Tau-66: evidence for a novel tau conformation in Alzheimer's disease.

We have characterized a novel monoclonal antibody, Tau-66, raised against recombinant human tau. Immunohistochemistry using Tau-66 reveals a somatic-neuronal stain in the superior temporal gyrus (STG) that is more intense in Alzheimer's disease (AD) brain than in normal brain. In hippocampus, Tau-66 yields a pattern similar to STG, except that neurofibrillary lesions are preferentially stained if present. In mild AD cases, Tau-66 stains plaques lacking obvious dystrophic neurites (termed herein 'diffuse reticulated plaques') in STG and the hippocampus. Enzyme-linked immunosorbent assay (ELISA) analysis reveals that Tau-66 is specific for tau, as there is no cross-reactivity with MAP2, tubulin, Abeta(1-40), or Abeta(1-42), although Tau-66 fails to react with tau or any other polypeptide on western blots. The epitope of Tau-66, as assessed by ELISA testing of tau deletion mutants, appears discontinuous, requiring residues 155-244 and 305-314. Tau-66 reactivity exhibits buffer and temperature sensitivity in an ELISA format and is readily abolished by SDS treatment. Taken together these lines of evidence indicate that the Tau-66 epitope is conformation-dependent, perhaps involving a close interaction of the proline-rich and the third microtubule-binding regions. This is the first indication that tau can undergo this novel folding event and that this conformation of tau is involved in AD pathology.

Oxidative regulation of fatty acid-induced tau polymerization.

Alzheimer's disease (AD) is characterized by the presence of amyloid-positive senile plaques and tau-positive neurofibrillary tangles. Aside from these two pathological hallmarks, a growing body of evidence indicates that the amount of oxidative alteration of vulnerable molecules such as proteins, DNA, and fatty acids is elevated in the brains of AD patients. It has been hypothesized that the elevated amounts of protein oxidation could lead directly to the formation of neurofibrillary tangles through a cysteine-dependent mechanism. We have tested this hypothesis in an in vitro system in which tau assembly is induced by fatty acids. Using sulfhydryl protective agents and site-directed mutagenesis, we found that cysteine-dependent oxidation of the tau molecule is not required for its polymerization and may even be inhibitory. However, by adjusting the oxidative environment of the polymerization reaction through the addition of a strong antioxidant or through the addition of an oxidizing system consisting of iron, adenosine diphosphate, and ascorbate, we found that oxidation does play a major role in our in vitro paradigm. The results indicated that fatty acid oxidation, the amount of which is found to be elevated in AD patients, can facilitate the polymerization of tau. However, "overoxidation" of the fatty acids can inhibit the process. Therefore, we postulate that specific fatty acid oxidative products could provide a direct link between oxidative stress mechanisms and the formation of neurofibrillary tangles in AD.

Nucleolar localization of the microtubule-associated protein tau in neuroblastomas using sense and anti-sense transfection strategies.

The Tau-1 monoclonal antibody was localized to the nucleolus of interphase cells and the nucleolar organizing regions (NORs) of acrocentric chromosomes in cultured human cells. Putative nucleolar and NOR tau was found in CG neuroblastoma cells which contain nucleolar tau and little cytoplasmic tau. To further establish the presence of tau in the nucleolus of these cells, sense and anti-sense transfection strategies were used. CG neuroblastoma cells were transfected with tau sense cDNA and immunostained with Tau-1. Cytoplasmic Tau-1 staining was greatly increased in CG cells which contain very little endogenous cytoplasmic tau. Nucleolar Tau-1 staining was also increased in certain CG cells indicating an increase in nucleolar tau in a subset of transfected cells. CG cells were also transfected with tau anti-sense cDNA which abolished Tau-1 staining in the nucleolus. These results contribute to a growing body of evidence defining tau as a multifunctional protein found in both the cytoplasm and nucleoli of primate cells.

Localization of specific epitopes on human microtubule-associated protein 2.

Microtubule-associated protein 2 (MAP-2) is an abundant neuronal cytoskeletal protein that binds to tubulin and stabilizes microtubules. Using fusion protein constructs we have defined the epitopes of 10 monoclonal antibodies (mAbs) to discrete regions of human MAP-2. Proteins were expressed in pATH vectors. After electrophoresis, immunoblotting was performed. By western blot analysis five of the mAbs (AP-14, AP-20, AP-21, AP-23, and AP-25) share epitopes with only the high molecular weight isoforms (MAP-2a, MAP-2b); two of the mAbs (AP-18 and tau 46) recognize MAP-2a, MAP-2b, and MAP-2c. Although AP-18 immunoreactivity was detected within heat-stable protein homogenates isolated from a human neuroblastoma cell line MSN, fusion protein constructs encompassing human MAP-2 were negative, suggesting that the AP-18 epitope is phosphorylated. Furthermore, AP-18 immunoreactivity was lost after alkaline phosphatase treatment of heat-stable protein preparations from MSN cells. Four of the mAbs (322, 636, 635, and 39) recognize epitopes located within amino acids 169-219 of human MAP-2. AP-21 maps to a region between amino acids 553 and 645. AP-23 maps between amino acids 645 and 993, whereas AP-20, AP-14, and AP-25 map between amino acids 995 and 1332. Expression of the region of MAP-2 between amino acids 1787 and 1824 was positive to tau 46.

Expression of the cytoskeletal-associated protein filamin in adult rat organs.

Filamin is a well-characterized actin-associated protein first isolated from chicken smooth muscle. Subsequently, this polypeptide and its nonmuscle homolog actin-binding protein have been shown to be expressed in avian muscle tissue, mammalian smooth muscle, mammalian macrophages and other blood cell types, as well as several cultured cell lines. In this report, the occurrence of this polypeptide in adult mammalian organs has been investigated. Immunoblot analysis using three anti-filamin monoclonal antibodies showed that this protein was largely detected in adult rat organs that possess a substantial smooth muscle component. Furthermore, the limited expression of filamin in smooth muscle tissue was corroborated by immunohistochemical analysis. In contrast to avian systems, filamin was never found in detectable quantities in either mammalian cardiac or skeletal muscle. Quantitative immunoblot analysis demonstrated that filamin amounts roughly correlated with the abundance of the smooth muscle component of a given organ, comprising as much as 16.5% of the total SDS-extractable protein in bovine aorta. Work in avian systems and cells in culture has suggested that filamin is a rather ubiquitous cytoskeletal element. By contrast, this work demonstrates that filamin is highly restricted in its expression in mammalian organ systems, in situ.

A novel tau transcript in cultured human neuroblastoma cells expressing nuclear tau.

We previously reported the presence of the microtubule-associated protein, tau in the nuclei of primate cells in culture. The present study confirms the existence of nuclear tau in two human neuroblastoma cells lines by indirect immunofluorescence and Western blot using mAbs to tau. Northern blot analysis of poly A+ mRNA detects a novel 2-kb tau transcript coexpressed with the 6-kb message in cultured human cells and human frontal cortex. PCR and cDNA sequencing demonstrate that the 2-kb message contains the entire tau coding region. Furthermore, actinomycin D transcription inhibition experiments indicate that the 2-kb message is not derived from the 6-kb message, but instead arises from the original tau transcript. One of the human neuroblastoma cell lines examined contains both nuclear and cytoplasmic tau as assayed by both Western blot and indirect immunofluorescence. Northern blot analysis of this cell line indicates that copious amounts of the 2-kb message are present while little of the 6-kb transcript is obvious. Immunofluorescence analysis of this cell line demonstrates that the cytoplasmic tau is not localized to microtubules. Together, these results indicate that the 2-kb tau message in humans may specify tau for non-microtubule functions in both the cytoplasm and the nucleus. We hypothesize that this is accomplished via a message targeting mechanism mediated by the untranslated regions of the tau messages.

Actin-binding protein is a component of bovine erythrocytes.

Actin-binding protein (ABP) is a well-characterized polypeptide capable of crosslinking filamentous actin. To date, this polypeptide has been shown to exist in a number of tissues and cultured cell lines. This report shows that by using a panel of three monoclonal antibodies for immunoblotting and immunofluorescence analysis, that ABP is present in bovine erythrocytes. Moreover, the data obtained suggest that this protein is a component of the erythrocyte membrane skeleton. Additionally, bovine erythrocyte ABP is shown to possess both an apparent molecular weight and an isoelectric point identical to that of bovine smooth muscle filamin, implying that these two polypeptides are identical.

Identification of the intermediate filament-associated protein gyronemin as filamin. Implications for a novel mechanism of cytoskeletal interaction.

In a previous paper, a monoclonal antibody (designated M1.4) that recognized a 240 kDa polypeptide was characterized. This antibody stained the intermediate filaments in several cell lines, and biochemical characteristics of the 240 kDa polypeptide led us to conclude that it was a novel intermediate filament-associated protein, which we termed gyronemin. Here we report that gyronemin is expressed in adult rat organs that contain a substantial smooth muscle component. Taking advantage of this observation, this protein was purified from bovine uterine tissue and, by biochemical, immunological and amino acid sequence analysis, found to be homologous to the actin-associated protein filamin. Three novel monoclonal antibodies raised using purified bovine gyronemin as the immunogen show this protein to be associated with actin-containing stress fibers, although our original M1.4 antibody continued to be localized along vimentin filaments. Since two-dimensional electrophoretic analysis did not demonstrate a difference in either relative molecular mass or isoelectric point of this polypeptide when associated with either filamentous system, we conclude that filamin is a bifunctional protein capable of associating with both the intermediate filament and actin cytoskeletal systems.

Identification of nuclear tau isoforms in human neuroblastoma cells.

The tau proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, tau has been shown to be an integral component of paired helical filaments, the principal constituent of the neurofibrillary tangles found in brains of patients with Alzheimer disease and of most aged individuals with Down syndrome (trisomy 21). We report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, we further demonstrate the existence of the entire tau molecule in the isolated nuclei of neuroblastoma cells. Nuclear tau proteins, like the tau proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that tau may function in processes not directly associated with microtubules and that highly insoluble complexes of tau may also play a role in normal cellular physiology.

Alzheimer disease proteins (A68) share epitopes with tau but show distinct biochemical properties.

Alz 50, a monoclonal antibody raised against Alzheimer brain homogenate, reacts with neurofibrillary tangles, microtubule-associated proteins tau, and Alzheimer brain proteins of molecular weight 70-60 kDa (A68). To study the relationship between A68 and normal human tau we compared the biochemical properties of these proteins and tested the reactivity of A68 with eight antibodies (Alz 50, Tau 60, Tau-2, Tau 14, Tau-1, Ab 636.7, NP14, Tau 46) that bind to various regions of tau molecule. On Western blots, all tau-reactive antibodies, except Tau-1, recognized A68. Pretreatment with alkaline phosphatase was required for the Tau-1 binding to A68. A68 consisted of three polypeptides of 68, 64, and 60 kDa, while tau contained 4-6 polypeptides of 50-65 kDa. A68 was less heterogenous than tau in the number of pI variants on two-dimensional gels. All A68 variants were more acidic (pI 5.5-6.5) than human tau (pI 6.5-8.5). Phosphatase treatment had only a minor effect on the pI and mobility of A68. Limited proteolysis of A68 with trypsin or chymotrypsin generated large fragments of 56-66 kDa (chymotrypsin) and 40-45 kDa (trypsin). While none of the fragments was recognized by Alz 50, the chymotryptic fragments were reactive with all the other tau antibodies, and the tryptic fragments were positive with five of the antibodies (Tau 14, Tau-1, Ab 636.7, NP14, and Tau 46). The peptide maps of A68 differed from that of tau in the number and the size of the peptide fragments. The differences in biochemical properties of these proteins and the sharing multiple epitopes suggest that A68 is a modified form of tau. The modification in part may be due to phosphorylation, although other changes rendering different isoelectrical properties and susceptibility to proteases need to be considered. The removal of the Alz 50 epitope by a cleavage of a 2-3 kDa fragment which does not contain the most C-terminal epitope (Tau 46) indicates that the Alz 50 epitope is located at the N-terminal periphery of the A68 molecule.

Identification and characterization of a novel mammalian intermediate filament-associated protein.

A novel monoclonal antibody, designated M1.4, recognizes the high molecular weight microtubule-associated protein MAP1A (ca. Mr 380 kD) in both bovine and rat brain. In HeLa cells, however, M1.4 binds to a 240 kD polypeptide on immunoblots and co-localizes with both vimentin and cytokeratin filaments using double-label immunofluorescence microscopy. Immunoelectron microscopy indicates that the 240 kD polypeptide localizes along bundled intermediate filaments in a periodic manner. Two-dimensional electrophoretic analysis indicates that the 240 kD polypeptide has a basic pI of 7.7. When HeLa cell intermediate filaments are isolated using standard non-ionic detergent/high-salt conditions the 240 kD polypeptide does not sediment with the intermediate filaments, unlike the established intermediate filament-associated protein plectin. Immunoblot analysis with M1.4 shows the 240 kD polypeptide is expressed in a number of mammalian cell lines. Additionally, double-label immunofluorescence shows the 240 kD polypeptide to associate with vimentin filaments in African Green Monkey kidney (CV-1) and JC neuroblastoma cells. Due to its unique biochemical and biological characteristics, the 240 kD polypeptide is clearly a novel intermediate filament-associated protein for which we have proposed the designation gyronemin (Gr. gyros: around; nemin: filament).

Estramustine binds a MAP-1-like protein to inhibit microtubule assembly in vitro and disrupt microtubule organization in DU 145 cells.

The twofold purpose of the study was (a) to determine if a MAP-1-like protein was expressed in human prostatic DU 145 cells and (b) to demonstrate whether a novel antimicrotubule drug, estramustine, binds the MAP-1-like protein to disrupt microtubules. SDS-PAGE and Western blots showed that a 330-kD protein was associated with microtubules isolated in an assembly buffer containing 10 microM taxol and 10 mM adenylylimidodiphosphate. After purification to homogeneity on an A5m agarose column, the 330-kD protein was found to promote 6 S tubulin assembly. Turbidimetric (A350), SDS-PAGE, and electron microscopic studies revealed that micromolar estramustine inhibited assembly promoted by the 330-kD protein. Similarly, estramustine inhibited binding of the 330-kD protein to 6-S microtubules independently stimulated to assemble with taxol. Immunofluorescent studies with beta-tubulin antibody (27B) and MAP-1 antibody (MI-AI) revealed that 60 microM estramustine (a) caused disassembly of MAP-1 microtubules in DU 145 cells and (b) removed MAP-1 from the surfaces of microtubules stabilized with 0.1 microM taxol. Taken together the data suggested that estramustine binds to a 330-kD MAP-1-like protein to disrupt microtubules in tumor cells.

Phylogenetic conservation of brain microtubule-associated proteins MAP2 and tau.

The major rat brain microtubule-associated proteins, MAP2 and tau, exhibit various properties that implicate them in the mechanisms underlying the growth of axons and dendrites during neuronal development. To determine if these properties represent fundamental morphogenetic mechanisms, we have examined the phylogenetic conservation of these proteins in Xenopus laevis, quail and rat with respect to their molecular form, cytological distribution and developmental expression. In all three species, the high-molecular weight form of MAP2 migrates as a pair of polypeptides (MAP2a and MAP2b); this doublet as well as the low-molecular weight form of MAP2 (MAP2c) and the tau proteins are markedly similar in size in the different classes of vertebrates. Immunohistochemical staining of the Xenopus and quail cerebellum showed that MAP2 is highly concentrated in dendrites whereas the tau proteins are predominantly confined to axons, exactly as they are in rat. The developmental regulation of these proteins in Xenopus and rat is also conserved. Between the larva and the adult (i.e. during metamorphosis) MAP2c undergoes a marked decrease while MAP2a undergoes a large increase. Thus, in both classes of vertebrates the timing of changes in MAP2 expression coincides with the maturation of neuronal morphology. Taken together, these conserved properties of MAP2 and tau in three phylogenetically divergent classes of vertebrates suggest that these proteins serve fundamental functions during neuronal morphogenesis.

Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology.

A monoclonal antibody to the microtubule-associated protein tau (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical filaments (PHF), in Alzheimer disease brain. The antibody also labeled isolated PHF that had been repeatedly washed with NaDodSO4. Dephosphorylation of the tissue sections with alkaline phosphatase prior to immunolabeling dramatically increased the number of tangles and plaques recognized by the antibody. The plaque core amyloid was not stained in either dephosphorylated or nondephosphorylated tissue sections. On immunoblots PHF polypeptides were labeled readily only when dephosphorylated. In contrast, a commercially available monoclonal antibody to a phosphorylated epitope of neurofilaments that labeled the tangles and the plaque neurites in tissue did not label any PHF polypeptides on immunoblots. The PHF polypeptides, labeled with the monoclonal antibody to tau, electrophoresed with those polypeptides recognized by antibodies to isolated PHF. The antibody to tau-labeled microtubules from normal human brains assembled in vitro but identically treated Alzheimer brain preparations had to be dephosphorylated to be completely recognized by this antibody. These findings suggest that tau in Alzheimer brain is an abnormally phosphorylated protein component of PHF.

The cytomatrix regulates "resolute" transport in erythrophores.

Light microscopic studies have indicated that most microtubule-directed transport is either saltatory or resolute in nature. The latter form of transport is an intriguing phenomenon, because it commonly involves the unidirectional bulk motion of an organelle(s) such as chromosomes in dividing cells or pigment granules in chromatophores. We have investigated the ultrastructural and biochemical basis for the resolute transport of pigment in chromatophores. Light and EM studies of erythrophores in situ have clearly shown that when the microtubules were completely removed with nocodazole, resolute transport continued and was stimulated by aggregating and dispersing agents. Light and electron microscopic studies of cultured erythrophores permeabilized with digitonin indicated that resolute motion was produced by a cytomatrix of 3 to 7 nm filaments. Immunofluorescent analysis with several monoclonal antibodies raised against MAP-2 further demonstrated that MAP-2 was an important component of the contractile cytomatrix that powers pigment aggregation and dispersion. We conclude that a microtubule-associated cytomatrix normally produces resolute pigment transport in chromatophores.

The distribution of tau in the mammalian central nervous system.

We have determined the biochemical and immunocytochemical localization of the heterogeneous microtubule-associated protein tau using a monoclonal antibody that binds to all of the tau polypeptides in both bovine and rat brain. Using immunoblot assays and competitive enzyme-linked immunosorbent assays, we have shown tau to be more abundant in bovine white matter extracts and microtubules than in extracts and microtubules from an enriched gray matter region of the brain. On a per mole basis, twice-cycled microtubules from white matter contained three times more tau than did twice-cycled microtubules from gray matter. Immunohistochemical studies that compared the localization of tau with that of MAP2 and tubulin demonstrated that tau was restricted to axons, extending the results of the biochemical studies. Tau localization was not observed in glia, which indicated that, at least in brain, tau is neuron specific. These observations indicate that tau may help define a subpopulation of microtubules that is restricted to axons. Furthermore, the monoclonal antibody described in this report should prove very useful to investigators studying axonal sprouting and growth because it is an exclusive axonal marker.

The periodic association of MAP2 with brain microtubules in vitro.

Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP1 (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/ml. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately 1 mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.