Identification of cDNA clones for the human microtubule-associated protein tau and chromosomal localization of the genes for tau and microtubule-associated protein 2

We have previously identified a partial human cDNA for MAP2, and we now report the isolation of human cDNA clones for tau. The RNA species recognized by the tau clones is a 6 kilobase (kb) message that is expressed in the human brain but not in other human tissues, and exhibits a developmental shift in size. We also report the human chromosomal localization of the MAP2 and tau genes. The MAP2 cDNA pKN7 was used to localize the MAP2 gene to chromosome 2q34-35. The tau cDNAs were used to confirm the presence of a tau gene on chromosome 17q21 and an additional region of homology on chromosome 6p21.     

A brief history of tau: the evolving view of the microtubule-associated protein tau in neurodegenerative diseases

The major historical milestones in tau-research are reviewed, with their implications for changing perspectives about the significance of tau-pathology in neurodegeneration. Abnormalities of tau-protein characterize the pathology of numerous neurodegenerative disorders, both sporadic and inherited. Over the years, opinions regarding the significance of tau in disease pathogenesis, particularly in Alzheimer's disease, have fluctuated. Early caution about the role of tau as a significant factor in neurodegenerative disease, especially Alzheimer's disease, has been superseded by acceptance of its key involvement in pathways which led to cell dysfunction and death. The discovery of familial "tauopathies", associated with tau-gene mutations, has confirmed that tau-dysmetabolism can independently lead to neurodegeneration. Debate about the centrality of its role remains, but current evidence makes it difficult to ignore the importance of tau in many neurodegenerative diseases. By examining the evolution of research on tau, related to advances in technology and the emergence of new diseases, the future developments needed to resolve remaining issues in the tau-story may be discerned.     

A mutation in the microtubule-associated protein tau in pallido-nigro-luysian degeneration.

We detected a missense mutation in exon 10 of tau that causes a substitution at codon 279 (N279K) in a Japanese patient with a familial background of parkinsonism and dementia originally described as pallido-nigro-luysian degeneration. This mutation is the same as one seen in a Caucasian family with pallido-ponto-nigral degeneration. The similarities between these two families suggest a common genetic mechanism that may account for the peculiar distribution of neuroglial degeneration with tauopathy.     

Light and electron microscope localization of the microtubule-associated tau protein in rat brain

We have studied the distribution of microtubule-associated tau proteins in rat brain using monoclonal and affinity-purified polyclonal antibodies. Tau staining is prominent in axons in white matter areas of brain, as reported by Binder et al. (1985). In addition, we also find tau protein in neuron cell bodies, especially in the brain stem and basal ganglia and in the cell bodies of interfascicular oligodendroglia. Using electron microscopy, tau antibodies and colloidal gold-labeled second antibodies, gold particles are found associated with microtubules in axons and in the cytoplasm of cell bodies, while the nuclei, mitochondria, and myelin remain unlabeled. In double-staining experiments, tau staining co-localizes with that of tubulin. Our studies indicate that tau proteins are more widely distributed in brain than previously reported and cannot be used as an exclusive marker for axons.     

Association of microtubule-associated protein tau gene polymorphisms with the risk of sporadic Alzheimer's disease: a meta-analysis

Background: Numerous epidemiological studies were published to investigate the role of microtubule-associated protein tau (MAPT) gene variations (rs7521G/A, rs242557G/A, rs1467967A/G, rs2471738C/T and rs3785883G/A) in SAD, and these genetic polymorphisms may be a risk factor for sporadic Alzheimer's disease (SAD). However, controversial results were revealed. Methods: The MEDLINE, Embase and HuGEnet databases were searched to identify eligible studies. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the genetic association of MAPT with the risk of SAD. Results: A total of 30 studies were included in this meta-analysis to assess the association between five single-nucleotide polymorphisms and susceptibility to SAD. The pooled results exhibited no significant association between rs1467967A/G, rs3785883G/A, rs2471738C/T and rs7521G/A polymorphisms and the risk of SAD. However, a lower risk of SAD was observed in the GG versus (GA + AA) model of rs242557G/A polymorphism (OR = 0.86, 95% CI = 0.751–0.983, P = 0.027). Conclusion: The present meta-analysis showed that rs1467967A/G, rs3785883G/A, rs2471738C/T and rs7521G/A polymorphisms were not associated with the risk of SAD. However, rs242557G/A genetic polymorphism was associated with susceptibility to SAD, and individuals with a GG genotype of rs242557G/A might be at a lower risk of SAD. Further studies with a larger sample size are required to validate these conclusions.     

Phosphorylation of microtubule-associated protein tau on Ser 262 by an embryonic 100 kDa protein kinase

This study examined the phosphorylation of tau on Ser 262, within the first microtubule-binding domain, by a developmentally regulated 100 kDa protein kinase exhibiting significantly greater activity in the embryonic rat brain than in the adult rat brain. This protein kinase co-purified with microtubules and co-immunoprecipitated with both tau and MAP-2. In addition to phosphorylating tau, MAP-2, and a Ser 262-containing peptide, the present protein kinase activity was shown to autophosphorylate as determined by the in-gel kinase assay in the absence of any protein or peptide polymerized into the matrix. Phosphorylation of tau with this protein kinase significantly reduced the tau–microtubule interaction, and the effect was significantly greater with microtubule-associated protein (MAP) preparations from embryonic brain than with preparations from the adult. Ser 262 is phosphorylated extensively in paired helical filament (PHF) tau from Alzheimer's disease (AD) brain, to a lesser extent in fetal tau, and only to a very minor extent in biopsy-derived human tau. Because the 100 kDa protein kinase activity phosphorylates Ser 262 and is higher in the fetal brain than the adult brain, it is hypothesized that an inappropriate re-expression and/or re-activation of this or a similar developmentally regulated protein kinase could contribute to the phosphorylation of Ser 262 in PHF-tau, and thus play a role in the pathogenesis of AD.     

Phosphorylation of human tau protein by microtubule-associated kinases: GSK3beta and cdk5 are key participants

Microtubules (MTs), primarily composed of alpha and beta tubulin polymers, must often work in concert with microtubule-associated proteins (MAPs) in order to modulate their functional demands. In a mature brain neuron, one of the key MAPs that resides primarily in the axonal compartment is the tau protein. Tau, in the adult human brain, is a set of six protein isoforms, whose binding affinity to MTs can be modulated by phosphorylation. In addition to the role that phosphorylation of tau plays in the "normal" physiology of neurons, hyperphosphorylated tau is the primary component of the fibrillary pathology in Alzheimer's disease (AD). Although many protein kinases are known to phosphorylate tau in vitro, the in vivo players contributing to the hyperphosphorylation of tau remain elusive. The experiments in this study attempt to define which protein kinases and protein phosphatases reside in the associated network of microtubules, thereby being strategically positioned to influence the phosphorylation of tau. Microtubule fractions are utilized to determine which of the microtubule-associated kinases most readily impacts the phosphorylation of tau at "AD-like" sites. Results from this study indicate that PKA, CK1, GSK3beta, and cdk5 associate with microtubules. Among the MT-associated kinases, GSK3beta and cdk5 most readily contribute to the ATP-induced "AD-like" phosphorylation of tau.     

Phosphorylated serine 199 of microtubule-associated protein tau is a neuronal epitope abundantly expressed in youth and an early marker of tau pathology

Microtubule-associated protein tau is abnormally phosphorylated in many neurodegenerative disorders, and is the major component of neurofibrillary degeneration, a degenerating process with many biochemical phenotypes. The serine 199 (S199) residue of tau is phosphorylated at early and late stages of Alzheimer's disease (AD). We studied the immunohistochemical distribution of this phosphorylated epitope in AD and other neurodegenerative disorders, as well as in controls of different ages. The phosphorylated S199 (S199P) epitope was observed in tau lesions from numerous diseases with neurofibrillary degeneration. This epitope was found to be abundantly expressed in the hippocampus formation in childhood and in young adult brain samples, and more specifically in subsets of neurons vulnerable to neurodegeneration. Interestingly, our data suggests that S199P is particularly resistant to phosphatase activity occurring during post-mortem delays. We suggest a peculiar and important role of the S199 residue as a qualitative indicator of the normal and pathological phosphorylation status of tau proteins.     

Acute rise in the concentration of free cytoplasmic calcium leads to dephosphorylation of the microtubule-associated protein tau

The objective of this study was to asses the response of the microtubule-associated protein tau to acute rise in the concentration of free cytoplasmic calcium ([Ca²⁺]_i) in rat cortical neurons and mouse cerebellar granule cells in culture. One-hour exposure to glutamate (100 μM), N-methyl-d-aspartate (100 μM), KCl (50 mM), and ionomycin (5 μM) led to tau protein dephosphorylation as indicated by an appearance of additional faster moving bands on Western immunoblots with a phosphorylation-independent antibody and an increase in the tau-1 immunoreactivity associated with the appearance of an additional faster moving band. Lowering the extracellular concentration of Ca²⁺ to less than 1 μM fully prevented the drug-induced tau protein dephosphorylation indicating a dependence on Ca²⁺ influx from the extracellular environment. Administration of okadaic acid (inhibitor of phosphatase 1/2A) simultaneously with the above mentioned drugs decreased the drug-mediated dephosphorylation. Pre-incubation with okadaic acid fully prevented the dephosphorylation. Treatment with cypermethrin (inhibitor of phosphatase 2B) was without effect when administered either alone, simultaneously with the drugs, or pre-incubated. These findings indicate that, independently of the influx pathway, [Ca²⁺]_i elevation leads to dephosphorylation of the microtubule-associated protein tau and implicate phosphatase 1 and/or 2A in the process.     

Molecular characterization of microtubule-associated proteins tau and MAP2.

Tau and MAP2 are two of the major microtubule-associated proteins in the vertebrate nervous system. They promote microtubule assembly and stability, and might be involved in the establishment and maintenance of neuronal polarity. In nerve cells immunohistochemistry shows complementary distributions, with tau being concentrated in axons and high molecular mass MAP2 being confined to dendrites. Each protein consists of multiple isoforms that contain three or four homologous tandem repeats near the carboxy-terminus, which constitute microtubule-binding domains. In humans, tau consists of at least six isoforms of related amino acid sequences that are produced from a single gene by alternative mRNA splicing and that are expressed in a stage- and cell type-specific manner. Tau is also a component of the paired helical filaments associated with Alzheimer's disease and other disorders of the CNS. Rat MAP2 consists of at least three isoforms produced from a single gene: high molecular mass MAP2a and MAP2b, and low molecular mass MAP2c. MAP2c is expressed only during early development and has so far been seen only in axons; MAP2a appears to replace MAP2c, whereas MAP2b is expressed throughout life. Messenger RNAs for MAP2 of high molecular mass are expressed both in cell bodies and in dendrites, consistent with the dendritic localization of the corresponding protein isoforms.     

Comparison of methods for the in vitro assembly of postmortem human brain microtubules that retain the microtubule-associated protein tau.

Several methods for the in vitro assembly of microtubules from postmortem human brain were compared for the purpose of obtaining microtubule preparations that best retained their microtubule-associated proteins. The polymerized microtubules from the preparations were examined by negative staining and electron microscopy and shown to consist of well-formed microtubules with varying amounts of abnormal assembly products that differed between methods. The microtubule protein was analyzed by SDS-polyacrylamide gel electrophoresis, quantitative densitometry, as well as trans-blotted onto membranes which were reacted with monoclonal antibodies to tubulin subunits and microtubule-associated proteins. All the preparations were found to contain both the alpha- and beta-tubulin subunits with quantitative differences, but they varied most, both quantitatively and qualitatively, in their content of microtubule-associated proteins. The optimal method for the assembly of soluble tubulin from postmortem human brain cytosol into intact microtubules which specifically retained most of their MAPs, especially tau, employed 4 M glycerol assembly buffer in the presence of 10 microM taxol and 1 mM GTP. The isolation methods were used to compare young and aged brains, and there were fewer microtubule-associated proteins, especially tau, associated with the microtubules in advanced age, in all preparations.     

Intracortical perfusion of glutamate in vivo induces alterations of tau and microtubule-associated protein 2 immunoreactivity in the rat

Modifications of microtubule-associated proteins (MAP) have been reported in both acute and chronic degenerative conditions, such as cerebral ischaemia and Alzheimer’s disease, and may be associated with cytoskeletal breakdown. Glutamate excitotoxicity has been implicated in the pathogenesis of both of these conditions and has been shown in some in vitro studies to induce changes in tau similar to those occurring in Alzheimer’s disease. This study examines the effects of high extracellular glutamate concentrations on the distribution of tau and MAP2 in vivo in order to determine whether glutamate induces similar changes in tau to those previously reported in vitro in the intact, adult central nervous system. Monosodium glutamate was perfused into the rat parietal cortex for 90 min using in vivo microdialysis and at 4 h after the start of perfusion the distribution of tau and MAP2 was determined by immunohistochemistry. At the core of the glutamate-induced lesion tau immunostaining, as detected with the Tau 1 antibody, was decreased in axons and increased within perikarya compared to controls. Increased immunostaining was not apparent with polyclonal antibodies raised against full-length tau or towards the N or C termini of the protein. In contrast, increased tau immunoreactivity was detected, with all the antibodies used in this study, within oligodendrocytes following either glutamate or sodium chloride perfusion. MAP2 immunoreactivity was increased within perikarya at the core of the glutamate-induced lesion, while dendritic immunoreactivity was reduced. These results suggest that glutamate excitotoxicity in vivo may not be involved in neurofibrillary tangle formation but may be important in the progression of cytoskeletal pathology following cerebral ischaemia. Received: 9 May 1995 / Revised: 12 December 1995 / Accepted: 26 February 1996     

Effects of microtubule-associated protein tau expression on neural stem cell migration after spinal cord injury

Our preliminary proteomics analysis suggested that expression of microtubule-associated protein tau is elevated in the spinal cord after injury. Therefore, the first aim of the present study was to examine tau expression in the injured spinal cord. The second aim was to determine whether tau can regulate neural stem cell migration, a critical factor in the successful treatment of spinal cord injury. We established rat models of spinal cord injury and injected them with mouse hippocampal neural stem cells through the tail vein. We used immunohistochemistry to show that the expression of tau protein and the number of migrated neural stem cells were markedly increased in the injured spinal cord. Furthermore, using a Transwell assay, we showed that neural stem cell migration was not affected by an elevated tau concentration in the outer chamber, but it was decreased by changes in intracellular tau phosphorylation state. These results demonstrate that neural stem cells have targeted migration capability at the site of injury, and that although tau is not a chemokine for targeted migration of neural stem cells, intracellular tau phosphorylation/dephosphorylation can inhibit cell migration.     

Mapping of the Alz 50 epitope in microtubule-associated proteins tau.

Alz 50 and seven other monoclonal antibodies have been shown to react with both tau and Alzheimer brain proteins of molecular mass 60-70 kDa. The location of some of the epitopes of these antibodies (Alz 50, Tau-2, NP14, Ab 636.7) on the tau molecule is unknown, whereas those of others (Tau 60, Tau 14, Tau-1, Tau 46) have recently been demonstrated in fetal human tau at amino acid residues 60-72, 83-120, 131-140, and 315-352. To determine the location of the unknown epitopes, human tau was digested with chymotrypsin and trypsin, and the bovine microtubule fraction was incubated with chymotrypsin. Comparison of the immunoblots of chymotryptic digested tau with those of untreated preparations showed that the Alz 50 epitope was more sensitive than other tau epitopes to proteolysis. Cleavage of a 3-4 kDa polypeptide from the periphery of tau was sufficient to remove the Alz 50 epitope, but not the epitopes of Tau 46 (C-end) or Tau 60 (N-end). The distribution of the Alz 50 epitope in endogenously degraded, chymotrypsin or trypsin digested tau fragments was different from that of the Tau 46 epitope known to be located within 38 residues from the C-terminus of the tau molecule. Based on these observations Alz 50 epitope was considered to be located within 3-4 kDa of the N-terminus of tau. A comparison of immunoblots of different tau-reactive antibodies showed similarities between Tau 60 and Tau-2, and between Tau 14, Tau-1, NP14, and Ab 636.7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arc interacts with microtubules/microtubule-associated protein 2 and attenuates microtubule-associated protein 2 immunoreactivity in the dendrites

Arc, activity-regulated cytoskeleton-associated gene, is an immediate early gene, and its expression is regulated by a variety of stimuli, such as electric stimulation and methamphetamine. The function of Arc, however, is unknown. To explore this function, we carried out expression experiments by transfecting green fluorescent protein (GFP)-Arc constructs or by using a protein transduction system in hippocampal cultured neurons. We found that the overexpression of Arc as well as Arc induction by seizure in vivo decreased microtubule-associated protein 2 (MAP2) staining in the dendrites by immunocytochemistry, although MAP2 content was not changed on Western blot. Furthermore, Arc interacted with newly polymerized microtubules and MAP2, leading to blocking of the epitope of MAP2. The data suggest that Arc increased by synaptic activities would trigger dendritic remodeling by interacting with cytoskeletal proteins.     

Alzheimer's disease: beta-Amyloid protein and tau

Research on the molecular pathogenesis of Alzheimer's disease (AD) has made great strides over the last decade. This progress is the result of protein chemical analysis of two extracellular and intracellular fibrillary lesions in AD brain conducted during the 1980s, which identified beta-amyloid protein (A beta) and tau as their major components, respectively. Linkage analysis of familial AD identified four responsible genes: three causative genes (beta-amyloid precursor protein, presenilin 1, and presenilin 2) and one susceptibility gene (apolipoprotein E epsilon 4). All those genes causing and predisposing to AD exhibit a common phenotype: an increased production of A beta 42, a longer, more amyloidogenic A beta species, and/or its enhanced deposition. This observation was substantiated when presenilins were shown to be directly involved in A beta production. Whereas A beta deposition is relatively specific for AD, tau deposition is observed in various neurodegenerative diseases and is assumed to be intimately associated with neuronal loss. The genetic analysis of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) revealed the presence of mutations in the tau gene in affected members. Thus, tau can lead to intracellular tau deposits and neuronal loss, although the mechanism remains to be clarified. Taken together, A beta might exert neurotoxicity through tau, leading to neuronal loss in the AD brain.