Abstract
Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of ~ 3 nm × 4 nm) is ~ 7 times larger than the β-strand distance (0.47 nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on TauFL or TauRD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused by a process distinct from assembly of TauRD filaments.
Highlights
Tau, a microtubule-associated protein (MAPT, Uniprot P10636), has an important role in microtubule assembly and stabilization
The proteins were expressed in E. coli, except Tau repeat domain with pro-aggregant mutation ΔK280 (TauRDΔK)-Green fluorescent protein (GFP) which could only be expressed in the baculovirus-Cell line from Spodoptera frugiperda (Sf9) cell system
The progression of Alzheimer Disease can be subdivided into several stages, as judged by abnormal changes in the neuronal protein Tau which spread in the brain with a predictable spatio-temporal sequence, following axonally connected pathways [13]
Summary
A microtubule-associated protein (MAPT, Uniprot P10636), has an important role in microtubule assembly and stabilization. Mostly basic composition, is natively unfolded and is highly soluble. Tau amyloidogenic aggregates characterize a wide range of neurodegenerative diseases known as Tauopathies [35, 45, 73] including Alzheimer Disease (AD). Mutations in the Tau gene alone are sufficient to cause neurodegeneration [36]. Biophysical and structural studies show that soluble monomeric Tau, upon nucleation by polyanionic cofactors like heparin or RNA, can form insoluble paired helical filaments (PHFs) in vitro [24, 38]. The pathways causing Tau aggregation in neurons and of Tau-induced neurodegeneration are not well understood
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