Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type

The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrPSc) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two-dimensional immunoblot profiles of detergent insoluble and proteinase K-resistant PrP species in a type of sporadic Creutzfeldt-Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full-length and truncated PrP forms present in the insoluble fractions were also separately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrPSc, but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent-insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two-dimensional blots of the proteinase K-resistant PrP fraction suggesting that different PrP species are selected for conversion to proteinase K-resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N-linked glycans. We also show that the over-representation of the highly glycosylated forms distinctive of the proteinase K-resistant PrPSc of vCJD in one-dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases.     

Cytosolic prion protein toxicity is independent of cellular prion protein expression and prion propagation

Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrP(Sc)), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrP(C) exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrP(C) is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(Prnp(+/+)) mice with PrP-ablated mice (TgPrnp(o/o)) to generate Tg1D4(Prnp(o/o)) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(Prnp(+/+)) and Tg1D4(Prnp(o/o)) mice, suggesting that cyPrP and PrP(C) function independently in the disease state. Additionally, Tg1D4(Prnp(o/o)) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrP(Sc). We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrP(C) and the generation of typical prion disease.     

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.     

Potential approaches for heterologous prion protein treatment of prion diseases

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are progressive, fatal neurodegenerative diseases with no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrP^C) into a protease resistant infectious form (PrP^res). The efficiency of this conversion is predicated upon a number of factors, most notably a strong homology between cellular PrP^C and PrP^res. In our recently published study, we infected mice with the RML-Chandler strain of scrapie and treated them with heterologous hamster prion proteins. This treatment was seen to reduce clinical signs of prion disease, to delay the onset of clinical symptoms and to prolong survival. In this current article we discuss potential mechanisms of action of treatment with heterologous prion proteins. We also discuss potential extensions of these studies using a heterologous rabbit PrP-based treatment strategy or a peptide based strategy, and improvement of treatment delivery including a lentiviral-based system.     

The prion protein

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders represented principally by Creutzfeldt-Jakob disease in humans, and by scrapie and bovine spongiform encephalopathy in animals. Also called prion diseases, these disorders have the property of being infectious, sporadic or genetic in origin. Although the nature of the responsible agent of these diseases is uncertain, it is clear that a protein called PrPSc has a central role in their pathology. PrPSc is a conformational variant of a normal protein called PrPC. PrPC is a glycoprotein expressed by most tissues and is attached on the cell membrane by a glycosyl-phosphatidylinositol anchor which would be consistent with roles in cell adhesion, ligand uptake, or transmembrane signaling. NMR studies revealed that the protein has a globular domain and a long amino-terminal tail that contains repeated octapeptide domains which bind metal ions with high affinities. PrPC is localized on the cell membrane in detergent resistant microdomains and may be part of functional complexes with other molecules. This is particularly relevant, knowing the possible role of the molecule in signal transduction, resistance to oxidative stress and neuronal survival. In conclusion, it appears that the understanding of the biology of PrP is essential for the understanding of the physiological function of the protein as well as for its pathological conversion considering that trafficking of this molecule governs generation of PrPSc.     

A prion primer

By biological and medical criteria, prions are infectious agents; however, many of their properties differ profoundly from those of conventional microbes. Prions are "encoded" by alterations in protein conformation rather than in nucleic acid or amino acid sequence. New epidemic prion diseases (bovine spongiform encephalopathy and new variant Creutzfeldt-Jakob disease) have recently emerged under the active surveillance of the modern world. The risk of contracting prion disease from blood products or other biologicals is now a focus of worldwide concern. Much has been discovered about prions and prion diseases, but much remains to be done.     

Subclinical prion infection

Prion diseases are transmissible neurodegenerative disorders that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt–Jakob disease (CJD) in humans. The principal component of the infectious agent responsible for these diseases appears to be an abnormal isoform of the host-encoded prion protein (PrP), designated PrP^Sc. Prion diseases are transmissible to the same or different mammalian species by inoculation with, or dietary exposure to, infected tissues. Although scrapie in sheep has been recognized for over 200 years, it is the recent epidemic of BSE that has centred much public and scientific attention on these neurodegenerative diseases. The occurrence of variant CJD in humans and the experimental confirmation that it is caused by the same prion strain as BSE has highlighted the need for intensive study into the pathogenesis of these diseases and new diagnostic and therapeutic approaches. The existence and implications of subclinical forms of prion disease are discussed.     

Mammalian prion proteins

The past two years have seen the extension of our knowledge on the cellular prion protein structure with new NMR data on both the hamster and human proteins. In addition, the folding dynamics of two cellular prion proteins have been elucidated. There are now several examples of recombinant prion proteins that are able to adopt different conformations in solution and recent work on the molecular basis of prion strains has done much to consolidate the protein-only hypothesis. Important advances in relating disease to structure have also been made through the identification of the minimal prion protein fragment that is capable of conferring susceptibility to and propagation of the scrapie agent.     

The role of the prion protein membrane anchor in prion infection

Normal cellular and abnormal disease-associated forms of prion protein (PrP) contain a C-terminal glycophosphatidyl-inositol (GPI) membrane anchor. The importance of the GPI membrane anchor in prion diseases is unclear but there are data to suggest that it both is and is not required for abnormal prion protein formation and prion infection. Utilizing an in vitro model of prion infection we have recently demonstrated that, while the GPI anchor is not essential for the formation of abnormal prion protein in a cell, it is necessary for the establishment of persistent prion infection. In combination with previously published data, our results suggest that GPI anchored PrP is important in the amplification and spread of prion infectivity from cell to cell.Key words: prion, GPI anchor, PrP, prion spread, scrapieIn transmissible spongiform encephalopathies (TSE or prion diseases) such as sheep scrapie, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, normally soluble and protease-sensitive prion protein (PrP-sen or PrP^C) is converted to an abnormal, insoluble and protease-resistant form termed PrP-res or PrP^Sc. PrP-res/PrP^Sc is believed to be the main component of the prion, the infectious agent of the TSE/prion diseases. Its precursor, PrP-sen, is anchored to the cell surface at the C-terminus by a co-translationally added glycophosphatidyl-inositol (GPI) membrane anchor which can be cleaved by the enzyme phosphatidyl-inositol specific phospholipase (PIPLC). The GPI anchor is also present in PrP-res, but is inaccessible to PIPLC digestion suggesting that conformational changes in PrP associated with PrP-res formation have blocked the PIPLC cleavage site.¹ Although the GPI anchor is present in both PrP-sen and PrP-res, its precise role in TSE diseases remains unclear primarily because there are data to suggest that it both is and is not necessary for PrP-res formation and prion infection.In tissue culture cells infected with mouse scrapie, PrP-res formation occurs at the cell surface and/or along the endocytic pathway^2–4 and may be dependent upon the membrane environment of PrP-sen. For example, localization via the GPI anchor to caveolae-like domains favors PrP-res formation⁵ while substitution of the GPI anchor addition site with carboxy termini favoring transmembrane anchored PrP-sen inhibits formation of PrP-res.^5,6 Other studies have shown that localization of both PrP-sen and PrP-res to lipid rafts, cholesterol and sphingolipid rich membrane microdomains where GPI anchored proteins can be located, is important in PrP-res formation.^6–9However, there are also data which suggest that such localization is not necessarily essential for PrP-res formation. Anchorless PrP-sen isolated from cells by immunoprecipitation or wild-type PrP-sen purified by immunoaffinity column followed by cation exchange chromatography are efficiently converted into PrP-res in cell-free systems.^10,11 Furthermore, recombinant PrP-sen derived from E. coli, which has no membrane anchor or glycosylation, can be induced to form protease-resistant PrP in vitro when reacted with prion-infected brain homogenates.^12–14 Finally, in at least one instance, protease-resistant recombinant PrP-res generated in the absence of infected brain homogenate was reported to cause disease when inoculated into transgenic mice.¹⁵The data concerning the role of the PrP-sen GPI anchor in susceptibility to TSE infection are similarly contradictory. Transgenic mice expressing anchorless mouse PrP-sen are susceptible to infection with mouse scrapie and accumulate both PrP-res and prion infectivity.¹⁶ Thus, the GPI anchor is clearly not needed for PrP-res formation or productive TSE infection in vivo. However, we recently published data demonstrating that, in vitro, anchored PrP-sen is in fact required to persistently infect cells.¹⁷ Given that anchorless PrP-sen is not present on the cell surface but is released into the cell medium, we speculated that the differences between the in vitro and in vivo data were related to the location of PrP-res formation. In the mice expressing anchorless PrP-sen, environments conducive to PrP-res formation are present in certain areas of the complex extracellular milieu of the brain where anchorless, secreted PrP-sen can accumulate and come into contact with PrP-res from the infectious inoculum. Since similar environments are missing in vitro, any PrP-res formation in cells expressing anchorless PrP-sen must be cell-associated. While this explanation addresses how extracellular PrP-res could be generated in an unusual transgenic mouse model of TSE infection, it does not really help to define how the GPI anchor is involved in normal prion infection of a cell.As with other infectious organisms such as viruses, TSE infection can be roughly divided into three steps: uptake, replication and spread. Over the last several years, data derived from new techniques as well as new cell lines susceptible to prion infection have increased our knowledge of some of the basic events that occur during each of these steps. In order to try to tease out the role of the GPI anchor in normal TSE pathogenesis, it is therefore useful to consider the process of TSE infection of a cell and how the GPI anchor might be involved in each stage.In a conventional viral infection, binding and uptake of the virus is essential to establish infection. Studying PrP-res uptake has been complicated by the lack of an antibody that can specifically distinguish PrP-res from PrP-sen in live cells and by the difficulty of detecting the input PrP-res from the PrP-res made de novo by the cell. Recently, however, several groups have been able to study PrP-res uptake using input PrP-res that was either fluorescently labeled^18–20 or tagged with the epitope to the monoclonal antibody 3F4,²¹ or cell lines that express little or no PrP-sen.^19,21–23 The data show that PrP-res uptake is independent of scrapie strain or cell type but is influenced by the PrP-res microenvironment as well as PrP-res aggregate size.²¹ Importantly, these studies demonstrated that PrP-sen expression was not required.^19,21–23 Given these data, it is clear that GPI anchored PrP-sen is not involved in the initial uptake of PrP-res into the cell.The next stage of prion infection involves replication of infectivity which is typically assayed by following cellular PrP-res formation. Once again, however, the issue of how to distinguish PrP-res in the inoculum from newly formed PrP-res in the cells has made it difficult to study the early stages of prion replication. To overcome this difficulty, we developed a murine tissue culture system that utilizes cells expressing mouse PrP-sen tagged with the epitope to the 3F4 antibody (Mo3F4 PrP-sen).²⁴ Wild-type mouse PrP does not have this epitope. As a result, following exposure to an infected mouse brain homogenate, de novo PrP-res formation can be followed by assaying for 3F4 positive PrP-res. Our studies showed that there were two stages of PrP-res formation: (1) an initial acute burst within the first 96 hours post-infection that was cell-type and scrapie strain independent and, (2) persistent PrP-res formation (i.e., formation of PrP-res over multiple cell passages) that was dependent on cell-type and scrapie strain and associated with long-term infection.²⁴ Acute PrP-res formation did not necessarily lead to persistent PrP-res formation suggesting that other cell-specific factors or processes are needed for PrP-res formation to persist.²⁴When cells expressing Mo3F4 PrP-sen without the GPI anchor (Mo3F4 GPI-PrP-sen) were exposed to mouse scrapie infected brain homogenates, GPI negative, 3F4 positive PrP-res (Mo3F4 GPI-PrP-res) was detected within 96 hours indicating that acute PrP-res formation had occurred.¹⁷ Thus, despite the fact that Mo3F4 GPI-PrP-sen is not expressed on the cell surface¹⁶ (Fig. 1A), it was still available for conversion to PrP-res. These results are consistent with data from cell-free systems and demonstrate that, at least acutely, membrane anchored PrP is not necessary for PrP-res formation in a cell.Open in a separate windowFigure 1Persistent infection of cells in vitro requires the expression of GPI-anchored cell surface PrP-sen. PrP knockout cells (CF10)²¹ were transduced with 3F4 epitope tagged mouse PrP-sen (Mo3F4), 3F4 epitope tagged mouse PrP-sen without the GPI anchor (Mo3F4 GPI-), or Mo3F4 GPI-PrP-sen plus wild-type, GPI anchored mouse PrP-sen (MoPrP). The cells were then exposed to the mouse scrapie strain 22L and passaged. (A) The presence of 3F4 epitope tagged, cell surface mouse PrP-sen was assayed by FACS analysis of fixed, non-permeabilized cells. CF10 cells expressing the following mouse PrP-sen molecules were assayed: Mo3F4 (solid line); Mo3F4 GPI⁻ (dashed line); Mo3F4 GPI⁻ + MoPrP (dotted and dashed line); Mo3F4 GPI⁻ + MoPrP infected with 22L scrapie (dotted line). Only cells expressing Mo3F4 PrP-sen were positive for cell surface, 3F4 epitope tagged PrP. (B) Persistent infection was analyzed by inoculating the cells intracranially into transgenic mice overexpressing MoPrP (Tga20 mice). Only cells expressing anchored mouse PrP-sen were susceptible to scrapie infection. Cells expressing anchorless mouse PrP-sen did not contain detectable infectivity in either the cells or the cellular supernatant (data not shown). Data in (B) are adapted from McNally 2009.¹⁷In terms of persistent PrP-res formation, however, our data suggest that the GPI anchor is important. Despite an initial burst of PrP-res formation within the first 96 hours post-infection, Mo3F4 GPI-PrP-res was not observed following passage of the cells nor did the cells become infected. This effect was not due either to resistance of the cells to scrapie infection or to an inability of the scrapie strain used to infect cells. When the same cells expressed anchored Mo3F4 PrP-sen and were exposed to the same mouse scrapie strain, both acute and persistent PrP-res formation were detected and the cells were persistently infected with scrapie (Fig. 1B).¹⁷ Taken together, these data demonstrate that cells expressing anchorless PrP-sen do not support persistent PrP-res formation. Furthermore, the data strongly suggest that GPI-anchored PrP-sen is required during the transition from acute to persistent scrapie infection. In support of this hypothesis, the resistance of cells expressing Mo3F4 GPI-PrP-sen to persistent prion infection could be overcome if wild-type GPI anchored PrP-sen was co-expressed in the same cell. When both forms of PrP-sen were expressed, anchored and anchorless forms of PrP-res were made and the cells became persistently infected (Fig. 1B).¹⁷ Thus, the data suggest that GPI anchored PrP is necessary to establish prion infection within a cell.How could GPI membrane anchored PrP be involved in the establishment and maintenance of persistent prion infection? Several studies have suggested that the GPI anchor is needed to localize PrP-sen to specific membrane environments where PrP-res formation is favored.^5–8 However, if this localization was essential for PrP-res formation, GPI-PrP-sen would presumably never form PrP-res. Lacking the GPI anchor, it would not be in the correct membrane environment to support conversion. As a result, neither acute nor persistent prion infection could occur. This is obviously not the case. Transgenic mice expressing only anchorless PrP-sen generate PrP-res and can be infected with scrapie even though (1) flotation gradients showed that anchorless PrP-sen was not in the same membrane environment as anchored PrP-sen and, (2) flow cytometry analysis demonstrated that anchorless PrP-sen was not present on the cell surface.¹⁶ Thus, the GPI anchor is not needed to target PrP-sen to a conversion friendly membrane environment.Consistent with the idea that the GPI anchor is not essential for PrP-res formation, in our studies anchorless PrP-sen could form PrP-res in cells acutely infected with scrapie despite the fact that it is processed differently than anchored PrP-sen, is not present on the cell surface (Fig. 1A), and is secreted.¹⁷ Persistent formation of anchorless PrP-res only occurred when both anchored and anchorless forms of PrP were expressed in the same cell.¹⁷ For this to happen both types of PrP must share a cellular compartment where PrP-res formation occurs, presumably either on the cell surface or in a specific location along the endocytic pathway^2,3 such as the endosomal recycling compartment.⁴ Analysis of infected and uninfected cells co-expressing Mo3F4 GPI-PrP-sen and wild-type PrP-sen demonstrated that Mo3F4 GPI-PrP-sen was not present on the cell surface (Fig. 1A). Thus, it is unlikely that GPI-PrP-res formation is occurring on the cell surface. We speculate that the anchored form of PrP-res encounters anchorless PrP-sen along either a secretory or endocytic pathway, allowing for the formation of anchorless PrP-res. Regardless of the precise location, the in vitro and in vivo data strongly suggest that the role of the anchor in persistent prion infection is not simply to localize PrP-sen to an environment compatible with PrP-res formation.However, the data are consistent with the idea that GPI anchored PrP is absolutely essential for the establishment of persistent infection in vitro. This is likely related to the spread of infectivity within a culture that is necessary for maintaining a persistent infection over time. Evidence suggests that PrP-res can be transferred between cells in a variety of ways including mother-daughter cell division,²⁵ cell-to-cell contact^26,27 and exosomes.²⁸ Tunneling nanotubes have also been hypothesized to be involved in intercellular prion spread¹⁹ and recent data suggest that spread can occur via these structures.²⁰ Any of these processes could involve the cell-to-cell transfer of PrP-res in membrane containing particles as has been observed in cell-free⁷ and cell-based systems.²⁹ If cell-to-cell contact were required, for example via simple physical proximity or perhaps tunneling nanotubes,^19,20 then the conversion of cell surface PrP-sen on the naïve cell by cell surface PrP-res on the infected cell would transfer infection to the naïve cell. In this instance, GPI membrane anchored, cell surface PrP-sen would be essential as it would allow for PrP-res formation on the cell surface. If spread is via cell division, then GPI-anchored, cell surface PrP-sen would be important for its role as a precursor to PrP-res formation.² In this instance, cell surface PrP-sen would be an essential intermediate in the continuous formation of PrP-res necessary for the accumulation and amplification of PrP-res within the cell. It would also help to cycle PrP between the cell surface and intracellular compartments where PrP-res can be formed.⁴ In either case, GPI-anchored PrP-sen would facilitate the accumulation of intracellular PrP-res to high enough levels to maintain both persistent infection in the mother cell and enable the transfer of organelles containing sufficient PrP-res to initiate infection in the daughter cell. Thus, we would suggest that efficient spread of infectivity requires not just the passive transfer of PrP-res from cell-to-cell but the concurrent initiation of conversion and amplification of PrP-res via cell surface, GPI anchored PrP-sen.In vivo, several lines of evidence suggest that the spread of scrapie infectivity also requires de novo PrP-res formation in the recipient cell and not simply transfer of PrP-res from one cell to another. For example, when neurografts from PrP expressing mice were placed in the brains of PrP knockout mice and the mice were challenged intracranially with scrapie, the graft showed scrapie pathology, but the surrounding tissue did not.³⁰ Furthermore, PrP-res from the graft migrated to the host tissue demonstrating that simple transfer of PrP-res was not sufficient and that PrP-sen expression was required for the spread of scrapie pathology.³⁰ In fact, these mice did not develop scrapie pathology following peripheral infection even when peripheral lymphoid tissues were reconstituted with PrP-sen expressing cells.³¹ Even though PrP-sen expressing cells were present in both the brain and spleen, in order for infectivity to spread from the lymphoreticular system to the central nervous system PrP-sen expression was also required in an intermediate tissue such as peripheral nerve.^31,32 Given that PrP-res uptake and trafficking do not require PrP-sen, the most obvious explanation for the requirement of PrP-sen in contiguous tissues is that de novo PrP-res formation in naïve cells is necessary for (1) infectivity to move from cell to cell within a tissue and, (2) infectivity to move from tissue to tissue.Another study demonstrated that peripheral expression of heterologous mouse PrP significantly increased the incubation time and actually prevented clinical disease in the majority of transgenic mice expressing hamster PrP in neurons of the brain.³³ Once again, if simple transfer and uptake of PrP-res were sufficient for spread, the presence of heterologous PrP molecules should not interfere because cellular uptake of PrP-res is independent of PrP-sen expression.^19,21–23 Clinical disease in these mice was likely prevented by the heterologous PrP molecule interfering with conversion of PrP-sen to PrP-res suggesting that prevention of de novo PrP-res formation inhibits spread of PrP-res and infectivity. These in vivo data, when combined with our recent in vitro data,¹⁷ provide evidence to support the importance of cell surface, and by extension GPI-anchored, PrP in the spread of prion infection.Our data demonstrate that the GPI anchor plays a role in the establishment of persistent scrapie infection in vitro. In our tissue culture system,²¹ as well as others where spread of infectivity by cell to cell contact appears to be limited,^25,34 the role of GPI anchored PrP-sen would be to amplify PrP-res to enable the efficient transfer of infectivity from mother to daughter cell. In cell systems where spread of prion infectivity may require cell to cell contact,^26,27 we propose that the role of GPI anchored PrP-sen is to facilitate the spread of prion infection via a chain of conversion from cell-to-cell, a “domino” type spread of infection that has been previously hypothesized.^35,36In vivo, such a mechanism might explain why neuroinvasion does not necessarily require axonal transport^32,37,38 and can occur independently of the axonal neurofilament machinery.³⁹ It would likely vary with cell type²⁷ and be most important in areas where infectivity is transferred from the periphery to the nervous system as well as in areas where cell division may be limited. It is also possible, if the location of PrP-res formation differs for different scrapie strains,⁴⁰ that the relative importance of a domino-like spread of infectivity in vivo would vary with the scrapie strain.Of course, spread of infectivity via a “wave” of GPI anchored, PrP mediated conversion would not preclude the spread of infectivity by other intracellular means such as axonal transport (reviewed in ref. ⁴¹). Furthermore, spread of infectivity may still also occur extracellularly such as in the unique case of mice which express anchorless PrP-sen,¹⁶ where our in vitro data would suggest that the cells themselves are not infected. In such a case, spread would require neither GPI anchored PrP-sen nor amplification of PrP-res in cells but would likely occur via other means such as blood⁴¹ or interstitial fluid flow.⁴²     

N-terminally tagged prion protein supports prion propagation in transgenic mice.

The eight amino acid sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, representing the FLAG peptide, was inserted after codons 22 or 88 of the mouse (Mo) prion protein (PrP) gene. Inclusion of the FLAG sequence at these locations interfered neither with the cellular processing of PrPC nor its conversion into PrPSc. Inclusion of the FLAG epitope at residue 22 but not at residue 88 facilitated immunodetection of tagged PrP by anti-FLAG monoclonal antibodies (mAbs). Inoculation of transgenic (Tg) mice expressing N-terminally tagged MoPrP with Mo prions resulted in abbreviated incubation times, indicating that the FLAG sequence was not deleterious to prion propagation. Immunopurification of FLAG-tagged MoPrPC in the brains of Tg mice was achieved using the calcium-dependent anti-FLAG M1 mAb and non-denaturing procedures. Although the function of PrPC remains unknown, our studies demonstrate that some modifications of PrPC do not inhibit the one biological activity that can be measured, i.e., conversion into PrPSc. Tagged PrP molecules may prove useful in the development of improved assays for prions as well as structural studies of the PrP isoforms.     

Accelerated high fidelity prion amplification within and across prion species barriers

Experimental obstacles have impeded our ability to study prion transmission within and, more particularly, between species. Here, we used cervid prion protein expressed in brain extracts of transgenic mice, referred to as Tg(CerPrP), as a substrate for in vitro generation of chronic wasting disease (CWD) prions by protein misfolding cyclic amplification (PMCA). Characterization of this infectivity in Tg(CerPrP) mice demonstrated that serial PMCA resulted in the high fidelity amplification of CWD prions with apparently unaltered properties. Using similar methods to amplify mouse RML prions and characterize the resulting novel cervid prions, we show that serial PMCA abrogated a transmission barrier that required several hundred days of adaptation and subsequent stabilization in Tg(CerPrP) mice. While both approaches produced cervid prions with characteristics distinct from CWD, the subtly different properties of the resulting individual prion isolates indicated that adaptation of mouse RML prions generated multiple strains following inter-species transmission. Our studies demonstrate that combined transgenic mouse and PMCA approaches not only expedite intra- and inter-species prion transmission, but also provide a facile means of generating and characterizing novel prion strains.     

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.     

The role of the prion protein membrane anchor in prion infection

In transmissible spongiform encephalopathies (TSE or prion diseases) such as sheep scrapie, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, normally soluble and protease-sensitive prion protein (PrP-sen or PrPC) is converted to an abnormal, insoluble and protease-resistant form termed PrP-res or PrPSc. PrP-res/PrPSc is believed to be the main component of the prion, the infectious agent of the TSE/prion diseases. Its precursor, PrP-sen, is anchored to the cell surface at the C-terminus by a co-translationally added glycophosphatidyl-inositol (GPI) membrane anchor which can be cleaved by the enzyme phosphatidyl-inositol specific phospholipase (PIPLC). The GPI anchor is also present in PrP-res, but is inaccessible to PIPLC digestion suggesting that conformational changes in PrP associated with PrP-res formation have blocked the PIPLC cleavage site. Although the GPI anchor is present in both PrP-sen and PrP-res, its precise role in TSE diseases remains unclear primarily because there are data to suggest that it both is and is not necessary for PrP-res formation and prion infection.     

Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC-GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC-GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.     

Propagation of prion strains through specific conformers of the prion protein.

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.     

Insoluble cellular prion protein and its association with prion and Alzheimer diseases

The soluble cellular prion protein (PrP^C) is best known for its association with prion disease (PrD) through its conversion to a pathogenic insoluble isoform (PrP^Sc). However, its deleterious effects independent of PrP^Sc have recently been observed not only in PrD but also in Alzheimer disease (AD), two diseases which mainly affect cognition. At the same time, PrP^C itself seems to have broad physiologic functions including involvement in cognitive processes. The PrP^C that is believed to be soluble and monomeric has so far been the only PrP conformer observed in the uninfected brain. In 2006, we identified an insoluble PrP^C conformer (termed iPrP^C) in uninfected human and animal brains. Remarkably, the PrP^Sc-like iPrPC shares the immunoreactivity behavior and fragmentation with a newly-identified PrP^Sc species in a novel human PrD termed variably protease-sensitive prionopathy. Moreover, iPrP^C has been observed as the major PrP species that interacts with amyloid β (Aβ) in AD. This article highlights evidence of PrP involvement in two putatively beneficial and deleterious PrP-implicated pathways in cognition, and hypothesizes first, that beneficial and deleterious effects of PrPC are attributable to the chameleon-like conformation of the protein and second, that the iPrP^C conformer is associated with PrD and AD.     

The fate of the prion protein in the prion/plasminogen complex

The cellular prion protein (PrP(c)) forms complexes with plasminogen. Here, we show that the PrP(c) in this complex is cleaved to yield fragments of PrP(c). The cleavage is accelerated by plasmin but does not appear to be dependent on it.     

Polythiophenes inhibit prion propagation by stabilizing prion protein (PrP) aggregates

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrP(C) (PrP(Sc)) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits.     

Porcine prion protein amyloid

ABSTRACT

Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.     

The prion protein family

Although the pivotal implication of the host-encoded Prion protein, PrP, in the neuropathology of transmissible spongiform encephalopathy is known for decades, its biological role remains mostly elusive. Genetic inactivation is one way to assess such issue but, so far, PrP-knockout mice did not help much. However, recent reports involving (1) further studies of these mice during embryogenesis, (2) knockdown experiments in Zebrafish and (3) knockdown of Shadoo, a protein with PrP-like functional domains, in PrP-knockout mice, all suggested a role of the Prion protein family in early embryogenesis. This view is challenged by the recent report that PrP/Shadoo knockout mice are healthy and fertile. Although puzzling, these apparently contradictory data may on the contrary help at deciphering the Prion protein family role through focusing scientific attention outside the central nervous system and by helping the identification of other loci involved in the genetic robustness associated with PrP.