Abstract

Results

First, we examined filaments from PSP, the most common tauopathy after Alzheimer’s disease, and belonging to the group of sporadic frontotemporal lobar degeneration disorders (FTLD-tau). Clinically, typical cases of PSP (Richardson’s syndrome; PSP-RS) are characterised by postural instability, supranuclear gaze palsy, behavioural and cognitive impairment, as well as bulbar symptoms (9,10). Neuropathologically, they are defined by abundant subcortical neurofibrillary tangles and neuropil threads, together with tufted astrocytes and oligodendroglial coiled bodies (11,12). Atypical forms of PSP are distinguished by differences in total tau load in specific brain regions (12). We determined structures, with resolutions of up to 2.7 Å, of tau filaments from the frontal cortex and thalamus of three cases of PSP-RS, the putamen and temporal cortex of two cases of PSP with predominant frontal presentation (PSP-F), the frontal cortex of one case with predominant parkinsonism (PSP-P) and the frontal cortex of one case with a predominant presentation of corticobasal syndrome (PSP-CBS).

In PSP-RS, PSP-CBS, PSP-P and PSP-F case 1, we observed tau filaments made of a single protofilament with an ordered core that spans residues 272-381. Although this is essentially the same region as that seen in the CBD core, it adopts a markedly different conformation, which we named the PSP fold (Figure 1b, Extended Data Figure 4a). Tau filament structures were identical between typical and atypical cases of PSP, consistent with the view that the initiating sites of tau pathology are similar in clinical subtypes, which are distinguished by different propagation patterns. When multiple tau seeds are found in a PSP brain (13), this may be indicative of co-pathology.

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Figure 1

Three-layered 4R tau filament structures

(a), Amino acid sequence of tau residues 272-387. Residues in R1-4 and in the C-terminal domain are coloured purple, blue, green, gold and orange, respectively; (b), Cryo-EM density map (in transparent grey) and atomic model for the PSP filaments. (c), As in (b), for GGT type 1, GGT type 2 and GGT type 3 filaments; (d) As in (b), for GPT type 1 and GPT type 2 filaments. Black arrows point to additional densities that are described in the main text.

In the PSP fold, repeats 2-4 (R2-4) form a 3-layer meander turning at the conserved PGGG motifs at the end of each repeat. R3 forms the central layer, which bends at G323 into a near-right angle. Following the outside of this bend, R2 packs against the entirety of R3. The R2-R3 interface contains two small cavities containing additional densities. One, between N279 and G323, and surrounded by hydrophobic side chains, is probably caused by molecules of a predominantly nonpolar nature, whereas the other, next to the salt bridge between K294 and D314, is most likely a solvent molecule. Most of R4 packs against the other side of R3. At the R3-R4 interface, there is a cavity between the positively charged K317, K321 and K340 that contains a bigger additional density, with a volume of ~30 ų, presumably corresponding to anionic molecules. This cavity is flanked by the negatively charged E338 and E342, reducing the net charge of the cavity to +1 per rung. The rest of the R3-R4 interface is of mixed chemical nature, containing hydrophobic interactions and a salt bridge between K311 and D348. The chain makes another hairpin turn at the PGGG motif of R4, with the C-terminal segment forming a short fourth layer covering the end of R2. On the outside of the PSP core, there are at least six additional densities that were conserved between cases, the most prominent being next to K280-K281 and H362.

We then examined tau filaments from GGT. Like PSP, GGT is a sporadic 4R tauopathy that belongs to the spectrum of FTLD-tau diseases; it is characterized by abundant globular glial tau inclusions (14–16). GGT is divided into three types (GGT-I, GGT-II and GGT-III), depending on the predominant involvement of glial cells in white or grey matter. Structures of tau filaments from the frontal cortex of previously described cases of GGT-I [case 1 in (17)] and GGT-II [case 1 in (18)], with resolutions of up to 2.9 Å, show a common, previously unknown three-layered protofilament fold, spanning residues 272-379 (Figure 1c, Extended Data Figure 4b). For GGT-I, we observed three filament types: type 1 comprises a single protofilament with the GGT fold; type 2 packs two protofilaments with approximate 21 screw symmetry; type 3 packs two protofilaments in an asymmetrical manner. For GGT-II, we only observed filaments of types 2 and 3. We were unable to solve the structures of tau filaments from the frontal cortex of a previously described case with GGT-III [case 1 in (19)], because of an absence of twist in the imaged filaments.

The GGT fold comprises essentially the same residues as the PSP fold and has a similar three-layered arrangement of R2-4. Again, the chain turns at the PGGG motifs, but each turn has a different conformation compared to its PSP counterpart. Moreover, the C-terminal domain points in the opposite direction and packs against the end of R4 in a hairpin-like structure that is almost identical to the equivalent part of the Pick fold. The only common substructure of the GGT and PSP folds comprises residues 273-285 and 322-330, extending to the additional densities in the small internal cavity between N279 and G323 and outside K280/K281. The cavity at the R3-R4 interface is bigger in the GGT than in the PSP fold and it contains a larger non-proteinaceous density, with a volume of ~50ų, surrounded by K317, K321 and K340. The cavity’s net positive charge is also greater, as E338 forms a salt bridge with K331.

Although the structures of tau filaments from the six cases of typical and atypical PSP described above were identical, we observed a different tau fold for filaments from the temporal cortex of case PSP-F2 (Figure 1d, Extended Data Figure 4c). Since this fold resembled both the GGT and PSP folds, we named it the GGT-PSP-Tau, or GPT, fold. We observed two types of GPT filaments: type 1 filaments comprise a single protofilament; type 2 filaments pack two protofilaments with approximate 2₁ screw symmetry, creating a large, solvent-filled cavity. Histologically, case PSP-F2 was different from other cases of PSP: it resembled cases with abundant spherical, 4R tau-immunoreactive, basophilic neuronal inclusions in limbic and other brain regions (Extended Data Figure 5) (20–22).

For PSP-F2, we used the same electron microscope that was used previously for structure determination to atomic resolution of an apoferritin test sample (23). Images from this microscope allowed classification into two alternative main chain conformations, one of which led to a reconstruction with a resolution of 1.9 Å (Extended Data Figure 6). This map allowed us to model 19 ordered water molecules per asymmetric unit, but it failed to provide further insights into the nature of the constituent molecules of the additional densities, probably because they did not follow the helical symmetry.

The GPT fold comprises the same residues as the PSP and GGT folds and has a similar three-layered structure. The GPT fold has two large substructures of closely similar conformations compared to the GGT fold: the hairpin formed by residues 356-378, as well as residues 273-294 and 312-346, including the small and large cavities with their associated internal and external additional densities. The relative orientation of the two substructures differs between the GPT and GGT folds, with different conformations of intervening sequences, in particular in the PGGG turn at the end of R2 and in the N-terminal part of R3 that have similar side chain orientations to PSP. In GPT type 2 filaments, the two symmetric inter-protofilament interfaces resemble the asymmetric interface of GGT type 3 filaments, increasing the overall similarity between GGT and GPT filaments (Extended Data Figure 6).

AGD is another 4R tauopathy belonging to the spectrum of sporadic FTLD-tau diseases (24–28). A defining feature is the presence of argyrophilic grains. Argyrophilic grains are rarely the sole pathological finding in cognitively impaired subjects and are most commonly found together with other tau pathologies, especially neurofibrillary tangles (26). We determined the structures of tau filaments from the nucleus accumbens of two cases of AGD, with resolutions up to 3.4 Å (Figure 2, Extended Data Figure 4d). We observed three types of filaments with a common protofilament core, the AGD fold, that adopts a four-layered ordered structure comprising residues 273-387 (type 1) or 279-381 (type 2), and resembles the CBD fold (5). Like CBD type I and type II filaments, AGD type 1 filaments comprise a single protofilament and AGD type 2 filaments pack two protofilaments with C2 symmetry. We were unable to solve the structure of AGD type 3 filaments to sufficient resolution for atomic modelling, but low-resolution cross-sections suggest an asymmetric packing of two protofilaments with the AGD fold. AGD case 1 also had Alzheimer PHFs and CTE type I filaments; AGD case 2 also had Alzheimer PHFs and SFs (Extended Data Figure 2d).

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Figure 2

Four-layered 4R tau filament structures

Cryo-EM density map and atomic model for AGD type 1 filaments and AGD type 2 filaments. Colours are as in Figure 1. The black arrow points to additional density discussed in the main text. The dotted lines indicate main chain connectivity for which cryo-EM density is missing.

Residues 293-357 of the AGD fold adopt the same conformation as in the CBD fold. However, the C-terminal segment (368-386) makes different interactions with R2, and the ordered core of the AGD fold is seven amino acids longer than that of the CBD fold. In addition, compared to the CBD cavity, the AGD cavity between R2 and the C-terminal segment is smaller and its net positive charge is less. It also contains a smaller additional density of unknown identity between K294 and K370. In AGD type 2 filaments, the turn at the end of R4, as well as the first and last six residues that form the ordered core of the AGD type 1 filaments, are disordered, and the termini point in a different direction compared to the AGD type 1 filaments.

Histologically, PSP-RS case 3 had also features of AGD, in particular the presence of argyrophilic grains in the entorhinal cortex (Extended Data Figure 7). Consistent with this, besides filaments with the PSP fold in thalamus, we also observed filaments of AGD type 2 and AGD type 3, as well as a minority of Alzheimer PHFs and SFs, in the entorhinal cortex from PSP-RS case 3 (Extended Data Figure 2e). By immunoblotting, the pattern of C-terminally truncated tau bands was like that of PSP in thalamus and like that of CBD in entorhinal cortex (Extended Data Figure 8). Thus, this case had concomitant PSP and AGD, as has been reported for other cases of PSP (26).

Astroglial 4R tau pathology in aging has been increasingly recognized; these cases have been subsumed under the umbrella term of ARTAG (29). Like AGD, ARTAG usually coexists with other pathologies. Structures of the majority species of tau filaments from the hippocampus of a case of ARTAG (30) were identical to AGD type 2 and type 3 filaments. Alzheimer PHFs and SFs were also observed (Extended Data Figure 2f).

Unlike the sporadic diseases described above, mutations +3 and +16 in intron 10 of MAPT give rise to a 4R tauopathy that belongs to the spectrum of inherited FTLD-tau (31–34). It is characterized by abundant filamentous tau inclusions in nerve cells and glial cells. Argyrophilic grains are present in entorhinal cortex, hippocampus and frontal cortex. Structures of tau filaments from the frontal cortex of two cases with the +3 mutation and one case with the +16 mutation all showed the AGD fold (Extended Data Figure 2g).

The differences between the structures of tau filaments from 4R tauopathies are consistent with results from immunoblots of sarkosyl-insoluble tau (Extended Data Figure 8). In PSP, CBD, GGT, AGD, ARTAG and MAPT intron 10 +3 and +16 mutation cases, tau bands of 64 and 68 kDa were in evidence, indicating the presence of full-length 4R tau. As reported previously (35–37), a strong C-terminal tau band of 33 kDa was also found in PSP and GGT, whereas a strong tau doublet of 37 kDa was seen in CBD, AGD and cases with mutation +16. The 37 kDa bands were also characteristic of ARTAG and cases with mutation +3. The 33 and 37 kDa bands correspond to N-terminally truncated tau at, or near, residues 186-187 and 168-169, respectively. Assembled full-length tau is probably cleaved in the fuzzy coat, with the size differences between the 33 and 37 kDa bands possibly resulting from different arrangements of the ends of the structured cores.

Whereas all diseases described above are 4R tauopathies, we also determined structures of tau filaments from two inherited types of 3R+4R tauopathies. FBD and FDD are caused by mutations in the integral membrane protein 2B gene (ITM2B) and are characterized by many extracellular inclusions of British amyloid (ABri) and Danish amyloid (ADan) that are associated with abundant tau-positive neurofibrillary tangles, neuropil threads and dystrophic neurites (38–41). Structures of tau filaments from the hippocampus of a previously described case of FBD (40) and the temporal cortex of a case of FDD (Extended Data Figure 2h) are identical to the PHFs that we previously observed for PART and Alzheimer’s disease (1,2,8). The hippocampus from the case of FBD also contained Alzheimer SFs, as well as CTE type I tau filaments, possibly as a result of head trauma.

Discussion

Clinical features are used to distinguish tauopathies. This is complemented and extended by post-mortem neuropathological examination. We now add a third layer of knowledge in the characterisation of these diseases. The cryo-EM structures presented here, together with those described previously (1–5,8), provide an overarching perspective that suggests a hierarchical classification of tauopathies based on their filament folds (Figure 3). We previously postulated that distinct conformers of filamentous tau define different tauopathies (5). This observation still holds. We now also show that some tauopathies share the same fold, and that multiple levels of similarity exist between folds.

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Figure 3

Structure-based Classification of Tauopathies

The dendrogram shows the proposed classification of tauopathies as described in the main text, with the corresponding folds displayed with the first β-strand in R3 oriented horizontally, except for the GGT and GPT folds, which are aligned to the PSP fold. Colours are as in Figure 1. Internal, non-proteinaceous densities are shown in black.

The first level of classification is based on the extent of the ordered cores, and coincides with the isoform compositions of tau inclusions in the corresponding diseases. All known tau folds from human diseases have a common ordered core region that comprises R3 and R4, as well as 10-13 amino acids of the C-terminal domain. However, the folds differ in their N-terminal extensions. The Alzheimer and CTE folds, which represent 3R+4R tauopathies, comprise only one or two amino acids from the C-termini of R1 and R2. The Pick fold, representing the only 3R tauopathy with known filament structures, comprises more than half of R1. Folds observed for 4R tauopathies comprise all of R2 and one or two residues of R1. As we previously observed for the CBD fold, incorporation of 3R tau isoforms is incompatible with the PSP, GPT, GGT and AGD folds, which agrees with the observation that only 4R tau isoforms assemble in these diseases. The presence of N279 from R2 in the fuzzy coat of the Alzheimer fold may explain why this residue is deamidated in tau assemblies from Alzheimer’s disease, but not from PSP or CBD (42)

At a second level, for the 3R+4R tauopathies, the CTE fold is distinct from the Alzheimer fold. However, whereas Alzheimer’s disease, FBD and FDD are distinct diseases that are characterized by different symptoms and/or neuropathology, they contain tau filaments with the Alzheimer fold. As exemplified by PART, which may be part of Alzheimer’s disease (43), the Alzheimer tau fold can form in the absence of Aβ deposits. By contrast, in cases of Alzheimer’s disease, FBD and FDD, the presence of extracellular amyloid deposits is accompanied by abundant intraneuronal tau inclusions with the Alzheimer fold. The Alzheimer fold has also recently been described in cases of prion disease with mutations Q160X and F198S in the PRNP gene, which are characterized by prion protein amyloid deposits and abundant 3R+4R tau inclusions (44).

Also at a second level, the 4R tauopathies are divided into two classes. The PSP, GPT and GGT folds comprise three-layered core regions, with R2 and R4 packing on either side of R3, whereas the packing of the C-terminal domain against part of R2 provides a fourth layer in the CBD and AGD folds. The division of 4R tauopathies based on three-layered and four-layered folds agrees with observations on post-translational modifications, which showed similarities between PSP and GGT, and between CBD and cases with mutation +16 in intron 10 of MAPT (45). A third level of classification for 4R tauopathies is provided by differences at the residue level between the three- and four-layered folds.

The AGD fold is also found in ARTAG, consistent with the presence of granular/fuzzy astrocytes in grey matter in both conditions. AGD and ARTAG can develop with age and coexist in a given human brain with the Alzheimer tau fold. The AGD fold also makes up the tau filaments found in cases with intron 10 mutations +3 and +16 of MAPT, consistent with the presence of argyrophilic grains in cases with the +3 mutation (Extended Data Figure 7). Assembled tau in AGD has been reported to lack acetylation (46). Features resembling AGD have also been described in cases of frontotemporal dementia caused by exon 10 mutations S305I and S305S in MAPT (47,48). These exonic and all known mutations in intron 10 destabilise the tau exon 10 splicing regulatory element RNA, resulting in a relative overproduction of 4R tau and the formation of 4R tau filaments (49). These filaments may have the AGD fold in common.

We present the first structures of tau filaments from cases with MAPT mutations. They form following the relative overproduction of 4R tau. Other MAPT mutations cause the assembly of mutant tau, without overproduction (7). Knowing the structures of tau filaments from cases with mutations at residue P301 (32,50–52) will be particularly important, since they have been used to produce the most widely used mouse models of human tauopathies (53,54). It will be interesting to compare the tau folds from mouse and human brains.

It is remarkable how well neuropathologically confirmed diagnoses correlate with particular tau folds. For instance, PSP and CBD have been grouped as clinically similar, but neuropathologically distinct 4R tauopathies (55). Their tau filament folds belong to different classes, confirming that they are separate disease entities. Prior to its definition, some cases of GGT were diagnosed as atypical PSP (56). Our observations that the tau filaments from GGT-I and GGT-II are distinct from those of PSP support the definition of GGT as a separate neuropathological entity. Moreover, the observation that tau filaments from GGT-III do not twist suggests that GGT-III itself may be a different disease from GGT-I and GGT-II. Likewise, the observation that the structures of tau filaments from PSP-F case 2 are different from those of PSP and GGT suggests that this individual may have suffered from a distinct disease. Histologically, this case was also different from typical and atypical PSP, with abundant spherical, 4R tau-immunoreactive, basophilic neuronal inclusions in limbic and other brain regions. We therefore name this disease Limbic-predominant Neuronal inclusion body 4R Tauopathy (LNT). Additional cases of LNT remain to be identified. It will be interesting to see if other tau folds exist that are intermediate between those of PSP and GGT.

The molecular mechanism that underlie tauopathies remain unknown. The presence of a specific tau fold in a given disease is consistent with its formation in a small number of brain cells, followed by the prion-like like spreading of tau inclusions (57). This may underlie the temporospatial staging of disease (12,27). Knowledge of the tau folds in the different diseases provides a new framework for studying tauopathies that will lead to a better understanding of disease pathogenesis. At a diagnostic level, the current findings will inform ongoing efforts to develop more specific and sensitive tau biomarkers.

Methods

No statistical methods were used to predetermine sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.

Extended Data

Extended Data Figure 1

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Tau immunohistochemistry

Representative tau staining of the brain regions used for cryo-EM structure determination (see Methods), using antibody AT8 (pS202/pT205 tau). Scale bars are 50 μm, except for GGT Type I, FBD and FDD, where they are 25 μm. For PSP-RS case 3, both the thalamus (Tha) and the entorhinal cortex (EC) are shown. Similar results were obtained using a minimum of six additional stained sections for each case.

Extended Data Figure 2

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Cryo-EM reconstructions

(a), Cryo-EM maps for tau filaments from six cases of PSP. For each map, a sum of the reconstructed density for several XY-slices is shown, corresponding to approximately 4.7 Å. The disease cases are referenced at the bottom of each image, the filament types at the top left and the percentages of a given filament type among the tau filaments in the data set at the top right. Scale bar 5 nm. The same scale applies to all panels, except (d-g) (b-h), As in (a), but a case of GGT-I and a case of GGT-II (b); a case diagnosed as PSP-F, but that contains filaments with the PGT fold (c); two cases of AGD (d); scale bar 5nm, the same scale applies to panels (d-g); entorhinal cortex of case PSP-RS3 (e); a case of ARTAG (f); three cases with mutations +16 or +3 in intron 10 of MAPT (g); a case of FBD and a case of FDD (h). Panels (d-f) contain blank squares to indicate the absence of AGD type I filaments in some cases. The inset with dashed lines shows 2D class average images of tau filaments from a case of GGT-III without apparent twist.

Extended Data Figure 3

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Cryo-EM resolution estimates

Fourier Shell Correlation (FSC) curves for cryo-EM maps and atomic structures of PSP filaments (from PSP-RS case 1); GGT filament types 1-3 (from GGT-I); GPT filament types 1a, 1b and 2 (from PSP-F case 2); and AGD filament types 1 and 2 (from AGD case 1 and the +16 case, repectively). FSC curves are shown for two independently refined cryo-EM half-maps (black); for the final refined atomic model against the final cryo-EM map (red); for the atomic model refined in the first half-map against that half-map (blue); and for the refined atomic model in the first half-map against the other half-map (yellow).

Extended Data Figure 4

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Schematics of tau filament folds

Schematics of the tau folds for PSP (a), GGT (b), GPT (c) and AGD (d). Negatively charged residues are shown in red, positively charged residues in blue, polar residues in green, apolar residues in white, sulfur-containing residues in yellow, prolines in purple and glycines in pink. Thick connecting lines with arrow heads are used to indicate β-strands; additional densities are shown in grey.

Extended Data Figure 5

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Tau pathology in Limbic-predominant Neuronal inclusion body 4R Tauopathy (LNT, PSP-F case 2)

a) Low power view of hippocampus stained with antibody AT8 (pS202/pT205 tau). b) Low power view of frontal cortex stained with AT8. c) Higher power view of hippocampus stained with AT8. d) AT8-positive globular astrocyte in hippocampus. e) AT8-positive neurons in hippocampus. f) Hippocampus stained with antibody RD4 (specific for 4R tau). g) Gallyas-Braak silver-positive neurons and glial cells in hippocampus. h) Hippocampus stained with antibody RD3 (specific for 3R tau). i) Higher power view of frontal cortex stained with AT8. j) AT8-positive globular astrocyte in frontal cortex. k) AT8-positive neurons in frontal cortex. l) Frontal cortex stained with RD4. m) Gallyas-Braak silver-positive neurons and glial cells in frontal cortex. n) Frontal cortex stained with RD3. Representative images are shown. Similar results were obtained using a minimum of six additional stained sections for each panel. Scale bars: 400 μm in (a); 200 μm in (b), 50 μm in (c, f, g, h, i, l, m, n); 10 μm in (d, e, j, k).

Extended Data Figure 6

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Structural comparisons

a) Comparison of two different main-chain conformations for GPT type 1 filaments (type 1a in purple; type 1b in green) and the main-chain conformation of GPT type 2 filaments (red). b) Comparison of the PSP (orange), GGT (blue) and GPT (purple) folds. c) Comparison of the inter-protofilament interfaces of GGT type 3 and GPT type 2 filaments. d) Comparison of the AGD type 1 (light blue), AGD type 2 (pink) and CBD (grey) folds.

Extended Data Figure 7

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Argyrophilic grains in the entorhinal cortex

Representative tau staining with antibodies RD4 (4R tau), RD3 (3R tau), AT8 (pS202/pT205 tau), as well as Gallyas-Braak silver, of the entorhinal cortex from cases 1 and 2 with mutation +3 in intron 10 of MAPT and from case 3 of PSP-RS. Similar results were obtained using a minimum of six additional stained sections for each case. Scale bar, 50 μm.

Extended Data Figure 8

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Immunoblot analysis of 4R tauopathies

Hyperphosphorylated full-length tau (64 and 68 kDa) and C-terminal fragments (33 kDa and 37 kDa) were detected in sarkosyl-insoluble fractions from the brain regions used for cryo-EM by anti-tau antibody T46. A prominent 33 kDa band was characteristic of PSP and GGT; strong 37 kDa bands were in evidence in AGD, ARTAG, cases with intron 10 mutations in MAPT (+3 and +16) and in CBD. PSP-RS case 3 had a strong 33 kDa band in thalamus (Tha) and strong 37 kDa bands in entorhinal cortex (EC), consistent with AGD co-pathology. Similar results were obtained in three independent experiments. The original, uncropped image is available in Supplementary Figure 1.

Supplementary Material

Acknowledgements

Footnotes

References