Structures of filaments from Pick's disease reveal a novel tau protein fold

Abstract

The ordered assembly of tau protein into abnormal filamentous inclusions underlies many human neurodegenerative diseases1. Tau assemblies seem to spread through specific neural networks in each disease2, with short filaments having the greatest seeding activity3. The abundance of tau inclusions strongly correlates with disease symptoms4. Six tau isoforms are expressed in the normal adult human brain-three isoforms with four microtubule-binding repeats each (4R tau) and three isoforms that lack the second repeat (3R tau)1. In various diseases, tau filaments can be composed of either 3R or 4R tau, or of both. Tau filaments have distinct cellular and neuroanatomical distributions5, with morphological and biochemical differences suggesting that they may be able to adopt disease-specific molecular conformations6,7. Such conformers may give rise to different neuropathological phenotypes8,9, reminiscent of prion strains10. However, the underlying structures are not known. Using electron cryo-microscopy, we recently reported the structures of tau filaments from patients with Alzheimer's disease, which contain both 3R and 4R tau11. Here we determine the structures of tau filaments from patients with Pick's disease, a neurodegenerative disorder characterized by frontotemporal dementia. The filaments consist of residues Lys254-Phe378 of 3R tau, which are folded differently from the tau filaments in Alzheimer's disease, establishing the existence of conformers of assembled tau. The observed tau fold in the filaments of patients with Pick's disease explains the selective incorporation of 3R tau in Pick bodies, and the differences in phosphorylation relative to the tau filaments of Alzheimer's disease. Our findings show how tau can adopt distinct folds in the human brain in different diseases, an essential step for understanding the formation and propagation of molecular conformers.

Conflict of interest statement

Author Information

The authors declare no competing interests.

Figures

Figure 1 |. Further characterization of the filamentous tau pathology of Pick’s disease

a, Staining of Pick bodies in the frontotemporal cortex of patient 4 by Bodian silver and antibody AT8 (pS202 and pT205 tau), but not by Gallyas-Braak silver. Nuclei are counterstained blue. Scale bars, 20 μm. b,c, Immunolabeling of the tau filaments extracted from the frontotemporal cortex of patient 4. Immunoblots (b) with antibodies BR133 (tau amino-terminus), BR134 (tau carboxy-terminus), RD3 (3R tau), Anti-4R (4R tau), AT8 (pS202 and pT205 tau) and 12E8 (pS262 tau and/or pS356 tau). Immunogold negative-stain electron micrographs (c) with antibodies BR133, BR134, 12E8 and MC1 of NPFs and WPFs with and without pronase treatment. Scale bar, 500 Å.

Figure 2 |. Narrow Pick filament (NPF) structure

a, Fourier shell correlation curves between two independently refined half-maps (black line) and between the cryo-EM reconstruction and refined atomic model (red line). b, Local resolution estimates for the NPF reconstruction. c, Helical axis views of the NPF reconstruction. d, Close-up views of the cryo-EM map with the atomic model overlayed. The top row shows the three PGGG motifs; the bottom row shows several amio acids with large side-chains.

Figure 3 |. Wide Pick filament (WPF) structure

a, Fourier shell correlation curves between two independently refined half-maps. b, Local resolution estimates for the WPF reconstruction. c, WPF density at high (light grey) and low (dark grey) threshold with densities for two NPFs overlaid (yellow and blue). The atomic models fitted to the NPF densities in the region of the protofilament interface are shown in the boxed out area. d, Cryo-EM images showing WPFs from patient 4 (false coloured red) where segments from one of the protofilaments have been lost. Scale bar, 500 Å. e, Negative-stain EM images of WPFs from patient 4 following incubation in 100 mM dithiothreitol for 20 h. Scale bar, 500 Å.

Figure 4:. Incompatibility of Pick tau filament fold with 4R tau

Atomic model of Pick fold with 4R tau sequence overlaid. The region formed by K254-K274 from R1 is replaced by the S285-V300 region from R2 in 4R tau. Residues that differ between these regions of R1 and R2 are coloured orange. The major discrepancies of lysine at position 294 in R2, instead of threonine at position 263 in R1, and valine at position 300 in R2, instead of glutamine at position 269 in R1, are highlighted with dashed red outlines. The minor discrepancy of weaker interactions of C291 of R2 with L357 and I360 than those formed by I260 of R1 is highlighted with a dashed yellow outline.

Figure 5 |. Seeded aggregation of full-length 3R, but not 4R, tau by the sarkosyl-insoluble fraction from Pick’s disease brain

a, Coomassie-stained SDS-PAGE of the 0N3R and 0N4R recombinant tau preparations used for seeded aggregation. Two additional recombinant tau preparations were carried out with similar results. b, Thioflavin T fluorescence measurements of 0N3R (red) and 0N4R (blue) recombinant tau following incubation with (triangles) or without (circles) the sarkosyl-insoluble fraction from the frontotemporal cortex of patient 4. The results are from three independent experiments using separate recombinant protein preparations. The sarkosyl-insoluble fraction from Pick’s disease brain efficiently seeded the aggregation of 3R, but not 4R, tau

Figure 6 |. Immunoblot analysis of additional Pick’s disease cases

a, Diagram of 2N4R tau showing the N-terminal inserts (N1, N2), the repeats (R1-R4) and the epitopes of antibodies BR133 (N-terminus), BR136 (R1), Anti-4R (R2), BR135 (R3), TauC4 (R4) and BR134 (C-terminus). b, Immunoblots of epitope-deletion recombinant tau constructs with the antibodies shown in a. Identical results were obtained in two independent repeats. c, Immunoblots using the antibodies BR136, Anti-4R, BR135 and TauC4 of tau filaments extracted from the frontotemporal cortex of 9 patients with Pick’s disease (patient 4 was used for cryo-EM). See Extended Data Table 1 for details of Pick’s disease patients.

Figure 7 |. Immunogold negative-stain EM analysis of additional Pick’s disease cases

a, Representative immunogold negative-stain electron microscopy with antibodies BR133 (tau amino-terminus), BR136 (tau R1), Anti-4R (tau R2), BR135 (tau R3), TauC4 (tau R4) and BR134 (tau carboxy-terminus) of NPFs and WPFs extracted from the frontotemporal cortex of patient 4. Scale bars, 100 nm. Similar results were obtained with tau filaments extracted from the frontotemporal cortices of eight additional patients with Pick’s disease b, Table summarizing results from a. Results for patient 4 (shown in a. and used for cryo-EM) are highlighted in yellow. See Extended Data Table 1 for details of Pick’s disease patients. Tick marks indicate antibody decoration of filaments, while crosses indicate that the antibodies did not decorate filaments. NPFs and WPFs were decorated by the antibodies against the N- and C-termini of tau, but not by the tau repeat-specific antibodies.

Figure 1 |. Filamentous tau pathology of Pick’s disease.

a, The brain used for cryo-EM (patient 4) showed atrophy of anterior frontal and temporal lobes of the cerebral cortex. Scale bar, 5 cm. b-d, Staining of Pick bodies in the frontotemporal cortex of patient 4 by antibody RD3 (3R tau; brown) (b), but not by antibodies Anti-4R (4R tau) (c) or 12E8 (pS262 tau and/or pS356 tau) (d). Nuclei were counterstained blue. Scale bars, 20 μm. e, Cryo-electron micrograph of tau filaments extracted from grey matter of the frontotemporal cortex of patient 4, in which narrow (NPFs; blue arrow) and wide (WPFs; red arrow) Pick filaments could be distinguished. Scale bar, 500 Å. f, Unsharpened cryo-EM density of NPF from patient 4. Scale bar, 25 Å. g, Unsharpened cryo- EM density of WPF from patient 4. Scale bar, 25 Å.

Figure 2 |. The Pick tau filament fold.

a, Sharpened, high-resolution cryo-EM map of the narrow Pick filament (NPF) with the atomic model of the Pick fold overlayed. b, Schematic view of the Pick fold. Amino acid numbering corresponds to the 441 amino acid human tau isoform, so residues 275–305 of R2 are not present.

Figure 3 |. Comparison of the Pick and Alzheimer tau filament folds.

a, Sequence alignment of the microtubule-binding repeats (R1-R4) with the observed nine β-strand regions (arrows) in the Pick fold and eight β-strand regions (arrows) in the Alzheimer fold, coloured from violet to red. b, Rendered view of the secondary structure elements in the Pick fold, depicted as three successive rungs. c, As in b, but in a view perpendicular to the helical axis, revealing the changes in height within a single molecule. d, Schematic of the secondary structure elements in the Pick and Alzheimer folds, depicted as a single rung. The positions of C322 and D348 in the two folds are highlighted. The symbols ‡ and * mark conserved turns of homologous regions in the Pick and Alzheimer folds.