A complex mechanism for inducer mediated tau polymerization

The accumulation of polymers of the microtubule associated protein tau is correlative with increased neurodegeneration in Alzheimer's disease and other related tauopathies. In vitro models have been developed in order to investigate molecular mechanisms that regulate the polymerization of tau. Arachidonic acid and heparin have been proposed to induce tau polymerization via a ligand dependent nucleation-elongation mechanism. However, certain aspects of these in vitro results are inconsistent with a classic nucleation-elongation mechanism. Using steady state and kinetic analyses of tau polymerization at a variety of protein and inducer concentrations, we have found that the thermodynamic barrier for nucleation in the presence of inducers is negligible, which was manifested by increases in protein polymerization at low tau concentrations and very rapid kinetics of polymerization. However, the mechanism of polymerization is complicated by the observation that high concentrations of inducer molecules result in the inhibition of tau fibril formation through different mechanisms for arachidonic acid and heparin. These observations indicate that the molar ratio of inducer to protein is a greater determinant of the rate and extent of tau polymerization than the concentration of tau itself. Our results are therefore not consistent with a canonical nucleation-elongation reaction but rather are more consistent with an allosteric regulation model in which the presence of small molecules induce a conformational change in the protein that decreases the thermodynamic barrier for polymerization essentially to zero.     

Differential assembly of human tau isoforms in the presence of arachidonic acid

Six tau isoforms arise from the alternative splicing of a single gene in humans. Insoluble, filamentous deposits of tau protein occur in a number of neurodegenerative diseases, and in some of these diseases, the deposition of polymers enriched in certain tau isoforms has been documented. Because of these findings, we have undertaken studies on the efficacy of fatty acid-induced polymerization of the individual tau isoforms found in the adult human CNS. The polymerization of each tau isoform in the presence of two concentrations of arachidonic acid indicated that isoforms lacking N-terminal exons e2 and e3 formed small, globular oligomers that did not go on to elongate into straight (SF) or paired helical (PHF) filaments under our buffer conditions. The polymerization of all isoforms containing e2 or e2 and e3 occurred readily at a high arachidonic acid concentration. Conversely, at a lower arachidonic acid concentration, only tau isoforms containing four microtubule binding repeats assembled well. Under all buffer conditions employed, filaments formed from three of the isoforms containing e2 and e3 resembled SFs in morphology but began to form PHF-like structures following extended incubation at 37 degrees C. These results indicate that polymerization of the intact tau molecule may be facilitated by e2 and e3. Moreover, tau isoforms containing three versus four microtubule binding repeats display different assembly properties depending on the solvent conditions employed.     

Tau phosphorylation at serine 396 and serine 404 by human recombinant tau protein kinase II inhibits tau's ability to promote microtubule assembly

In Alzheimer's disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly. Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimer's disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615-622). We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau. Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, we show that phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization. Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitro results suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules.     

Ligand-dependent tau filament formation: implications for Alzheimer's disease progression.

The mechanism through which arachidonic acid induces the polymerization of tau protein into filaments under reducing conditions was characterized through a combination of fluorescence spectroscopy and electron microscopy. Results show that polymerization follows a ligand-mediated mechanism, where binding of arachidonic acid is an obligate step preceding tau-tau interaction. Homopolymerization begins with rapid (on the order of seconds) nucleation, followed by a slower elongation phase (on the order of hours). Although essentially all synthetic filaments have straight morphology at early time points, they interact with thioflavin-S and monoclonal antibody Alz50 much like authentic paired helical filaments, suggesting that the conformation of tau protein is similar in the two filament forms. Over a period of days, synthetic straight filaments gradually adopt paired helical morphology. These results define a novel pathway of tau filament formation under reducing conditions, where oxidation may contribute to final paired helical morphology, but is not a necessary prerequisite for efficient nucleation or elongation of tau filaments.     

Two separate 18-amino acid domains of tau promote the polymerization of tubulin

Tau is a heat-stable microtubule-associated protein which promotes tubulin polymerization. The assembly promoting region of tau was localized using synthetic peptides modeled after domains found in both human and mouse tau. The design of these synthetic peptides was based on the triple repeat motif found in mouse tau. The first peptide, Tau-(187-204), and the second peptide, Tau-(218-235), are capable of promoting the polymerization of tubulin into microtubules, at concentrations above 100 microM. Two other peptides tested, TauR and Tau-(250-267), were not able to promote the assembly of tubulin over a range of concentrations up to 800 microM. TauR is a random analog of Tau-(187-204). Although TauR is unable to promote polymerization, it can modify Tau-(187-204)-induced tubulin assembly.     

In vitro polymerization of tau protein monitored by laser light scattering: method and application to the study of FTDP-17 mutants

Tau polymerization into the filaments that compose neurofibrillary tangles is seminal to the development of many neurodegenerative diseases. It is therefore important to understand the mechanisms involved in this process. However, a consensus method for monitoring tau polymerization in vitro has been lacking. Here we demonstrate that illuminating tau polymerization reactions with laser light and measuring the increased scattering at 90 degrees to the incident beam with a digital camera results in data that closely approximate the mass of tau polymer formation in vitro. The validity of the technique was demonstrated over a range of tau concentrations and through multiple angle scattering measurements. In addition, laser light scattering data closely correlated with quantitative electron microscopy measurements of the mass of tau filaments. Laser light scattering was then used to measure the efficiency with which the mutant tau proteins found in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) form filamentous structures. Several of these mutant proteins display enhanced polymerization in the presence of arachidonic acid, suggesting a direct role for these mutations in tau the filament formation that characterizes FTDP-17.     

The natural osmolyte trimethylamine N-oxide (TMAO) restores the ability of mutant tau to promote microtubule assembly

Coding region and intronic mutations in the gene for microtubule-associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Most coding region mutations effect a reduced ability of tau protein to interact with microtubules and lead to the formation of a filamentous pathology made of hyperphosphorylated tau. Here we show that trimethylamine N-oxide (TMAO) restores the ability of tau with FTDP-17 mutations to promote microtubule assembly. To mimic phosphorylation, serine and threonine residues in tau were singly or multiply mutated to glutamic acid, resulting in a reduced ability of tau to promote microtubule assembly. With the exception of the most heavily substituted protein (27 glutamic acid residues), TMAO increased the ability of mutant tau to promote microtubule assembly. However, it had no significant effect on heparin-induced assembly of tau into filaments.     

Molecular cloning and functional characterization of chicken brain tau: isoforms with up to five tandem repeats

Tau is a major microtubule-associated protein in mammalian brain, where it exists as multiple isoforms that are produced from a single gene by alternative mRNA splicing. Here we present the first report on the structure and function of tau protein from a nonmammalian vertebrate. In the adult chicken brain, five main tau isoforms are expressed. One isoform has three tandem repeats, two isoforms have four repeats each, and two isoforms have five repeats each. Similar to mammalian tau, some chicken tau isoforms contain an amino-terminal insert of 53 amino acids. Unlike mammalian tau, a 34 amino acid insert in the proline-rich region upstream of the repeats is alternatively spliced in chicken tau. It is preceded by a constitutively expressed sequence of 17 amino acids that is absent in tau from human and rodent brains. The expression of chicken tau isoforms and their phosphorylation are developmentally regulated, similar to what has been described in mammalian brain. Functionally, chicken tau isoforms with five repeats have the greatest ability to promote microtubule assembly, followed by isoforms with four and three repeats, respectively. The 34 amino acid insert positively influences both the rate and the extent of microtubule assembly, whereas the 53 amino acid insert only influences the extent of assembly.     

Regulation of microtubule protein levels during cellular morphogenesis in nerve growth factor-treated PC12 cells

Nerve growth factor induces neurite process formation in pheochromacytoma (PC12) cells and causes the parallel increase in levels of the microtubule-associated proteins, tau and MAP1, as well as increases in tubulin levels. Mechanisms to insure balanced accumulation of microtubule proteins and make their levels highly responsive to nerve growth factor were investigated. The effects on tau, MAP1, and tubulin are due to changes in protein synthesis rates, which for tau and tubulin we could show are due in part to changes in the mRNA levels. Whereas tubulin shows feedback regulation to modulate synthesis up or down, tau protein synthesis is not affected in a straightforward way by microtubule polymerization and depolymerization. The degradation of tau, MAP1, and both tubulin polypeptides, however, are stimulated by microtubule depolymerization caused by colchicine, or nerve growth factor removal. Combined feedback on synthesis and stability make tubulin levels highly responsive to assembly states. In addition, the linkage of tau and MAP1 turnover with the state of microtubule polymerization amplifies any change in their rate of synthesis, since tau and MAP1 promote microtubule polymerization. This linkage lends itself to rapid changes in the state of the system in response to nerve growth factor.     

Tau protein function in living cells

Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The injected tau protein increases tubulin polymerization and stabilizes microtubules against depolymerization. This increased polymerization does not, however, cause major changes in cell morphology or microtubule arrangement. Thus, tau protein acts in vivo primarily to induce tubulin assembly and stabilize microtubules, activities that may be necessary, but not sufficient, for neuronal morphogenesis.     

In vitro assembly of tau protein: mapping the regions involved in filament formation

Unraveling the mechanism of self-assembly of the protein tau into paired helical filaments (PHFs) is a crucial step toward the understanding of Alzheimer's and other neuropathological diseases at the molecular level. In an effort to map the role of different regions of tau in the mechanism of self-assembly, we have studied the polymerization ability of different tau fragments using an in vitro assay. Our results indicate that the N-terminal domain interferes with tau's ability to polymerize in vitro. The effect seems to be size dependent. Particularly, an isoform of tau from the peripheral nervous system, which has a much larger N-terminal domain, was found unable to form filaments in our in vitro assay. This finding can explain why in Alzheimer's patients PHFs only accumulate in the neurons from the central nervous system. We also report that a short segment of tau located in the third microtubule binding repeat (residues 317 to 335, peptide 1/2R) is probably the minimal segment of that region able to grow into filaments in vitro and in the presence of heparin. In contrast with whole peptide 1/2R, peptides corresponding to either the N-terminal or C-terminal halves of this segment were unable to form filaments. Finally, our polymerization studies of peptides from the C-terminal domain reveal a short sequence spanning residues 391 to 407 that grows into filaments in vitro. This tau segment forms filaments regardless of whether is incubated with heparin. Moreover, such filaments differ in diameter and morphology, suggesting a different mechanism of self-assembly.     

Contrasting roles of tau and microtubule-associated protein 2 in the vinblastine-induced aggregation of brain tubulin

Two different proteins, tau and microtubule-associated protein 2 (MAP 2), are able to stimulate tubulin polymerization into microtubules in vitro, but it is not certain if both proteins act by the same mechanism. We have examined the effects of tau and MAP 2 on the vinblastine-induced polymerization of tubulin into spiral filaments. In the presence of tau, vinblastine induced extensive aggregation of tubulin as shown by a large increase in turbidity. The increase in turbidity was accompanied by the formation of large numbers of spirals composed of a filament 40-60 A in diameter. The rate and extent of this aggregation into spirals were dependent on the concentrations of tubulin, tau, and vinblastine. Unlike normal microtubule assembly, this type of aggregation was not inhibited by colchicine or podophyllotoxin. In contrast, MAP 2, even at high concentrations, was less effective than tau at promoting the vinblastine-induced increase in turbidity of tubulin. In fact, MAP 2 strongly inhibited the effect of tau. These results indicate that tau and MAP 2 interact differently with the tubulin molecule in the presence of vinblastine and suggest that the two proteins may play different roles in regulating or promoting microtubule assembly. Vinblastine may thus be a useful probe in analyzing the modes of interactions of tau and MAP 2 with tubulin.     

Purification of tau,a microtubule-associated protein that induces assembly of microtubules from purified tubulin

Tau, a microtubule-associated protein which copurifies with tubulin through successive cycles of polymerization and depolymerization, has been isolated from tubulin by phosphocellulose chromatography and purified to near homogeneity. The purified protein is seen to migrate during electrophoresis on acrylamide gels as four closely spaced bands of apparent molecular weights between 55,000 and 62,000. Specific activity for induction of microtubule formation from purified tubulin has been assayed by quantitative electron microscopy and is seen to be enhanced three- to fourfold in the purified tau when compared with the unfractionated microtubule-associated proteins. Nearly 90% of available tubulin at 1 mg/ml is found to be polymerizable into microtubules with elevated levels of tau. Moreover, the critical concentration for polymerization of the reconstituted tau + tubulin system is seen to be a function of tau concentration and may be lowered to as little as 30 μg of tubulin per ml. Under depolymerizing conditions, 50% of the tubulin at only 1 mg/ml may be driven into ring structures. A separate purification procedure for isolation of tau directly from cell extracts has been developed and data from this purification suggest that tau is present in the extract in roughly the same proportion to tubulin as is found in microtubules purified by cycles of assembly and disassembly. Tau is sufficient for both nucleation and elongation of microtubules from purified tubulin and hence the reconstituted tau + tubulin system defines a complete microtubule assembly system under standard buffer conditions. In an accompanying paper (Cleveland et al., 1977) the physical and chemical properties of tau are discussed and a model by which tau may function in microtubule assembly is presented.     

Heparin-induced conformational change in microtubule-associated protein Tau as detected by chemical cross-linking and phosphopeptide mapping

In Alzheimer's disease, microtubule-associated protein tau becomes abnormally phosphorylated and aggregates into paired helical filaments. Sulfated glycosaminoglycans such as heparin and heparan sulfate were shown to accumulate in pretangle neurons, stimulate in vitro tau phosphorylation, and cause tau aggregation into paired helical filament-like filaments. The sulfated glycosaminoglycan-tau interaction was suggested to be the central event in the development of neuropathology in Alzheimer's disease brain (Goedert, M., Jakes, R., Spillantini, M. G., Hasegawa, M., Smith, M. J., and Crowther, R. A. (1996) Nature 383, 550-553). The biochemical mechanism by which sulfated glycosaminoglycans stimulate tau phosphorylation and cause tau aggregation remains unclear. In this study, disuccinimidyl suberate (DSS), a bifunctional chemical cross-linker, cross-linked tau dimers, tetramers, high molecular size aggregates, and two tau species of sizes 72 and 83 kDa in the presence of heparin. In the absence of heparin only dimeric tau was cross-linked by DSS. Fast protein liquid chromatography gel filtration revealed that 72- and 83-kDa species were formed by intramolecular cross-linking of tau by DSS. These observations indicate that heparin, in addition to causing aggregation, also induces a conformational change in tau in which reactive groups are unmasked or move closer leading to the DSS cross-linking of 72- and 83-kDa species. Heparin-induced structural changes in tau molecule depended on time of heparin exposure. Dimerization and tetramerization peaked at 48 h, whereas conformational change was completed within 30 min of heparin exposure. Heparin exposure beyond 48 h caused an abrupt aggregation of tau into high molecular size species. Heparin stimulated tau phosphorylation by neuronal cdc2-like kinase (NCLK) and cAMP-dependent protein kinase. Phosphopeptide mapping and phosphopeptide sequencing revealed that tau is phosphorylated by NCLK on Thr212 and Thr231 and by cAMP-dependent protein kinase on Ser262 only in the presence of heparin. Heparin stimulation of tau phosphorylation by NCLK showed dependence on time of heparin exposure and correlated with the heparin-induced conformational change of tau. Our data suggest that heparin-induced conformational change exposes new sites for phosphorylation within tau molecule.     

Cysteine oxidation of tau and microtubule-associated protein-2 by peroxynitrite: modulation of microtubule assembly kinetics by the thioredoxin reductase system

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative conditions including Alzheimer and Parkinson diseases. We report that peroxynitrite- and hydrogen peroxide-induced disulfides in the neuron-specific microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the ubiquitous thioredoxin reductase system composed of thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH. Tau and microtubule-associated protein-2 cysteine oxidation and reduction were quantitated by monitoring the incorporation of 5-iodoacetamidofluorescein, a thiol-specific labeling reagent. Cysteine oxidation of tau and microtubule-associated protein-2 to disulfides altered the ability of the proteins to promote the assembly of microtubules from purified porcine tubulin. Treatment of tau and microtubule-associated protein-2 with either the thioredoxin reductase system or small molecule reductants fully restores the ability of the MAPs to promote microtubule assembly. Thus changes in the redox state of microtubule-associated proteins may regulate microtubule polymerization in vivo.     

Inhibition of tau polymerization by its carboxy-terminal caspase cleavage fragment

Abnormal aggregation of the microtubule-associated protein, tau, occurs in many neurodegenerative diseases, making it important to understand the mechanisms of tau polymerization. Previous work has indicated that the C-terminal region of tau inhibits polymerization in vitro, and a growing body of evidence implicates caspase cleavage of tau at Asp 421 in the C-terminus as an important inducer of tau polymerization in Alzheimer's disease. In the present study, we provide evidence that the C-terminal peptide fragment produced by caspase cleavage inhibits tau polymerization, suggesting that caspase cleavage of tau enhances its polymerization by removing the inhibitory control element. Moreover, we provide evidence that the peptide assumes an alpha-helical configuration and inhibits tau assembly by interacting with residues 321-375 in the microtubule binding repeat region. These findings indicate that formation of the fibrillar pathologies during the course of Alzheimer's disease may be driven or sustained by apoptotic events leading to caspase activation.     

Role of cysteine-291 and cysteine-322 in the polymerization of human tau into Alzheimer-like filaments.

Filamentous tau pathology is central to a large number of dementing disorders, including Alzheimer's disease in which polymerized tau is hyperphosphorylated. Previous studies on heparin-dependent tau polymerization, using recombinant tau isoforms lacking Cys-291, suggest that tau dimerization via Cys-322 is critical for initiation of assembly of soluble tau into filaments. We report heparin-dependent in vitro polymerization of human recombinant tau (1-383 isoform), containing both Cys-291 and Cys-322, into paired helical filaments as characterized by electron microscopy. Tau polymerization, under physiological tau concentrations in the presence of dithiothreitol (DTT), was followed by a Thioflavine S fluorescence assay. To understand the molecular basis for heparin-induced tau polymerization, we expressed and purified C291A, C322A, and C291A/C322A tau mutants. The DTT requirement for tau polymerization was abolished using either the C291A or C322A tau mutant and polymerization was not observed with the C291A/C322A tau double mutant. Analysis by sodium dodecyl sulfate gel electrophoresis showed that, unlike wild type tau, a significant amount of the C291A mutant and the C322A mutant is present as a disulfide bonded dimer. Taken together these results suggest that, in isoforms containing both Cys-291 and Cys-322, a dimeric tau with an intermolecular disulfide bond through either Cys-291 or Cys-322 is presumably acting as a seed for initiation of tau polymerization.     

Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly.

This paper describes the physical and chemical properties of purified tau, a protein which is associated with brain microtubules and which induces assembly of microtubules from tubulin. Purified tau is composed of four polypeptides which migrate at positions equivalent to molecular weights between 55,000 and 62,000 during electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. These polypeptides are shown to be closely related by peptide mapping and by amnio acid analysis. A comparison by various techniques of the high molecular weight microtubule-associated proteins with the tau polypeptides indicates no apparent relationship. Tau is found by analytical ultracentrifugation and by sedimentation equilibrium to have a sedimentation coefficient of 2.6 S and a native molecular weight of 57,000. Tau, therefore, must be highly asymmetric (an axial ratio of 20:1 using a prolate ellipsoid model), and yet possess little α-helical structure as indicated by circular dichroism. Isoelectric focusing shows tau to be a neutral or slightly basic protein. Tau is also seen to be phosphorylated by a protein kinase which copurifies with microtubules.In the assembly process, tau apparently regulates the formation of longitudinal oligomers from tubulin dimers, and hence promotes ring formation under depolymerizing conditions and microtubule formation under polymerizing conditions. The known asymmetry of the tau molecule suggests that tau induces assembly by binding to several tubulin molecules per tau molecule, thereby effectively increasing the local concentration of tubulin and inducing the formation of longitudinal filaments. The role of tau is discussed in light of reports of polymerization induced by particular non-physiological conditions and by various polycations. The formation of normal microtubules over a wide range of tubulin and tau concentrations under mild buffer conditions suggests that tau and tubulin define a complete in vitro assembly system under conditions which approach physiological.     

Relationship between lipemia and sensitivity to arachidonic acid in rabbits]

The hypotensive effect of arachidonic acid in the rabbit increases simultaneously with the fall of the plasmatic unesterified fatty acids (NEFA), after treatment with nicotinic acid or heparin. In the case of nicotinic acid sensitization, which occurs in 10 minutes, the fall of NEFA occurs immediately. The sensitization by heparin appears only after 40 minutes following the injection; during this latency, the NEFA are enhanced; after the fall of NEFA, the rabbit becomes more sensitive to arachidonic acid. Mechanisms of heparinic sensitization are discussed.     

Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies

The intracellular polymerization of cytoskeletal proteins into their supramolecular assemblies raises many questions regarding the regulatory patterns that control this process. Binding experiments using the ELISA solid phase system, together with protein assembly assays and electron microscopical studies provided clues on the protein-protein associations in the polymerization of tubulin and actin networks. In vitro reconstitution experiments of these cytoskeletal filaments using purified tau, tubulin, and actin proteins were carried out. Tau protein association with tubulin immobilized in a solid phase support system was inhibited by actin monomer, and a higher inhibition was attained in the presence of preassembled actin filaments. Conversely, tubulin and assembled microtubules strongly inhibited tau interaction with actin in the solid phase system. Actin filaments decreased the extent of in vitro tau-induced tubulin assembly. Studies on the morphological aspects of microtubules and actin filaments coexisting in vitro, revealed the association between both cytoskeletal filaments, and in some cases, the presence of fine filamentous structures bridging these polymers. Immunogold studies showed the association of tau along polymerized microtubules and actin filaments, even though a preferential localization of labeled tau with microtubules was revealed. The studies provide further evidence for the involvement of tau protein in modulating the interactions of microtubules and actin polymers in the organization of the cytsokeletal network.