Search

synucleinopathies are diseases characterized by the aggregation of α-synuclein (α-syn), which forms fibrils through misfolding and accumulates in a prion-like manner. To detect the presence of these α-syn aggregates in clinical samples, seed amplification assays (SAAs) have been developed. These SAAs are capable of amplifying the α-syn seeds, allowing for their detection. αSyn-SAAs have been reported under the names ‘protein misfolding cyclic amplification’ (αSyn-PMCA) and ‘real-time quaking-induced conversion'α-Syn-RT-QuIC. The α-Syn RT-QuIC, in particular, has been adapted to amplify and detect α-syn aggregates in various biospecimens, including cerebrospinal fluid (CSF), skin, nasal brushing, serum and saliva. The α-syn RT-QuIC assay has demonstrated good sensitivity and specificity in detecting pathological α-syn, particularly in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) cases, with an accuracy rate of up to 80 %. Additionally, differential diagnosis between DLB and PD, as well as PD and multiple system atrophy (MSA), can be achieved by utilizing certain kinetic thioflavin T (ThT) parameters and other parameters. Moreover, the positive detection of α-syn in the prodromal stage of synucleinopathies provides an opportunity for early intervention and management. In summary, the development of the α-syn RT-QuIC assay has greatly contributed to the field of synucleinopathies. Therefore, we review the development of α-syn RT-QuIC assay and describe in detail the recent advancements of α-syn RT-QuIC assay for detecting pathological α-syn in synucleinopathies.

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrP(Sc)) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrP(Sc) propagation involves conversion from its normal isoform, PrP(C), by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrP(Sen)) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrP(Sc) (PrP(Res)). Scrapie brain dilutions up to 10(-8) and 10(-13) were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrP(Res) levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrP(C). Although the brains of 263K-affected mice had little immunoblot-detectable PrP(Res), RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrP(Res) strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrP(Res) levels. We also found that eQuIC, which incorporates a PrP(Sc) immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrP(Sc).

Adult-onset neurodegenerative disorders, like Parkinson's disease (PD) and dementia with Lewy bodies (DLB), that share the accumulation of aggregated α-synuclein (αSyn agg ) as their hallmark molecular pathology are collectively known as α-synucleinopathies. Diagnosing α-synucleinopathies requires the post-mortem detection of αSyn agg in various brain regions. Recent efforts to measure αSyn agg in living patients include quantifying αSyn agg in different biofluids as a biomarker for PD. We adopted the real-time quaking-induced conversion (RT-QuIC) assay to detect very low levels of αSyn agg . We first optimized RT-QuIC for sensitivity, specificity, and reproducibility by using monomeric recombinant human wild-type αSyn as a substrate and αSyn agg as the seed. Next, we exposed mouse microglia to αSyn pre-formed fibrils (αSyn PFF ) for 24 h. RT-QuIC assay revealed that the αSyn PFF is taken up rapidly by mouse microglia, within 30 min, and cleared within 24 h. We then evaluated the αSyn RT-QuIC assay for detecting αSyn agg in human PD, DLB, and Alzheimer's disease (AD) post-mortem brain homogenates (BH) along with PD and progressive supranuclear palsy (PSP) cerebrospinal fluid (CSF) samples and then determined protein aggregation rate (PAR) for αSyn agg . The PD and DLB BH samples not only showed significantly higher αSyn agg PAR compared to age-matched healthy controls and AD, but RT-QuIC was also highly reproducible with 94% sensitivity and 100% specificity. Similarly, PD CSF samples demonstrated significantly higher αSyn agg PAR compared to age-matched healthy controls, with 100% sensitivity and specificity. Overall, the RT-QuIC assay accurately detects αSyn agg seeding activity, offering a potential tool for antemortem diagnosis of α-synucleinopathies and other protein-misfolding disorders. Graphical Abstract A schematic representation of αSyn RT-QuIC assay.

Background: Parkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal α-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either α-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization of α-synuclein or tau aggregates in the brain. Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM)., Methods: We have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for OM analysis. In particular, by using human recombinant α-synuclein as substrate of reaction, we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induce α-synuclein aggregation, and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP., Results: Our results showed that a significant percentage of MSA and PD samples induced α-synuclein aggregation with high efficiency, but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination., Conclusions: Our study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities for α-synuclein. Additional studies are required for (i) estimating sensitivity and specificity of the technique and for (ii) evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms. RT-QuIC analyses of OM and cerebrospinal fluid (CSF) can be combined with the aim of increasing the overall diagnostic accuracy of these diseases, especially in the early stages., Competing Interests: Competing interestsThe authors declare that they have no competing interests.

The diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) can be difficult, but the real-time quaking-induced conversion (RT-QuIC) assays have made a considerable impact on its clinical diagnosis. This technique exploits the ability of the misfolded pathological form of prion protein (PrP Sc ) found in cerebrospinal fluid (CSF) to induce conversion of normal PrP to the misfolded form, which subsequently aggregates. The formation of these aggregates of misfolded PrP is monitored in real time using fluorescent dyes. The current sensitivity of CSF RT-QuIC undertaken at the UK National CJD Research & Surveillance Unit is 92% and the specificity is 100%. The interpretation of the RT-QuIC traces is affected by the presence of raised CSF red and white cells counts and elevated total protein concentrations. We recommend that CSF samples for RT-QuIC analysis are clear and colourless with a white cell count of <10 x10^6/L and have a total protein concentration of <1 g/L., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Prion real-time quaking-induced conversion (RT-QuIC) is emerging as the most potent assay for the in vivo diagnosis of Creutzfeldt–Jakob disease (CJD), but its full application, especially as a screening test, is limited by suboptimal substrate availability, reagent costs, and incomplete assay standardization. Therefore, the search for the most informative cerebrospinal fluid (CSF) surrogate biomarker is still of primary importance. We compared the diagnostic accuracy of CSF protein 14-3-3, measured with both western blot (WB) and enzyme-linked immunosorbent assay (ELISA), total (t)-tau and neurofilament light chain protein (NfL) alone or in combination with RT-QuIC in 212 subjects with rapidly progressive dementia in which we reached a highly probable clinical diagnosis at follow-up or a definite neuropathological diagnosis. T-tau performed best as surrogate CSF biomarker for the diagnosis of CJD (91.3% sensitivity and 78.9% specificity). The 14-3-3 ELISA assay demonstrated a slightly higher diagnostic value compared to the WB analysis (76.9% vs. 72.2%), but both methods performed worse than the t-tau assay. NfL was the most sensitive biomarker for all sCJD subtypes (> 95%), including those with low values of t-tau or 14-3-3, but showed the lowest specificity (43.1%). When ELISA-based biomarkers were adopted as screening tests followed by RT-QuIC, t-tau correctly excluded a higher number of non-CJD cases compared to NfL and 14-3-3 ELISA. Our study showed that among the CSF surrogate biomarkers of potential application for the clinical diagnosis of CJD, t-tau performs best either alone or as screening test followed by RT-QuIC as a second-level confirmatory test. [ABSTRACT FROM AUTHOR]

We applied RT‐QuIC assay to detect α‐synuclein aggregates in cerebrospinal fluid (CSF) of patients with suspected Creutzfeldt–Jakob disease who had a neuropathological diagnosis of dementia with Lewy bodies (DLB) (n = 7), other neurodegenerative diseases with α‐synuclein mixed pathology (n = 20), or without Lewy‐related pathology (n = 49). The test had a sensitivity of 92.9% and specificity of 95.9% in distinguishing α‐synucleinopathies from non‐α‐synucleinopathies. When performed in the CSF of patients with DLB (n = 36), RT‐QuIC was positive in 17/20 with probable DLB, 0/6 with possible DLB, and 0/10 with Alzheimer disease. These results indicate that RT‐QuIC for α‐synuclein is an accurate test for DLB diagnosis. [ABSTRACT FROM AUTHOR]

The diagnosis of chronic wasting disease (CWD) relies on demonstration of the disease-associated misfolded CWD prion protein (PrPCWD) in brain or retropharyngeal lymph node tissue by immunodetection methods, e.g. ELISA and immunohistochemistry (IHC). The success of these methods relies on a quality sample of tissues, which requires both anatomical knowledge and considerable dissection to collect. As the prevalence of CWD continues to increase globally, the development of fast and cost-effective methods to detect the disease is vital to facilitate CWD detection and surveillance. To address these issues, we have evaluated third eyelids from CWD-infected deer and elk using real-time quaking induced conversion (RT-QuIC). We identified prion seeding activity in third eyelids in 24 of 25 (96%) CWD-infected white-tailed deer (Odocoileus virginianus). We detected RT-QuIC positivity in the third eyelid as early as 1 month after experimental CWD exposure. In addition, we identified prion seeding activity in third eyelids of 18 of 25 (72%) naturally exposed asymptomatic CWD-positive rocky mountain elk (Cervus canadensis nelson). We compared CWD detection by RT-QuIC and IHC in third eyelid, retropharyngeal lymph node, and brain in 10 deer in early symptomatic stage of disease. IHC detected PrPCWD deposition in third eyelid lymphoid follicles in 5 of 10 deer (50%) whereas third eyelids of all 10 animals were positive by RT-QuIC. This difference reflected in part a lower requirement for lymphoid follicle presence for seeding activity detection by RT-QuIC. In conclusion, RT-QuIC analysis of the third eyelid, an easily accessed tissue, has potential to advance CWD detection and testing compliance. [ABSTRACT FROM AUTHOR]

Sporadic Creutzfeldt-Jakob disease (sCJD) is a transmissible, rapidly progressive and fatal neurodegenerative disease. The transmissible agent linked to sCJD is composed of the misfolded form of the host-encoded prion protein. The combination of histopathological and biochemical analyses has allowed the identification and sub-classification of six sCJD subtypes. This classification depends on the polymorphic variability of codon 129 of the prion protein gene and the PrPres isotype, and appears to be associated with neuropathological and clinical features. Currently, sCJD subtyping is only fully achievable post mortem. However, a rapid and non-invasive method for discriminating sCJD subtypes in vita would be invaluable for the clinical management of affected individuals, and for the selection of participants for clinical trials. The CSF analysis by Real Time Quaking Induced Conversion (RT-QuIC) reaction is the most sensitive and specific ante mortem sCJD diagnostic test available to date, and it is used by a number of laboratories internationally. RT-QuIC takes advantage of the natural replication mechanisms of prions by template-induced misfolding, employing recombinant prion protein as reaction substrate. We asked whether epitope mapping, of the RT-QuIC reaction products obtained from seeding RT-QuIC with brain and CSF samples from each of the six molecular subtypes of sCJD could be employed to distinguish them and therefore achieve in vita sCJD molecular subtyping. We found that it is possible to distinguish the RT-QuIC products generated by sCJD biological samples from the ones generated by spontaneous conversion in the negative controls, but that different sCJD subtypes generate very similar, if not identical RT-QuIC reaction products. We concluded that whilst RT-QuIC has demonstrable diagnostic value it has limited prognostic value at this point in time. [ABSTRACT FROM AUTHOR]

Background: Leucine‐rich kinase 2 (LRRK2)‐linked Parkinson's disease (PD) is clinically indistinguishable from idiopathic PD (IPD). A pleiotropic neuropathology has been recognized but the majority of studies in LRRK2 p.G2019S patients reveal Lewy‐type synucleinopathy as its principal histological substrate. To date no in vivo biomarkers of synucleinopathy have been found in LRRK2 mutation carriers. Objectives: We used real‐time quaking‐induced conversion (RT‐QuIC) technique to assess the presence of alpha‐synuclein (a‐syn) aggregates in cerebrospinal fluid (CSF) of LRRK2 p.G2019S carriers. Methods: CSF samples of 51 subjects were analyzed: 15 LRRK2 p.G2019S PD, 10 IPD, 16 LRRK2 p.G2019S nonmanifesting carriers (NMC) and 10 healthy controls. The presence of parkinsonism and prodromal symptoms was assessed in all study subjects. Results: Forty percent (n = 6) LRRK2‐PD, and 18.8% (n = 3) LRRK2‐NMC had a positive a‐syn RT‐QuIC response. RT‐QuIC detected IPD with 90% sensitivity and 80% specificity. No clinical differences were detected between LRRK2‐PD patients with positive and negative RT‐QuIC. A positive RT‐QuIC result in LRRK2‐NMC occurred in a higher proportion of subjects meeting the Movement Disorder Society research criteria for prodromal PD. Interpretation: RT‐QuIC detects a‐syn aggregation in CSF in a significant number of patients with LRRK2‐PD, but less frequently than in IPD. A small percentage of LRRK2‐NMC tested also positive. If appropriately validated in long‐term studies with large number of mutation carriers, and hopefully, postmortem or in vivo confirmation of histopathology, RT‐QuIC could contribute to the selection of candidates to receive disease modifying drugs, in particular treatments targeting a‐syn deposition. [ABSTRACT FROM AUTHOR]

Background: Sporadic Creutzfeldt-Jakob disease (sCJD) is a human prion disease that is a relatively common differential diagnosis in dementia patients. Therefore it needs a good diagnostic tool. Brain autopsy is the golden standard for the diagnosis of CJD; however, a less invasive technique is 14-3-3 protein measurement in the cerebrospinal fluid (CSF). In this systematic review, we compared the diagnostic value of the 14-3-3 protein measurement to the newer RT-QuIC test and a variant of RT-QuIC where nasal brushing is used to collect the samples., Methods: The search via MeSH terms and quality assessment was carried out by two individual researchers., Results: In 14-3-3 and RT-QuIC the sensitivity was comparable, respectively, 88% and 86%. Specificity however was higher in RT-QuIC 99.5% compared to 80% in 14-3-3. Nasal brushing showed the best results with a sensitivity of 97% and a specificity of 100%., Conclusion: Nasal brushing, despite being the best diagnostic tool according to the data, needs more study since there has only been a few studies regarding the technique. It is safe to say that due to the high specificity, RT-QuIC is superior to 14-3-3 testing.

Chronic wasting disease (CWD) is a highly contagious prion disease affecting captive and free-ranging cervid populations. CWD has been detected in United States, Canada, South Korea and, most recently, in Europe (Norway, Finland and Sweden). Animals with CWD release infectious prions in the environment through saliva, urine and feces sustaining disease spreading between cervids but also potentially to other non-cervids ruminants (e.g. sheep, goats and cattle). In the light of these considerations and due to CWD unknown zoonotic potential, it is of utmost importance to follow specific surveillance programs useful to minimize disease spreading and transmission. The European community has already in place specific surveillance measures, but the traditional diagnostic tests performed on nervous or lymphoid tissues lack sensitivity. We have optimized a Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting CWD prions with high sensitivity and specificity to try to overcome this problem. In this work, we show that bank vole prion protein (PrP) is an excellent substrate for RT-QuIC reactions, enabling the detection of trace-amounts of CWD prions, regardless of prion strain and cervid species. Beside supporting the traditional diagnostic tests, this technology could be exploited for detecting prions in peripheral tissues from live animals, possibly even at preclinical stages of the disease.

Chronic wasting disease (CWD) is a fatal prion disease which infects deer, elk and moose. CWD was first described as a wasting syndrome in captive deer in Colorado and Wyoming wildlife facilities from 1967 to 1979. Currently, CWD has been reported in 26 states of the USA, three Canadian provinces, South Korea, Norway and Finland. Since human consumption of cervids is common, it is critical to determine if CWD can infect humans. Published research, including epidemiologic studies and transmission studies using animal models, including transgenic mice that express human prion protein, have suggested existence of a strong species barrier between cervid CWD and humans. In the current study, we tested CWD transmission into two additional strains of transgenic mice (tg66 and tgRM). These mice over-express human prion protein at high levels and are highly sensitive to infection by human-tropic prions. One hundred and eight mice were inoculated intracerebrally with three different sources of CWD. After long periods of observation, brain tissues from CWD-inoculated mice were screened for evidence of prion infection by RT-QuIC, immunohistochemistry (IHC) and immunoblot. No IHC or immunoblot evidence was found to suggest transmission had occurred, and most mice were negative by RT-QuIC assay. However, four mice with inconsistent positive RT-QuIC reactions were detected. The seeding activity detected in these mice may represent a low level of CWD agent, suggesting a possible transfer of CWD infection. Alternatively, these results might be due to false positive reactions or residual CWD inoculum.

The abnormal assembly of tau, α-synuclein (αSyn), or prion protein into oligomers and multimers underpins the molecular pathogenesis of multiple neurodegenerative diseases. Such pathological aggregates can often grow by seeded polymerization mechanisms. We and others have taken advantage of these mechanisms to amplify seeding activities in vitro and devise ultrasensitive, specific and quantitative assays for these etiological biomarkers. Real-time quaking-induced conversion (RT-QuIC) assays are performed in multiwell plates with fluorescent readouts, facilitating efficient throughput. Prion RT-QuIC assays on cerebrospinal fluid (CSF) samples are being widely used for antemortem diagnosis of human prion diseases. Recently, we have also described a tau RT-QuIC prototype that has been optimized for Pick disease (with predominant 3R tau pathology) that detects 3R tau seeds in postmortem CSF, and brain tissue dilutions as extreme as a billion-fold. αSyn RT-QuIC prototypes have also been developed, providing ~92% diagnostic sensitivity and 100% specificity for Parkinson's disease and dementia with Lewy bodies using antemortem CSF. Here we provide detailed protocols for our 3R tau and αSyn RT-QuIC assays and refer the reader to published up-to-date protocols for prion RT-QuIC assays (Orru et al. Methods Mol Biol 1658:185-203, 2017; Schmitz et al. Nat Protoc 11:2233-2242, 2016).

The development of the real-time quaking-induced conversion (RT-QuIC), an in vitro protein misfolding amplification assay, was an innovation in the scientific field of protein misfolding diseases. In prion diseases, these types of assays imitate the pathological conversion of the cellular prion protein (PrPC) into a protease-resistant and/or amyloid form of PrP, called PrP resistant (PrPRes). The RT-QuIC is an automatic assay system based on real-time measuring of thioflavin-T (Th-T) incorporation into amyloid fibrils using shaking for disaggregation. It has already been applied in diagnostics, drug pre-screening, and to distinguish between different prion strains. The seeded conversion efficiency and the diagnostic accuracy of the RT-QuIC assay strongly depend on the kind of recombinant PrP (rec PrP) substrate. The DNA sequences of different substrates may originate from different species, such as human, bank vole, and hamster, or from a combination of two species, e.g., hamster-sheep chimera. In routine use, either full-length (FL) or truncated substrates are applied which can accelerate the conversion reaction, e.g., to a more sensitive version of RT-QuIC assay. In the present review, we provide an overview on the different types of PrP substrates (FL and truncated forms), recapitulate the production and purification process of different rec PrP substrates, and discuss the diagnostic value of CSF RT-QuIC in human prion disease diagnostics. [ABSTRACT FROM AUTHOR]

The accumulation of misfolded proteins as amyloid fibrils in the brain is characteristic of most neurodegenerative disorders. These misfolded proteins are capable of self-amplifying through protein seeding mechanisms, leading to accumulation in the host. First shown for PrP prions and prion diseases, it is now recognized that self-propagating misfolded proteins occur broadly in neurodegenerative diseases and include amyloid-β (Aβ) and tau in Alzheimer's disease (AD), tau in chronic traumatic encephalopathy (CTE), Pick's disease (PiD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP), and α-synuclein (α-syn) in Parkinson's disease (PD) and Lewy body dementias (LBD). Techniques able to directly measure these bioactive protein seeds include the real-time quaking-induced conversion (RT-QuIC) assays. Initially developed for the detection of PrP prions and subsequently for the detection of other misfolded protein seeds, these assays take advantage of the mechanism of protein-based self-propagation to result in exponential amplification of the initial protein seeds from biospecimens. Disease-specific "protein seeds" recruit and template the misfolding of native recombinant protein substrates to elongate amyloid fibrils. The amplification power of these assays allows for detection of minute amounts of disease-specific protein seeds to better support early and accurate diagnosis. In addition to the diagnostic capabilities, assay readouts have been shown to reveal biochemical, structural, and kinetic information of protein seed self-propagation. This review examines the various protein seed amplification assays currently available for distinct neurodegenerative diseases, with a focus on RT-QuIC assays, along with the insights their readouts provide into protein seed structures and strain differences. [ABSTRACT FROM AUTHOR]

Real-time quaking-induced conversion (RT-QuIC) is a cell-free abnormal form of prion protein (PrPSc) amplification method using recombinant prion protein from Escherichia coli that can measure prion seeding activity in samples with high sensitivity. The advantages of this method are that it is much more sensitive than Western blotting, which is usually used to detect PrPSc, and that prion seeding activity can be easily quantified by combining it with endpoint dilution of the sample, and that it can be amplified in most species and prion strains. A decade has passed since the development of RT-QuIC, and many studies have been reported that take advantage of its characteristics. In particular, its usefulness in the diagnosis of sporadic CJD has been clarified, and it is recommended to be one of the diagnostic criteria. Future challenges include the establishment of a method to differentiate prion strains and application of RT-QuIC to early diagnosis of prion diseases and determination of treatment efficacy. [ABSTRACT FROM AUTHOR]