Molecular characterization of the minimal protease resistant tau unit of the Alzheimer's disease paired helical filament.

The Alzheimer's disease paired helical filament (PHF), after digestion with Pronase, retains its characteristic morphological features. We term this the protease resistant core PHF. A 12 kDa tau fragment can be released from the core as an essentially pure preparation. Sequence analysis of this fragment revealed six distinct N-termini beginning in the repeat region of tau. The precise C-terminus is unknown, but the fragment is approximately 100 residues long. A monoclonal antibody, mAb 423, which recognizes the core PHF and the 12 kDa tau fragment, does not recognize normal full-length tau. We describe cDNA synthesis and expression of candidate 12 kDa tau analogues which permit the mapping of the mAb 423 epitope. mAb 423 recognizes all and only those analogues which terminate at Glu391, which lies beyond the homology repeat region. Addition or removal of a single residue at the C-terminus abolishes immunoreactivity. Therefore, mAb 423, together with knowledge of the N-terminus, can be used to measure the precise extent of 12 kDa PHF core tau fragment which we term the minimal protease resistant tau unit of the core PHF. This unit is 93-95 residues long, which is equivalent to three repeats, but is 14-16 residues out of phase with respect to the maximum homology organization of the repeat region. mAb 423 labels isolated PHFs prior to Pronase digestion and intracellular granular and neurofibrillary degeneration in Alzheimer's disease tissues. The constraints which determine endogenous truncation at Glu391 appear to be characteristic of an assembled configuration of tau, either within the PHF or its precursor.     

The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis

In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.     

Structural and functional differences between 3-repeat and 4-repeat tau isoforms. Implications for normal tau function and the onset of neurodegenetative disease

Tau, MAP2, and MAP4 are members of a microtubule-associated protein (MAP) family that are each expressed as "3-repeat" and "4-repeat" isoforms. These isoforms arise from tightly controlled tissue-specific and/or developmentally regulated alternative splicing of a 31-amino acid long "inter-repeat:repeat module," raising the possibility that different MAP isoforms may possess some distinct functional capabilities. Consistent with this hypothesis, regulatory mutations in the human tau gene that disrupt the normal balance between 3-repeat and 4-repeat tau isoform expression lead to a collection of neurodegenerative diseases known as FTDP-17 (fronto-temporal dementias and Parkinsonism linked to chromosome 17), which are characterized by the formation of pathological tau filaments and neuronal cell death. Unfortunately, very little is known regarding structural and functional differences between the isoforms. In our previous analyses, we focused on 4-repeat tau structure and function. Here, we investigate 3-repeat tau, generating a series of truncations, amino acid substitutions, and internal deletions and examining the functional consequences. 3-Repeat tau possesses a "core microtubule binding domain" composed of its first two repeats and the intervening inter-repeat. This observation is in marked contrast to the widely held notion that tau possesses multiple independent tubulin-binding sites aligned in sequence along the length of the protein. In addition, we observed that the carboxyl-terminal sequences downstream of the repeat region make a strong but indirect contribution to microtubule binding activity in 3-repeat tau, which is in contrast to the negligible effect of these same sequences in 4-repeat tau. Taken together with previous work, these data suggest that 3-repeat and 4-repeat tau assume complex and distinct structures that are regulated differentially, which in turn suggests that they may possess isoform-specific functional capabilities. The relevance of isoform-specific structure and function to normal tau action and the onset of neurodegenerative disease are discussed.     

Structural transitions in tau k18 on micelle binding suggest a hierarchy in the efficacy of individual microtubule‐binding repeats in filament nucleation

The protein tau is found in an aggregated filamentous state in the intraneuronal paired helical filament deposits characteristic of Alzheimer's disease and other related dementias and mutations in tau protein and mRNA cause frontotemproal dementia. Tau isoforms include a microtubule‐binding domain containing either three or four imperfect tandem microtubule binding repeats that also form the core of tau filaments and contain hexapaptide motifs that are critical for tau aggregation. The tau microtubule‐binding domain can also engage in direct interactions with detergents, fatty acids, or membranes, which can greatly facilitate tau aggregation and may also mediate some tau functions. Here, we show that the alternatively spliced second microtubule‐binding repeat exhibits significantly different structural characteristics compared with the other three repeats in the context of the intact repeat domain. Most notably, the PHF6* hexapeptide motif located at the N‐terminus of repeat 2 has a lower propensity to form strand‐like structure than the corresponding PHF6 motif in repeat 3, and unlike PHF6 converts to partially helical structure in the micelle‐bound state. Interestingly, the behavior of the Module‐B motif, located at the beginning of repeat 4, resembles that of PHF6* rather than PHF6. Our observations, combined with previous results showing that PHF6* and Module‐B are both less effective than PHF6 in nucleating tau aggregation, suggest a hierarchy in the efficacy of these motifs in nucleating tau aggregation that originates in differences in their intrinsic propensities for extended strand‐like structure and the resistance of these propensities to changes in tau's environment.     

Molecular cloning of XTP, a tau-like microtubule-associated protein from Xenopus laevis tadpoles

The microtubules of the mammalian nervous system are stabilised by several microtubule-associated proteins (MAPs), including the tau and MAP-2 protein families. The most prominent feature of mammalian tau and MAP-2 proteins is a common and highly homologous microtubule-binding region consisting of three or four imperfect tandem repeats. In this paper we report the cloning and characterisation of a Xenopus laevis tau-like protein (XTP) from tadpole tails. This protein encompasses two isoforms of 673 or 644 amino acids with four tandem repeats that are highly homologous to mammalian tau repeats. Both isoforms share a common amino terminal half, whereas the carboxyl terminus downstream of the repeat region is unique for each isoform. Northern blot analysis revealed that both isoforms are preferentially expressed in the tail of X. laevis tadpoles, whereas a shorter version of XTP is expressed in the head. Recombinant proteins of both XTP isoforms were able to bind microtubules. The longest isoform, however, was more effective at promoting tubulin polymerisation, indicating that sequences downstream of the repeat region affect the microtubule assembling capacity. These results demonstrate that tau-like proteins are found in non-mammalian vertebrate species, where they may support the stability of microtubules.     

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.     

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.     

Modulation of microtubule dynamics by tau in living cells: implications for development and neurodegeneration

The neural microtubule-associated protein tau binds to and stabilizes microtubules. Because of alternative mRNA splicing, tau is expressed with either 3 or 4 C-terminal repeats. Two observations indicate that differences between these tau isoforms are functionally important. First, the pattern of tau isoform expression is tightly regulated during development. Second, mutation-induced changes in tau RNA splicing cause neuronal cell death and dementia simply by altering the isoform expression ratio. To investigate whether 3- and 4-repeat tau differentially regulate microtubule behavior in cells, we microinjected physiological levels of these two isoforms into EGFP-tubulin-expressing cultured MCF7 cells and measured the effects on the dynamic instability behavior of individual microtubules by time-lapse microscopy. Both isoforms suppressed microtubule dynamics, though to different extents. Specifically, 4-repeat tau reduced the rate and extent of both growing and shortening events. In contrast, 3-repeat tau stabilized most dynamic parameters about threefold less potently than 4-repeat tau and had only a minimal ability to suppress shortening events. These differences provide a mechanistic rationale for the developmental shift in tau isoform expression and are consistent with a loss-of-function model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal cell death and dementia.     

Three- and four-repeat Tau coassemble into heterogeneous filaments: an implication for Alzheimer disease

Tau filaments are the pathological hallmark of numerous neurodegenerative diseases including Alzheimer disease, Pick disease, and progressive supranuclear palsy. In the adult human brain, six isoforms are expressed that differ by the presence or absence of the second of four semiconserved repeats. As a consequence, half of the tau isoforms have three repeats (3R tau), whereas the other half of the isoforms have four repeats (4R tau). Tauopathies can be characterized based on the isoform composition of their filaments. Alzheimer disease filamentous inclusions contain all isoforms. Pick disease filaments contain 3R tau. Progressive supranuclear palsy filaments contain 4R tau. Here, we used site-directed spin labeling of recombinant tau in conjunction with electron paramagnetic resonance spectroscopy to obtain structural insights into these filaments. We find that filaments of 4R tau and 3R tau share a highly ordered core structure in the third repeat with parallel, in-register arrangement of β-strands. This structure is conserved regardless of whether full-length isoforms (htau40 and htau23) or truncated constructs (K18 and K19) are used. When mixed, 3R tau and 4R tau coassemble into heterogeneous filaments. These filaments share the highly ordered core in the third repeat; however, they differ in their overall composition. Our findings indicate that at least three distinct types of filaments exist: homogeneous 3R tau, homogeneous 4R tau, and heterogeneous 3R/4R tau. These results suggest that individual filaments found in Alzheimer disease are structurally distinct from those in the 3R and 4R tauopathies.     

Functional differences of tau isoforms containing 3 or 4 C-terminal repeat regions and the influence of oxidative stress.

We report functional differences between tau isoforms with 3 or 4 C-terminal repeats and a difference in susceptibility to oxidative conditions, with respect to the regulation of microtubule dynamics in vitro and tau-microtubule binding in cultured cells. In the presence of dithiothreitol in vitro, a 3-repeat tau isoform promotes microtubule nucleation, reduces the tubulin critical concentration for microtubule assembly, and suppresses dynamic instability. Under non-reducing conditions, threshold concentrations of 3-repeat tau and tubulin exist below which this isoform still promotes microtubule nucleation and assembly but fails to reduce the tubulin critical concentration or suppress dynamic instability; above these threshold concentrations, amorphous aggregates of 3-repeat tau and tubulin can be produced at the expense of microtubule formation. A 4-repeat tau isoform is less sensitive to the oxidative potential of the environment, behaving under oxidative conditions similarly to the 3-repeat isoform under reducing conditions. Under conditions of oxidative stress, in Chinese hamster ovary cells stably expressing either 3- or 4-repeat tau, 3-repeat tau disassociates from microtubules more readily than the 4-repeat isoform, and tau-containing high molecular weight aggregates are preferentially observed in lysates from the Chinese hamster ovary cells expressing 3-repeat tau, indicating greater susceptibility of 3-repeat tau to oxidative conditions, compared with 4-repeat tau in vivo.     

Variations in filament conformation dictate seeding barrier between three- and four-repeat tau

Tau filaments are the pathological hallmark of >20 neurodegenerative diseases including Alzheimer's disease. Six tau isoforms exist that can be grouped into 4-repeat (4R) tau and 3-repeat (3R) tau based on the presence or absence of the second of four microtubule binding repeats. Recent evidence suggests that tau filaments can transfer between cells and spread through the brain. Here we demonstrate in vitro that seeded filament growth, a prerequisite for tau spreading, is crucially dependent on the isoform composition of individual seeds. Seeds of 3R tau and 3R/4R tau recruit both types of isoforms. Seeds of 4R tau recruit 4R tau, but not 3R tau, establishing an asymmetric barrier. Conformational templating of 4R tau onto 3R tau seeds eliminates this barrier, giving rise to a new type of tau filament. These findings provide fundamental mechanistic insights into the seeding, propagation, and diversification of tau filaments.     

Insertions, substitutions, and the origin of microsatellites

This paper uses data from the Human Gene Mutation Database to contrast two hypotheses for the origin of short DNA repeats: substitutions and insertions that duplicate adjacent sequences. Because substitutions are much more common than insertions, they are the dominant source of new 2-repeat loci. Insertions are rarer, but over 70% of the 2-4 base insertion mutations are duplications of adjacent sequences, and over half of these generate new repeat regions. Insertions contribute fewer new repeat loci than substitutions, but their relative importance increases rapidly with repeat number so that all new 4-5-repeat mutations come from insertions, as do all 3-repeat mutations of tetranucleotide repeats. This suggests that the process of repeat duplication that dominates microsatellite evolution at high repeat numbers is also important very early in microsatellite evolution. This result sheds light on the puzzle of the origin of short tandem repeats. It also suggests that most short insertion mutations derive from a slippage-like process during replication.     

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.     

Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: differential expression of tau protein mRNAs in human brain.

We have isolated cDNA clones encoding a 383-amino acid isoform of the human microtubule-associated protein tau. It differs from previously determined tau sequences by the presence of an additional repeat of 31 amino acids, giving four, rather than three, tandem repeats in its carboxy-terminal half. The extra repeat is encoded by a separate exon. Probes derived from cDNA clones encoding the three (type I) and four repeat (type II) tau protein isoforms detected mRNAs for both forms in all adult human brain areas examined. However, in foetal brain only type I mRNA was found. Type I and type II mRNAs were present in pyramidal cells in cerebral cortex. In the hippocampal formation, type I mRNA was found in pyramidal and granule cells; type II mRNA was detected in most, though not all, pyramidal cells but not in granule cells. These observations indicate that tau protein mRNAs are expressed in a stage- and cell-specific manner. Tau protein is found in the protease-resistant core of the paired helical filament, the major constituent of the neurofibrillary tangle in Alzheimer's disease. Taken in conjunction with previous findings, the present results indicate that both the three and four repeat-containing tau protein isoforms are present in the core of the paired helical filament.     

Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau.

Tau (tau) is a major constituent of paired helical filaments (PHF) found in Alzheimer's disease. The current study examines the possibility that the distinct properties of PHF-associated tau proteins (tau PHF) result from post-translational modifications of normal soluble tau (tau s). Following hydrofluoric acid (HF) treatment, tau PHF proteins are heat- and acid-stable, soluble in 2-(N-morpholino)ethanesulfonic acid buffers and display the same molecular weight, pI, and immunochemical properties as normal tau s. Alkaline phosphatase treatment of dissociated PHF results in similar, although less extensive, electrophoretic changes and a reduction in PHF-1 immunoreactivity. Therefore, phosphorylation of normal tau s appears to be responsible for the distinct properties of tau PHF. Although our results suggest that all of the normal tau isoforms are in PHF, the relative abundance of individual tau species differs in HF-treated PHF and tau s samples. Moreover, the loss of PHF following HF treatment suggests that post-translational modifications contribute to the structural stability of PHF.     

Differential effect of three-repeat and four-repeat tau on mitochondrial axonal transport

Tau protein is present in six different splice forms in the human brain and interacts with microtubules via either 3 or 4 microtubule binding repeats. An increased ratio of 3 repeat to 4 repeat isoforms is associated with neurodegeneration in inherited forms of frontotemporal dementia. Tau over-expression diminishes axonal transport in several systems, but differential effects of 3 repeat and 4 repeat isoforms have not been studied. We examined the effects of tau on mitochondrial transport and found that both 3 repeat and 4 repeat tau change normal mitochondrial distribution within the cell body and reduce mitochondrial localization to axons; 4 repeat tau has a greater effect than 3 repeat tau. Further, we observed that the 3 repeat and 4 repeat tau cause different alterations in retrograde and anterograde transport dynamics with 3 repeat tau having a slightly stronger effect on axon transport dynamics. Our results indicate that tau-induced changes in axonal transport may be an underlying theme in neurodegenerative diseases associated with isoform specific changes in tau's interaction with microtubules.     

Structure,stability, and aggregation of paired helical filaments from tau protein and FTDP-17 mutants probed by tryptophan scanning mutagenesis

By using tryptophan scanning mutagenesis, we observed the kinetics and structure of the polymerization of tau into paired helical filaments (PHFs) independently of exogenous reporter dyes. The fluorescence exhibits pronounced blue shifts due to burial of the residue inside PHFs, depending on Trp position. The effect is greatest near the center of the repeat domain, showing that the packing is tightest near the beta-structure inducing hexapeptide motifs. The tryptophan response allows measurement of PHF stability made by different tau isoforms and mutants. Unexpectedly, the stability of PHFs is quite low (denaturation half-points approximately 1.0 m GdnHCl), implying that incipient aggregation should be reversible and that the observed high stability of Alzheimer PHFs is due to other factors. The stability increases with the number of repeats and with tau mutants promoting beta-structure, arguing for a gain of toxic function in frontotemporal dementias. Fluorescence resonance energy transfer (FRET) was used to analyze the distances of Tyr(310) to tryptophans in different positions. The degree of FRET in the soluble protein was position-dependent, with highest signals within the second and third repeats but low or no signals further away. In PHFs most mutants showed FRET, indicating that tight packing results from assembly of tau into PHFs.     

Tau aggregation is driven by a transition from random coil to beta sheet structure

The abnormal aggregation of the microtubule associated protein tau into paired helical filaments (PHFs) is one the hallmarks of Alzheimer's disease. The soluble protein is one of the longest natively unfolded proteins, lacking significant amounts of secondary structure over a sequence of 441 amino acids in the longest isoform. Furthermore, the unfolded character is consistent with some notable features of the protein like stability towards heat and acid treatment. It is still unclear how these characteristics support the physiological function of binding to and stabilization of microtubules. We review here some recent studies on how an unfolded protein such as tau can adopt beta-structure, which then leads to the highly ordered morphology of the PHFs. The core sequence for both microtubule binding and PHF formation is the microtubule binding domain containing three or four repeats. This region alone is sufficient for PHF formation and mostly unfolded in the soluble state. A search for sequence motifs within this region crucial for PHF building revealed two hexapeptides in the second and the third repeat. Some of the genetically linked cases of FTDP-17 show missense mutations in or adjacent to these hexapeptide motifs. Proteins containing the P301L and the DeltaK280 mutations exhibit accelerated aggregation. The importance of the two hexapeptides stems from their capacity to undergo a conformational change from a random coil to a beta sheet structure. The increase of beta sheet structure is a typical feature of an amyloidogenic protein and is the basis of other characteristics like a decreased sensitivity towards proteolytic degradation and Congo red binding. PHFs aggregated in vitro and in vivo contain beta-sheet structure, as judged by circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction.     

Generation of VNTRs and heteroplasmy by sequence turnover in the mitochondrial control region of two elephant seal species

We describe an unusual repetitive DNA region located in the 3′ end of the light (L)-strand in the mitochondrial control region of two elephant seal species. The array of tandem repeats shows both VNTR (variable-number tandem repeat) and sequence variation and is absent from 12 compared mammalian species, except for the occurrence in the same location of a distinct repetitive region in rabbit mtDNA and a similar repeat in the harbor seal. The sequence composition and arrangement of the repeats differ considerably between the northern elephant seal (Mirounga angustirostris) and the southern species (M. leonina) despite an estimated divergence time of 1 MY (based on an mtDNA-RNA gene and the nonrepetitive control region). Analysis of repeat sequence relationships within and between species indicate that divergence in sequence and structure of repeats has involved both slippagelike and unequal crossingover processes of turnover, generating very high levels of divergence and heteroplasmy. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992