Ultrasensitive RT-QuIC Seed Amplification Assays for Disease-Associated Tau, α-Synuclein, and Prion Aggregates

Saijo, Eri; Groveman, Bradley R.; Kraus, Allison; Metrick, Michael A.; Orrú, Christina D.; Hughson, Andrew G.; Caughey, Byron

doi:10.1007/978-1-4939-8820-4_2

Cited by 63 publications

(52 citation statements)

References 29 publications

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“…The formation of amplified amyloid products in such reactions can be detected over time in multi-well plates using the amyloid-sensitive dye thioflavin T (ThT) [9,33]. For example, real time quaking-induced conversion (RT-QuIC) assays have been adapted to the ultrasensitive detection of prions [2, 18, 24-26, 32, 33] as well as pathologic forms of α-synuclein [4,8,13,19,30] and tau [21,23,28]. These assays are providing a basis for accurate molecular diagnosis of the associated neurodegenerative diseases [5,18,35].…”

Section: Introductionmentioning

confidence: 99%

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

Metrick

Ferreira²,

Saijo³

et al. 2020

acta neuropathol commun

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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.

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Section: Introductionmentioning

confidence: 99%

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

Metrick

Ferreira²,

Saijo³

et al. 2020

acta neuropathol commun

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“…RT-QuIC (real-time quaking-induced conversion) is a highly sensitive biochemical assay originally developed in the prion field to detect minute amounts of scrapie prion protein (PrP Sc ) in brain tissue or body fluids [21,22]. In the RT-QuIC assay, a mixture of test specimen and recombinant prion protein in a multiwell plate is intermittently shaken to amplify prion protein aggregates in a seed-dependent manner [23]. Real-time monitoring of fluorescence signal of thioflavin T (ThT) that binds to β-sheet-rich structures in amyloid fibrils allows the detection of prion seeding activity in tested samples [24].…”

Section: Introductionmentioning

confidence: 99%

RT-QuIC-based detection of alpha-synuclein seeding activity in brains of dementia with Lewy Body patients and of a transgenic mouse model of synucleinopathy

Han

Jang

Green

et al. 2020

Prion

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RT-QuIC is a shaking-based cyclic amplification technique originally developed in the prion field to detect minute amounts of scrapie prion protein (PrP Sc). In this study, we applied the RT-QuIC assay to investigate a-synuclein (a-syn) seeding activity in brains of Dementia with Lewy Body (DLB) patients and in brains of G2-3 transgenic mice expressing human a-syn with A53T mutation. The results show that a-syn seeding activity varies between patients with detectable dilutions ranging from 10 −3 to 10 −8 dilutions of brain tissue and is stable under exposures to the cycles of freezing, thawing and sonication. A53T a-syn aggregates from G2-3 transgenic mice greatly favoured A53T recombinant human a-syn as substrates in comparison to wild-type a-syn, suggesting that conformations for wild-type a-syn to be able to adopt are not compatible with that of A53T aggregates from G2-3.

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“…Undoubtedly, cell-free systems for prion amplification will continue to evolve and provide better results, and their implementation in clinical practice worldwide in a near future is foreseeable. Moreover, their adaptation to other protein misfolding-related neurodegenerative diseases may also revolutionize diagnosis of other, much more prevalent disorders, such as Parkinson's disease [36]. Together, with other indicators, such as neuroimaging or surrogate biomarker analysis, detection of PrP Sc in pre-clinical phases of the disease using prion propagation techniques has the potential to become the gold standard in TSE diagnosis, being based on a highly specific, pathognomonic biomarker.…”

Section: Discussionmentioning

confidence: 99%

“…Although some genetic TSE cannot be detected yet using this approach, the development of new substrates and modifications in the methodology might solve these problems soon [42,106]. In fact, RT-QuIC based on CSF samples is being adapted to other protein-misfolding based neurodegenerative disorders such as some synucleinopathies and tauopathies [36]. Given the success of the RT-QuIC, the almost full specificity and high sensitivity achieved and its ease of use in clinical settings, it has surpassed other methods, such as PMCA for the detection of PrP Sc in CSF.…”

Section: Prp Sc In Csf In Human Prion Diseasesmentioning

confidence: 99%

Detection of Pathognomonic Biomarker PrPSc and the Contribution of Cell Free-Amplification Techniques to the Diagnosis of Prion Diseases

et al. 2020

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Transmissible spongiform encephalopathies or prion diseases are rapidly progressive neurodegenerative diseases, the clinical manifestation of which can resemble other promptly evolving neurological maladies. Therefore, the unequivocal ante-mortem diagnosis is highly challenging and was only possible by histopathological and immunohistochemical analysis of the brain at necropsy. Although surrogate biomarkers of neurological damage have become invaluable to complement clinical data and provide more accurate diagnostics at early stages, other neurodegenerative diseases show similar alterations hindering the differential diagnosis. To solve that, the detection of the pathognomonic biomarker of disease, PrP Sc , the aberrantly folded isoform of the prion protein, could be used. However, the amounts in easily accessible tissues or body fluids at pre-clinical or early clinical stages are extremely low for the standard detection methods. The solution comes from the recent development of in vitro prion propagation techniques, such as Protein Misfolding Cyclic Amplification (PMCA) and Real Time-Quaking Induced Conversion (RT-QuIC), which have been already applied to detect minute amounts of PrP Sc in different matrixes and make early diagnosis of prion diseases feasible in a near future. Herein, the most relevant tissues and body fluids in which PrP Sc has been detected in animals and humans are being reviewed, especially those in which cell-free prion propagation systems have been used with diagnostic purposes.

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Ultrasensitive RT-QuIC Seed Amplification Assays for Disease-Associated Tau, α-Synuclein, and Prion Aggregates

Cited by 63 publications

References 29 publications

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

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

RT-QuIC-based detection of alpha-synuclein seeding activity in brains of dementia with Lewy Body patients and of a transgenic mouse model of synucleinopathy

Detection of Pathognomonic Biomarker PrPSc and the Contribution of Cell Free-Amplification Techniques to the Diagnosis of Prion Diseases

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