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.     

Developmental Changes in τ Phosphorylation: Fetal τ Is Transiently Phosphorylated in a Manner Similar to Paired Helical Filament-τ Characteristic of Alzheimer's Disease

Rat and human fetal brain τ were probed with a panel of monoclonal antibodies (tau-1, AT8, 8D8, RT97, SMI31, SMI34) that distinguish between paired helical filament (PHF)-τ of Alzheimer's disease and normal adult brain τ. These antibodies discriminate between normal and PHF-τ because their epitopes are phosphorylated in PHF-τ. Although only one molecular isoform of τ was shown to be expressed in fetal brain, two fetal τ species could be distinguished on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the slower migrating species was recognized by all of the PHF-τ-specific antibodies. Moreover, this immunoreactivity was shown to be phosphorylation dependent. Our observations suggest that the abnormal phosphorylation of τ in Alzheimer's disease may be the result of reactivation of pathways governing the phosphorylation of τ in the developing brain.     

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.     

Microheterogeneity of Micro tubule-Associated τ Proteins Is Due to Differences in Phosphorylation

We have studied the heterogeneity of the microtubule-associated tau proteins using tau-specific antibodies and two-dimensional electrophoresis. Both monoclonal and polyclonal antibodies to tau proteins recognize five bands in cow brain microtubule proteins run on sodium dodecyl sulfate (SDS)-polyacrylamide gels, with apparent molecular weights between 56,000 and 66,000. Immunoblots of cow brain microtubules separated on two-dimensional gels, using nonequilibrium pH gradient electrophoresis in the first dimension and SDS-gel electrophoresis in the second, reveal that greater than 30 isoforms of tau exist. The tau proteins vary in pI from 6.5 to 8.5, with the higher-molecular-weight forms being more acidic. The microheterogeneity of tau is not induced by cycling of microtubules, because two-dimensional immunoblots of tau from total brain are almost identical to those of tau from cycled tubules. Adult rat brain tau, which appears as three doublet bands on SDS gels, also exhibits considerable isoelectric heterogeneity, as does tau from 7-day-old rats, which appears as only one band on SDS gels. After dephosphorylation of cow brain tau with alkaline phosphatase, the highest-molecular-weight band disappears on SDS gels. On two-dimensional gels, the number of tau variants decreases by more than half after dephosphorylation, and the more basic species increase greatly in intensity. Preliminary experiments with tau labeled in vivo with 32PO4 also indicate that the more acidic tau proteins are the more highly phosphorylated forms. Thus, isoelectric heterogeneity of tau proteins exists at all ages and is due, at least in part to differences in the state of phosphorylation of tau isoforms.     

Differential Axonal Transport of Soluble and Insoluble τ in the Rat Sciatic Nerve

Abstract: Axonal transport of microtubule-associated protein τ was studied in the motor fibers of the rat sciatic nerve 1–4 weeks after labeling of the spinal cord with [³⁵S]methionine. As 60–70% of low molecular weight τ in this system was found to be insoluble in 1% Triton-containing buffer, labeled proteins in 6-mm consecutive nerve segments were first separated into Triton-soluble and insoluble fractions. Two-dimensional gel electrophoresis and immunoblotting with anti-tau antibody confirmed the presence of τ among labeled, transported proteins in both fractions. Isoform composition of labeled τ was similar to that of bulk axonal τ, the most acidic species with apparent molecular mass of 66 kDa being the major component. Transport profiles obtained by measuring radioactivities associated with this major isoform showed that soluble and insoluble τ were transported at different rates. Insoluble τ, which contained the majority of τ-associated radioactivity, was transported at 1.7 mm/day in slow component a (SCa), whereas soluble τ was transported faster, at 3 mm/day, corresponding to the rate of slow component b (SCb). Cotransport of insoluble τ with insoluble tubulin in SCa suggests its association with stable microtubules.     

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.     

A τ-Related Protein of 130 kDa Is Present in Alzheimer Brain

Two abnormal entities of 69 and 130 kDa, immunologically related to the microtubule-associated tau proteins, are present in the hippocampus and the frontal cortex of the Alzheimer brain, which contain a large number of neurofibrillary tangles (NFTs), but are absent in the cerebellum, which does not contain these structures. Epitope mapping with antibodies spanning domains present in the N-terminal, middle, and C-terminal tau sequence demonstrated that the 69- and 130-kDa entities belong to the tau family. Both the 69- and the 130-kDa proteins were found in an insoluble form and were the major tau species present in purified NFTs. A procedure was devised that allowed us to prepare from Alzheimer hippocampi two NFT fractions differing in size (20 and 3 microns), both of which contained the tau entities of 130 and 69 kDa.     

Amino Acid Residues 226–240 of τ Which Encompass the First Lys-Ser-Pro Site of τ, Are Partially Phosphorylated in Alzheimer Paired Helical Filament-τ

Abstract: A synthetic peptide corresponding to residues 226–240 (E9 peptide) of human τ, which contains an Lys-Ser-Pro motif, was used to raise a polyclonal antibody. The antibody, E9, was 10-fold less reactive with phospho-E9 peptide than with native E9 peptide. E9 antibody was used to study the extent of phosphorylation in a modified form of τ (PHF-τ) that is found in Alzheimer's disease brain and is incorporated into paired helical filaments (PHFs). E9 immunolabeled Alzheimer's disease neurofibrillary tangles and abnormal neurites in brain sections intensely, with increased immunoreactivity detected after pretreatment of sections with phosphatase. On immunoblots and ELISA, E9 reacted with PHF-τ and recombinant human τ but not with the high and middle molecular weight neurofilament proteins. Phosphatase treatment of PHF-τ improved the E9 immunoreactivity by 30–50%. Dephosphorylated high but not middle molecular weight neurofilament protein became reactive with E9. These results indicate that <50% of the PHF-T is phosphorylated in the subregion corresponding to residues 226–240 of τ and suggest that the phosphorylation of this region may not be essential for PHF formation.     

Localization and Developmental Changes of τ Protein Kinase I/Glycogen Synthase Kinase-3β in Rat Brain

Abstract: τ protein kinase I (TPKI) purified from bovine brain extract has been shown to phosphorylate τ and to form paired helical filament (PHF) epitopes and was found recently to be identical to glycogen synthase kinase-3β (GSK-3β). Before elucidating a role of TPKI/GSK-3β in PHF formation, it is necessary to investigate the normal function of the enzyme. To study the distribution and developmental changes of the enzyme, specific polyclonal antibodies were prepared against TPKI and GSK-3α. Immunoblot analysis demonstrated that TPKI was nearly specifically localized in the brain of adult rats. The level of TPKI in the rat brain was high at gestational day 18, peaked on postnatal day 8, and then decreased rapidly to a low level, which was sustained up to 2 years. Immunohistochemistry indicated primarily neuronal localization of TPKI. Growing axons were stained most intensely in the developing cerebellum, but the immunoreactivity became restricted to the gray matter in the mature tissue. Parallel fibers had a high level of TPKI and also stained intensely for τ. These findings indicate that τ is one of the physiological substrates of TPKI and suggest that the enzyme plays an important role in the growth of axons during development of the brain.     

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.     

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.     

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.     

Reactivating Kinase/p38 Phosphorylates τ Protein In Vitro

Abstract: Neurofibrillary tangles, one of the major pathological hallmarks of Alzheimer-diseased brains, consist primarily of aggregated paired helical filaments (PHFs) of hyperphosphorylated τ protein. τ from normal brain and especially from foetal brain is also phosphorylated on some of the sites phosphorylated in PHFs, mainly at serines or threonines followed by prolines. A number of protein kinases can phosphorylate τ in vitro; those that require or accept prolines include GSK3 and members of the mitogen-activated protein (MAP) kinase family, ERK1, ERK2, and SAP kinase-β/JNK. In this report, we show that another member of the MAP kinase family, the stress-activated kinase p38/RK, can phosphorylate τ in vitro. Western blots with phosphorylation-sensitive antibodies showed that p38, like ERK2 and SAP kinase-β/JNK, phosphorylated τ at sites found phosphorylated physiologically (Thr¹⁸¹, Ser²⁰², Thr²⁰⁵, and Ser³⁹⁶) and also at Ser⁴²², which is phosphorylated in neurofibrillary tangles but not in normal adult or foetal brain. These findings support the possibility that cellular stress might contribute to τ hyperphosphorylation during the formation of PHFs, and hence, to the development of τ pathology.     

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.     

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.     

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.     

Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Human τ Detection

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.