁹⁶ in juvenile τ at around postnatal day 8. By immunohistochemistry on the hippocampus and neocortex of 3–11-day-old rats, phosphorylated Ser³⁹⁶ was found in young axonal tracts and neuropil, where TPKI immunoreactivity was also detected. TPKI and phospho-Ser¹⁹⁹ immunoreactivities were also detected in the perikarya of pyramidal neurons. TPKI immunoreactivity had declined to a low level and phosphorylated serine immunoreactivities were undetectable in the sections of adult brain. These findings implicate TPKI in paired helical filament-like phosphorylation of juvenile form of τ in the developing brain.     

Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

Abstract: Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of β-amyloid is mediated via free radicals, and as the deposition of β-amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein τ that resemble those found in PHF-τ. Contrary to causing an increase in τ phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of τ and so we conclude that oxidative stress is not the direct cause of τ hyperphosphorylation and hence of PHF formation.     

Alzheimer's Disease Neurofibrillary Tangles Contain Mitosis-Specific Phosphoepitopes

Abstract: Paired helical filaments (PHFs) are the major components of neurofibrillary lesions present in Alzheimer's disease (AD). PHFs are composed of the microtubule-associated protein (MAP) τ, which is abnormally phosphorylated in AD. Normal fetal τ is also phosphorylated and shares certain phosphoepitopes with PHF-τ. The abnormal phosphorylation of PHF-τ is considered to be involved in the formation of PHFs and subsequent degeneration of AD neurons. We have previously shown that other neuronal MAPs, such as MAP1B, contain mitosis-specific phosphoepitopes. In addition to mitotic cells, these epitopes are also expressed in fetal brain and PC12 cells during differentiation and neurite outgrowth. One hypothesis regarding the etiology of AD involves the reactivation of a fetal-like state and mitotic conditions in selected neurons. To determine if similar mitosis-associated phosphoepitopes appeared in AD, sections of hippocampal tissue were stained for immunoreactivity with antibodies recognizing both τ and mitotic phosphoepitopes. Both the MPM2 mitotic phosphoepitope antibody and the AT8 PHF-τ antibody stained neurofibrillary lesions and colocalized to pyramidal neurons in AD samples. In addition, PHFs isolated from an AD brain reacted with both antibodies. The MPM2 antibody specifically reacted with τ in the isolated PHF fraction but not normal adult τ. In addition, MPM2 failed to react with normal fetal or adult τ obtained from rat brains. The MPM2 antibody also recognized human MAP1B; however, MAP1B was not present in the PHF fraction. Our results indicate that MPM2 recognized a phosphoepitope present on PHF-τ. Because normal fetal or adult rat brain τ did not express the MPM2 epitope, it is likely that this phosphoepitope is specific for the disease state.     

τ Protein from Alzheimer's Disease Patients Is Glycated at Its Tubulin-Binding Domain

Abstract: Glycated residues of τ protein from paired helical filaments isolated from the brains of Alzheimer's disease patients were localized by doing a proteolytic cleavage of the protein, fractionation of the resulting peptides, and identification of those peptides using specific antibodies. The most suitable residues for glycation, lysines, present at the tubulin-binding motif of τ protein, seem to be preferentially modified compared with those lysines present at other regions. Among these modified lysines, those located in the sequence comprising residues 318–336 (in the largest human τ isoform) were found to be glycated, as determined by the reaction with an antibody that recognizes a glycated peptide containing this sequence. Because those lysines are present in a tubulin binding motif of τ protein, its modification could result in a decrease in the interaction of τ with tubulin.     

Protein Phosphatase 2A Is the Major Enzyme in Brain that Dephosphorylates τ Protein Phosphorylated by Proline-Directed Protein Kinases or Cyclic AMP-Dependent Protein Kinase

Abstract: The paired helical filament (PHF), which makes up the major fibrous component of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated and abnormally phosphorylated microtubule-associated protein τ. Previous studies have identified serine and threonine residues phosphorylated in PHF-τ and have shown that τ can be phosphorylated at several of these sites by proline-directed protein kinases and cyclic AMP-dependent protein kinase. Here we have investigated which protein phosphatase activities can dephosphorylate recombinant τ phosphorylated with mitogen-activated protein kinase, glycogen synthase kinase-3β, neuronal cdc2-like kinase, or cyclic AMP-dependent protein kinase. We show that protein phosphatase 2A is by far the major protein phosphatase activity in brain that dephosphorylates τ phosphorylated in this manner.     

τ Phosphorylation in Human, Primate, and Rat Brain: Evidence that a Pool of τ Is Highly Phosphorylated In Vivo and Is Rapidly Dephosphorylated In Vitro

Abstract: The extent of τ phosphorylation is thought to regulate the binding of τ to microtubules: Highly phosphorylated τ does not bind to tubules, whereas dephosphorylated τ can bind to microtubules. It is interesting that the extent of τ phosphorylation in vivo has not been accurately determined. τ was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that τ isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated τ was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of τ were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of τ in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive τ was present as slow-mobility (66- and 68-kDa) forms of τ. The percentage of immunoreactive τ in faster-mobility pools (42–54 kDa) increased in proportion to the decrease in content of 66–68-kDa τ as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on τ is rapid and depends on neuronal phosphatases. Furthermore, τ is highly phosphorylated in normal-appearing human, primate, and rodent brain. The presence of a highly phosphorylated pool of τ in adult brain may modify the present hypotheses on how paired helical filaments of Alzheimer's disease are formed.     

τ Protein Kinase II Is Involved in the Regulation of the Normal Phosphorylation State of τ Protein

Abstract: To study the phosphorylation state of τ in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for τ protein kinase II. Immunoblot experiments using these antisera demonstrated that τ in microtubule-associated proteins is phosphorylated at Ser¹⁴⁴ and at Ser³¹⁵. Almost all τ variants separated on two-dimensional gel electrophoresis were phosphorylated at Ser¹⁴⁴ and nearly one-half of them at Ser³¹⁵. Phosphorylation at Ser¹⁴⁴ and at Thr¹⁴⁷ of τ isolated from heat-stable brain extracts was shown to be developmentally regulated, with the highest level of phosphorylation found at postnatal week 1. In vitro phosphorylation of τ by τ protein kinase I, a kinase responsible for abnormal phosphorylation of τ found in paired helical filaments of patients with Alzheimer's disease, was enhanced by prior phosphorylation of τ by τ protein kinase II. Thus, we suggest that τ protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of τ in neuronal cells.     

Stress-Activated Protein Kinase/c-Jun N-Terminal Kinase Phosphorylates τ Protein

Abstract: A proportion of the neuronal microtubule-associated protein (MAP) τ is highly phosphorylated in foetal and adult brain, whereas the majority of τ in the neurofibrillary tangles of Alzheimer's patients is hyperphosphorylated; many of the phosphorylation sites are serines or threonines followed by prolines. Several kinases phosphorylate τ at such sites in vitro. We have now shown that purified recombinant stress-activated protein kinase/c-Jun N-terminal kinase, a proline-directed kinase of the MAP kinase extended family, phosphorylates recombinant τ in vitro on threonine and serine residues. Western blots using antibodies to phosphorylation-dependent τ epitopes demonstrated that phosphorylation occurs in both of the main phosphorylated regions of τ protein. Unlike glycogen synthase kinase-3, the c-Jun N-terminal kinase readily phosphorylates Thr²⁰⁵ and Ser⁴²², which are more highly phosphorylated in Alzheimer τ than in foetal or adult τ. Glycogen synthase kinase-3 may preferentially phosphorylate the sites found physiologically, in foetal and to a smaller extent in adult τ, whereas stress-activated/c-Jun N-terminal kinase and/or other members of the extended MAP kinase family may be responsible for pathological proline-directed phosphorylations. Inflammatory processes in Alzheimer brain might therefore contribute directly to the pathological formation of the hyperphosphorylated τ found in neurofibrillary tangles.     

Polymerization of τ into Filaments in the Presence of Heparin: The Minimal Sequence Required for τ - τ Interaction

Abstract: Paired helical filaments isolated from the brains of patients with Alzheimer's disease are composed of a major protein component, the microtubule-associated protein termed τ, together with other nonprotein components, including heparan, a glycosaminoglycan, the more extensively sulfated form of which is heparin. As some of these nonprotein components may modulate the assembly of τ into filamentous structures, we have analyzed the ability of the whole τ protein or some of its fragments to self-assemble in the presence of heparin. Different τ fragments, all of them containing some sequences of the tubulin-binding motif, can assemble in vitro into filaments. We have also found formation of polymers with the 18-residue-long peptide corresponding to the third tubulin-binding motif of τ. This suggests that the ability of τ for self-assembly could be localized in a short sequence of amino acids present in the tubulin-binding repeats of the τ molecule.     

τ Self-Association: Stabilization with a Chemical Cross-Linker and Modulation by Phosphorylation and Oxidation State

Abstract: τ is a major component of paired helical filaments found in the neurofibrillary tangles of Alzheimer's diseased brain. However, the mechanism or mechanisms responsible for the association of τ to form these aggregates remains unknown. In this study, the role of intermolecular disulfide bonds in the formation of higher order oligomers of bovine τ and the human recombinant τ isoform T3 was examined using the chemical cross-linking agent disuccinimidylsuberate (DSS). In addition, the role of phosphorylation and oxidation state on the in vitro self-association of τ was studied using this experimental model. Stabilization of τ-τ interactions with DSS indicated that intermolecular disulfide bonds probably play a predominant role in dimer formation, but the formation of higher order oligomers of τ cannot be attributed to these bonds alone. τ-τ interactions were significantly decreased either by blocking Cys residues or by exposing the τ to a reducing (nitrogen and dithiothreitol), instead of an oxidizing, environment. τ self-association was also significantly decreased by prior phosphorylation with calcium/calmodulin-dependent protein kinase II. Phosphorylation by cyclic AMP-dependent protein kinase or dephosphorylation by alkaline phosphatase did not alter τ self-assembly. These data suggest a role for several factors that may modulate τ self-association in vivo.     

τ Regulation of Microtubule-Microtubule Spacing and Bundling

Abstract: τ proteins are microtubule-associated proteins that promote microtubule polymerization in vitro and in vivo. They are a family of neuronal proteins with apparent molecular weights in the range 50,000–68,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recently, a new member of this family has been described and its cDNA has been cloned. It has an apparent molecular weight of 116,000 and has been called high-molecular-weight τ (HMW τ). All the τ proteins are encoded by a single gene, which undergoes complex alternative splicing. In the present study, we have cloned into the baculovirus a cDNA fully encoding HMW τ as well as a truncated cDNA encoding a protein beginning 13 amino acids in front of the τ microtubule-binding domain. HMW τ-recombinant-virus-infected Sf9 cells overexpressed HMW τ, which induced the polymerization of microtubules and the formation of long cellular processes similar to those induced by low-molecular-weight τ (LMW τ) overexpression. Process cross sections revealed a larger spacing (≈35 nm) between microtubules when induced by HMW τ than when induced by LMW τ (≈20 nm). The truncated construct also induces processes, where microtubules were packed far more closely together (≈10 nm). Although branching did not occur in processes induced by intact τs, 10% of the processes induced by the truncated τ protein branched.     

A τ Fragment Containing a Repetitive Sequence Induces Bundling of Actin Filaments

Abstract: Much indirect evidence suggests that the interconnections of actin microfilaments with the microtubule system are mediated by microtubule-associated proteins (MAPs). In this study we provide new data to support the interaction of a specific tubulin-binding domain on τ with actin in vitro. In actin polymerization assays, the synthetic peptide VRSKIGSTENLKHQPGGG, corresponding to the first repetitive sequence of τ protein, increased turbidity at 320 nm in a dose-dependent fashion. A salient feature of the τ peptide-induced assembly process is the formation of a large amount of actin filament bundles, as revealed by electron microscopic analysis. An increase in the τ peptide concentration resulted in a proportional increase in the bundling of actin filaments. It is interesting that a gradual decrease of pH within the range 7.6–4.7 resulted in a higher effect of τ peptide in promoting bundles of actin filaments. A similar pH-dependent effect was observed for τ protein-induced bundling. An analysis of the mechanisms that operate in the peptide induction of actin filament bundles suggests the involvement of electrostatic forces, because the neutralization of ɛ-aminolysyl residues by selective carbamoylation resulted in a complete loss of the peptide induction of actin bundles. The data suggest that a τ repetitive sequence (also found in MAP-2 and MAP-4) containing a common tubulin binding motif may constitute a functional domain on τ for the dynamics of the interconnections between actin filaments and microtu-bules.     

Okadaic Acid Induces Hyperphosphorylation of τ Independently of Mitogen-Activated Protein Kinase Activation

Abstract: Hyperphosphorylation of the microtubule-associated protein τ is a characteristic of Alzheimer brain tissue. Recent in vitro data suggest that mitogen-activated protein kinase (MAPK), a proline-directed protein kinase, phosphorylates the sites on τ common to Alzheimer's disease. Using an okadaic acid-induced τ hyperphosphorylation model, we have tested the requirement for MAPK activity, using a specific inhibitor {PD098059 [2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one]} of the MAPK activator Mek1. Mobility shift, phosphoepitope analysis, and direct measurement of kinase activity indicated that the Mek1 inhibitor dose-dependently blocked basal and okadaic acid-induced MAPK activation. Despite a block of MAPK activation by this inhibitor, robust τ hyperphosphorylation was observed in response to okadaic acid. In addition, activation of MAPK by phorbol 12-myristate 13-acetate did not result in τ phosphorylation, indicating that in primary cultures of cortical neurons elevated MAPK activity is not sufficient to induce τ hyperphosphorylation.     

Phosphorylation of τ In Situ: Inhibition of Calcium-Dependent Proteolysis

Abstract: In this study, the in situ phosphorylation and subsequent calcium-activated proteolysis of τ protein were examined in human neuroblastoma (LA-N-5) cells, which were differentiated into a neuronal phenotype. The phosphorylation of τ was increased by treating the cells with forskolin and rolipram, which elevate cyclic AMP levels, by treating with the phosphatase inhibitor okadaic acid, or by treating with a combination of both treatments. Phosphorylated τ migrated slightly slower on sodium dodecyl sulfate-polyacrylamide gels than τ from untreated cells. Immunostaining with the phosphate-sensitive monoclonal antibody Tau-1 was also decreased in cells treated with okadaic acid, indicating an increase in the phosphorylation of specific Ser-Pro motifs within the molecule. Calcium-dependent, in situ proteolysis of τ protein was induced by treating the cells with the calcium ionophore A23187. τ protein was proteolyzed to a significantly lesser extent in cells treated with forskolin and rolipram, okadaic acid, or both than in cells in which phosphorylation was not increased. Partially purified τ protein from cells treated with a combination of forskolin, rolipram, and okadaic acid was also more resistant to proteolysis by calpain in vitro compared with τ isolated from control cells. These data suggest a possible role for phosphorylation in the regulation of τ metabolism and in pathological conditions in which the balance between protein kinases and phosphatases is disrupted.     

DMAP-85: A τ-Like Protein from Drosophila melanogaster Larvae

Abstract: Microtubule-associated proteins (MAPs) play major regulatory roles in the organization and integrity of the cytoskeletal network. Our main interest in this study was the identification and the analysis of structural and functional aspects of Drosophila melanogaster MAPs. A novel MAP with a relative molecular mass of 85 kDa from Drosophila larvae was found associated with taxol-polymerized microtubules. In addition, this protein bound to mammalian tubulin in an overlay assay and coassembled with purified bovine brain tubulin in microtubule sedimentation experiments. The estimated stoichiometry of 85-kDa protein versus tubulin in the polymers was 1:5.3 ± 0.2 mol/mol. It was shown that the 85-kDa protein bound specifically to an affinity column of Sepharose-βII-(422–434) tubulin peptide, which contains the sequence of the MAP binding domain on βII-tubulin. Affinity-purified 85-kDa protein enhanced microtubule assembly in a concentration-dependent manner. This effect was significantly decreased by the presence of the βII-(422–434) peptide in the assembly assays, thus confirming the specificity of the 85-kDa protein interaction with the C-terminal domain on tubulin. Furthermore, this protein also exhibited a strong affinity for calmodulin, based on affinity chromatographic assays. Monoclonal and polyclonal anti-τ antibodies, including sequence-specific probes that recognize repeated microtubule-binding motifs on τ, MAP-2, and MAP-4 and specific N-terminal sequences of τ, cross-reacted with the 85-kDa protein from Drosophila larvae. These results suggest that τ and Drosophila 85-kDa protein share common functional and structural epitopes. We have named this protein as DMAP-85 for Drosophila MAP. The finding on a Drosophila protein with functional homology and structural similarities to mammalian τ opens new perspectives to understand the cellular roles of MAPs.     

Alzheimer's Disease Abnormally Phosphorylated τ Is Dephosphorylated by Protein Phosphatase-2B (Calcineurin)

Abstract: Abnormally hyperphosphorylated τ is the major protein subunit of paired helical filaments in Alzheimer brains. We have examined its site-specific dephosphorylation by different protein phosphatases. Dephosphorylation of τ was monitored by its interaction with several phosphorylation-dependent antibodies. Alzheimer τ was dephosphorylated by brain protein phosphatase-2B at the abnormally phosphorylated sites Ser⁴⁶, Ser¹⁹⁹, Ser²⁰², Ser²³⁵, Ser³⁹⁶, and Ser⁴⁰⁴, and its relative mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis shifted to that of normal τ. Protein phosphatases-1 and -2A could dephosphorylate only some of the above six phosphorylation sites. These results indicate that protein phosphatase-2B might be involved in hyperphosphorylation of τ in Alzheimer's disease.