| Modeling Induction of Neurofibrillary Tangle Formation in Alzheimer’s Disease Jeff Kuret1; 1OHIO STATE UNIVERSITY, Columbus, United States; PAPER: 402/Medicine/Plenary (Oral) SCHEDULED: 11:30/Tue. 29 Nov. 2022/Ballroom B ABSTRACT: Tauopathies are neurodegenerative disorders characterized by the intracellular accumulation of filamentous aggregates composed of tau protein, with Alzheimer’s disease (AD) being the most prevalent example [1]. Recent cryo-electron microscopy (CryoEM) reports have revealed the polymorphic nature of these filaments, where tau protomers adopt disease-specific folds within the amyloid cores of each conformer [2]. Despite this conformational heterogeneity, each polymorph core consists of the microtubule repeat region of tau and contains at least one hexapeptide motif (i.e., PHF6 and PHF6* motifs) essential for initiating aggregation through a nucleation-dependent mechanism [3]. In addition to tau, the majority of disease-derived conformers copurify with undefined non-proteinaceous substances [2]. These interact with specific amino acids side chains, especially Lys, implying they have poly-anionic character. Whether non-proteinaceous materials influence tau nucleation and polymorphism or are merely trapped during aggregation is unknown. To clarify how anionic substances foster tau aggregation, full-length recombinant human 2N4R tau was fibrillized in the presence of Geranine G, a small-molecule dye. Resulting aggregates were then subjected to sedimentation, spectrophotometic and CryoEM analyses. The role of Lys residues in Geranine G-mediated induction was interrogated by Lys-to-Ala mutagenesis and in vitro aggregation assay. Sedimentation and spectrophotometric analysis showed that Geranine G remained stably associated with tau aggregates after induction (8.7 ± 0.6 mol/mol stoichiometry). Upon CryoEM analysis, 2D classification revealed the presence of multiple polymorphs. One species corresponding to a tau protofilment was solved at 3.3 Å resolution. The core region of this tau aggregate was not a perfect replica of any known polymorph but most closely resembled the heparin-induced synthetic protofilament and the kernal of three-layer tau aggregates isolated from human brain (e.g., globular glial tauopathy). The density map also captured densities for seven GG molecules in complex with it. Interactions were primarily through Lys residues via solvent separated ion pairing. Lys-to-Ala mutagenesis identified the binding pose associated with Lys317/Lys321 as making the greatest contribution to Geranine-G induced tau aggregation propensity. We propose two mechanisms for Geranine G-mediated induction of 2N4R tau aggregation. First, GG neutralizes the net positive charge of tau protein. This effect is likely nonspecific for individual Lys residues. Second, GG specifically stabilizes the heterosteric zipper formed by the PHF6* motif through bridging Lys residues within its interacting segment. This segment frequently associates with nonproteinaceous substances in tauopathy polymorphs (i.e., in AD and all three-layer polymorphs) consistent with a potential role in promoting aggregation and modulating formation of disease conformers. References: [1] Limorenko G., Lashuel H.A. Chem Soc Rev. 51 (2022) 513-565. [2] Shi Y. et al. Nature 598 (2021) 359-363. [3] Yang J., Agnihotri M.V., Huseby C.J., Kuret J., Singer S.J. Biophys J. 120 (2021) 1396-1416. |
