Concentration of Disease-Associated Prion Protein with Silicon Dioxide

Reagents that can precipitate the disease-associated prion protein (PrP^Sc) are vital for the development of high sensitivity tests to detect low levels of this disease marker in biological material. Here, a range of minerals are shown to precipitate both ovine cellular prion protein (PrP^C) and ovine scrapie PrP^Sc. The precipitation of prion protein with silicon dioxide is unaffected by PrP^Sc strain or host species and the method can be used to precipitate bovine BSE. This method can reliably concentrate protease-resistant ovine PrP^Sc (PrP^res) derived from 1.69 μg of brain protein from a clinically infected animal diluted into either 50 ml of buffer or 15 ml of plasma. The introduction of a SiO₂ precipitation step into the immunological detection of PrP^res increased detection sensitivity by over 1,500-fold. Minerals such as SiO₂ are readily available, low cost reagents with generic application to the concentration of diseases-associated prion proteins.     

Evaluation of non-immunoaffinity methods for isolation of cellular prion protein from bovine brain

Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases that affect the central nervous system of many animals, including humans. Research suggests that TSEs are caused by conversion of the cellular prion protein (PrP^C), which is encoded in many tissues, especially brain, to the pathological form (PrP^Sc). This conversion affects PrP^Sc structure, conferring different biochemical properties, such as the increased resistance to proteinase K, that have been widely used for its purification. By contrast, PrP^C is less resistant and its isolation is more challenging. Here, we propose a purification strategy to efficiently recover PrP^C from healthy bovine brain using conventional non-immunoaffinity methods. The applicability of extraction using detergents, size exclusion chromatography, diafiltration with molecular weight cutoff (MWCO) filters, and immobilized metal affinity chromatography (IMAC) using Western blot (WB) analysis to detect the presence of PrP^C is discussed in detail.     

Identification of an Intracellular Site of Prion Conversion

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP^C), denoted PrP^Sc, which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrP^C into PrP^Sc and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrP^C and PrP^Sc and measured PrP^Sc levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.     

Evaluation of the Possible Transmission of BSE and Scrapie to Gilthead Sea Bream (Sparus aurata)

In transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders affecting many species, the key event in disease pathogenesis is the accumulation of an abnormal conformational isoform (PrP^Sc) of the host-encoded cellular prion protein (PrP^C). While the precise mechanism of the PrP^C to PrP^Sc conversion is not understood, it is clear that host PrP^C expression is a prerequisite for effective infectious prion propagation. Although there have been many studies on TSEs in mammalian species, little is known about TSE pathogenesis in fish. Here we show that while gilthead sea bream (Sparus aurata) orally challenged with brain homogenates prepared either from a BSE infected cow or from scrapie infected sheep developed no clinical prion disease, the brains of TSE-fed fish sampled two years after challenge did show signs of neurodegeneration and accumulation of deposits that reacted positively with antibodies raised against sea bream PrP. The control groups, fed with brains from uninfected animals, showed no such signs. Remarkably, the deposits developed much more rapidly and extensively in fish inoculated with BSE-infected material than in the ones challenged with the scrapie-infected brain homogenate, with numerous deposits being proteinase K-resistant. These plaque-like aggregates exhibited congophilia and birefringence in polarized light, consistent with an amyloid-like component. The neurodegeneration and abnormal deposition in the brains of fish challenged with prion, especially BSE, raises concerns about the potential risk to public health. As fish aquaculture is an economically important industry providing high protein nutrition for humans and other mammalian species, the prospect of farmed fish being contaminated with infectious mammalian PrP^Sc, or of a prion disease developing in farmed fish is alarming and requires further evaluation.     

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Background

Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrP^Sc, a protease-resistant misfolded isoform of the cellular prion protein PrP^C. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrP^Sc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrP^Sc in BSE-affected cattle therefore remains unknown.

Methodology/Principal Findings

We report here that PrP^Sc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP^Sc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrP^Sc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrP^Sc in blood was not substantiated in the BSE-affected cattle examined.

Conclusions/Significance

The distribution of PrP^Sc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrP^Sc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.     

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans^9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrP^C) and the infectious protein (PrP^Sc), an abnormally-folded isoform of PrP^{C 8}.One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation¹³. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrP^Sc are poorly characterized due to their unusual conformation and aggregation states.PrP^Sc can seed the conversion of PrP^C to PrP^Scin vitro¹⁴. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrP^Sc, at an accelerated rate, from a system containing excess amounts of PrP^C and minute amounts of the PrP^Sc seed¹⁹. This technique has proven to effectively recapitulate the species and strain specificity of PrP^Sc conversion from PrP^C, to emulate prion strain interference, and to amplify very low levels of PrP^Sc from infected tissues, fluids, and environmental samples^6,7,16,23 .This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.     

Biochemical Characteristics and PrPSc Distribution Pattern in the Brains of Cattle Experimentally Challenged with H-type and L-type Atypical BSE

Besides the classical form of bovine spongiform encephalopathy (BSE) that has been known for almost three decades, two atypical forms designated H-type and L-type BSE have recently been described. While the main diagnostic feature of these forms is the altered biochemical profile of the accumulated PrP^Sc, it was also observed in the initial analysis that L-type BSE displays a distribution pattern of the pathological prion protein (PrP^Sc), which clearly differs from that observed in classical BSE (C-type). Most importantly, the obex region in the brainstem is not the region with the highest PrP^Sc concentrations, but PrP^Sc is spread more evenly throughout the entire brain. A similar distribution pattern has been revealed for H-type BSE by rapid test analysis. Based on these findings, we performed a more detailed Western blot study of the anatomical PrP^Sc distribution pattern and the biochemical characteristics (molecular mass, glycoprofile as well as PK sensitivity) in ten different anatomical locations of the brain from cattle experimentally challenged with H- or L-type BSE, as compared to cattle challenged with C-type BSE. Results of this study revealed distinct differences in the PrP^Sc deposition patterns between all three BSE forms, while the biochemical characteristics remained stable for each BSE type among all analysed brain areas.     

Rapid, high-throughput detection of PrPSc by 96-well immunoassay

Prion diseases are emerging infectious disorders that affect several mammalian species including humans. The transmissible agent is comprised of PrP^Sc, a misfolded isoform of the normal host-encoded prion protein PrP^C. Immunodetection of PrP^Sc is often utilized for prion disease diagnosis and tracking spread of the infectious agent through the host. We have developed a rapid, high-throughput 96-well immunoassay, which is specific for the detection of PrP^Sc. This assay has PrP^Sc detection limits similar to western blot and is advantageous because of its comparatively shorter running time, smaller start-up and operation costs and large sample capacity.Key words: prion disease, immunodetection, PrP^Sc     

Abnormalities in Stress Proteins in Prion Diseases

1. Prion diseases include kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker disease (GSS), and fatal familia insomnia (FFI) of humans, as well as scrapie and bovine spongiform encephalopathy (BSE) of animals.2. All these disorders involve conversion of the normal, cellular prion protein (PrP^C) into the corresponding scrapie isoform (PrP^Sc). PrP^C adopts a structure rich in -helices and devoid of -sheet, in contrast to PrP^Sc, which has a high -sheet content and is resistant to limited digestion by proteases. That a conformational transition features in the conversion of PrP^C into PrP^Sc implies that prion diseases are disorders of protein conformation.3. This concept has been extended by our studies with heat shock proteins (Hsp), many of which are thought to function as molecular chaperones. We found that the induction of some Hsps but not others was profoundly altered in scrapie-infected cells and that the distribution of Hsp73 is unusual in these cells.4. Whether the conversion of PrP^C into PrP^Sc is assisted by molecular chaperones, or if the accumulation of the abnormally folded PrP^Sc is complexed with Hsps remains to be established.     

Establishment of bovine prion peptide-based monoclonal antibodies for identifying bovine prion

To obtain high titer monoclonal antibodies (McAbs) which can react with mammalian prion protein (PrP), Balb/C mice were immunized with bovine (Bo) PrP peptide (BoPrP 209—228 aa) coupled to keyhole limpet hemocyanin (KLH). The hybridoma cell lines secreting monoclonal antibodies against the pep-tide were established by cell fusion and cloning. The obtained McAbs were applied to detect recombi-nant human, bovine and hamster PrP, cellular prion protein (PrP^c) in normal bovine brain and patho-genic scrapie prion protein (PrP^Sc) accumulated in the medulla oblongata of bovine spongiform en-cephalopathy(BSE)specimen with Western blot and immunohistochemical detection, respectively. The current procedure might offer a simple, feasible method to raise high titer antibodies for studying bio-logical features of PrP in mammals, as well as detection of transmissible spongiform encephalopathy (TSE) and diagnosis of BSE, in particular.     

Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle

A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24). To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP). The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 199.7 ± 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 ± 13.5 days). Further, C-BSE was transmitted to wild-type mice with the incubation period of about 409 days, whereas BSE/JP24 prion inoculated mice showed no clinical signs up to 649 days. Severe vacuolation and a widespread and uniform distribution of PrP^Sc were pathologically observed in the brain of BSE/JP24 prion affected TgBoPrP mice. The molecular weight and glycoform ratio of PrP^Sc in BSE/JP24 were different from those in C-BSE, and PrP^Sc in BSE/JP24 exhibited weaker proteinase K resistance than that in C-BSE. These findings revealed that the BSE/JP24 prion has distinct biological and biochemical properties reported for that of C-BSE. Interestingly, a shorter incubation period was observed at the subsequent passage of the BSE/JP24 prion to TgBoPrP mice (152.2 ± 3.1 days). This result implies that BSE/JP24 prion has newly emerged and showed the possibility that L-type BSE prion might be classified into multiple strains.Key words: prion, atypical BSE, PrP^Sc     

The N-Terminal Sequence of Prion Protein Consists an Epitope Specific to the Abnormal Isoform of Prion Protein (PrPSc)

The conformation of abnormal prion protein (PrP^Sc) differs from that of cellular prion protein (PrP^C), but the precise characteristics of PrP^Sc remain to be elucidated. To clarify the properties of native PrP^Sc, we attempted to generate novel PrP^Sc-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP^Sc purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP^Sc from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP^C from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31–39 and 41–47, respectively. This indicates that a PrP^Sc-specific epitope exists in the N-terminal region of PrP^Sc, and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP^Sc. We found that the ratio of proteinase K (PK)-sensitive PrP^Sc to PK-resistant PrP^Sc was constant throughout the disease time course.     

Subcritical Water Hydrolysis Effectively Reduces the In Vitro Seeding Activity of PrPSc but Fails to Inactivate the Infectivity of Bovine Spongiform Encephalopathy Prions

The global outbreak of bovine spongiform encephalopathy (BSE) has been attributed to the recycling of contaminated meat and bone meals (MBMs) as feed supplements. The use of MBMs has been prohibited in many countries; however, the development of a method for inactivating BSE prions could enable the efficient and safe use of these products as an organic resource. Subcritical water (SCW), which is water heated under pressure to maintain a liquid state at temperatures below the critical temperature (374°C), exhibits strong hydrolytic activity against organic compounds. In this study, we examined the residual in vitro seeding activity of protease-resistant prion protein (PrP^Sc) and the infectivity of BSE prions after SCW treatments. Spinal cord homogenates prepared from BSE-infected cows were treated with SCW at 230–280°C for 5–7.5 min and used to intracerebrally inoculate transgenic mice overexpressing bovine prion protein. Serial protein misfolding cyclic amplification (sPMCA) analysis detected no PrP^Sc in the SCW-treated homogenates, and the mice treated with these samples survived for more than 700 days without any signs of disease. However, sPMCA analyses detected PrP^Sc accumulation in the brains of all inoculated mice. Furthermore, secondary passage mice, which inoculated with brain homogenates derived from a western blotting (WB)-positive primary passage mouse, died after an average of 240 days, similar to mice inoculated with untreated BSE-infected spinal cord homogenates. The PrP^Sc accumulation and vacuolation typically observed in the brains of BSE-infected mice were confirmed in these secondary passage mice, suggesting that the BSE prions maintained their infectivity after SCW treatment. One late-onset case, as well as asymptomatic but sPMCA-positive cases, were also recognized in secondary passage mice inoculated with brain homogenates from WB-negative but sPMCA-positive primary passage mice. These results indicated that SCW-mediated hydrolysis was insufficient to eliminate the infectivity of BSE prions under the conditions tested.     

Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure

Prions are molecular pathogens, able to convert a normal cellular prion protein (PrP^C) into a prion (PrP^Sc). The information necessary for this conversion is contained in the conformation of PrP^Sc. Mass spectrometry (MS) and small-molecule covalent reactions have been used to study prions. Mass spectrometry has been used to detect and quantitate prions in the attomole range (10^?18 mole). MS-based analysis showed that both possess identical amino acid sequences, one disulfide bond, a GPI anchor, asparagine-linked sugar antennae, and unoxidized methionines. Mass spectrometry has been used to define elements of the secondary and tertiary structure of wild-type PrP^Sc and GPI-anchorless PrP^Sc. It has also been used to study the quaternary structure of the PrP^Sc multimer. Small molecule reagents react differently with the same lysine in the PrP^C conformation than in the PrP^Sc conformation. Such differences can be detected by Western blot using mAbs with lysine-containing epitopes, such as 3F4 and 6D11. This permits the detection of PrP^Sc without the need for proteinase K pretreatment and can be used to distinguish among prion strains. These results illustrate how two important chemical tools, mass spectrometry and covalent modification by small molecules, are being applied to the detection and structural study of prions. Furthermore these tools are or can be applied to the study of the other protein misfolding diseases such as Alzheimer Disease, Parkinson Disease, or ALS.     

Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrP^C) to the infectious scrapie form (PrP^Sc). However, the mechanism that exogenous PrP^Sc infects cells and where pathologic conversion of PrP^C to the PrP^Sc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrP^C to the pathologic PrP^Sc form in N2a cells exposed to strain RML PrP^Sc infected brain homogenates, suggesting that a critical determinant of PrP^C conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP^Sc infects cells by lipid raft dependent, macropinocytosis.     

Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC

The mammalian prions replicate by converting cellular prion protein (PrP^C) into pathogenic conformational isoform (PrP^Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP^Sc on conversion of PrP^C in vitro using PrP^Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP^Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP^Sc. The tight correlation between conversion potency of small oligomers of human sPrP^Sc observed in vitro and duration of the disease suggests that sPrP^Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.     

Direct Detection of Disease Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N Terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.Key Words: BSE, scrapie and vCJD Diagnosis, DELFIA®, novel N terminal antibodies     

Novel amplification mechanism of prions through disrupting sortilin-mediated trafficking

Conformational conversion of the cellular prion protein, PrP^C, into the abnormally folded isoform of prion protein, PrP^Sc, which leads to marked accumulation of PrP^Sc in brains, is a key pathogenic event in prion diseases, a group of fatal neurodegenerative disorders caused by prions. However, the exact mechanism of PrP^Sc accumulation in prion-infected neurons remains unknown. We recently reported a novel cellular mechanism to support PrP^Sc accumulation in prion-infected neurons, in which PrP^Sc itself promotes its accumulation by evading the cellular inhibitory mechanism, which is newly identified in our recent study. We showed that the VPS10P sorting receptor sortilin negatively regulates PrP^Sc accumulation in prion-infected neurons, by interacting with PrP^C and PrP^Sc and trafficking them to lysosomes for degradation. However, PrP^Sc stimulated lysosomal degradation of sortilin, disrupting the sortilin-mediated degradation of PrP^C and PrP^Sc and eventually evoking further accumulation of PrP^Sc in prion-infected neurons. These findings suggest a positive feedback amplification mechanism for PrP^Sc accumulation in prion-infected neurons.     

Species and Strain Glycosylation Patterns of PrPSc

Background

A key event in transmissible spongiform encephalopathies (TSEs) is the conversion of the soluble, protease-sensitive glycosylated prion protein (PrP^C) to an abnormally structured, aggregated and partially protease-resistant isoform (PrP^Sc). Both PrP isoforms bear two potential glycosylation sites and thus in a typical western blot with an anti-PrP antibody three distinct bands appear, corresponding to the di-, mono- or unglycosylated forms of the protein. The relative intensity and electrophoretic mobility of the three bands are characteristic of each TSE strain and have been used to discriminate between them.

Methodology/Principal Findings

In the present study we used lectin-based western blotting to evaluate possible variations in composition within sugar chains carried by PrP^Sc purified from subjects affected with different TSEs. Our findings indicate that in addition to the already well-documented differences in electrophoretic mobility and amounts of the glycosylated PrP^Sc forms, TSE strains also vary in the abundance of specific N-linked sugars of the PrP^Sc protein.

Conclusions/Significance

These results imply that PrP glycosylation might fine-tune the conversion of PrP^C to PrP^Sc and could play an accessory role in the appearance of some of the characteristic features of TSE strains. The differences in sugar composition could also be used as an additional tool for discrimination between the various TSEs.