A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrPC to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD50). Brain tissue from 263K scrapie-affected hamsters gave SD50 values of 1011-1012/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD50 values of 103.5–105.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD50 values of 102.0–102.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.

The development of technologies for the in vitro amplification of the abnormal conformers of prion protein (PrP Sc ) has generated the potential for a novel diagnostic assay for prion disease. Previously, we developed a new PrP Sc amplification assay designated quaking-induced conversion (QUIC), which involves intermittent, automated shaking of the substrate, soluble recombinant PrP. We further improved the rapidity and practicality of this method by combining it with thioflavin T fluorescence to monitor the amyloid fibril formation. This assay, termed "real-time QUIC (RT-QUIC)", allows within 48 h, the detection of ≥1 fg of PrP Sc in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects, and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the ante-mortem evaluation of suspected CJD. Definitive ante-mortem confirmation of CJD requires the detection of PrP Sc in patient biopsy specimens, the practice of which is discouraged because it is both invasive and poses risks to health care personnel. Recently, however, in vitro PrP Sc amplification techniques, including protein misfolding cyclic amplification (PMCA) 5-7 , the amyloid seeding assay 8 , as well as QUIC have been reported to enable the direct and highly sensitive detection of PrP Sc in various tissues, including cerebrospinal fluid (CSF). QUIC assays involve the use of soluble recombinant PrP (rPrP-sen) as a substrate, which is seeded with PrP Sc , and then subjected to intermittent automated shaking. This technique can be performed more easily than the PMCA, which requires repeated sonication. Previous studies have demonstrated that QUIC assays correctly discriminated between normal and scrapie-infected CSF samples in both hamster and sheep prion disease models 9,10 . However, ultrasensitive PrP Sc detection in CSF from CJD subjects has not yet been accomplished. Accordingly, we further refined the QUIC 5 assay to improve its sensitivity and practicability, and then applied the technique in a blind pilot study to detect PrP Sc in CJD-CSF specimens.Given that a correlation between protease-resistant rPrP aggregate (rPrP-res) levels and thioflavin T (ThT) fluorescence had been shown previously 7 , we sought to determine the relative kinetics of rPrP-res formation by monitoring levels of ThT fluorescence in the QUIC assay. This was intended to minimize the time needed to detect rPrP-res. We first tested whether PrP Sc -dependent rPrP-res (rPrP-res (Sc) ) formation could be induced using a microplate reader with intermittent shaking. Human rPrP-sen (rHuPrP-sen) and a 10 -7 dilution of CJD (molecular subtype MM1) brain homogenate (BH) were used as the substrate and "seed", respectively. We conducted QUIC reactions at various concentrations (0, 0.25, 0.5 and 1.0 M) of guanidine-HCl (GdnHCl), because it h...

IntroductionThe definitive diagnosis of genetic prion diseases (gPrD) requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau) protein in the cerebrospinal fluid (CSF), but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann-Sträussler-Scheinker syndrome (GSS). We recently developed a new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC)”, to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt-Jakob disease (sCJD) patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT-QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined.Method/Principal Findings56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N), and 24 cases of genetic CJD (gCJD), comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%), FFI (100%), gCJD E200K (87%), and gCJD V203I (100%). On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%), FFI (0%), gCJD E200K (73%), and gCJD V203I (67%). Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI.Conclusion/SignificanceRT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patient's family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.

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Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. in this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNP Sc ) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as Lc3-ii, ATG12-ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNP Sc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNP Sc in addition to attenuation of microgliosis and neuroprotection.

The prion-like seeding of misfolded α-synuclein (αSyn) involved in the pathogenesis of Lewy body diseases (LBD) remains poorly understood at the molecular level. Using the real-time quaking-induced conversion (RT-QUIC) seeding assay, we investigated whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, can convert Escherichia coli-derived recombinant αSyn (r-αSyn) to fibrils. Diffuse neocortical DLB yielded 50% seeding dose (SD50) values of 107~1010/g brain. Limbic DLB was estimated to have an SD50 value of ~105/g brain. Furthermore, RT-QUIC assay discriminated DLB from other neurological and neurodegenerative disorders. Unexpectedly, the prion-like seeding was reconstructed in reactions seeded with oligomer-like species, but not with insoluble aggregates of r-αSyn, regardless of Ser129 phosphorylation status. Our findings suggest that RT-QUIC using r-αSyn can be applied to detect seeding activity in LBD, and the culprit that causes prion-like seeding may be oligomeric forms of αSyn.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-017-0624-1) contains supplementary material, which is available to authorized users.

We recently developed a new in vitro amplification technology, designated "real-time quaking-induced conversion (RT-QUIC)," for detection of the abnormal form of prion protein (PrP Sc ) in easily accessible specimens such as cerebrospinal fluid (CSF). After assessment of more than 200 CSF specimens from Japanese and Australian patients, we found no instance of a false positive, and more than 80% accuracy for the correct diagnosis of sporadic CreutzfeldtJakob disease (sCJD). Furthermore, the RT-QUIC can be applied to other prion diseases, including scrapie, chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE), and is able to quantify prion seeding activity when combined with an end-point dilution of samples. These results indicate that the RT-QUIC, with its high sensitivity and specificity, will be of great use as an early, rapid and specific assay for prion diseases. Diagnosis of Creutzfeldt-Jakob Disease: The Current SituationHuman prion diseases, including Creutzfeldt-Jakob disease (CJD), are incurable neurodegenerative disorders characterized by progressive spongiform changes and the accumulation of abnormal prion protein (PrP Sc ) in the central nervous system. 1 The majority of CJD cases (approximately 85%) are sporadic in nature, but the remaining cases comprise genetic and infectious forms. Iatrogenic CJD is the consequence of inadvertent transmission during medical procedures

SUMMARY Correlations between changes in regional, cortical, cerebral blood flow (rCBF) and histological changes in the corresponding brain regions were examined following middle cerebral arterial occlusion in 24 cats. In all animals, the duration of arterial occlusion was 2 hours followed by 2 hours of recirculation. The animals were divided into 2 groups according to the severity of the observed histological damage. Severe cortical damage was observed in 8 cats (Group A), and, in the remaining 16 cats, little or no cortical damage was seen (Group B). There was a statistically significant difference between these 2 groups in the average rCBF values during ischemia. During recirculation, there was a prompt and uniform recovery of rCBF in animals in group B but a marked diversity of rCBF ranging from hyperemia to oligemia in animals in group A. This diversity of rCBF reflects inhomogeneous blood flow. This study indicates potential hazards for surgical revascularization in the acute stage of stroke when brain damage has progressed beyond a certain level.