Seeding selectivity and ultrasensitive detection of tau aggregate conformers of Alzheimer disease

Abstract

Alzheimer disease (AD) and chronic traumatic encephalopathy (CTE) involve the abnormal accumulation in the brain of filaments composed of both three-repeat (3R) and four-repeat (4R) (3R/4R) tau isoforms. To probe the molecular basis for AD’s tau filament propagation and to improve detection of tau aggregates as potential biomarkers, we have exploited the seeded polymerization growth mechanism of tau filaments to develop a highly selective and ultrasensitive cell-free tau seed amplification assay optimized for AD (AD real-time quaking-induced conversion or AD RT-QuIC). The reaction is based on the ability of AD tau aggregates to seed the formation of amyloid fibrils made of certain recombinant tau fragments. AD RT-QuIC detected seeding activity in AD (n = 16) brains at dilutions as extreme as 107–1010-fold, but was 102–106-fold less responsive when seeded with brain from most cases of other types of tauopathy with comparable loads of predominant 3R or 4R tau aggregates. For example, AD brains had average seeding activities that were orders of magnitude higher than Pick disease brains with predominant 3R tau deposits, but the opposite was true using our previously described Pick-optimized tau RT-QuIC assay. CTE brains (n = 2) had seed concentrations comparable to the weakest of the AD specimens, and higher than 3 of 4 specimens with 3R/4R primary age-related tauopathy. AD seeds shared properties with the tau filaments found in AD brains, as AD seeds were sarkosyl-insoluble, protease resistant, and reactive with tau antibodies. Moreover, AD RT-QuIC detected as little as 16 fg of pure synthetic tau fibrils. The distinctive seeding activity exhibited by AD and CTE tau filaments compared to other types of tauopathies in these seeded polymerization reactions provides a mechanistic basis for their consistent propagation as specific conformers in patients with 3R/4R tau diseases. Importantly, AD RT-QuIC also provides rapid ultrasensitive quantitation of 3R/4R tau-seeding activity as a biomarker.Electronic supplementary materialThe online version of this article (10.1007/s00401-018-1947-3) contains supplementary material, which is available to authorized users.

Human Tau Isoform Aggregation and Selective Detection of Misfolded Tau from Post-Mortem Alzheimer’s Disease Brains

“…To our knowledge, no studies have been reported to selectively detect misfolded tau seeds from AD brains using recombinant full-length tau isoforms as substrates. Our study complements a recent investigation describing ultrasensitive detection of tau aggregate conformer of AD with a mixture of two significantly shortened tau fragments as dual substrates (Kraus et al, 2019). The successful seeding activities of AD brain homogenates with individual tau isoforms as a substrate further validated the predicted presence of all six misfolded tau isoforms in AD brains (Goedert et al, 1992;Lee et al, 1999).…”

“…RT-QuIC assay has been used for detection of seeding activities of misfolded proteins, first for prions and then for aSyn and tau in vitro (Atarashi et al, 2008;Orru et al, 2017;Groveman et al, 2018;Sano et al, 2018;Saijo et al, 2017;Kraus et al, 2019). To determine whether various recombinant wild-type human tau isoforms can be converted by the tau aggregate seeds from AD brain samples, we detected autopsied AD and non-AD brain samples from neuropathologically confirmed cadavers (Table 1).…”

“…RT-QuIC Analysis. Tau RT-QuIC assays were conducted as previously described with minor modifications (Orru et al, 2017;Saijo et al, 2017;Kraus et al, 2019;Wang et al, 2019). In brief, each recombinant tau isoform was thawed at room temperature and filtered with a 100 kDa spin column filter (Millipore) to remove unwanted tau aggregates.…”

“…Two methods to amplify misfolded proteins in vitro, the real-time quaking-induced conversion (RT-QuIC) assay and the protein misfolding cyclic amplification protocol (PMCA) are used for ultrasensitive detection in a number of neurodegenerative diseases, such as prion disease (Moda et al, 2014;Orru et al, 2017;Wang et al, 2019), Ab oligomers in AD (Salvadores et al, 2014), and a-synuclein in Parkinson's disease (Fairfoul et al, 2016;Shahnawaz et al, 2017;Groveman et al, 2018). Specifically for misfolded tau detection in AD and other tauopathies such as Pick's disease, detection of the prion-like tau seeding activities by RT-QuIC assays was successfully developed using engineered short fragments of tau or a mixture of tau fragments with mutations as a substrate (Saijo et al, 2017;Kraus et al, 2019). However, detection of AD and related tauopathies with recombinant full-length human tau isoforms that match tau isoforms in vivo has not been reported.…”

Propagation of Tau Pathology: Integrating Insights From Postmortem and In Vivo Studies

“…A recently described technique called real-time quaking-induced conversion, which exploits the ability of prion protein to induce self-aggregation, has been used to develop a diagnostic CSF test for Creutzfeldt-Jakob disease (145). This method has now also been developed into a test to detect pathological forms of tau in CSF from patients with AD or other tauopathies (146,147). Seedcompetent high-molecular-weight tau species have been detected in human CSF (78), but there is yet no established method in clinical laboratory practice.…”