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...

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.

Influenza virus matrix protein 1 (M1) has been shown to play a crucial role in the virus replication, assembly and budding. We identified heat shock cognate protein 70 (Hsc70) as a M1 binding protein by immunoprecipitation and MALDI-TOF MS. The C terminal domain of M1 interacts with Hsc70. We found that Hsc70 does not correlate with the transport of M1 to the nucleus, however, it does inhibit the nuclear export of M1 and NP, thus resulting in the inhibition of viral production. This is the first demonstration that Hsc70 is directly associated with M1 and therefore is required for viral production.

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of prion protein (PrPC). To date, there is no effective treatment for the disease. The accumulated PrP, termed PrPSc, forms amyloid fibrils and could be infectious. It has been suggested that PrPSc is abnormally folded and resistant to proteolytic degradation, and also inhibits proteasomal functions in infected cells, thereby inducing neuronal death. Recent work indicates that the ubiquitin-proteasome system is involved in quality control of PrPC. To reveal the significance of prion protein ubiqitination, we focused on ubiquitin-specific protease 14 (USP14), a deubiqutinating enzyme that catalyzes trimming of polyubiquitin chains and plays a role in regulation of proteasomal processes. Results from the present study showed that treatment with a selective inhibitor of USP14 reduced PrPC, as well as PrPSc, levels in prion-infected neuronal cells. Overexpression of the dominant negative mutant form of USP14 reduced PrPSc, whereas wildtype USP14 increased PrPSc in prion-infected cells. These results suggest that USP14 prevents degradation of both normal and abnormal PrP. Collectively, a better understanding about the regulation of PrPSc clearance caused by USP14 might contribute greatly to the development of therapeutic strategies for prion diseases.