A single ultrasensitive assay for detection and discrimination of tau aggregates of Alzheimer and Pick diseases

Abstract

Multiple neurodegenerative diseases are characterized by aggregation of tau molecules. Adult humans express six isoforms of tau that contain either 3 or 4 microtubule binding repeats (3R or 4R tau). Different diseases involve preferential aggregation of 3R (e.g Pick disease), 4R (e.g. progressive supranuclear palsy), or both 3R and 4R tau molecules [e.g. Alzheimer disease and chronic traumatic encephalopathy]. Three ultrasensitive cell-free seed amplification assays [called tau real-time quaking induced conversion (tau RT-QuIC) assays] have been developed that preferentially detect 3R, 4R, or 3R/4R tau aggregates in biospecimens. In these reactions, low-fg amounts of a given self-propagating protein aggregate (the seed) are incubated with a vast excess of recombinant tau monomers (the substrate) in multi-well plates. Over time, the seeds incorporate the substrate to grow into amyloids that can then be detected using thioflavin T fluorescence. Here we describe a tau RT-QuIC assay (K12 RT-QuIC) that, using a C-terminally extended recombinant 3R tau substrate (K12CFh), enables sensitive detection of Pick disease, Alzheimer disease, and chronic traumatic encephalopathy seeds in brain homogenates. The discrimination of Pick disease from Alzheimer disease and chronic traumatic encephalopathy cases is then achieved through the quantitative differences in K12 RT-QuIC assay thioflavin T responses, which correlate with structural properties of the reaction products. In particular, Fourier transform infrared spectroscopy analysis of the respective K12CFh amyloids showed distinct β-sheet conformations, suggesting at least partial propagation of the original seed conformations in vitro. Thus, K12 RT-QuIC provides a single assay for ultrasensitive detection and discrimination of tau aggregates comprised mainly of 3R, or both 3R and 4R, tau isoforms.

Towards an improved early diagnosis of neurodegenerative diseases: the emerging role of in vitro conversion assays for protein amyloids

“…Using similar RT-QuIC conditions, but employing different tau constructs, the same group adapted the RT-QuIC for the detection of tau misfolded species from mixed (3R/4R) tauopathies, such as AD and chronic traumatic encephalopathy patients, or 4R tauopathies, such as PSP and CBD [49,79]. While the former assay, once again, only worked with a brain homogenate as seed, the 4R-specific construct also revealed promising results in the samples collected in vivo, although the accuracy was significantly lower in the CSF samples obtained in vivo compared to those collected post-mortem [49,57,78,79]. In summary, tau RT-QuIC embodies enormous potential but needs to be refined and tested in a large cohort of patients before being proposed as a valuable diagnostic tool.…”

From Seeds to Fibrils and Back: Fragmentation as an Overlooked Step in the Propagation of Prions and Prion-Like Proteins

“…Specifically, 40 rounds of 5s sonication pulses increased the detection limit for synthetic K18 tau seeds from 0.1% to 0.00001% of monomer equivalent. Further refinements made by others have enabled researchers to not only detect misfolded tau, but to also distinguish between different tauopathies based on the different kinds of tau aggregates involved [ 32 , 33 , 34 ].…”

Defining the Protein Seeds of Neurodegeneration using Real-Time Quaking-Induced Conversion Assays

“…Additional assays have been developed for the detection of 4R tau aggregates [ 46 ], as well as a second assay capable of detecting 3R/4R (including AD) but also 3R tau conformers (of PiD), known as the K12 RT-QuIC assay [ 71 ]. The 4R RT-QuIC demonstrates preferential selectivity for 4R tauopathies including PSP, CBD, and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).…”

“…4R tau seeds are also detectable in PSP and corticobasal syndrome (CBS)/CBD post- and premortem CSF, however with much less sensitivity than that demonstrated for brain [ 46 ]. The K12 RT-QuIC demonstrates similar sensitivity to that of the AD RT-QuIC, and is able to detect both 3R seeds, in the case of PiD cases, and 3R/4R tau seeds of AD, CTE, and PART [ 71 ]. Both the 4R and K12 RT-QuIC assays are capable of discriminating templating subtypes of disease-specific seeds using readouts of ThT amplitudes and fibrillar assay products.…”