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.     

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.     

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.     

Toward a unified scheme for the aggregation of tau into Alzheimer paired helical filaments

Alzheimer's disease is characterized by aggregates of tau protein. Attempts to study the conditions for aggregation in vitro have led to different experimental systems, some of which appear mutually exclusive (e.g., oxidative vs reductive conditions, induction by polyanions vs fatty acids). We show here that different approaches and pathways can be viewed in a common framework, and that apparent differences can be explained by variations in the kinetics of subreactions. A unified view of PHF aggregation should help to analyze the causes of PHF aggregation and devise methods to prevent it.     

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.     

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.     

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.     

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.     

Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro

Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood. Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli. All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking. The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding. One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores. Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue. A similar self-assembly occurs with the chemically cross-linked dimers. In both cases there is no need for phosphorylation of the protein.     

A distinct form of tau is selectively incorporated into Alzheimer's paired helical filaments

Tau, a microtubule-associated phosphoprotein, was identified as a definite component of paired helical filaments which progressively accumulate in Alzheimer's disease brain. To learn more about tau in the aged brain, we have isolated and sequenced a cDNA clone encoding tau from a cDNA library of an aged human brain. The cloned cDNA sequence included a new insert of 93 nucleotides, which added a fourth repeat to the three-repeat type of tau already reported. Perhaps, this four-repeat type of tau is predominant in normal aged brain. In contrast, the sequence analysis of paired helical filaments showed that the integrated tau is of three-repeat type. This indicates that a distinct form of tau is selectively incorporated into paired helical filaments.     

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.     

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.     

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimer's disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of beta-structure around the hexapeptide motif PHF6 ((306)VQIVYK(311)) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif ((275)VQIINK(280)) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of beta-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, DeltaK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for beta-structure around the hexapeptide motifs.     

Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments

One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. We have studied the relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs. The proline-directed kinases MAPK and GSK3 are known to phosphorylate most Ser-Pro or Thr-Pro motifs in the regions flanking the repeat domain of tau: they induce the reaction with several antibodies diagnostic of Alzheimer PHFs, but this type of phosphorylation has only a weak effect on tau-microtubule interactions and on PHF assembly. By contrast, MARK and PKA phosphorylate several sites within the repeats (notably the KXGS motifs including Ser262, Ser324, and Ser356, plus Ser320); in addition PKA phosphorylates some sites in the flanking domains, notably Ser214. This type of phosphorylation strongly reduces tau's affinity for microtubules, and at the same time inhibits tau's assembly into PHFs. Thus, contrary to expectations, the phosphorylation that detaches tau from microtubules does not prime it for PHF assembly, but rather inhibits it. Likewise, although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly. This implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylation protects tau against aggregation.     

Tau-related protein present in paired helical filaments has a decreased tubulin binding capacity as compared with microtubule-associated protein tau

We have isolated, after exhaustive detergent treatments, a 33 kDa tau-related protein isolated from paired helical filaments from Alzheimer's disease patient brains. The N-terminal sequence of the 33 kDa protein begins at residue 71 of the sequence described for human fetal tau protein. This truncated form of tau is not the consequence of the translation of a tau RNA lacking a region at its 5' end, as measured by primer extension analyses, suggesting that the 33 kDa protein must be generated by proteolysis of previously synthesized tau. This tau-related protein has only one blocked cysteine residue and also has a decreased tubulin binding capacity as compared with that of tau protein.     

Deamidation and isoaspartate formation in smeared tau in paired helical filaments. Unusual properties of the microtubule-binding domain of tau

An extensive loss of a selected population of neurons in Alzheimer's disease is closely related to the formation of paired helical filaments (PHFs). The most striking characteristic of PHFs upon Western blotting is their smearing. According to a previously described protocol (Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1993) Neuron 10, 1151-1160), smeared tau was purified, and its peptide map was compared with that of soluble (normal) tau. A CNBr fragment from soluble tau (CN5; residues 251-419 according to the 441-residue isoform) containing the microtubule-binding domain migrated at 15 and 18 kDa on SDS-polyacrylamide gel electrophoresis, whereas that from smeared tau exhibited two larger, unusually broad bands at approximately 30 and approximately 45 kDa, presumably representing dimers and trimers of CN5. In the peptide map of smeared tau-derived CN5, distinct peaks eluting at unusual locations were noted. Amino acid sequence and mass spectrometric analyses revealed that these distinct peptides bear isoaspartate at Asn-381 and Asp-387. Because no unusual peptides other than aspartyl or isoaspartyl peptide were found in the digests of smeared tau-derived CN5, it is likely that site-specific deamidation and isoaspartate formation are involved in its dimerization and trimerization and thus in PHF formation in vivo.     

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.     

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.     

Structural stability of paired helical filaments requires microtubule-binding domains of tau: a model for self-association.

Highly purified and SDS-soluble paired helical filaments (PHFs) were immunogold labeled and immunoblotted with antibodies to tau: Tau 14 (N-terminal half), AH-1 (microtubule-binding domain), and Tau 46 (C-terminal end). The main component of PHFs was modified tau of 68, 64, and 60 kd, also called A68 or PHF-tau. Trypsin digestion reduced the maximum width of PHFs by 10%-20%, increased aggregation of filaments, and abolished the binding of Tau 14, but had no effect on the binding of AH-1. The smallest tau-reactive tryptic fragments were 13 and 7-8 kd, positive with AH-1, and negative with Tau 46. Our results and the model of Crowther and Wischik suggest that by self-association and anti-parallel arrangement of the microtubule-binding domains, PHF-tau forms the backbone of PHFs.