Sequestration of free cholesterol in cell membranes by prions correlates with cytoplasmic phospholipase A<Subscript>2</Subscript>activation

Background  

The transmissible spongiform encephalopathies (TSEs), otherwise known as the prion diseases, occur following the conversion of the normal cellular prion protein (PrP^C) to an alternatively folded isoform (PrP^Sc). The accumulation of PrP^Sc within the brain leads to neurodegeneration through an unidentified mechanism. Since many neurodegenerative disorders including prion, Parkinson's and Alzheimer's diseases may be modified by cholesterol synthesis inhibitors, the effects of prion infection on the cholesterol balance within neuronal cells were examined.     

Glimepiride Reduces the Expression of PrPC,Prevents PrPSc Formation and Protects against Prion Mediated Neurotoxicity

Background

A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrP^C) into a disease related, alternatively folded isoform (PrP^Sc). The accumulation of PrP^Sc within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases.

Principal Findings

Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrP^C from the surface of prion-infected neuronal cells. The cell surface is a site where PrP^C molecules may be converted to PrP^Sc and glimepiride treatment reduced PrP^Sc formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82–146. Glimepiride treatment significantly reduce both the amount of PrP82–146 that bound to neurones and PrP82–146 induced activation of cytoplasmic phospholipase A₂ (cPLA₂) and the production of prostaglandin E₂ that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrP^C expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC.

Conclusions

Collectively, these results indicate that glimepiride may be a novel treatment to reduce PrP^Sc formation and neuronal damage in prion diseases.     

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     

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.     

Observation of intermediate states of the human prion protein by high pressure NMR spectroscopy

Background  

Prions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrP^Sc, of the cellular prion protein, PrP^C. The conversion and thus the propensity of PrP^C to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool to study the physico-chemical properties of proteins as well as the dynamics and structure of folding intermediates.     

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.     

The Octarepeat Region of the Prion Protein Is Conformationally Altered in PrPSc

Background

Prion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrP^C. Little is known about the details of the structural rearrangement of physiological PrP^C into a still-elusive disease-associated conformation termed PrP^Sc. Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59–89), though not essential, play a role in modulating prion replication and disease presentation.

Methodology/Principal Findings

Here, we report that trypsin digestion of PrP^Sc from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrP^Sc fragment including amino acids ∼49–231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrP^Sc are exposed in PrP^C.

Conclusions/Significance

We conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrP^Sc. This phenomenon may be relevant to the mechanism by which the amino terminus of PrP^C participates in PrP^Sc conversion, and may also be exploited for diagnostic purposes.     

Identification and structural analysis of C-terminally truncated collapsin response mediator protein-2 in a murine model of prion diseases

Background  

Prion diseases are fatal neurodegenerative disorders that accompany an accumulation of the disease-associated form(s) of prion protein (PrP^Sc) in the central nervous system. The neuropathological changes in the brain begin with focal deposits of PrP^Sc, followed by pathomorphological abnormalities of axon terminal degeneration, synaptic loss, atrophy of dendritic trees, and eventual neuronal cell death in the lesions. However, the underlying molecular basis for these neuropathogenic abnormalities is not fully understood.     

De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype

Prions are the proteinaceous infectious agents responsible for Transmissible Spongiform Encephalopathies. Compelling evidence supports the hypothesis that prions are composed exclusively of a misfolded version of the prion protein (PrP^Sc) that replicates in the body in the absence of nucleic acids by inducing the misfolding of the cellular prion protein (PrP^C). The most common form of human prion disease is sporadic, which appears to have its origin in a low frequency event of spontaneous misfolding to generate the first PrP^Sc particle that then propagates as in the infectious form of the disease. The main goal of this study was to mimic an early event in the etiology of sporadic disease by attempting de novo generation of infectious PrP^Sc in vitro. For this purpose we analyzed in detail the possibility of spontaneous generation of PrP^Sc by the protein misfolding cyclic amplification (PMCA) procedure. Under standard PMCA conditions, and taking precautions to avoid cross-contamination, de novo generation of PrP^Sc was never observed, supporting the use of the technology for diagnostic applications. However, we report that PMCA can be modified to generate PrP^Sc in the absence of pre-existing PrP^Sc in different animal species at a low and variable rate. De novo generated PrP^Sc was infectious when inoculated into wild type hamsters, producing a new disease phenotype with unique clinical, neuropathological and biochemical features. Our results represent additional evidence in support of the prion hypothesis and provide a simple model to study the mechanism of sporadic prion disease. The findings also suggest that prion diversity is not restricted to those currently known, and that likely new forms of infectious protein foldings may be produced, resulting in novel disease phenotypes.     

PrP Expression,PrPSc Accumulation and Innervation of Splenic Compartments in Sheep Experimentally Infected with Scrapie

Background

In prion disease, the peripheral expression of PrP^C is necessary for the transfer of infectivity to the central nervous system. The spleen is involved in neuroinvasion and neural dissemination in prion diseases but the nature of this involvement is not known. The present study undertook the investigation of the spatial relationship between sites of PrP^Sc accumulation, localisation of nerve fibres and PrP^C expression in the tissue compartments of the spleen of scrapie-inoculated and control sheep.

Methodology/Principal Findings

Laser microdissection and quantitative PCR were used to determine PrP mRNA levels and results were compared with immunohistochemical protocols to distinguish PrP^C and PrP^Sc in tissue compartments of the spleen. In sheep experimentally infected with scrapie, the major sites of accumulation of PrP^Sc in the spleen, namely the lymphoid nodules and the marginal zone, expressed low levels of PrP mRNA. Double immunohistochemical labelling for PrP^Sc and the pan-nerve fibre marker, PGP, was used to evaluate the density of innervation of splenic tissue compartments and the intimacy of association between PrP^Sc and nerves. Some nerve fibres were observed to accompany blood vessels into the PrP^Sc-laden germinal centres. However, the close association between nerves and PrP^Sc was most apparent in the marginal zone. Other sites of close association were adjacent to the wall of the central artery of PALS and the outer rim of germinal centres.

Conclusions/Significance

The findings suggest that the degree of PrP^Sc accumulation does not depend on the expression level of PrP^C. Though several splenic compartments may contribute to neuroinvasion, the marginal zone may play a central role in being the compartment with most apparent association between nerves and PrP^Sc.     

A novel real-time ultrasonic method for prion protein detection using plasminogen as a capture molecule

Background  

High resolution ultrasonography (HR-US) can monitor the molecular changes and biochemical interactions between proteins in real-time. The aim of this study was to use HR-US to characterize the real-time interactions between plasminogen coated beads and PrP^Sc and to determine if this approach could be applied to the identification of animals affected by prion diseases. Plasminogen, immobilized to beads, was used as a capturing tool for PrP^Sc in brain homogenates from scrapie affected sheep and the binding reaction was monitored in real-time in an ultrasonic cell.     

Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein

Background

The accumulation of protease resistant conformers of the prion protein (PrP^res) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific.

Methodology/Principal Finding

In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP^res formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrP^C substrate was found to specifically prevent PrP^res formation seeded by mouse derived PrP^Sc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP^res formation, while having no effect on PrP^res formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans.

Conclusions/Significance

Cofactor requirements for PrP^res formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.     

Infectivity-associated PrPSc and disease duration-associated PrPSc of mouse BSE prions

ABSTRACT

Disease-related prion protein (PrP^Sc), which is a structural isoform of the host-encoded cellular prion protein, is thought to be a causative agent of transmissible spongiform encephalopathies. However, the specific role of PrP^Sc in prion pathogenesis and its relationship to infectivity remain controversial. A time-course study of prion-affected mice was conducted, which showed that the prion infectivity was not simply proportional to the amount of PrP^Sc in the brain. Centrifugation (20,000 ×g) of the brain homogenate showed that most of the PrP^Sc was precipitated into the pellet, and the supernatant contained only a slight amount of PrP^Sc. Interestingly, mice inoculated with the obtained supernatant showed incubation periods that were approximately 15 d longer than those of mice inoculated with the crude homogenate even though both inocula contained almost the same infectivity. Our results suggest that a small population of fine PrP^Sc may be responsible for prion infectivity and that large, aggregated PrP^Sc may contribute to determining prion disease duration.     

In vitro amplification of H-type atypical bovine spongiform encephalopathy by protein misfolding cyclic amplification

The in vitro amplification of prions by serial protein misfolding cyclic amplification has been shown to detect PrP^Sc to levels at least as sensitive as rodent bioassay but in a fraction of the time. Bovine spongiform encephalopathy is a zoonotic prion disease in cattle and has been shown to occur in 3 distinct forms, classical BSE (C-BSE) and 2 atypical BSE forms (L-BSE and H-BSE). Atypical forms are usually detected in asymptomatic, older cattle and are suggested to be spontaneous forms of the disease. Here, we show the development of a serial protein misfolding cyclic amplification method for the detection of H-BSE. The assay could detect PrP^Sc from 3 distinct experimental isolates of H-BSE, could detect PrP^Sc in as little as 1×10^?12 g of brain material and was highly specific. Additionally, the product of serial protein misfolding cyclic amplification at all dilutions of seed analyzed could be readily distinguished from L-BSE, which did not amplify, and C-BSE, which had PrP^Sc with distinct protease K-resistance and protease K-resistant PrP^Sc molecular weights.     

Immunopurification of Pathological Prion Protein Aggregates

Background

Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP^C) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrP^Sc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP^C molecules. A great deal of effort has been devoted to developing protocols for purifying PrP^Sc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP^Sc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases.

Principal Findings

We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP^Sc and mutant PrP aggregates at electrophoretic homogeneity. PrP^Sc purified from prion-infected mice was able to seed misfolding of PrP^C in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons.

Significance

The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.     

Peroxiredoxin 6 promotes upregulation of the prion protein (PrP) in neuronal cells of prion-infected mice

Background

It has been widely established that the conversion of the cellular prion protein (PrP^C) into its abnormal isoform (PrP^Sc) is responsible for the development of transmissible spongiform encephalopathies (TSEs). However, the knowledge of the detailed molecular mechanisms and direct functional consequences within the cell is rare. In this study, we aimed at the identification of deregulated proteins which might be involved in prion pathogenesis.

Findings

Apolipoprotein E and peroxiredoxin 6 (PRDX6) were identified as upregulated proteins in brains of scrapie-infected mice and cultured neuronal cell lines. Downregulation of PrP gene expression using specific siRNA did not result in a decrease of PRDX6 amounts. Interestingly, selective siRNA targeting PRDX6 or overexpression of PRDX6 controlled PrP^C and PrP^Sc protein amounts in neuronal cells.

Conclusions

Besides its possible function as a novel marker protein in the diagnosis of TSEs, PDRX6 represents an attractive target molecule in putative pharmacological intervention strategies in the future.     

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrP^Sc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion.

Case presentation

We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrP^Sc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrP^Sc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine.

Conclusions

A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

     

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.     

Generation of antisera to purified prions in lipid rafts

Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrP^C into alternately folded PrP^Sc. Immunoassays toward pre-clinical detection of infectious PrP^Sc have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrP^Sc selective antibodies. We report a method to purify infectious PrP^Sc from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrP^Sc was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM >20-fold and purified DRM immunogen >40-fold) relative to 1% crude brain homogenate. Purification of PrP^Sc from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrP^Sc antigen was performed using wild-type (wt) and Prnp^0/0 mice, both on Balb/cJ background. A robust immune response against PrP^Sc was observed in all inoculated Prnp^0/0 mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrP^C and PrP^Sc, while binding to other brain-derived protein was not observed. In contrast, the PrP^Sc inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.Key words: prion, scrapie, Prnp0/0 mice, purification methodology, antibody, antisera, lipid-rafts, detergent resistant membranes, neuroscience, immunization, diagnostic     

The Distribution of Prion Protein Allotypes Differs Between Sporadic and Iatrogenic Creutzfeldt-Jakob Disease Patients

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrP^Sc). The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrP^C) into infectious PrP^Sc. By contrast, human growth hormone-associated cases of iatrogenic CJD (iCJD) in the United Kingdom (UK) are associated with exposure to an exogenous source of PrP^Sc. In both forms of CJD, heterozygosity at residue 129 for methionine (M) or valine (V) in the prion protein gene may affect disease phenotype, onset and progression. However, the relative contribution of each PrP^C allotype to PrP^Sc in heterozygous cases of CJD is unknown. Using mass spectrometry, we determined that the relative abundance of PrP^Sc with M or V at residue 129 in brain specimens from MV cases of sCJD was highly variable. This result is consistent with PrP^C containing an M or V at residue 129 having a similar propensity to misfold into PrP^Sc thus causing sCJD. By contrast, PrP^Sc with V at residue 129 predominated in the majority of the UK human growth hormone associated iCJD cases, consistent with exposure to infectious PrP^Sc containing V at residue 129. In both types of CJD, the PrP^Sc allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Therefore, factors other than PrP^Sc allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD.