Tau and Alzheimer paired helical filaments

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The molecular structure of PHFs is still unknown but determining it represents one of the major goals in the field, since this would greatly aid in the development of methods and drugs to prevent pathological aggregation. From a structural point of view, there was a long gap between Alzheimer's discovery of neurofibrillary tangles (Alzheimer 1907a) and the identification of PHFs as their basic elements, made possible by the advances in electron microscopy (Kidd 1963; Terry 1963). Another two decades passed with attempts to find suitable conditions for the isolation and characterization of PHFs (e.g., Ihara et al. 1983; Wisniewski et al. 1984). One important outcome was the reconstruction of PHFs from negatively stained electron micrographs, which showed each half of a PHF to be composed of three protein densities, with overall dimensions of ~ 8 nm x 20 nm (Crowther and Wischik 1985); Subsequent work showed an analogous doubly tripartite structure for "straight filaments," a minor variant of Alzheimer PHF preparations (Crowther 1991). These variants appear to be caused by subtle changes in charge distribution around the ^-structure forming motifs in the repeat domain at the beginning of R3 and R4 (DeTure et al. 2002).

With the development of specific antibodies against PHFs and microtubule proteins, the search for the protein composition of PHFs yielded tau protein as the major subunit (Brion et al. 1985d; Grundke-Iqbal et al. 1986b; Kosik et al. 1988). Shortly thereafter, molecular cloning resulted in the elucidation of tau sequences from mouse and humans and confirmed that the protein from Alzheimer PHFs was indeed tau (Lee etal. 1988; Wischik et al. 1988a; Goedert etal. 1988,1989). This finding set the stage for the expression of recombinant tau protein and the structural and biochemical analysis of tau and PHFs.

Ageneral difficultyinstudyingthe formationofPHFsfromsoluble tauwas thehigh solubility of the protein that counteracts assembly; the second problem was to derive criteria for the in vitro generation of bona fide PHFs. These problems were overcome by a search for appropriate tau constructs and assembly conditions, including the dimerization of the protein by disulfide crosslinking, which accelerates PHF assembly (Wille et al. 1992). The resulting fibers had the typical PHF morphology with an ~ 80 nm crossover repeat (Fig. 4). A further step in accelerating PHF assembly and making it amenable for structural studies was the discovery that polyanionic molecules greatly facilitate PHF assembly. These include molecules such as sulfated glycosaminoclycans, heparin, RNA, acidic peptides, or fatty acid micelles (Goedert et al. 1996; Perez et al. 1996; Kampers et al. 1996; Wilson and Binder 1997).

An important issue concerning the substructure of PHFs was the question of whether they should be considered as "amyloid". The current definition of an amyloid, evolving from the analysis of several pathologically aggregating proteins, is that of an aggregating fibril whose backbone consists of P-sheets whose strands are oriented across (i.e., perpendicular to) the fiber axis ("cross-P-structure"). The distance between successive strands is ~ 0.47 nm, so X-ray fiber diffraction patterns reveal a sharp meridional 0.47-nm reflection. Using this criterion, Kirschner et al. (1986) suggested a cross-P structure for both types of fibers isolated from Alzheimer brains, from amyloid plaques (containing the Ap peptide) and neurofibrillary tangles (PHFs containing tau). In the case of PHFs, the diagnostic 0.47-nm reflection was weak, the purity of the preparation was somewhat uncertain, and later studies failed to confirm the reflection

Phf Morphology
Fig. 4. Electron micrographs of paired helical filaments (PHF) isolated from Alzheimer brain (left) and reassembled in vitro from recombinant (rec) tau (repeat domain with pro-aggregation mutant AK280). Note the typical twisted appearance with crossover repeats of ~ 80 nm (arrowheads)

in Alzheimer PHFs (Schweers et al. 1994). Instead, other types of axial repeats were reported that suggested a non-amyloid packing of subunits (e.g., 3 nm; Crowther and Wischik 1986).

The resolution of this puzzle came with the realization that the aggregation of tau was based on very short motifs in the repeat domain (hexapeptide motifs 275VQIINK280 and 306VQIVYK311 at the beginning of R2 and R3, respectively; von Bergen et al. 2000, 2001; Fig. 1). These "aggregation motifs" tend to interact with a cross-P structure, forming the core of PHFs, whereas the bulk of the protein remains largely disordered. Thus the amyloid character of tau is poorly visible with full-length tau, but it becomes apparent with peptides derived from the aggregation motifs and/or improved procedures of specimen preparation (von Bergen et al. 2001; Gianetti et al. 2000; Berriman et al. 2003; Inouye et al. 2006; Fig. 5). The aggregation motifs coincide with sequences where nascent P-structure can already be detected in soluble tau by NMR spectroscopy, and indeed this region reveals a very low mobility, compared with the fuzzy coat (Mukrasch et al.2005; Sillen et al. 2005). The role of the hexapeptide motifs is further underscored by proline mutations that interrupt p-structure and thus inhibit aggregation ("anti-aggregation" mutants), or, conversely, by mutations that enhance the propensity for P-structure (e.g., P301L or AK280, both described for frontotemporal dementias) and thus promote aggregation ("pro-aggregation" mutants; Barghorn et al. 2000). On the basis of these data, it is possible to draw a rough outline of the steps involved in PHF aggregation (Fig. 6).

The next steps would be the determination of the packing of tau subunits within PHFs and their folding at high resolution. These goals have not yet been achieved, but they will likely occur in three stages:

Paired Helical Filaments

Fig. 5. Principle of PHF aggregation by forming cross-p structure. Left, X-ray fiber diagram of PHFs reassembled in vitro (from repeat domain, AK280 mutant). Note the meridional reflection indicating the 0.47-nm spacing typical of adjacent strands in a p-sheet, and the equatorial reflection at ~ 1 nm typical of the separation between p-sheets. Right, illustration of a cross-p structure, with p-strands (short gray arrows) oriented perpendicular to the fiber axis (vertical arrow)

Fig. 5. Principle of PHF aggregation by forming cross-p structure. Left, X-ray fiber diagram of PHFs reassembled in vitro (from repeat domain, AK280 mutant). Note the meridional reflection indicating the 0.47-nm spacing typical of adjacent strands in a p-sheet, and the equatorial reflection at ~ 1 nm typical of the separation between p-sheets. Right, illustration of a cross-p structure, with p-strands (short gray arrows) oriented perpendicular to the fiber axis (vertical arrow)

- First, since the backbone of PHFs consists of cross-P-structure, the analogy with other amyloid-forming proteins (e.g., Ap peptide or peptides from yeast prion protein; for review, see Nelson and Eisenberg 2006) makes it likely that there will be protofibrils that are made up of pairs of juxtaposed P-sheets that interact axially by hydrogen bonding between their main chain strands and laterally through the sidechains across the sheets. This presumably includes hydrophobic interactions, as suggested by the nature of the pro-aggregation motifs. Since these residues are near one another, it is likely that their distances and interactions can be determined by spectroscopic methods, such as NMR or EPR. One example is the EPR study of Mar-gittai and Langen (2004), who concluded that residues 301-320 in R3 must lie close to the corresponding residues in a neighboring molecule. This could be achieved by placing this stretch of residues in adjacent strands of a cross-P-sheet structure.

- The second level will be the arrangement of protofibrils within a PHF. Their number and interaction are currently unknown, but there are several constraints on possible arrangements: 1) The mass-per-length of the PHF core, determined by scanning transmission electron microscopy (STEM), is ~ 60-70 kDa/nm, equivalent to roughly 3.5-4.5 repeat domain molecules per nm (Wischik et al. 1988a; von Bergen et al. 2006; for variations among PHFs, see Ksiezak-Reding and Wall 2005). Note, for comparison, that adjacent molecules in cross-P-structure are spaced 0.47 nm apart, equivalent to ~ 2 molecules per nm, which would allow only ~ 2 protofibrils. 2) The overall cross-sectional dimensions of the PHF core (comprising mainly the repeat domain) are ~ 8 nmx ~ 20 nm. This area is divided up into two halves, each containing three density peaks (and intervening valleys of lower density), so that the effective area is estimated at ~ 80 nm2 (Crowther 1991). These features, combined

Tau Structure

Fig. 6. Model of steps in PHF aggregation. The disordered tau monomer (lower left) initially dimerizes (upper row; this step can be enhanced by disulfide crosslinking), then partially converts to incipient p-structure around the hexapeptide motifs, followed by subunit addition to form a PHF nucleus and then a fiber with cross-p structure and several protofibrils. These steps can be accelerated by polyanions. If p-structure is prevented, e.g., by proline mutations in the hexapeptide motifs or by tau inhibitor compounds, the aggregation process is interrupted (lower row)

Fig. 6. Model of steps in PHF aggregation. The disordered tau monomer (lower left) initially dimerizes (upper row; this step can be enhanced by disulfide crosslinking), then partially converts to incipient p-structure around the hexapeptide motifs, followed by subunit addition to form a PHF nucleus and then a fiber with cross-p structure and several protofibrils. These steps can be accelerated by polyanions. If p-structure is prevented, e.g., by proline mutations in the hexapeptide motifs or by tau inhibitor compounds, the aggregation process is interrupted (lower row)

with the typical density of compact protein domains of ~ 0.8 kDa/nm3, represent boundary conditions that models of tau folding in PHFs will have to meet.

- The third and least well-defined aspect of PHF structure is the "fuzzy coat" surrounding the core (Wischik et al. 1988a). PHFs assembled from full-length tau and from the repeat domain have similar dimensions by electron microscopy, suggesting that the non-repeat parts of tau, comprising ~ 70% of the protein (roughly residues 1-240, 370-441), make only a minor contribution to the apparent images, presumably because they retain their natively unfolded character (Barghorn et al. 2004). The extent of the fuzzy coat is best visualized by immunogold labelling, where antibody-binding sites can extend away from the center of the PHF. Nevertheless, a substantial fraction of tau molecules in PHFs must have a folded conformation, because PHFs can be immunopurified with antibody MC-1 whose epitope comprises tau residues near the N-terminus and within the repeat domain (Jicha et al. 1997).

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