Abstract
Tau protein fibrillization is implicated in the pathogenesis of several neurodegenerative diseases collectively known as Tauopathies. For decades, investigating Tau fibrillization in vitro has required the addition of polyanions or other co-factors to induce its misfolding and aggregation, with heparin being the most commonly used. However, heparin-induced Tau fibrils exhibit high morphological heterogeneity and a striking structural divergence from Tau fibrils isolated from Tauopathies patients’ brains at ultra- and macro-structural levels. To address these limitations, we developed a quick, cheap, and effective method for producing completely co-factor-free fibrils from all full-length Tau isoforms and mixtures thereof. We show that Tau fibrils generated using this ClearTau method – ClearTau fibrils – exhibit amyloid-like features, possess seeding activity in biosensor cells and hiPSC-derived neurons, retain RNA-binding capacity, and have morphological properties and structures more reminiscent of the properties of the brain-derived Tau fibrils. We present the proof-of-concept implementation of the ClearTau platform for screening Tau aggregation-modifying compounds. We demonstrate that these advances open opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies.
Introduction
The microtubule-binding protein (MAP) Tau is an intrinsically disordered protein (IDP) that is most prominently associated with the dynamic regulation and stabilization of cytoskeletal and mitotic microtubules1. In neurons, Tau is also important for regulating axon outgrowth and maintaining axonal transport and cytoskeletal integrity2. However, factors such as post-translational modifications (PTMs)3, mutations in the protein sequence4, interaction with other proteins5,6 and changes to the biochemistry of its surrounding environment, such as pH or the presence of drugs7, may result in the lowered affinity, weaker interaction, or full dissociation of Tau from microtubules (for a recent review see8). Tau may then accumulate and aggregate into higher molecular weight species, such as fibrils associated with pathology. Increasing evidence points to Tau aggregation and PTMs as central events in the pathogenesis of Alzheimer’s disease (AD) and Tauopathies, events that investigators strive to faithfully model in the laboratory (for a recent review see9). In addition to amyloid plaques composed of β-amyloid, a classic hallmark of AD, another prominent pathological feature of AD is hyperphosphorylated Tau which is found in neuronal cell bodies or neurites in the form of paired helical filaments (PHFs) and straight filaments (SFs)10. Tau aggregates and fibrillar structures are also found in the brain of individuals afflicted by other neurodegenerative diseases (NDs), collectively known as Tauopathies, which include Pick’s disease (PiD) and progressive supranuclear palsy (PSP)11,12,13,14,15,16,17,18. Tau exists as six isoforms in the human central nervous system, designated 4R2N, 4R1N, 4R0N, 3R2N, 3R1N, and 3R0N. Where the Tau isoform compositions of the Tau fibrils are known13, Tauopathies are classified into predominantly 3 R (i.e., PiD), predominantly 4 R (i.e, PSP), or mixed (3 R + 4 R; i.e., AD).
Full-length Tau isoforms are highly soluble and notoriously resistant to aggregation on their own, in contrast to other amyloid-forming proteins, such as α-synuclein19, β-amyloid peptide20 or amylin21, which misfold, aggregate, and form amyloid fibrils simply by incubation at 37 °C. The molecular and cellular factors that trigger Tau misfolding and aggregation, and drive the Tau fibrillization processes remain unclear. Therefore, to study Tau fibrillization in vitro, investigators have adopted Tau aggregation systems using various negatively-charged co-factor molecules or protein modifications, such as truncations or phosphorylation, which are used to induce or accelerate the full-length Tau fibrillization process. Negatively-charged polysaccharide free-floating heparin (FFH) has thus far been the most commonly used Tau aggregation co-factor, although others including RNA, anionic lipids, or small proteins are occasionally used (reviewed in22). The mechanisms by which anionic co-factors induce Tau aggregation are thought to include electrostatic charge neutralization of highly positively-charged regions on Tau, promoting changes in the local and global Tau polypeptide shape22. This allows the folding of the aggregation-promoting Tau regions PHF6* and PHF6 into β-sheet-containing conformation, leading to Tau fibrillization and assembly into higher-order Tau fibrils. In addition, structural and biophysical studies have shown a high affinity of specific positively-charged Tau residues for heparin, notably the interfibrillar interface-forming residues in the core of the fibril folds.
To be clinically relevant, in vitro Tau aggregation systems must recapitulate some of the morphological, biochemical, and structural features of Tau aggregates and fibrils found in human pathologies. Several high-resolution structures of Tau fibrils composed of different Tau isoforms have been recently solved using cryoelectron microscopy (cryo-EM; see23), including Tau filaments isolated from post-mortem patient brain tissues. These structures combined with other experimental observations suggest that heparin or other co-factors used to induce Tau aggregation in vitro introduce several limitations that preclude biologically-relevant studies from elucidating the role of Tau aggregation in the pathogenesis of Tauopathies (Fig. 1). First, recent studies demonstrated that in vitro FFH-induced Tau fibrils differ from pathological patient-derived fibrils at the biochemical (lack of PTM patterns due to the use of the unmodified isoforms) and structural (amyloid core conformation) levels24. In addition, the mature Tau fibrils in AD, corticobasal degeneration (CBD), and chronic traumatic encephalopathy (CTE) are composed of doublet Tau filaments with the interfilament interfaces differing between these disorders, which manifests in various twisted morphologies of the higher-order Tau fibrils. In contrast, conventional in vitro-produced Tau fibrils extensively comprise a single-filament and are structurally and morphologically divergent from any Tau fibrillar structures derived from ND patients’ brains, recently illustrated by cryo-EM-solved FFH-induced fibril structures24. Second, the addition of heparin to Tau results in heterogeneous fibril populations, whereas the ultrastructures of pathological Tau fibrils are consistent between patients and are a signatory of the specific Tauopathies. Third, heparin and other co-factors have been shown to bind strongly and remain associated with the Tau fibril, which could interfere with their interaction with other small molecules and ligands (e.g., RNAs, small-molecule compounds)25,26. The Tau fibrils induced by RNA also show no similarity to any known pathological Tau fibril structures27. These properties preclude the use of FFH- and RNA-induced Tau fibrils to investigate the structural basis of Tau aggregation, cellular uptake, and toxicity or their use for the development and testing of Tau-targeting and binding molecules, as well as positron emission tomography (PET) tracers….
