Structure-based inhibitors halt prion-like seeding by Alzheimer's disease-and tauopathy-derived brain tissue samples

Abstract

In Alzheimer's disease (AD) and tauopathies, tau aggregation accompanies progressive neurodegeneration. Aggregated tau appears to spread between adjacent neurons and adjacent brain regions by prion-like seeding. Hence, inhibitors of this seeding offer a possible route to managing tauopathies. Here, we report the 1.0 Å resolution micro-electron diffraction structure of an aggregation-prone segment of tau with the sequence SVQIVY, present in the cores of patient-derived fibrils from AD and tauopathies. This structure illuminates how distinct interfaces of the parent segment, containing the sequence VQIVYK, foster the formation of distinct structures. Peptide-based fibril-capping inhibitors designed to target the two VQIVYK interfaces blocked proteopathic seeding by patient-derived fibrils. These VQIVYK inhibitors add to a panel of tau-capping inhibitors that targets specific polymorphs of recombinant and patient-derived tau fibrils. Inhibition of seeding initiated by brain tissue extracts differed among donors with different tauopathies, suggesting that particular fibril polymorphs of tau are associated with certain tauopathies. Donors with progressive supranuclear palsy exhibited more variation in inhibitor sensitivity, suggesting that fibrils from these donors were more polymorphic and potentially vary within individual donor brains. Our results suggest that a subset of inhibitors from our panel could be specific for particular disease-associated polymorphs, whereas inhibitors that blocked seeding by extracts from all of the tauopathies tested could be used to broadly inhibit seeding by multiple disease-specific tau polymorphs. Moreover, we show that tau-capping inhibitors can be transiently expressed in HEK293 tau biosensor cells, indicating that nucleic acid-based vectors can be used for inhibitor delivery.

Keywords: amyloid; crystal structure; fibril; inhibitor; neurodegeneration; prion; protein aggregation; protein structure; seeding; structural biology; tau protein; tauopathy; zipper interface.

Figure 1.

Top, outline of workflow used to design and test inhibitors of tau seeding. Figure citations are of figures in this paper, and lozenges are colored to correspond to A–E in this figure. A, ZipperDB (20) prediction of the aggregation-driving peptide segments in tau identifies segments VQIINK and VQIVYK. Sequences with scores exceeding an empirical threshold of −23 kcal/mol on the vertical axis (colored red) report hexapeptide segments with energetically favorable steric zipper scores. B, nanocrystals of the SVQIVY peptide segment predicted from the ZipperDB plot in A to have greater steric zipper–forming propensity compared with the parent segment, VQIVYK. C, electron diffraction collected by micro-ED from representative SVQIVY nanocrystals shown in B. D, model of a fibril-capping inhibitor (magenta) designed to block elongation by binding to the tip of a fibril (gray). The example shown is of a VQIINK capping inhibitor, WMINK, designed to inhibit aggregation from three interfaces formed by different polymorphs of the VQIINK steric zipper, labeled interface A, B, and C (19). E, seeding inhibition assay, carried out by transfecting tau biosensor cells (32) with tau fibrils (recombinant or brain-derived, as indicated) that were pretreated with capping inhibitor.

Figure 2.

Design of SVQIVYK-based capping inhibitors and classification of seeding inhibition on recombinant tau fibrils in tau biosensor cells. A, VQIVYK Class 3 interface formed by the left and center β-sheets colored green and cyan and Class 1 interface formed by the right and center β-sheets colored orange and cyan. Lysine 311, which is absent from the SVQIVY peptide, was modeled using coordinates from the previously determined VQIVYK crystal structure (Protein Data Bank entry 2ON9). Numbering along the backbone corresponds to residues modified in B to create different capping inhibitors that are listed in Table 1. B, yellow, a composite capping inhibitor that contains tryptophan substitutions at positions 2, 3, 4, and 5 (numbering corresponding to A) to show modeled steric clashes with the VQIVYK steric zipper structure, colored gray. Steric overlaps were mapped in PyMOL (23) using the show_bumps script. Green dots and disks show favorable van der Waals contacts, and red disks show steric clashes. Larger discs represent more severe clashes. C, seeding inhibition by VQIVYK-based capping inhibitors derived from peptides with tryptophan substitutions at the indicated positions (numbering corresponding to A). Seeding (expressed as 10⁻³ power) and inhibition were measured using recombinant fibrils of tau40 and HEK293 tau biosensor cells that stably express P301S 4R1N tau fused to YFP (32). Seeding inhibition was determined by counting the number of fluorescent puncta as a function of inhibitor concentration. IC₅₀ values were calculated from dose–response plots, except for cases indicated as not determined (ND). Dashed magenta lines, 50% inhibition. Inhibitor concentrations that reduce seeding by >50% are colored yellow with magenta data points. D and E, representative images of seeded tau-4R1N cells from C with no inhibitor (D) or 50 μm VY-WIW (E). Representative tau-4R1N cells containing aggregated tau puncta are marked with red arrows, and cells without are marked with white arrows. F, seeding inhibition by VQIINK-based capping inhibitors, otherwise as in C. G, seeding in tau-K18–expressing biosensor cells that were first transiently transfected with vector encoding the VY-WIW capping inhibitor peptide or a scrambled peptide as a control. Error bars, S.D.

Figure 3.

Inhibition of seeding by AD-derived brain extracts using the VQIVYK and VQIINK panel of capping inhibitors. A and B, seeding in tau-K18 biosensor cells 6 days after the addition of crude brain extract from AD donor 1 (AD1) (A), or 1 day after seeding with fibrils purified from AD1 (B). Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor. C, negative-stain electron micrograph of fibrils used for seeding in B. D, representative images from seeding inhibition experiments in A and B (seeded and inhibition of crude brain extract and purified AD fibrils). Red arrows point to representative cells containing seeded tau aggregates, and white arrows point to cells lacking aggregated tau. E, comparison of seeding by crude brain extract from AD donor 2 (AD2) with overnight versus no inhibitor preincubation. The left plot colored gray on the left shows seeding in tau-K18 biosensor cells and inhibition following overnight inhibitor preincubation. The experiment plotted on the right was performed using exactly the same conditions, except overnight inhibitor preincubation. Dashed magenta line, 50% inhibition; dotted green line, 25% inhibition. Inhibitors that reduce seeding by >50% are colored yellow with magenta data points, and inhibitors that reduce seeding by >25% but <50% are colored green with magenta data points. Error bars, S.D.

Figure 4.

Seeding produces fibrils of recombinant tau that have morphologies and inhibitor sensitivities that are similar to the AD brain–derived polymorph. A–C, electron micrographs of recombinant tau-K19+ fibrils (A), AD brain-derived fibrils (B), and recombinant tau-K19+ seeded by AD brain-derived fibrils (C). Orange arrows, representative ribbon-like fibrils characteristic of the recombinant fibril polymorph; purple arrows, fibrils with helical symmetry that are characteristic of the AD brain–derived PHF polymorph. D, sensitivity of AD-derived and recombinant tau-K19+ fibrils to a panel of VQIVYK-based inhibitors. Seeding was measured in tau-K18 biosensor cells and normalized for each fibril polymorph relative to seeding without inhibitor. Note that recombinant tau-K19+ fibrils seeded by an AD brain–derived specimen exhibit inhibitor sensitivities that are more similar to AD brain–derived fibrils than to the recombinant tau-K19+ fibril polymorph. Error bars, S.D.

Figure 5.

Inhibitor sensitivity of CTE-derived tau seeds. A–C, seeding by crude brain extract from the temporal cortex (A), recombinant tau-K18+ fibrils that were seeded with CTE-derived tau seeds (B), or recombinant tau-K18+ fibrils that were aggregated in the presence of heparin (C). Seeding inhibition measurements for the CTE-seeded recombinant fibril polymorph in B were carried out in tau biosensor cells after three sequential rounds of in vitro seeding. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor. In A–C, magenta arrows were used to mark capping inhibitors that were effective at blocking seeding by CTE-derived tau from crude brain extracts, green arrows mark inhibitors effective at blocking seeding by recombinant tau-K18 fibrils, and the blue arrow marking IN-W3 in B emphasizes that it is the only one of the inhibitors that blocks seeding by both the CTE-derived tau and recombinant tau fibrils. D–F, representative images from A, B, and C, respectively, showing seeding and inhibition in tau-K18 biosensor cells. Red arrows, representative cells containing seeded tau aggregates; white arrows, representative cells lacking aggregated tau. Error bars, S.D.

Figure 6.

Inhibitor profiling in biosensor cells seeded by brain extract from four different PSP donors. A, tissue sections from donors 1 and 2 were harvested from the midbrain and from the locus coeruleus for donor 3. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor. VQIINK inhibitors showing >70% inhibition are highlighted on bar graphs with a red outline. B, seeding by extracts from PSP donors 1, 2, and 3 after treatment with the capping peptide VY-W4. C, representative images showing seeding and inhibition in tau biosensor cells. Red arrows, representative cells containing seeded tau aggregates; white arrows, representative cells lacking aggregated tau. D and E, as in A, except tissue sections came from two different brain regions, the cerebellum (D) or frontal cortex (E), of a fourth PSP donor. F–H, representative images from D. I–K, representative images from E. Error bars, S.D.

Figure 7.

Top, locations of segments in tau targeted by different inhibitors of the panel and crystal structures of corresponding seeding interfaces. The filled boxes of the table below show efficacies of inhibitors for each donor tested in this study. For this analysis, inhibitors were scored as effective (filled box) if seeding was inhibited by 50% or more. Otherwise, inhibitors were scored as ineffective (open box).