Cryo-EM structures of amyloid-β 42 filaments from human brain

Abstract

Filament assembly of amyloid-β peptides ending at residue 42 (Aβ42) is a central event in Alzheimer’s disease. We report the cryo-EM structures of Aβ42 filaments from human brain. Two structurally related S-shaped protofilament folds give rise to two types of filaments. Type I filaments were found mostly in the brains from individuals with sporadic Alzheimer’s disease and Type II filaments in individuals with familial Alzheimer’s disease and other conditions. The structures of Aβ42 filaments from brain differ from those of filaments assembled in vitro. By contrast, in App ^NL-F knock-in mice, Aβ42 deposits were made of Type II filaments. Knowledge of Aβ42 filament structures from human brain may lead to the development of inhibitors of assembly and improved imaging agents.

Alzheimer’s disease is defined by the simultaneous presence of two different filamentous amyloid inclusions in brain: abundant extracellular plaques of Aβ and intraneuronal neurofibrillary tangles of tau (1). Genetic evidence has indicated that Aβ is key to the pathogenesis of Alzheimer’s disease (2,3). Multiplications of the APP gene encoding the Aβ precursor protein, as well as mutations in APP and in PSEN1 and PSEN2, the presenilin genes, cause familial Alzheimer’s disease. Presenilins form part of the γ-secretase complex that is required for the production of Aβ from APP. Although variability in γ-secretase cleavage results in Aβ peptides that vary in size, those of 40 (Aβ40) and 42 (Aβ42) amino acids are the most abundant. Mutations associated with familial Alzheimer’s disease increase the ratios of Aβ42 to Aβ40 (4,5), the concentration of Aβ42 (6), and/or the assembly of Aβ42 into filaments (7).

Three major types of Aβ inclusions are typical of the brain in Alzheimer’s disease (8-11): diffuse and focal deposits in the parenchyma, as well as vascular deposits. Diffuse deposits, which contain loosely packed Aβ filaments, are found in several brain regions, including entorhinal cortex, pre-subiculum, striatum, brainstem, cerebellum and subpial area. Focal deposits, in the form of dense core plaques, contain a spherical core of tightly packed Aβ filaments surrounded by more loosely packed filaments. Dense core plaques are found mostly in hippocampus and cerebral cortex. In advanced cases of Alzheimer’s disease, diffuse and focal Aβ deposits are widespread. In around 80% of cases of Alzheimer’s disease, Aβ deposits are also found in the walls of blood vessels (cerebral amyloid angiopathy). Electron cryomicroscopy (cryo-EM) provided the structures of Aβ40 aggregates from the meninges of Alzheimer’s disease brain (12). Meningeal deposits have a high Aβ40 and a low Aβ42 content and are morphologically distinct from parenchymal plaques.

Diffuse plaques and the loosely packed material of dense core plaques consist mainly of filamentous Aβ42, whereas plaque cores and blood vessel deposits are made of both Aβ40 and Aβ42. Aβ42 aggregates faster than Aβ40 and is the major species in plaques, despite the proteolytic processing of APP generating more soluble Aβ40 (4,8,13).

Aβ deposition appears to follow spatiotemporal spreading, suggesting that pathology may propagate through seeded aggregation, similar to prions (14-16). A prion-like mechanism may also explain the formation of Aβ deposits in cerebral blood vessels in some adults who received intramuscular injections of contaminated human growth hormone preparations as children and in individuals who were given dura mater grafts or underwent neurosurgery (17-19), even though they did not have the symptoms of Alzheimer’s disease. Besides Alzheimer’s disease, Aβ42 deposits can also be present as copathology in a number of other conditions, especially as a function of age (10). Despite their importance for disease pathogenesis, the structures of Aβ42 filaments from brain are not known.

Here we used cryo-EM to determine the structures of Aβ42 filaments extracted from the brains of ten individuals (Figs. 1, S1, Table S1). When using a sarkosyl extraction method developed for α-synuclein filaments (20,21), we found abundant Aβ42 filaments alongside other amyloids. By contrast, we only observed tau filaments (22,23), when extracting frontal cortex from individuals with Alzheimer’s disease using the standard sarkosyl extraction method (24). Five individuals had Alzheimer’s disease, with three sporadic and two familial (mutation in APP encoding V717F and mutation in PSEN1 encoding F105L) cases. Five individuals had other conditions, aging-related tau astrogliopathy (ARTAG), Parkinson’s disease dementia (PDD), dementia with Lewy bodies (DLB), familial frontotemporal dementia (FTD) caused by a GRN mutation and pathological aging (PA).

Figure 1. Cryo-EM maps of Type I, Type Ib and Type II Aβ42 filaments from brain.

Five cases of Alzheimer’s disease [three sporadic (sAD cases 1-3) and two familial (fAD case 1, mutation in APP encoding V717F; fAD case 2, mutation in PSEN1 encoding F105L)]; other human diseases [aging-related tau astrogliopathy (ARTAG), Parkinson’s disease dementia (PDD), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD) caused by a GRN mutation and pathological aging (PA)]; and homozygous mice of the App ^NL-F knock-in line. For each map, a sum of the reconstructed densities for several XY-slices, approximating one β-rung, is shown. Filament types (Type I, Type Ib and Type II) are indicated on the top left; the percentages of a given filament type among Aβ42 filaments in the dataset are shown on the top right. The same scales apply to all panels.

Type I Aβ42 Filaments from Human Brain

For individuals with sporadic Alzheimer’s disease, we observed a majority of twisted Aβ filaments, which we named Type I filaments (Figs. 1, 2A, B, D). They are made of two identical S-shaped (a double curve resembling the letter S or its reverse) protofilaments embracing each other with extended arms. The 2.5 Å resolution map of Type I filaments from sporadic Alzheimer’s disease case 1 was used to build the atomic model (Fig. S2A). The ordered core of each protofilament extends from G9-A42 with the N-terminal arm consisting of residues 9-18 and the S-shaped of residues 19-42. The secondary structure of protofilaments comprises five β-strands, which are each made of three or more residues. The S-shaped domain folds around two hydrophobic clusters: the N-terminal part around the side chains of F19, F20, V24 and I31, and the C-terminal part around the side chains of A30, I32, M35, V40 and A42 (Figs. 2B, D, S2C).

Figure 2. Structures of Type I and Type II Aβ42 filaments from brain.

(A) Amino acid sequence of Aβ1-42. Type I filaments (in orange) extend from G9-A42; Type II filaments (in blue) from V12-A42. Thick connecting lines with arrowheads indicate β-strands (β1-β5 and β1-β4). (B,C) Cryo-EM density map (in transparent grey) and atomic models for Type I (B) and Type II (C) filaments. Each filament type is made of two identical protofilaments shown in orange (Type I) and blue (Type II). The density maps are displayed using the zone feature in ChimeraX at a distance of 2 Å. Associated solvent molecules are shown in white and putative metals in teal (B) and purple (C). (D,E) Schematics of Type I (D) and Type II (E) Aβ42 folds. They were produced using atom2svg.py (https://doi.org/10.5281/zenodo.4090924). Negatively charged residues are shown in red, positively charged residues in blue, polar residues in green, non-polar residues in white, sulfur-containing residues in yellow and glycines in pink. Thick connecting lines with arrowheads indicate β-strands.

The two protofilaments pack against each other with pseudo-2₁ symmetry (Fig. S2E). They form a predominantly hydrophobic interface involving the side chains of L34, V36, V39 and I41 on the outer surface of the S-shaped domain, and the side chains of Y10, V12, Q15 and L17 in the N-terminal arm. In sporadic Alzheimer’s disease cases 1 and 3, we also observed a minority of Type Ib filaments, in which two Type I filaments run side by side and are held together by polar interactions, including salt bridges between K16 and E22 (Figs. 1, S3).

Several additional densities, attributed to ordered solvent molecules, are resolved in the 2.5 Å resolution cryo-EM map (Figs. 2B, 3A). One of these, located adjacent to the negatively charged carboxyl groups of E22 and D23 on the filament surface, most likely corresponds to a bound metal ion (Fig. 3B, C), as the conformations of both acidic residues are restrained, and the binding of metal ions would alleviate the electrostatic repulsion between their negatively charged carboxyl groups. Charged solvent molecules have been proposed to act as cofactors for filament formation by neutralisation of charges on in-register parallel β-sheets in amyloids (25). By contrast, there are no additional densities associated with an ordered grid of imidazole groups formed on the surface of Type I filaments by H13 and H14. Their side chains are held together by a hydrogen bond, with H13 making a second hydrogen bond with the side chain of E11 in the next Aβ42 molecule.

Figure 3. Protofilament folds and putative metal ion-binding sites of Type I and Type II Aβ42 filaments.

(A) Superposition of the structures of F20-V24 arcs overlaid on the corresponding section of the 2.5 Å electron density map of Type I filaments. Putative metal ions are shown as teal and purple spheres. (B,C) Side views of putative metal ion-binding sites in Type I (teal) and Type II (purple) protofilaments, superimposed on the corresponding density maps. (D) Superposition of Type I (orange) and Type II (blue) protofilaments, based on the central layer of their S-shaped domains. (E,F) Side views of Type I (E) and Type II (F) protofilaments along the central β3 strand. The centre layer monomers in five successive rungs are shown in cartoon, with β-strands shown as arrows.

Type II Aβ42 Filaments from Human Brain

For individuals with familial Alzheimer’s disease and other conditions, we observed a major, twisted filament type, distinct from Type I, which we named Type II (Figs. 1, 2A, C, E). In case 3 of sporadic Alzheimer’s disease, 17% of filaments were Type II, whereas in case 2 of familial Alzheimer’s disease, 24% of filaments were Type I. The atomic model of Type II filaments, built using the 2.8 Å resolution map obtained for the case of pathological aging (Fig. S2B), revealed that the ordered core extends from 12-42 and comprises four β-strands. Residues 20-42 adopt an S-shaped fold similar to that of Type I filaments, with the same side chain orientations. Differences between folds are mostly limited to the orientations of a few peptide groups affecting secondary structure assignments. Peptides G25-S26 and V36-G37 are flipped by approximately 180° in the Type II fold. The flipped G25-S26 peptide results in a slight expansion of the N-terminal hydrophobic cluster, which faces outwards in Type II filaments by accommodating the side chains of L17 and V18 instead of F19 (Figs. 3D, S2D). The reorientation of the second peptide leads to a shift of the C-terminal segment of the Type II fold along the helical axis by approximately one Aβ peptide, compared to its position in the Type I fold (Fig. 3E, F).

When compared to the Type I protofilament interface, that of Type II protofilaments is smaller and is formed by the opposite side of the S-shaped fold. Type II protofilaments pack against each other with C2 symmetry (Fig. S2F). The protofilament interface is primarily stabilised by electrostatic interactions between the amino group of K28 from one protofilament and the C-terminal carboxyl group of A42 from the other, and vice versa (Fig. 2C). Unlike Type I filaments, hydrophobic residues on the outer surfaces of the S-shaped domains remain exposed, forming non-polar patches on the surface of Type II filaments (Fig. S2D). There are fewer additional densities for ordered solvent molecules in the 2.8 Å map of Type II filaments than in the 2.5 Å map of Type I filaments, but the density for the putative metal ion bound to E22 and D23 is prominent in the equivalent location (Figs. 2B,C, 3E,F).

Comparison with Known Structures

Type I and Type II filaments have a left-handed twist and are structurally different from Aβ40 aggregates from the meninges of individuals with Alzheimer’s disease, which comprise two identical protofilaments with an unrelated C-shaped fold and a right-handed twist (12). They also differ from the cryo-EM structures of left-handed Aβ40 filaments, which were derived from the cerebral cortex of an individual with Alzheimer’s disease by seeded filament growth (26), but share with them a common substructure (Fig. 4A). In the seeded Aβ40 filaments, which comprised two extended protofilaments, residues G25-G37 adopted virtually the same conformation as in the middle of the S-shaped fold of Type I and Type II filaments. Structures of Aβ42 filaments assembled in vitro, obtained by cryo-EM (27) and solid-state NMR (28-30), each have a single or two identical protofilaments with an S-shaped domain like that of Type I and Type II filaments (Fig. 4B). In two NMR structures, the inter-protofilament packing also resembled that of Type I filaments. However, when examined at the single residue level, none of the Aβ42 filaments assembled in vitro displayed the same side chain orientations and contacts or the same inter-protofilament packing as in Type I and Type II filaments. The structures of in vitro assembled filaments of Aβ40 with the Osaka mutation (deletion of codon 693 in APP, corresponding to E22 in Aβ), based on a large number of unambiguous intra- and intermolecular solid-state NMR distance restraints, are most similar to those of Type I Aβ42 filaments (Fig. 4C) (31).

Figure 4. Comparison of protofilaments and filaments of brain Aβ42 with those of seeded recombinant Aβ40, recombinant Aβ42 and recombinant Aβ40ΔE22.

(A) Comparison of the cryo-EM structures of human brain Type I and Type II Aβ42 protofilaments with the cryo-EM structure of seeded recombinant Aβ40 protofilaments. Type I is in orange; Type II is in blue; seeded Aβ40 (PDB 6W0O) is in grey. (B) Comparison of cryo-EM structures of human brain Type I and Type II protofilaments with cryo-EM and NMR structures of recombinant protofilaments. PDBs and colour codes for recombinant Aβ42 filaments: 5OQV, wheat; 2NAO, dark grey; 2MXU, grey; 5KK3, light grey. (C) Comparison of cryo-EM structures of human brain Type I filaments and NMR structures of recombinant Aβ40ΔE22 filaments. Human brain Type I is in orange; recombinant Aβ40ΔE22 (PDB 2MVX) is in grey, with residues around ΔE22 shown in green. (D) Comparison of cryo-EM structures of Aβ42 filaments from the brains of mouse knock-in line App ^NL-F with human brain Type II filaments. Mouse brain filaments are in green; human brain Type II filaments are in blue.

Reconstructions of Type I and Type II filaments show strong densities for residues 41 and 42, indicating that the majority of molecules corresponds to Aβ42. In agreement, immunoblotting (Fig. S4) and mass spectrometry (Fig. S5) of extracted filaments showed that Aβ42 was the majority species in all cases, with variable amounts of Aβ40.

We performed immunohistochemistry on the contralateral sides of the brain regions used for cryo-EM, immunoblotting and mass spectrometry (Figs. S6, S7). Deposits of Aβ42 were also more numerous than those of Aβ40, with sporadic Alzheimer’s disease cases 1 and 3, familial Alzheimer’s disease case 1, as well as the cases of ARTAG, PDD, DLB and PA showing almost exclusively Aβ42 deposits. Most deposits of Aβ40 were present in sporadic and familial Alzheimer’s disease cases 2. By immunohistochemistry, Aβ40 deposits were also abundant in FTD. This difference with immunoblotting and mass spectrometry may reflect a hemispheric asymmetry in Aβ deposition. Plaque cores were most numerous in sporadic Alzheimer’s disease cases 1-3 and blood vessel deposits of Aβ40 were found in sporadic and familial Alzheimer’s disease cases 2. In all cases, diffuse deposits of Aβ were more numerous than focal and blood vessel deposits.

It is possible that low levels of Aβ40, or shorter peptides, may be incorporated in Type I and Type II filaments. The inter-protofilament salt bridge between K28 of one protofilament and the C-terminal carboxyl of A42 of the other, in Type II, but not Type I, filaments, suggests that it is more likely that hybrid Aβ42/Aβ40 filaments are of Type I. This is supported by the structural similarities of Type I Aβ42 filaments with filaments of Aβ40 with the Osaka mutation (Fig. 4C) (31). We did not find evidence for filaments composed predominantly of Aβ40. However, we cannot exclude that such filaments were present in low amounts, or were not extracted as dispersed filaments suitable for cryo-EM reconstruction.

Depending on the filament type, 8 or 11 residues are disordered at the amino-terminus. The β-site APP cleaving enzyme 1 (BACE1) generates the amino-terminus of Aβ (32). BACE1 mainly cleaves at residue 1 of Aβ, but some cleavage at residues 11 or 12 also occurs. Structures of Type I and Type II filaments from brain are compatible with the incorporation of shorter peptides. However, by immunoblotting and mass spectrometry (Figs. S4, S5), the bulk of Aβ42 in the extracted filaments was full-length. It follows that the amino-terminal residues that are not present in Type I or Type II filament cores form the fuzzy coat of Aβ42 filaments. This is supported by the decoration of Type I and Type II filaments using antibodies specific for the amino-terminal region of Aβ (Fig. S8). Tau filaments were unlabelled. The fuzzy coat of Aβ42 filaments thus comprises around 20% of the molecule, with the core making up 80%. By contrast, the fuzzy coat of tau filaments from Alzheimer’s disease amounts to over 80% (22,23).

In vitro aggregation is essential for studying the molecular mechanisms that underlie amyloid formation. However, available methods for the assembly of recombinant tau and α-synuclein yield filament structures that are different from those of filaments extracted from human brain (21,22,23,33,34). The same appears to be true of Aβ42 filaments, which only partially reproduce the structures from human brain.

Type II Aβ42 Filaments from App ^NL-F Mouse Brain

Animal models provide another tool for studying the molecular mechanisms of Alzheimer’s disease. App ^NL-F knock-in mice express humanized Aβ and harbour the Swedish double mutation (KM670/671NL), as well as the Beyreuther/Iberian mutation (I716F) in App (35). They develop abundant deposits of wild-type human Aβ42, neuroinflammation and memory impairment, without requiring the overexpression of APP. To further study the relevance of this mouse model for human disease, we determined the cryo-EM structures of Aβ42 filaments from the brains of 18-month-old homozygous App ^NL-F mice (Figs. 1, 4D). They were identical to Type II filaments from human brain, providing a mouse experimental system with filament structures like those from human brain. It is possible that cofactors required for the formation of Type II filaments are present in the brains of App ^NL-F knock-in mice, but missing from in vitro experiments.

Discussion

Type I and Type II Aβ42 filaments from brain are each made of two identical protofilaments, but the protofilaments of Type I filaments differ from those of Type II. This is unlike tau assembly in human brain, where a single protofilament gives rise to two or more types of filaments (36) and α-synuclein in multiple system atrophy, where four protofilaments give rise to two different filaments (21). Here, Type I filaments were limited to cases of sporadic Alzheimer’s disease that had also the largest number of plaque cores. A majority of Type II filaments was present in cases with abundant diffuse deposits of Aβ and a smaller number of focal plaques with cores. This included cases of familial Alzheimer’s disease, as well as cases of ARTAG, PDD, DLB, FTD and PA. Cases of Alzheimer’s disease with a majority of Type I filaments were older at death than other Alzheimer and non-Alzheimer cases with a majority of Type II filaments in neocortex. There was no correlation between Aβ42 filament type and APOE genotype. The relevance of these differences between Type I and Type II filaments is not known. Because positron emission tomography compound PiB (Pittsburgh compound B) visualizes Ab deposits in both sporadic and familial cases of Alzheimer’s disease, it probably labels both filament types (37).

Like V717F, the mutation in APP encoding I716F, increases the ratio of Aβ42 to Aβ40 (4,38,39). This may explain the presence of Type II Aβ42 filaments in App ^NL-F mice and in human cases with F717 APP. Line App ^NL-F may therefore be a model for some cases of familial Alzheimer’s disease, but not necessarily of sporadic disease.

Differential labelling by luminescent conjugated oligothiophene amyloid ligands suggested substantial heterogeneity in the molecular architecture of Aβ deposits from the brains of patients with Alzheimer’s disease (40). Our findings indicate that this heterogeneity is not the result of differences in the structures of Aβ42 filaments. As suggested for Aβ40 (19,41), a single Aβ42 filament type predominated in a given Alzheimer’s disease brain. Together with a second filament type, it accounted for the Aβ42 filaments from different cases of Alzheimer’s disease, ARTAG, PDD, DLB, FTD and PA. Knowledge of the structures of Aβ42 filaments from brain may lead to the development of better in vitro and animal models for these diseases, inhibitors of Aβ42 assembly, and imaging agents with increased specificity and sensitivity.

Once sentence summary.

In Alzheimer’s disease and other conditions, two structurally related protofilaments form Type I and Type II Aβ42 filaments.

Funding

This work was supported by the U.K. Medical Research Council (MC_UP_1201/25, to B.F.; MC_UP_A025_1013, to S.H.W.S.; MC_U105184291, to M.G.), Alzheimer’s Research U.K. (ARUK-RS2019-001, to B.F.), the Rainwater Charitable Foundation (to M.G.), the U.S. National Institutes of Health (P30-AG010133, UO1-NS110437, RF1-AG071177, to B.G. and R.V.) and the Department of Pathology and Laboratory Medicine, Indiana University School of Medicine (to B.G., K.L.N. and R.V.). W.Z. was supported by a Foundation that prefers to remain anonymous. G.G.K. was supported by the Safra Foundation and the Rossy Foundation.

Data and materials availability

Maps have been deposited in the Electron Microscopy Data Bank (EMDB) with the accession codes EMDB 13800 and 13809. Atomic coordinates have been deposited in the Protein Data Bank under accession codes 7Q4B and 7Q4M. Please address requests for materials to the corresponding authors.