Reconstitution of the Alzheimer’s Disease Tau Core Structure from Recombinant Tau^297–391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM

Abstract

The protein tau misfolds into disease-specific fibrillar structures in more than 20 neurodegenerative diseases collectively referred to as tauopathies. To understand and prevent disease-specific mechanisms of filament formation, in vitro models for aggregation that robustly yield these different end point structures will be necessary. Here, we used cryo-electron microscopy (cryo-EM) to reconstruct fibril polymorphs taken on by residues 297−391 of tau under conditions previously shown to give rise to the core structure found in Alzheimer's disease (AD). While we were able to reconstitute the AD tau core fold, the proportion of these paired helical filaments (PHFs) was highly variable, and a majority of filaments were composed of PHFs with an additional identical C-shaped protofilament attached near the PHF interface, termed triple helical filaments (THFs). Since the impact of filament layer quaternary structure on the biological properties of tau and other amyloid filaments is not known, the applications for samples of this morphology are presently uncertain. We further demonstrate the variation in the proportion of PHFs and PHF-like fibrils compared to other morphologies as a function of shaking time and AD polymorph-favoring cofactor concentration. This variation in polymorph abundance, even under identical experimental conditions, highlights the variation that can arise both within a lab and in different laboratory settings when reconstituting specific fibril polymorphs in vitro.

Post-Translational Modifications Control Phase Transitions of Tau

“…We hypothesized that AD-specific PTMs can facilitate the nucleation of Tau paired helical filaments in vitro . The Tau region of interest was selected based on the published study that identified Tau(297-391) as the sequence that recapitulates the AD fold in vitro, 13–17 and our prior synthetic work which indicated a strategically advantageous synthetic disconnection at C291 to produce a peptide fragment Tau(291-391). 45 We selected three PTMs that are linked to Tau physiology and disfunction: Acetylation of Tau filaments occurs in the region that forms the rigid fibril core and usually overlaps with ubiquitination sites.…”

“…10–12 Only recently aided by high-throughput cryo-EM, short fragments of Tau have been shown to self-assemble into the AD and CTE filaments, but these peptides also form many other structures under similar conditions (Figure 1). 13–17…”

“…fragments of Tau have been shown to self-assemble into the AD and CTE filaments, but these peptides also form many other structures under similar conditions (Figure 1). [13][14][15][16][17] The factors that assist or control folding of full-length Tau into single disease specific polymorphs remain unclear. In understanding the structural polymorphism of Tau, posttranslational modifications (PTMs) that alter the charge and hydrophobicity of amino acid side chains need to be considered.…”