Ryuichiro Atarashi

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

The development of technologies for the in vitro amplification of abnormal conformations of prion protein (PrPSc) has generated the potential for sensitive detection of prions. Here we developed a new PrPSc amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows the detection of ≥1 fg of PrPSc 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 antemortem evaluation of suspected CJD.

Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein.

The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects ≥50 ag of hamster PrPSc (≈0.003 lethal dose) within 2–3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-μl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.

Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking

To the editor: A key problem in managing prion diseases is the lack of a rapid, practical assay for prions (infectivity) at low-level infectious, or sub-infectious, amounts. Prion diseases involve the accumulation of a pathological, typically protease-resistant form of prion protein, termed PrPSc, which appears to propagate itself in infected hosts by inducing the conversion of its normal hostencoded precursor, PrP-sen, into additional PrPSc (refs. 1–4). In crude brain homogenates, PrPSc and infectivity can be amplified from endogenous PrP-sen during multiple rounds of intermittent sonication and serial dilution into fresh normal brain homogenate2,4. This ultrasensitive assay, termed PMCA, allows detection of ~1 ag of PrPSc in ~3 weeks5. To improve the speed and practicality of prion detection assays, we recently developed a cell-free conversion reaction that supports sustained PrPSc-seeded conversion of recombinant PrP-sen (rPrP-sen) to specific protease-resistant (rPrP-res) forms. This method (which we previously reported in Nature Methods), called rPrP-PMCA, uses periodic sonication and serial reaction rounds of the PMCA method, but is faster6. To circumvent problems associated with sonication in the PMCA and rPrP-PMCA methods (see Supplementary Results online), we have now developed a new prion assay, abbreviated QUIC for quaking-induced conversion, which uses rPrP-sen as a substrate and automated tube shaking rather than sonication. This assay can detect about one lethal prion dose within a day, and is faster and simpler than previous described PMCA6 and rPrP-PMCA5 assays. Initial testing of QUIC reaction conditions revealed that periodic shaking enhanced PrPSc-seeded conversion of hamster rPrP-sen (residues 23–231) into PK-resistant conversion products (rPrP-res(Sc), where (Sc) refers to seeding by PrPSc; Supplementary Fig. 1 and Supplementary Methods online). Consistent with our previous observations with rPrP-PMCA reactions6, the rPrP-res(Sc) reaction products had 17-, 13-, 12and 11-kDa fragments, which represented different C-terminal PrP fragments (Supplementary Fig. 2 online). These results showed that periodic shaking could substitute for sonication in promoting rPrP-res(Sc) formation. Additional experiments revealed that rPrP-res(Sc) generation was also sensitive to rPrP-sen concentration, reaction volume (Supplementary Fig. 1), reaction time (Supplementary Fig. 2), number of serial reactions (Supplementary Fig. 3 online), temperature (Supplementary Fig. 4 online) and shaking cycle (Supplementary Results). In QUIC reactions performed at 45 °C, we observed rPrP-res(Sc) formation in single 46-h QUIC reactions seeded with ≥100 ag of PrPSc (Fig. 1a). In contrast, 21 negative control reactions seeded with comparable dilutions of normal brain homogenate or buffer alone produced no rPrP-res (Fig. 1b). We obtained results similar to those shown in Figure 1a,b in an independent repeat experiment done in triplicate (data not shown). When we diluted products of PrPSc-seeded reactions 1,000-fold into fresh rPrP-sen to seed the subsequent reaction rounds, we observed strong propagation of rPrP-res(Sc) through at least 4 serial reactions (Supplementary Fig. 5 online). Elevation of QUIC reaction temperatures accelerated rPrP-res(Sc) formation. At 55 °C, we detected rPrP-res(Sc) in single 8-h reactions seeded with as little as 10 fg PrPSc (~2 lethal intracerebral doses; Supplementary Fig. 4). We detected 1 fg in 18-h reactions (Supplementary Fig. 6 online). At 65 °C, we detected 100 fg PrPSc seed with a 4-h reaction (Supplementary Fig. 4). However, at 65 °C, there was also 25 20

Mammalian Prions Generated from Bacterially Expressed Prion Protein in the Absence of Any Mammalian Cofactors

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that are associated with the conformational conversion of a normal prion protein, PrPC, to a misfolded aggregated form, PrPSc. The protein-only hypothesis asserts that PrPSc itself represents the infectious TSE agent. Although this model is supported by rapidly growing experimental data, unequivocal proof has been elusive. The protein misfolding cyclic amplification reactions have been recently shown to propagate prions using brain-derived or recombinant prion protein, but only in the presence of additional cofactors such as nucleic acids and lipids. Here, using a protein misfolding cyclic amplification variation, we show that prions causing transmissible spongiform encephalopathy in wild-type hamsters can be generated solely from highly purified, bacterially expressed recombinant hamster prion protein without any mammalian or synthetic cofactors (other than buffer salts and detergent). These findings provide strong support for the protein-only hypothesis of TSE diseases, as well as argue that cofactors such as nucleic acids, other polyanions, or lipids are non-obligatory for prion protein conversion to the infectious form.

Physiological Expression of the Gene for PrP-Like Protein, PrPLP/Dpl, by Brain Endothelial Cells and its Ectopic Expression in Neurons of PrP-Deficient Mice Ataxic Due to Purkinje Cell Degeneration

Recently, a novel gene encoding a prion protein (PrP)-like glycoprotein, PrPLP/Dpl, was identified as being expressed ectopically by neurons of the ataxic PrP-deficient (PRNP(-/-)) mouse lines exhibiting Purkinje cell degeneration. In adult wild-type mice, PrPLP/Dpl mRNA was physiologically expressed at a high level by testis and heart, but was barely detectable in brain. However, transient expression of PrPLP/Dpl mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth. In situ hybridization paired with immunohistochemistry using anti-factor VIII serum identified brain endothelial cells as expressing the transcripts. Moreover, in the neonatal wild-type mice, the PrPLP/Dpl mRNA colocalized with factor VIII immunoreactivities in spleen and was detectable on capillaries in lamina propria mucosa of gut. These findings suggested a role of PrPLP/Dpl in angiogenesis, in particular blood-brain barrier maturation in the central nervous system. Even in the ataxic Ngsk PRNP(-/-) mice, the physiological regulation of PrPLP/Dpl mRNA expression in brain endothelial cells was still preserved. This strongly supports the argument that the ectopic expression of PrPLP/Dpl in neurons, but not deregulation of its physiological expression in endothelial cells, is involved in the neuronal degeneration in ataxic PRNP(-/-) mice.

Early detection of abnormal prion protein in genetic human prion diseases now possible using real-time QUIC assay.

Introduction The 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 Findings 56 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/Significance RT-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 patients family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.

Identification of a novel gene encoding a PrP-like protein expressed as chimeric transcripts fused to PrP exon 1/2 in ataxic mouse line with a disrupted PrP gene.

Abstract1. Mouse lines lacking prion protein (PrPC) have a puzzling phenotypic discrepancy. Some, but not all, developed late-onset ataxia due to Purkinje cell degeneration.2. Here, we identified aberrant mRNA species in the brain of Ngsk Prnp0/0 ataxic, but not in nonataxic Zrch Prnp0/0 mouse line. These mRNAs were chimeric between the noncoding exons 1 and 2 of the PrP gene (Prnp) and the novel sequence encoding PrP-like protein (PrPLP), a putative membrane glycoprotein with 23% identity to PrPC in the primary amino acid structure. The chimeric mRNAs were generated from the disrupted Prnp locus of Ngsk Prnp0/0 mice lacking a part of the Prnp intron 2 and its splice acceptor signal.3. In the brain of wild-type and Zrch Prnp0/0 mice, PrPLP mRNA was barely detectable. In contrast, in the brain of Ngsk Prnp0/0 mice, PrP/PrPLP chimeric mRNAs were expressed in neurons, at a particularly high level in hippocampus pyramidal cells and Purkinje cells under the control of the Prnp promoter.4. In addition to the functional loss of PrPC, ectopic PrPLP expression from the chimeric mRNAs could also be involved in the Purkinje cell degeneration in Ngsk Prnp0/0 mice.

Distinct structures of scrapie prion protein (PrPSc)-seeded versus spontaneous recombinant prion protein fibrils revealed by hydrogen/deuterium exchange.

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrPSc) are largely unknown. PrPSc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrPC). One manifestation of PrPSc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrPSc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrPSc. Here, we compare the structures of PrPSc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrPSc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrPSc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrPSc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.

Real-time quaking-induced conversion

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 (PrPSc) 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 Creutzfeldt–Jakob 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.

Prion Protein Is Necessary for Latent Learning and Long-Term Memory Retention

Abstract1. The cellular prion protein, designated PrPc, is a key molecule in the prion diseases but its physiological function remains unknown. To elucidate whether PrPc plays some role in the central nervous system, we established a line of mice in which the PrP gene had been disrupted and subsequently conducted long-term observations.2. Performance in latent learning and passive avoidance was evaluated using water-finding and step-through tests, respectively.3. PrP–/– mice showed impaired performance in the water-finding test, indicating a disturbance in latent learning, at 23 weeks of age. In the step-through test, although the PrP–/– mice showed normal learning ability and short-term memory retention, they evidenced a significant disturbance in long-term memory retention.4. These results indicate that PrPc is needed for certain types of learning and memory and that the loss of function of this protein may contribute to the pathogenesis of prion diseases.