Tau filaments with the Alzheimer fold in human MAPT mutants V337M and R406W

Abstract

Frontotemporal dementia (FTD) and Alzheimer's disease (AD) are the most common forms of early-onset dementia. Unlike AD, FTD begins with behavioral changes before the development of cognitive impairment. Dominantly inherited mutations in MAPT, the microtubule-associated protein tau gene, give rise to cases of FTD and parkinsonism linked to chromosome 17. These individuals develop abundant filamentous tau inclusions in brain cells in the absence of β-amyloid deposits. Here, we used cryo-electron microscopy to determine the structures of tau filaments from the brains of human MAPT mutants V337M and R406W. Both amino acid substitutions gave rise to tau filaments with the Alzheimer fold, which consisted of paired helical filaments in all V337M and R406W cases and of straight filaments in two V337M cases. We also identified another assembly of the Alzheimer fold into triple tau filaments in a V337M case. Filaments assembled from recombinant tau (297-391) with substitution V337M had the Alzheimer fold and showed an increased rate of assembly.

Fig. 1. Mutation encoding V337M in MAPT: cryo-EM cross-sections of tau filaments and immunolabelling.

a, Cross-sections through the cryo-EM reconstructions, perpendicular to the helical axis and with a projected thickness of approximately one rung, are shown for the frontal cortex from cases 1–3. Resolutions (in Å) and percentages of filament types are indicated at the bottom left and top right, respectively. Scale bar, 10 nm. b, Immunoblotting of sarkosyl-insoluble tau from the frontal cortex of cases 1–3 with substitution V337M. Phosphorylation-independent anti-tau antibody BR134 was used. Source data

Fig. 2. Mutation encoding V337M in MAPT: cryo-EM structures of tau filaments.

a, Cryo-EM density map and atomic model of PHF. Two identical protofilaments extend from G273/G304 to E380. Inset, zoomed-in view showing that both wild-type (V) and mutant (M) residues can fit into the density at position 337. b, Backbone representation of the overlay of PHFs extracted from the frontal cortex of case 3 with the mutation encoding V337M in MAPT (blue) and PHFs extracted from the frontal cortex of an individual with sporadic AD (white; PDB 5O3L). The r.m.s.d. between Cα atoms of the two structures was 0.78 Å. c, Cryo-EM density map and atomic model of SF. Two asymmetrically packed protofilaments A and B extend from G273/G304 to E380. d, Overlay of SF extracted from the frontal cortex of case 1 with substitution V337M (protofilament A, dark green; protofilament B, light green) and SF extracted from the frontal cortex of an individual with AD (PDB 5O3T) (protofilament A, black; protofilament B, gray). In protofilament A, strand β4 (residues 336–341) is shifted along the helical axis by 3 Å. Protofilament B adopts the same structure as in AD. e, Cryo-EM density map and atomic model of TF. Three identical protofilaments (A, B and C) extend from G273/G304 to E380. An additional nonproteinaceous density at the filament’s three-fold axis is labeled with a red arrow. Inset, zoomed-in view showing one of the three identical protofilament interfaces and K331 residues from each protofilament coordinating the additional density. f, Overlay of individual protofilaments from TFs with substitution V337M (orange), PHFs with substitution V337M (blue) and PHFs from AD (white), viewed at a 45° angle to the filament axes, as in d. The r.m.s.d. between Cα atoms of TFs and V337M tau PHFs was 0.653 Å and that between Cα atoms of TFs and AD PHFs was 0.872 Å.

Fig. 3. In vitro assembly of V337M tau (297–391).

a, In vitro assembly assay monitored by ThT fluorescence of V337M tau (297–391) (magenta), wild-type tau (297–391) (blue) and a 50:50 mixture of V337M tau (297–391) and wild-type tau (297–391) (green). b, Cross-sections through the cryo-EM reconstructions, perpendicular to the helical axis and with a projected thickness of approximately one rung, are shown for assembled V337M tau (297–391). c, Cryo-EM density map and atomic model of PHF. Two identical protofilaments extend from V306 to R379. Inset, zoomed-in view showing the mutant methionine at position 337. d, Overlay of PHFs assembled from recombinant V337M tau (297–391) (magenta) and extracted from the frontal cortex of an individual with mutation V337M (blue). The r.m.s.d. between Cα atoms was 0.80 Å with a 9° rotation of the β-helix region relative to the rest of the ordered core being the main difference between the two structures. Source data

Fig. 4. Mutation encoding R406W in MAPT: cryo-EM cross-sections of tau filaments and immunoblotting.

a, Cross-sections through the EM reconstructions, perpendicular to the helical axis and with a projected thickness of approximately one rung, are shown for the temporal cortex, parietal cortex and hippocampus of case 1 and for the frontal, temporal and parietal cortices of case 2. Resolutions (in Å) and percentages of filament types are indicated at the bottom left and top right, respectively. Scale bar, 10 nm. b, Immunoblotting of sarkosyl-insoluble tau from the temporal cortex, parietal cortex and hippocampus of case 1 with substitution R406W and from the frontal, temporal and parietal cortices of case 2 with substitution R406W. Phosphorylation-independent anti-tau antibody BR134 was used. Source data

Fig. 5. Mutation encoding R406W in MAPT: cryo-EM structures of tau filaments.

a, Cryo-EM density and atomic model of PHF from the frontal cortex of case 2. Two identical protofilaments extend from G273/G304 to E380. b, Overlay of PHFs extracted from the frontal cortex of case 2 (blue) and the frontal cortex of a case of sporadic AD (black). The r.m.s.d. between Cα atoms of the two structures was 0.78 Å.

Extended Data Fig. 1. MAPT mutation encoding V337M: immunohistochemical localization of tau inclusions in frontal cortex and hippocampus.

A. a-f, Tau pathology in grey matter of frontal cortex. Higher magnifications of tissue areas within the insets in a-c are shown in d-f. Intraneuronal inclusions (blue arrows) and neuropil threads (red arrows) are in evidence. Antibody: AT8. Scale bars, 200 µm (a-c); 40 µm (d-f). B.a,b, Low magnification views of tau pathology in grey matter of hippocampus. c,d, Neurofibrillary tangles and neuropil threads in the pyramidal cell layer. Antibody: AT8. Scale bars, 1,000 µm (a,b) and 40 µm (c,d).

Extended Data Fig. 2. Mass spectrometry of tau from the sarkosyl-insoluble fractions of cases with MAPT mutation encoding V337M.

MALDI mass spectra of the frontal cortex from cases 1–3 (a-c). Wild-type (V337) and mutant (M337) tau peptides were detected. Source data

Extended Data Fig. 3. Comparison of V337M tau filament structures to those from other conditions.

Tau paired helical filaments (PHFs) (a) and straight filaments (SFs) (b) from the frontal cortex of individuals with missense mutation V337M in MAPT were compared to those of PHFs and SFs from Alzheimer’s disease (AD), primary age-related tauopathy (PART), familial British dementia (FBD), familial Danish dementia (FDD), posterior cortical atrophy (PCA), Gerstmann-Sträussler-Scheinker disease (GSS), cerebral amyloid angiopathy with prion protein deposits (PrP-CAA) and Down’s syndrome (DS).

Extended Data Fig. 4. MAPT mutation encoding R406W: immunohistochemical localization of tau in temporal cortex, parietal cortex and hippocampus of case 1.

a,c,e, Tau-immunopositive nerve cell bodies (arrows) and neuropil threads (arrowheads) are shown in temporal cortex; b,d, parietal cortex; f, hippocampus. c,d,e,f, Labelled astrocytes (double arrowheads). Panel (d) shows plaques composed of numerous threads, corresponding probably to the processes of neurons and astrocytes. Antibodies: AT8 (a,b,d,f); RD4 (c); RD3 (e). Scale bar, 25 µm.

Extended Data Fig. 5. MAPT mutation encoding R406W: immunohistochemical localization of tau inclusions in the hippocampus of case 1.

The pyramidal layer is shown. Tau-immunopositive intracellular neuronal inclusions (single headed arrows) and extracellular ghost inclusions (double headed arrows), as well as neuropil threads (single arrowheads) and astrocytic inclusions (double arrowheads) are indicated. Antibodies: AT8 (a), RD4 (b), anti-4R (c), RD3 (d). Scale bar, 25 µm.

Extended Data Fig. 6. MAPT mutation encoding R406W: immunohistochemical localization of tau inclusions in case 2.

a, Mild atrophy of the frontal cortex, severe atrophy of the temporal cortex and underlying white matter, with marked reduction in bulk of the hippocampus. Anterior and temporal horns of the lateral ventricle are dilated; b, tau pathology in the anterior frontal cortex; c, some stained cells resemble tufted or thorn-shaped astrocytes; d, abundant neuronal inclusions and neuropil threads; e, astrocytic plaque; f,g, structures in-between astrocytic plaques and tufted astrocytes; h, tufted astrocyte; i,k, Tau pathology in the lateral temporal cortex was similar to that in the anterior frontal cortex; l, CA4 region of the hippocampus; m, dentate gyrus; j, subpial astrocytic tau pathology at the depth of a sulcus in the lateral temporal cortex. n, Low-power view of the midbrain; o, subpial astrocytic tau pathology; p, neuronal tau staining in the substantia nigra. AT8 antibody. Scale bar: b, 750 µm; c,k, 70 µm; d, 40 µm; e,f,g,h, 30 µm; i, 670 µm; j, 50 µm; l. 100 µm; m, 110 µm; n, 5.5 µm; o,p, 90 µm.

Extended Data Fig. 7. Representative mass spectrometry of tau from the sarkosyl-insoluble fractions of cases with MAPT mutation encoding R406W.

MALDI mass spectra of parietal cortex from case 1 (a) and frontal cortex from case 2 (b). Wild-type (R406) and mutant (W406) peptides were detected in parietal cortex from case 1. Only mutant peptides (W406) were detected in frontal cortex from case 2. Similarly, only mutant peptides were detected in temporal cortex and hippocampus from case 1, as well as in temporal cortex, parietal cortex and hippocampus from case 2. Source data

Extended Data Fig. 8. Representative 2D class averages. 2D class averages of filaments from three cases with MAPT mutation encoding V337M (a) and two cases with MAPT mutation encoding R406W (b).

Scale bar, 10 nm.

Extended Data Fig. 9. Cryo-EM image processing workflow (V337M case3).

a, A representative motion-corrected micrograph; the scale bar, 50 nm. b, Representative 2D classes; the box size is 843 Å. c, The overview of the cryo-EM imaging process. Angular distribution histogram of the final reconstruction is represented at the bottom. d, Local resolution maps of PHF, SF and TF, estimated using RELION.

Extended Data Fig. 10. Fourier shell correlation (FSC) curves.

FSC curves of cryo-EM maps (left panel) and model to map validation (right panel). a, PHF from V337M tau mutant; b, SF from V337M tau mutant; c, TF from V337M tau mutant; d, PHF from R406W tau mutant; e, PHF assembled from recombinant V337M tau (297–391). Source data