Tau factor polymers are similar to paired helical filaments of Alzheimer's disease

Tau factor, upon urea treatment, is able to polymerize in vitro. These polymers are composed of tau factor as shown by immunogold staining. The structure of tau polymers is very similar to that of paired helical filaments (PHFs) of Alzheimer's disease in their dimensions as well as in their periodicity. Metal shadowing of both polymers shows a similar twisting. Also, similar peptide maps were found for tau factor and a 33 kDa protein that is the main component of our PHF preparations.     

Tau paired helical filaments from Alzheimer's disease brain and assembled in vitro are based on beta-structure in the core domain

Tau protein, a neuronal microtubule-associated protein, forms insoluble fibers ("paired helical filaments") in Alzheimer's disease and other tauopathies. Conflicting views on the structure of the fibers have been proposed recently, ranging from mainly alpha-helical structure to mainly beta-sheet, or a mixture of mostly random coil and beta-sheet. We have addressed this issue by studying tau fibers immunopurified from Alzheimer brain tissue by a conformation-specific antibody and comparing them with fibers reassembled from recombinant tau or tau constructs in vitro, using a combination of electron microscopy and spectroscopic methods. Brain-derived fibers and reassembled fibers both exhibit a typical twisted appearance when examined by electron microscopy. The soluble tau protein is a natively unfolded protein dominated by random coil structure, whereas Alzheimer PHFs and reassembled fibers show a shift toward an increase in the level of beta-structure. The results support a model in which the repeat domain of tau (which lies within the core of PHFs) adopts an increasing level of beta-structure during aggregation, whereas the N- and C-terminal domains projecting away from the PHF core are mostly random coil.     

Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms.

Preparations of dispersed paired helical filaments (PHFs) from the brains of Alzheimer's disease and Down's syndrome patients display on gels three principal bands corresponding to abnormally modified forms of the microtubule-associated protein tau. Interpretation of the pattern is difficult because there are six tau isoforms in normal brain and phosphorylation changes their mobility. By enzymatic dephosphorylation at high temperature, we have shifted the three abnormal bands obtained from dispersed PHFs to align with the six nonphosphorylated tau isoforms. By using antibodies specific for some of the inserts that distinguish the various isoforms and label PHFs, we have established a correspondence between PHFs, abnormal bands, and isoforms. This identification of isoforms is a necessary step in unravelling the molecular pathogenesis of PHFs.     

Proteolytic fragments of Alzheimer's paired helical filaments

We found that at least a part of Alzheimer's paired helical filaments (PHF) was cleaved by proteases to release three major polypeptides, the apparent molecular weights of which were 10K, 26K, and 36K daltons. These proteolytic fragments were strongly labeled with the antibodies specific to PHF. Absorption with highly purified PHF eliminated this labeling. The monospecific antibodies bound to the 10K daltons protein, the most intensely immunolabeled one, stained isolated neurofibrillary tangles composed of PHF. From these observations, we conclude that these polypeptides released by proteases, especially the 10K daltons protein, are derived from PHF themselves.     

Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments.

From bovine brain microtubules we purified tau protein kinase I (TPKI, Mr 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tau protein kinase II (TPKII) whose activity was attributed to a 30-kDa protein on SDS-PAGE by affinity-labeling using an ATP analog. Both kinases were activated by tubulin. TPKII, but not TPKI, phosphorylated tau fragment peptides previously used for detection of a Ser/ThrPro kinase activity. Therefore, TPKII was considered to be the Ser/ThrPro kinase. TPKI was more effective than TPKII for producing the decrease of tau-1 immunoreactivity and mobility shift of tau on SDS-PAGE. Moreover, TPKI, but not TPKII nor other well-known protein kinases, generated an epitope present on paired helical filaments. These findings suggested that tau phosphorylated by TPKI resembled A-68, a component of paired helical filaments.     

Alzheimer''s paired helical filaments share epitopes with neurofilament side arms.

A panel of monoclonal antibodies to neurofilaments have been investigated with regard to the location of their respective epitopes on neurofilament polypeptides and their ability to label the neurofibrillary tangles and paired helical filaments (PHF) which are characteristic of Alzheimer's disease. All of the neurofilament monoclonal antibodies that label tangles and PHF are directed against epitopes in the side arm domains of the two larger neurofilament polypeptides, NF-H and NF-M, and do not recognise the alpha-helical rod domains of these proteins. Immuno-electron microscopy demonstrates that the neurofilament antibodies label the constituent PHF per se and do not simply stain neurofilaments that might be admixed with PHF. These neurofilament epitopes are differentially retained by PHF, following isolation. Thus, antibody labelling of PHF is not simply due to the presence of normal neurofilament polypeptides. We propose that in tangle-bearing neurons, neurofilaments are degraded by proteases and that it is fragments of the side arms which contribute to the composition of PHF.     

Subunit structure of paired helical filaments in Alzheimer''s disease

The neurofibrillary tangles that occur in the brain in cases of senile dementia of the Alzheimer type contain a distinctive type of filament, the paired helical filament (PHF). We have developed a method for isolating the tangles postmortem in sufficient yield for structural study of PHFs by electron microscopy of negatively stained and shadowed preparations. This material shows the characteristic helical structure seen in sectioned embedded material. In addition, two striking fragmentation patterns are observed. (a) Some filaments show sharp transverse breaks at apparently random positions along the filament. (b) In a few PHFs one strand is missing for a variable length, whereas the other appears to maintain its structural integrity. The shadowed specimens show the PHF to be wound in a left-handed manner. These observations indicate that the PHF consists of subunits of very limited axial extent arranged along two left-handed helical strands. The visualization of the substructure within the PHFs is rather variable and a model building approach has therefore been adopted, which has allowed the main features seen in the images to be interpreted. The subunit appears to have at least two domains in a radial direction and an axial extent of less than 5 nm. The whole structure can best be described as a twisted ribbon and indeed alkali treatment does untwist PHFs to give flat ribbons. The nature of the proposed model makes it most unlikely that the PHF is formed by a simple collapse of normal cytoskeletal elements, such as neurofilaments.     

The carboxyl third of tau is tightly bound to paired helical filaments

To obtain definitive evidence that tau is a component of paired helical filaments (PHF) in Alzheimer's disease, we fractionated and sequenced PHF-derived peptides according to a previously described procedure. In the PHF digest, we found four independent tau peptides that were located in the carboxyl third of tau. Subsequent extensive analysis of the PHF digest did not provide any other tau peptides. The conventional PHF antiserum and a new antiserum directed toward formic acid-denatured PHF reacted with the distinct CNBr fragments of tau localized on the carboxy-terminal portion of tau by protein sequencing. From these observations, we conclude that the carboxyl third of tau is tightly bound to PHF.     

Structure of tau protein and assembly into paired helical filaments

Over the past few years the systematic investigation of paired helical filament assembly from tau protein in vitro has become feasible. We review our current understanding of the structure and conformations of tau protein and how this affects tau's assembly into the pathological paired helical filaments in Alzheimer's disease.     

Epitopes that span the tau molecule are shared with paired helical filaments

Tau protein has been shown to be an integral component of Alzheimer paired helical filaments (PHF). However, the extent to which tau is incorporated into PHF has not been clear because the antibodies used to label PHF generally do not have precisely defined epitopes. Here we define the antigenic sites for five monoclonal antibodies that react with tau and cross-react with SDS-extracted neurofibrillary tangles. The reactive sites were determined by screening a lambda gt11 sublibrary expressing small fragments of the tau sequence. The mapped epitopes were found to span almost the entire length of tau, suggesting that PHF contains tau in its entirety or nearly in its entirety. One antibody was found to cross-react with microtubule-associated protein 2, implying some degree of homology between the two proteins.     

beta-Amyloid induces paired helical filament-like tau filaments in tissue culture

Paired helical filaments (PHF) are the principal pathologic components of neurofibrillary tangles in Alzheimer's disease (AD). To reproduce the formation of PHF in tissue culture, we stably expressed human tau with and without pathogenic mutations in human SH-SY5Y cells and exposed them for 5 days to aggregated synthetic beta-amyloid peptide (A beta 42). This caused a decreased solubility of tau along with the generation of PHF-like tau-containing filaments. These were 20 nm wide and had periodicities of 130-140 nm in the presence of P301L mutant tau or 150-160 nm in the presence of wild-type tau. Mutagenesis of the phosphoepitope serine 422 of tau prevented both the A beta 42-mediated decrease in solubility and the generation of PHF-like filaments, suggesting a role of serine 422 or its phosphorylation in tau filament formation. Together, our data underscore a role of A beta 42 in the formation of PHF-like filaments. Our culture system will be useful to map phosphoepitopes of tau involved in PHF formation and to identify and characterize modifiers of the tau pathology. Further adaptation of the system may allow the screening and validation of compounds designed to prevent PHF formation.     

The conformations of filamentous and soluble tau associated with Alzheimer paired helical filaments

Paired helical filaments (PHF) occur in Alzheimer's diseased brains and are known to be composed of the microtubule-associated protein, tau. In the present report, circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM) were used to characterize PHF suspended in Tris-buffered saline (TBS), sodium acetate buffer, and water. In TBS the CD spectrum of PHF was observed to have a spectral pattern consistent with 31-37% alpha-helix, 15-20% beta-sheet, 20-23% turn, and 26-29% unordered structure. The TBS sample was found to undergo a cooperative thermal transition between 70 and 75 degrees C, consistent with the changes observed in filament morphology, and it suggests that filamentous tau in the PHF (PHF-tau) makes a substantial contribution to the overall CD. Observed changes in the CD spectrum following removal of PHF by centrifugation suggest that PHF-tau possesses a higher fraction of alpha-helical structure than soluble tau. In acetate buffer, where only straight filaments were observed, the CD was consistent with a marked decrease in the fraction of alpha-helix and an increase in the fraction of beta-sheet relative to the sample in TBS. In water, where only rudimentary filaments remain, the CD was consistent with a Type II or II' beta-turn conformation. Only noncooperative thermal transitions were observed for the PHF samples in acetate buffer and water, consistent with the presence of a heterogeneous population of folded structures. Taken cumulatively, the results are consistent with immunological data showing the presence of folded forms of tau and suggest that phosphorylation or nonproteinaceous components are able to induce conformations of tau other than the random coil conformation previously reported for cloned or purified human tau.     

Microtubule-associated protein tau. A component of Alzheimer paired helical filaments

Microtubule-associated protein tau was purified from bovine brain microtubules by either (1) phosphocellulose chromatography, (2) heat treatment at pH 6.4, (3) heat treatment at pH 2.7, (4) heat treatment at pH 2.7 followed by extraction with perchloric acid and precipitation with glycerol, or (5) by precipitation with ammonium sulfate followed by extraction with perchloric acid. All of these tau preparations reacted specifically with antibodies to Alzheimer paired helical filaments. Affinity purified antibodies to tau labeled both Alzheimer neurofibrillary tangles and plaque neurites but not amyloid in Alzheimer brain tissue sections and labeled paired helical filament polypeptides on Western blots. Human brain tau and paired helical filament polypeptides co-migrated on sodium dodecyl sulfate-polyacrylamide gels. These results suggest that tau is a major component of Alzheimer paired helical filaments.     

Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease

The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation.     

One of the antigenic determinants of paired helical filaments is related to tau protein

Paired helical filaments (PHF) are unusual neuronal fibers which accumulate progressively in the brain in Alzheimer's disease (AD). The insolubility of PHF in various kinds of solvents enabled us to obtain highly purified PHF, but prevented the application of conventional analytical methods to identify their components. Here we report that antibodies against purified PHF recognize tau protein, a brain-specific microtubule-associated protein, suggesting that a portion of PHF is tau protein.     

The natively unfolded character of tau and its aggregation to Alzheimer-like paired helical filaments

The abnormal aggregation of the microtubule-associated protein Tau into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer disease (AD). Tau in solution behaves as a natively unfolded or intrinsically disordered protein while its aggregation is based on the partial structural transition from random coil to beta-structure. Our aim is to understand in more detail the unfolded nature of Tau, to investigate the aggregation of Tau under different conditions and the molecular interactions of Tau in filaments. We show that soluble Tau remains natively unfolded even when its net charge is minimized, in contrast to other unfolded proteins. The CD signature of the random-coil character of Tau shows no major change over wide variations in charge (pH), ionic strength, solvent polarity, and denaturation. Thus there is no indication of a hydrophobicity-driven collapse, neither in the microtubule-binding repeat domain constructs nor in full-length Tau. This argues that the lack of hydrophobic residues but not the net charge accounts for unfolded nature of soluble Tau. The aggregation of the Tau repeat domain (that forms the core of PHFs) in the presence of nucleating polyanionic cofactors (heparin) is efficient in a range of buffers and pH values between approximately 5 and 10 but breaks down beyond that range, presumably because the pattern of charged interactions disappears. Similarly, elevated ionic strength attenuates aggregation, and the temperature dependence is bell-shaped with an optimum around 50 degrees C. Reporter dyes ThS and ANS record the aggregation process but sense different states (cross-beta-structure vs hydrophobic pockets) with different kinetics. Preformed PHFs are surprisingly labile and can be disrupted by denaturants at rather low concentration ( approximately 1.0 M GdnHCl), much less than required to denature globular proteins. Partial disaggregation of Tau filaments at extreme pH values monitored by CD and EM indicate the importance of salt bridges in filament formation. In contrast, Tau filaments are remarkably resistant to high temperature and high ionic strength. Overall, the stability of PHFs appears to depend mainly on directed salt bridges with contributions from hydrophobic interactions as well, consistent with a recent structural model of the PHF core derived from solid state NMR (Andronesi, O. C., von Bergen, M., Biernat, J., Seidel, K., Griesinger, C., Mandelkow, E., and Baldus, M. (2008) Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy.     

Association of the carboxy-terminus of beta-amyloid protein precursor with Alzheimer paired helical filaments.

We investigated whether a peptide fragment from the C-terminus of beta-amyloid protein precursor is associated with Alzheimer paired helical filaments (PHFs). Antiserum BR188, to the last 20 amino acids of the precursor, did not cross-react with tau protein, known to be in PHFs. It did react with all five pronase-treated PHF preparations assayed by ELISA and immunogold-labelled the same PHF fibrils that a PHF-specific tau antibody labelled. Neither antibody labelled beta/A4 fibrils. These results suggest that a fragment from the C-terminus of beta-amyloid precursor protein copurifies with pronase-treated PHFs and may play a role in their molecular pathogenesis.     

Residual structure in the repeat domain of tau: echoes of microtubule binding and paired helical filament formation

The microtubule-associated protein tau is found aggregated into paired helical filaments in the intraneuronal neurofibrillary tangle deposits of victims of Alzheimer's disease (AD) and other related dementias. Tau contains a repeat domain consisting of three or four 31-32-residue imperfect repeats that forms the core of tau filaments and is capable of self-assembling into filaments in vitro. We have used high-resolution NMR spectroscopy to characterize the structural properties of the three-repeat domain of tau at the level of individual residues. We find that three distinct regions of the polypeptide corresponding to previously mapped microtubule interaction sites exhibit a preference for helical conformations, suggesting that these sites adopt a helical structure when bound to microtubules. In addition, we directly observe a marked preference for extended or beta-strand-like conformations in a stretch of residues between two of the helical regions, which corresponds closely to a region previously implicated as an early site of beta-strand structure formation and intermolecular interactions leading to paired helical filament (PHF) formation. This observation supports the idea that this region of the protein plays a crucial role in the formation of tau aggregates. We further show that disulfide-bond-mediated dimer formation does not affect and is not responsible for the observed structural preferences of the protein. Our results provide the first high-resolution view of the structural properties of the protein tau, are consistent with an important role for beta structure in PHF formation, and may also help explain recent reports that tau filaments contain helical structure.     

Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease

Antisera to paired helical filaments (PHF) were found to contain a significant amount of tau antibodies specific for a phosphorylated form, but only a negligible amount of those specific for a non-phosphorylated form. Also, the phosphorylated tau-specific antibodies, but not the non-phosphorylated tau-specific ones, labeled neurofibrillary tangles isolated in the presence of sodium dodecyl sulfate (SDS) and stained both tangles and senile plaque neuritis in fixed tissue sections in a very similar way to as the whole antiserum did. Taken together, these results strongly suggest that a major antigenic determinant of PHF is phosphorylated tau itself.