Highly efficient protein misfolding cyclic amplification

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrP(C) into PrP(Sc) in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP(C) may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrP(C) into PrP(Sc) from ～10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrP(Sc) by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrP(C) susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.     

A sensitive filter retention assay for the detection of PrP(Sc) and the screening of anti-prion compounds

A hallmark of prion diseases is the accumulation of an abnormally folded prion protein, denoted PrP(Sc). Here we describe a new and highly sensitive method for the detection of PrP(Sc) in brain and other tissue samples that utilizes both PrP(Sc) diagnostic criteria in combination; protease resistance and aggregation. Upon filtration of tissue extracts derived from scrapie- or bovine spongiform encephalopathy-infected animals, PrP(Sc) is retained and detected on the membranes. Laborious steps such as SDS-PAGE and Western blotting are avoided with concomitant gain in sensitivity and reliability. The new procedure also proved useful in a screen for anti-prion compounds in a scrapie-infected cell culture model.     

Detection of prions in blood

Prion diseases are caused by an unconventional infectious agent termed prion, composed mainly of the misfolded prion protein (PrP(Sc)). The development of highly sensitive assays for biochemical detection of PrP(Sc) in blood is a top priority for minimizing the spread of the disease. Here we show that the protein misfolding cyclic amplification (PMCA) technology can be automated and optimized for high-efficiency amplification of PrP(Sc). We show that 140 PMCA cycles leads to a 6,600-fold increase in sensitivity over standard detection methods. Two successive rounds of PMCA cycles resulted in a 10 million-fold increase in sensitivity and a capability to detect as little as 8,000 equivalent molecules of PrP(Sc). Notably, serial PMCA enables detection of PrP(Sc) in blood samples of scrapie-afflicted hamsters with 89% sensitivity and 100% specificity. These findings represent the first time that PrP(Sc) has been detected biochemically in blood, offering promise for developing a noninvasive method for early diagnosis of prion diseases.     

Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin

Disease-related PrP(Sc) [pathogenic PrP (prion protein)] is classically distinguished from its normal cellular precursor, PrP(C)(cellular PrP) by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease-resistant fragments of PrP(Sc) using PK (proteinase K), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrP(Sc) in its full-length form. In the present study, we show that thermolysin can degrade PrP(C) while preserving both PK-sensitive and PK-resistant isoforms of disease-related PrP in both rodent and human prion strains. For mouse RML (Rocky Mountain Laboratory) prions, the majority of PK-sensitive disease-related PrP isoforms do not appear to contribute significantly to infectivity. In vCJD (variant Creutzfeldt-Jakob disease), the human counterpart of BSE (bovine spongiform encephalopathy), up to 90% of total PrP present in the brain resists degradation with thermolysin, whereas only approximately 15% of this material resists digestion by PK. Detection of PK-sensitive isoforms of disease-related PrP using thermolysin should be useful for improving diagnostic sensitivity in human prion diseases.     

Detection and localisation of PrP(Sc) in the liver of sheep infected with scrapie and bovine spongiform encephalopathy

Prions are largely contained within the nervous and lymphoid tissue of transmissible spongiform encephalopathy (TSE) infected animals. However, following advances in diagnostic sensitivity, PrP(Sc), a marker for prion disease, can now be located in a wide range of viscera and body fluids including muscle, saliva, blood, urine and milk, raising concerns that exposure to these materials could contribute to the spread of disease in humans and animals. Previously we demonstrated low levels of infectivity in the liver of sheep experimentally challenged with bovine spongiform encephalopathy. In this study we show that PrP(Sc) accumulated in the liver of 89% of sheep naturally infected with scrapie and 100% of sheep challenged with BSE, at both clinical and preclinical stages of the disease. PrP(Sc) was demonstrated in the absence of obvious inflammatory foci and was restricted to isolated resident cells, most likely Kupffer cells.     

Methionine sulfoxides on PrPSc: a prion-specific covalent signature

Prion diseases are fatal neurodegenerative disorders believed to be transmitted by PrP (Sc), an aberrant form of the membrane protein PrP (C). In the absence of an established form-specific covalent difference, the infectious properties of PrP (Sc) were uniquely ascribed to the self-perpetuation properties of its aberrant fold. Previous sequencing of the PrP chain isolated from PrP(27-30) showed the oxidation of some methionine residues; however, at that time, these findings were ascribed to experimental limitations. Using the unique recognition properties of alphaPrP mAb IPC2, protein chemistry, and state of the art mass spectrometry, we now show that while a large fraction of the methionine residues in brain PrP (Sc) are present as methionine sulfoxides this modification could not be found on brain PrP (C) as well as on its recombinant models. In particular, the pattern of oxidation of M213 with respect to the glycosylation at N181 of PrP (Sc) differs both within and between species, adding another diversity factor to the structure of PrP (Sc) molecules. Our results pave the way for the production of prion-specific reagents in the form of antibodies against oxidized PrP chains which can serve in the development of both diagnostic and therapeutic strategies. In addition, we hypothesize that the accumulation of PrP (Sc) and thereafter the pathogenesis of prion disease may result from the poor degradation of oxidized aberrantly folded PrP.     

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 (PrP(Sc)) has generated the potential for sensitive detection of prions. Here we developed a new PrP(Sc) amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows 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 antemortem evaluation of suspected CJD.     

Aberrant metal binding by prion protein in human prion disease

Human prion diseases are characterized by the conversion of the normal prion protein (PrP(C)) into a pathogenic isomer (PrP(Sc)). Distinct PrP(Sc) conformers are associated with different subtypes of prion diseases. PrP(C) binds copper and has antioxidation activity. Changes in metal-ion occupancy can lead to significant decline of the antioxidation activity and changes in conformation of the protein. We studied the trace element status of brains from patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found a decrease of up to 50% of copper and an increase in manganese of approximately 10-fold in the brain tissues from sCJD subjects. We have also studied the metal occupancy of PrP in sCJD patients. We observed striking elevation of manganese and, to a lesser extent, of zinc accompanied by significant reduction of copper bound to purified PrP in all sCJD variants, determined by the PrP genotype and PrP(Sc) type, combined. Both zinc and manganese were undetectable in PrP(C) preparations from controls. Copper and manganese changes were pronounced in sCJD subjects homozygous for methionine at codon 129 and carrying PrP(Sc) type-1. Anti-oxidation activity of purified PrP was dramatically reduced by up to 85% in the sCJD variants, and correlated with increased in oxidative stress markers in sCJD brains. These results suggest that altered metal-ion occupancy of PrP plays a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sCJD variants, they may contribute to the diversity of PrP(Sc) and disease phenotype in sCJD. Finally, this study also presented two potential approaches in the diagnosis of CJD; the significant increase in brain manganese makes it potentially detectable by MRI, and the binding of manganese by PrP in sCJD might represent a novel diagnostic marker.     

Immunoreactivity enhancement with chelators for increasing the detection sensitivity of human PrPSc by Western blotting

Prion diseases are neurodegenerative disorders affecting humans as Creutzfeldt-Jakob disease. The host-encoded prion protein (PrP(C)) will be converted into a structurally altered isoform (PrP(Sc)). PrP(Sc) differ in sizes and glycoform patterns and can be identified using molecular typing with Western blotting. The electrophoretic mobility of PrP(Sc) changes on treatment with metal ions or chelators prior to digestion with proteases. The effects of chelators applied to PrP(Sc) after protease digestion had not been examined in detail, we investigated these effects in this study. Application of EDTA, NTA and DTPA, and to a lesser extent EGTA, significantly enhanced PrP(Sc) signals in immunoblots. PrP(Sc) intensities increased two- to three-fold compared with untreated PrP(Sc). Since the immunoblot method is highly specific, sensitivity is the limiting factor. Enhancing sensitivity might be important in the determination of PrP(Sc) at levels close to or just below the limits of detection. It is to be expected that application of chelators to digested protein samples will increase the sensitivity of PrP(Sc) detection using the Western blot technique.     

Isolation of proteinase K-sensitive prions using pronase E and phosphotungstic acid

Disease-related prion protein, PrP(Sc), is classically distinguished from its normal cellular precursor, PrP(C), by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrP(Sc) using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrP(C), while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrP(C).     

In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).     

Disease-associated prion protein elicits immunoglobulin M responses in vivo

Prion diseases such as Creutzfeldt-Jakob disease are believed to result from the misfolding of a widely expressed normal cellular prion protein, PrPc. The resulting disease-associated isoforms, PrP(Sc), have much higher beta-sheet content, are insoluble in detergents, and acquire relative resistance to proteases. Although known to be highly aggregated and to form amyloid fibrils, the molecular architecture of PrP9Sc) is poorly understood. To date, it has been impossible to elicit antibodies to native PrP(Sc) that are capable of recognizing PrP(Sc) without denaturation, even in Pm-P(o/o) mice that are intolerant of it. Here we demonstrate that antibodies for native PrPc and PrP(Sc) can be produced by immunization of Pm-P(o/o) mice with partially purified PrPc and PrP(Sc) adsorbed to immunomagnetic particles using high-affinity anti-PrP monoclonal antibodies (mAbs). Interestingly, the polyclonal response to PrP(Sc) was predominantly of the immunoglobulin M (IgM) isotype, unlike the immunoglobulin G (IgG) responses elicited by PrP(c) or by recombinant PrP adsorbed or not to immunomagnetic particles, presumably reflecting the polymeric structure of disease-associated prion protein. Although heat-denatured PrP(Sc) elicited more diverse antibodies with the revelation of C-terminal epitopes, remarkably, these were also predominantly IgM suggesting that the increasing immunogenicity, acquisition of protease sensitivity, and reduction in infectivity induced by heat are not associated with dissociation of the PrP molecules in the diseased-associated protein. Adsorbing native proteins to immunomagnetic particles may have general applicability for raising polyclonal or monoclonal antibodies to any native protein, without attempting laborious purification steps that might affect protein conformation.     

Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice

There is increasing concern over the extent to which bovine spongiform encephalopathy (BSE) prions have been transmitted to humans, as a result of the rising number of variant Creutzfeldt-Jakob disease (vCJD) cases. Toward preventing new transmissions, diagnostic tests for prions in livestock have been developed using the conformation-dependent immunoassay (CDI), which simultaneously measures specific antibody binding to denatured and native forms of the prion protein (PrP). We employed high-affinity recombinant antibody fragments (recFab) reacting with residues 95-105 of bovine (Bo) PrP for detection and another recFab that recognizes residues 132-156 for capture in the CDI. We report that the CDI is capable of measuring the disease-causing PrP isoform (PrP(Sc)) in bovine brainstems with a sensitivity similar to that of end-point titrations in transgenic (Tg) mice expressing BoPrP. Prion titers were approximately 10(7) ID(50) units per gram of bovine brainstem when measured in Tg(BoPrP) mice, a figure approximately 10 times greater than that determined by bioassay in cattle and approximately 10,000x greater than in wild-type mice. We also report substantial differences in BoPrP(Sc) levels in different areas of the obex region, where neuropathology has been consistently observed in cattle with BSE. The CDI was able to discriminate between PrP(Sc) from BSE-infected cattle and Tg(BoPrP) mice as well as from chronic wasting disease (CWD)-infected deer and elk. Our findings argue that applying the CDI to livestock should considerably reduce human exposure to animal prions.     

A protease-resistant prion protein isoform is present in urine of animals and humans affected with prion diseases.

Prion protein (PrP)(Sc), the only known component of the prion, is present mostly in the brains of animals and humans affected with prion diseases. We now show that a protease-resistant PrP isoform can also be detected in the urine of hamsters, cattle, and humans suffering from transmissible spongiform encephalopathies. Most important, this PrP isoform (UPrP(Sc)) was also found in the urine of hamsters inoculated with prions long before the appearance of clinical signs. Interestingly, intracerebrally inoculation of hamsters with UPrP(Sc) did not cause clinical signs of prion disease even after 270 days, suggesting it differs in its pathogenic properties from brain PrP(Sc). We propose that the detection of UPrP(Sc) can be used to diagnose humans and animals incubating prion diseases, as well as to increase our understanding on the metabolism of PrP(Sc) in vivo.     

Detection of Bovine Spongiform Encephalopathy-Specific PrPSc by Treatment with Heat and Guanidine Thiocyanate

The conversion of a ubiquitous cellular protein (PrP(C)), an isoform of the prion protein (PrP), to the pathology-associated isoform PrP(Sc) is one of the hallmarks of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE). Accumulation of PrP(Sc) has been used to diagnose BSE. Here we describe a quantitative enzyme-linked immunosorbent assay (ELISA) that involves antibodies against epitopes within the protease-resistant core of the PrP molecule to measure the amount of PrP in brain tissues from animals with BSE and normal controls. In native tissue preparations, little difference was found between the two groups. However, following treatment of the tissue with heat and guanidine thiocyanate (Gh treatment), the ELISA discriminated BSE-specific PrP(Sc) from PrP(C) in bovine brain homogenates. PrP(Sc) was identified by Western blot, centrifugation, and protease digestion experiments. It was thought that folding or complexing of PrP(Sc) is most probably reversed by the Gh treatment, making hidden antigenic sites accessible. The digestion experiments also showed that protease-resistant PrP in BSE is more difficult to detect than that in hamster scrapie. While the concentration of PrP(C) in cattle is similar to that in hamsters, PrP(Sc) sparse in comparison. The detection of PrP(Sc) by a simple physicochemical treatment without the need for protease digestion, as described in this study, could be applied to develop a diagnostic assay to screen large numbers of samples.     

An N-terminal polybasic domain and cell surface localization are required for mutant prion protein toxicity

There is evidence that alterations in the normal physiological activity of PrP(C) contribute to prion-induced neurotoxicity. This mechanism has been difficult to investigate, however, because the normal function of PrP(C) has remained obscure, and there are no assays available to measure it. We recently reported that cells expressing PrP deleted for residues 105-125 exhibit spontaneous ionic currents and hypersensitivity to certain classes of cationic drugs. Here, we utilize cell culture assays based on these two phenomena to test how changes in PrP sequence and/or cellular localization affect the functional activity of the protein. We report that the toxic activity of Δ105-125 PrP requires localization to the plasma membrane and depends on the presence of a polybasic amino acid segment at the N terminus of PrP. Several different deletions spanning the central region as well as three disease-associated point mutations also confer toxic activity on PrP. The sequence domains identified in our study are also critical for PrP(Sc) formation, suggesting that common structural features may govern both the functional activity of PrP(C) and its conversion to PrP(Sc).     

Glycosylphosphatidylinositol anchor-dependent stimulation pathway required for generation of baculovirus-derived recombinant scrapie prion protein

The pathogenic isoform (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrP(Sc) seed catalyzes the structural conversion of endogenous PrP(C) into nascent PrP(Sc) in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrP(res)) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrP(Sc) by PMCA. However, the biological properties of PrP(Sc) obtained by in vivo transmission of recPrP(res) are unique or different from those of PrP(Sc) used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrP(Sc). In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrP(C) substrate for amplification of the mouse scrapie prion strain Chandler, and PrP(Sc) that accumulated in mice inoculated with Bac-PrP(res) had biochemical and pathological properties very similar to those of the PrP(Sc) seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrP(res), the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrP(Sc). Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.     

Allelic origin of protease-sensitive and protease-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease with the P102L mutation

Gerstmann-Str?ussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP(Sc), consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrP(Sc) isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrP(Sc) has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrP(Sc) allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrP(Sc) molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrP(Sc) quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.     

Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein

Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrP(C)-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrP(C) than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrP(Sc), in the agent-inoculated PrP-overexpressing cells, while no PrP(Sc) was detectable in the untransfected cells after 3 passages. Production of PrP(Sc) in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrP(Sc)-A strains Chandler, 139A, and 22L but not to PrP(Sc)-B strains 87V and 22A. We further demonstrate the successful transmission of PrP(Sc) from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrP(C) and the sequence of PrP(Sc). This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.     

Molecular biology of prion diseases

What is the nature of the transmissible agent responsible for neurodegenerative diseases such as scrapie and mad-cow disease in animals and Creutzfeldt-Jakob disease in man? There is now weighty evidence that PrP(Sc), a modified version of the ubiquitously expressed host protein PrP(C), is responsible for pathogenesis of these diseases and that conversion of PrP(C) into PrP(Sc) under the influence of PrP(Sc) is the process leading to the propagation of PrP(Sc) and disease progression.