Proteopathic tau seeding predicts tauopathy in vivo

Abstract

Transcellular propagation of protein aggregates, or proteopathic seeds, may drive the progression of neurodegenerative diseases in a prion-like manner. In tauopathies such as Alzheimer's disease, this model predicts that tau seeds propagate pathology through the brain via cell-cell transfer in neural networks. The critical role of tau seeding activity is untested, however. It is unknown whether seeding anticipates and correlates with subsequent development of pathology as predicted for a causal agent. One major limitation has been the lack of a robust assay to measure proteopathic seeding activity in biological specimens. We engineered an ultrasensitive, specific, and facile FRET-based flow cytometry biosensor assay based on expression of tau or synuclein fusions to CFP and YFP, and confirmed its sensitivity and specificity to tau (∼300 fM) and synuclein (∼300 pM) fibrils. This assay readily discriminates Alzheimer's disease vs. Huntington's disease and aged control brains. We then carried out a detailed time-course study in P301S tauopathy mice, comparing seeding activity versus histological markers of tau pathology, including MC1, AT8, PG5, and Thioflavin S. We detected robust seeding activity at 1.5 mo, >1 mo before the earliest histopathological stain. Proteopathic tau seeding is thus an early and robust marker of tauopathy, suggesting a proximal role for tau seeds in neurodegeneration.amyloid | neuropathology | dementia | aging P rotein aggregation characterizes many neurodegenerative disorders, including Alzheimer's disease (AD) and the related tauopathies. These disorders feature the accumulation of fibrillar deposits of the microtubule-associated protein tau with progressive deterioration of the central nervous system. Tau pathology and its associated brain atrophy do not appear randomly throughout the brain, but rather progress along distinct neural networks (1-5). This aspect suggests a role for transcellular spread of a pathogenic agent via neural connections. Our laboratory and others have previously hypothesized that tau aggregates-or seeds-serve as this agent of spread, transmitting the aggregated state from cell to cell via prion-like mechanisms (6-15).Mounting fundamental insights support this hypothesis. Tau seeds applied to the outside of cells bind the cell surface by attaching to heparan sulfate proteoglycans, triggering uptake by macropinocytosis (13). Upon internalization, tau seeds nucleate the fibrillization of endogenous tau monomer via templated conformational change, or seeding (8, 10). Tau seeding requires a critical unit of size for activity, as only particular species propagate the aggregated state (16). In vivo studies have described tau protein spreading from local sites to distant regions, presumably via transsynaptic movement (11,12,(17)(18)(19). Finally, our laboratory and another recently demonstrated that tau propagates discrete amyloid conformations through the brains of animals that give rise to unique neuropathologies (18,20).Despite this evidence, it remains unclear wheth...

Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies

“…Seeding properties of many FTDP-17 mutants had yet to be examined, and current cultured cell tau seeding models tend to utilize either mutated truncated repeat domain (RD) tau (29,44,47), which can intrinsically aggregate in these models (56, 57), or full-length tau mutants at the P301 site (30). WT tau has been reported to aggregate with seeding in some cell culture studies, albeit only at low levels, especially when compared to P301L tau or RD tau (29,30,48).…”

“…Despite this, there is a lack of data in the field comparing the seeding propensities of multiple tau mutants and much of the current data utilizes specific mutants P301L and P301S, or truncated repeat domain (RD) tau (29,30,(44)(45)(46)(47). However, disease phenotypes in FTDP-17 vary significantly between mutants and even within the same family (13).…”

Tau protein liquid–liquid phase separation can initiate tau aggregation

“…We found that tauHMW was able to initiate tau aggregation in HEK TauRDP301S‐CFP/YFP cells (Holmes et al , 2014; Fig 5D), and, excitingly, showed rapid (1–2 min) phase separation into droplets as well as aggregation when applying crowding conditions (10% PEG, Fig 5E). Furthermore, by immunohistological labeling of phospho‐tau in post‐mortem human brain tissue with mild AD pathology (Braak stage III; Braak & Braak, 1995), we could find spherical phospho‐tau accumulations in hippocampal neurons (Fig EV6).…”

“…HEK293 cells stably expressing the repeat domain (TauRD, aa243‐372) of human mutant Tau441P301S tagged with CFP and YFP (HEK TauRDP301S‐CFP/YFP (Holmes et al , 2014)] were treated with preformed p‐tau441 droplets, soluble p‐tau441 [0.5 μM tau, 0.8% lipofectamine (LifeTechnologies)], or TauHMW from human AD brain lysates (20 ng tau, 0.8% lipofectamine). Treating these cells with tau material competent of seeding tau aggregation results in intracellular aggregation of TauRDP301S‐CFP/YF, which can be detected as small fluorescent FRET‐positive protein aggregates.…”

C-Terminally Truncated Forms of Tau, But Not Full-Length Tau or Its C-Terminal Fragments, Are Released from Neurons Independently of Cell Death

“…Animal (Clavaguera et al, 2009;Frost et al, 2009;de Calignon et al, 2012;Kfoury et al, 2012) and cell culture studies suggest that tau aggregation can propagate from neuron to neu-ron (Frost et al, 2009, Holmes et al, 2014 and there is growing interest in how tau is released and taken up by cells.…”