Structure-specific Mini-Prion Model for Alzheimer's Disease Tau Fibrils

Abstract

A critical discovery of the past decade is that tau protein fibrils adopt disease-specific hallmark structures in each tauopathy. The faithful generation of synthetic fibrils adopting hallmark structures that can serve as targets for developing diagnostic and/or therapeutic strategies remains a grand challenge. We report on a rational design of synthetic fibrils built of a short peptide that adopts a critical structural motif in tauopathy fibrils found in Alzheimer's Disease (AD) and Chronic Traumatic Encephalopathy (CTE). They serve as minimal prions with exquisite seeding competency, in vitro and in tau biosensor cells, for recruiting tau constructs ten times larger its size en route to AD or CTE fibril structures. We demonstrate that the generation of AD and CTE-like fibril structures is dramatically catalyzed in the presence of mini-AD prions and further influenced by salt composition in solution. Double Electron-Electron Resonance studies confirmed the preservation of AD-like folds across multi-generational seeding. Fibrils formed with the full AD/CTE-like core show strong seeding competency, with their templating effect dominating over the choice of salt composition that tunes the initial selection of AD- and CTE-like fibril populations. The mini-AD prions serve as a potent catalyst with templating capabilities that offer a novel strategy to design pathological tau fibril models.

Figure 1.

(a) Schematic overview of tau constructs used in this study. The top panel shows the domain organization of the full-length 2N4R isoform of tau with distinct color coding for the N-terminal inserts (N1, N2), proline-rich regions (P1, P2), microtubule-binding repeats (R1–R4), and the C-terminal domain. The constructs used in this study—0N4R-P301L, Tau187-P301L (with the P301L mutation indicated by a yellow mark), and dGAE—are shown below, aligned with the same domain color scheme. Regions corresponding to the sequences that form Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE) fibril folds are also indicated. (b) Cryo-EM-derived intramolecular folding of tau in CTE type I (PDB: 6NWP) and AD paired helical filament (PHF) folds (PDB: 5O3L), with the mini-AD motif highlighted. (c) Amino acid sequence of the mini-AD peptide and its predicted structure from AlphaFold2. (d) Thioflavin T (ThT) fluorescence assay showing the fibrillization kinetics of mini-AD. (e) Negative-stain TEM (ns-TEM) images of mini-AD fibrils, revealing twisted fibrillar morphology (Scale bar: 100 nm).

Figure 2.

(a) Distance distribution between spin labels at the termini of the mini-AD fold: prediction from the AF2 model using DEERPREdict (dark blue) versus experimental distribution from cw-EPR measurements of spin-labeled mini-AD fibrils analyzed with ShortDistances (cyan). (b) Top: cw-EPR spectrum of mini-AD fibrils showing the experimental signal (pale cyan), simulated fit (dark cyan), and contributions from distinct spectral components: mobile (green, 2%), immobile (yellow, 8%), and broadened (bright-pink, 90%). Bottom: Corresponding structural models for each spectral component are shown using the same color scheme, with red dots indicating spin label positions and annotated end-to-end distances. (c) Cryo-EM density map of mini-AD fibrils, viewed along the fibril axis, revealing a dimer-of-dimers architecture composed of four U-shaped mini-AD folds. (d) Comparison of the cryo-EM density map for a single mini-AD unit (middle) within the cryo-EM density map of mini AD with (i) a simulated 2.4 Å resolution map (top) of the AF2-predicted trimer structure and (ii) the corresponding density (bottom) of an AD-like fibril protofilament assembled in vitro (pale green density, PDB: 7QKK, shown as a representative map for all AD/CTE-like fibrils containing mini-AD motif) (e) sidechain orientation of the PHF6 region and ³⁷⁴HKLTF³⁷⁸ sequence of AF2 structure of mini-AD overlaid on the cryo-EM density of a single mini-AD fold suggesting cross-β interactions as predicted.

Figure 3.

(a) Schematic illustrating the proposed mechanism by which mini-AD seeds promote the templated aggregation of dGAE monomers into AD-like fibrils. Sky-blue denotes stacked arrangement single hairpin structural unit of the mini-AD seed, while pale green represents the full fold adopted by dGAE. (b) ThT fluorescence kinetics for the AD-reaction in the absence (grey) and presence (green) of mini-AD seeds. (c) Representative ns-TEM image of fibrils formed in the AD-reaction seeded with mini-AD seeds (AD-dGAE_ms). (d) Paired helical filament (PHF) morphology of AD-fibrils, characterized by an ~80 nm crossover distance. (e) (Top) Schematic depiction of examples of protofilament packing arrangements adopted by AD-like folds of dGAE fibrils(16). (Bottom) Ns-TEM image of AD-dGAE_ms with distinct arrows highlighting the observed morphological heterogeneity: white arrows indicate PHF-like structures, while black and blue arrows denote other higher-order assemblies such as THF- or QHF-like morphologies. (f) ThT fluorescence kinetics for the CTE-reaction with (red-orange) and without (grey) mini-AD seeds. (g) Corresponding ns-TEM image of mini-AD–seeded fibrils formed under CTE reaction conditions (CTE-dGAE_ms). 200 nm scale bars are shown in each ns-TEM image.

Figure 4.

(a) Schematic representation of the DEER experiment used to track structural ensembles seeded by mini-AD. A mixture of 5% spin-labeled dGAE monomers (MTSL labels shown as red dots at positions Q351 and T373) and 95% unlabeled dGAE is incubated with mini-AD seeds. Three major structural classes that can be templated by mini-AD—AD-like (yellow), CTE-like (cyan), and IM_J-like (pink)—are illustrated, each showing distinct quaternary arrangements and intramolecular folding patterns, with the relative separation between spin-label sites indicated. (b) Simulated inter-spin distance distributions for the three expected structures (top panel: AD-like in pale yellow, CTE-like in pale cyan, IM_J-like in pale pink) compared to experimental DEER data from mini-AD–seeded dGAE fibrils under CTE-reaction (CTE-dGAE_ms, middle panel, red-orange) and AD-reaction conditions (AD-dGAE_ms, bottom panel, green) showing shifts in populations.

Figure 5.

(a, b) Normalized ThT fluorescence kinetics (left) and ns-TEM images (right) of fibrils formed by (a) AD-dGAE_ms seeding dGAE monomers under AD-reaction conditions (light green), and (b) CTE-dGAE_ms seeding dGAE monomers under CTE-reaction conditions (orange). 200 nm scale bars are shown in each ns-TEM image. (c, d) DEER-derived distance distributions (P(r)) for fibrils generated by multiple rounds of seeding under (c) AD and (d) CTE-reaction conditions. The legend for each P(r) distribution indicates the fibrils for which the distribution is shown. Only the mean distributions are shown; for full datasets including error estimates, see Figures S7–10. Approximate population percentages (bar graphs on the right of the P(R) distribution) were estimated from the relative peak areas within the predicted distance ranges for the three expected structures: AD-like (pale yellow), CTE-like (pale cyan), and IM_J-like (pale pink). The expected distance distribution range for each structure is shown in the P(r) distribution plots with the same colors. The overlapping regions of expected distances for AD and CTE is shown in pale green and that of CTE and IM_J are shown is dark pink in the P(r) distribution plots.

Figure 6.

Tau aggregate formation, propagation, and their characteristics in cells seeded with mini-AD seeds. (a) H4 cell stably expressing mClover3–0N4R-P301L, seeded with mini-AD fibrils, imaged at 0- and 12-h post addition of mini-AD seeds (Scale bar 20 μm). Puncta formation is observed in the latter. (b) (Top) Schematic representation of the plasmid construct used to express two copies of Tau187. The construct includes mRuby3-Tau187(3R) (red), followed by a self-cleaving P2A sequence, which enables expression of mClover3-Tau187(4R)-P301L (green) within the same cell. (Bottom) Confocal fluorescence images of H4 neuroglioma cells following fibril treatment. Cells expressing the Tau187 dual-isoform construct were incubated for 24 hours with mini-AD fibrils, patient-derived AD fibrils, AD-dGAE_ms or jR2R3-P301L fibrils. Mini-AD, AD-dGAE_ms and patient-derived AD fibrils induced puncta formation in both the 3R (green) and 4R (red) channels, demonstrating incorporation of both tau isoforms. In contrast, jR2R3-P301L fibrils selectively seeded the 4R isoform, with no detectable puncta formation in the 3R channel (Scale bar: 20 μm).