Prion protein self-interaction in prion disease therapy approaches

Transmissible spongiform encephalopathies (TSEs) or prion diseases are unique disorders that are not caused by infectious micro-organisms (bacteria or fungi), viruses or parasites, but rather seem to be the result of an infectious protein. TSEs are comprised of fatal neurodegenerative disorders affecting both human and animals. Prion diseases cause sponge-like degeneration of neuronal tissue and include (among others) Creutzfeldt-Jacob disease in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. TSEs are characterized by the formation and accumulation of transmissible (infectious) disease-associated protease-resistant prion protein (PrP(Sc)), mainly in tissues of the central nervous system. The exact molecular processes behind the conversion of PrP(C) into PrP(Sc) are not clearly understood. Correlations between prion protein polymorphisms and disease have been found, however in what way these polymorphisms influence the conversion processes remains an enigma; is stabilization or destabilization of the prion protein the basis for a higher conversion propensity? Apart from the disease-associated polymorphisms of the prion protein, the molecular processes underlying conversion are not understood. There are some notions as to which regions of the prion protein are involved in refolding of PrP(C) into PrP(Sc) and where the most drastic structural changes take place. Direct interactions between PrP(C) molecules and/or PrP(Sc) are likely at the basis of conversion, however which specific amino acid domains are involved and to what extent these domains contribute to conversion resistance/sensitivity of the prion protein or the species barrier is still unknown.     

An overview of transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders of humans and animals associated with an accumulation of abnormal isoforms of prion protein (PrP) in nerve cells. The pathogenesis of TSEs involves conformational conversions of normal cellular PrP (PrP(c)) to abnormal isoforms of PrP (PrP(Sc)). While the protein-only hypothesis has been widely accepted as a causal mechanism of prion diseases, evidence from more recent research suggests a possible involvement of other cellular component(s) or as yet undefined infectious agent(s) in PrP pathogenesis. Although the underlying mechanisms of PrP strain variation and the determinants of interspecies transmissibility have not been fully elucidated, biochemical and molecular findings indicate that bovine spongiform encephalopathy in cattle and new-variant Creutzfeldt-Jakob disease in humans are caused by indistinguishable etiological agent(s). Cumulative evidence suggests that there may be risks of humans acquiring TSEs via a variety of exposures to infected material. The development of highly precise ligands is warranted to detect and differentiate strains, allelic variants and infectious isoforms of these PrPs. This article describes the general features of TSEs and PrP, the current understanding of their pathogenesis, recent advances in prion disease diagnostics, and PrP inactivation.     

Prion agent diversity and species barrier

Mammalian prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSE), a group of fatal, neurodegenerative diseases, affecting both domestic animals and humans. The most widely accepted view to date is that these agents lack a nucleic acid genome and consist primarily of PrP(Sc), a misfolded, aggregated form of the host-encoded cellular prion protein (PrP(C)) that propagates by autocatalytic conversion and accumulates mainly in the brain. The BSE epizooty, allied with the emergence of its human counterpart, variant CJD, has focused much attention on two characteristics that prions share with conventional infectious agents. First, the existence of multiple prion strains that impose, after inoculation in the same host, specific and stable phenotypic traits such as incubation period, molecular pattern of PrP(Sc) and neuropathology. Prion strains are thought to be enciphered within distinct PrP(Sc) conformers. Second, a transmission barrier exists that restricts the propagation of prions between different species. Here we discuss the possible situations resulting from the confrontation between species barrier and prion strain diversity, the molecular mechanisms involved and the potential of interspecies transmission of animal prions, including recently discovered forms of TSE in ruminants.     

Susceptibility of transgenic mice expressing chimeric sheep,bovine and human PrP genes to sheep scrapie

The use of Transgenic (Tg) mice expressing chimeric sheep/mouse (Sh/Mo) prion protein (PrP) and chimeric bovine/mouse (Bo/Mo) PrP genes was evaluated as a sheep scrapie model. We also investigated the potential for the transmission of sheep scrapie to a human/mouse (Hu/Mo) PrP Tg mouse line. The Sh/Mo PrP and Bo/Mo PrP Tg Prnp(+/+) or Prnp(0/0) mouse lines were inoculated intracerebrally with brain homogenates from three sheep with natural scrapie (KU, Y5 or S2). Incubation periods were slightly shorter in Sh/Mo PrP Tg Prnp(+/+), than in non-Tg mice inoculated with KU brain homogenate. In contrast, the incubation period was significantly prolonged (p<0.05) in Bo/Mo PrP Tg Prnp(+/+) mice inoculated with KU brain homogenate. The incubation period was significantly longer in all Tg Prnp(+/+) and Prnp(0/0), than in non-Tg mice (p<0.01) inoculated withY5 brain homogenate. None of the Tg Prnp(0/0) mice inoculated with S2 brain homogenate developed clinical signs and PrP(Sc) was undetectable in their brains. These results suggested that expression of the Sh/Mo PrP or Bo/Mo PrP transgenes does not confer susceptibility to sheep prions upon mice, and thus none of the Tg mouse lines could be a suitable model of sheep scrapie. Hu/Mo PrP Tg Prnp(0/0) mice inoculated with natural and experimental scrapie or mouse prions did not develop clinical signs of scrapie and PrP(Sc) was undetectable. These results suggested that neither sheep nor mouse strains of scrapie are highly transmissible to humans.     

Healthy goats naturally devoid of prion protein

Prion diseases such as scrapie in small ruminants, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in man, are fatal neurodegenerative disorders. These diseases result from the accumulation of misfolded conformers of the host-encoded prion protein (PrP) in the central nervous system. To date naturally-occurring PrP free animals have not been reported. Here we describe healthy non-transgenic animals, Norwegian Dairy Goats, lacking prion protein due to a nonsense mutation early in the gene. These animals are predicted to be resistant to prion disease and will be valuable for research and for production of prion-free products.     

Comparison of brain PrPd distribution in ovine BSE and scrapie

Scrapie and bovine spongiform encephalopathy (BSE) are both prion diseases affecting ruminants, and these diseases do not share the same public health concerns. Surveillance of the BSE agent in small ruminants has been a great challenge, and the recent identification of diverse prion diseases in ruminants has led to the development of new methods for strain typing. In our study, using immunohistochemistry (IHC), we assessed the distribution of PrP(d) in the brains of 2 experimentally BSE-infected sheep with the ARQ/ARQ genotype. Distribution of PrP(d) in the brain, from the spinal cord to the frontal cortex, was remarkably similar in the 2 sheep despite different inoculation routes and incubation periods. Comparatively, overall PrP(d) brain distribution, evaluated by IHC, in 19 scrapie cases with the ARQ/ARQ, ARQ/VRQ, and VRQ/VRQ genotypes, in some cases showed similarities to the experimentally BSE-infected sheep. There was no exclusive neuroanatomical site with a characteristic and specific PrP(d) type of accumulation induced by the BSE agent. However, a detailed analysis of the topography, types, and intensity of PrP(d) deposits in the frontal cortex, striatum, piriform cortex, hippocampus, mesencephalon, and cerebellum allowed the BSE-affected sheep group to be distinguished from the 19 scrapie cases analyzed in our study. These results strengthen and emphasize the potential interest of PrP(d) brain mapping to help in identifying prion strains in small ruminants.     

Cell models of prion infection

Due to recent renewal of interest and concerns in prion diseases, a number of cell systems permissive to prion multiplication have been generated in the last years. These include established cell lines, neuronal stem cells and primary neuronal cultures. While most of these models are permissive to experimental, mouse-adapted strains of prions, the propagation of natural field isolates from sheep scrapie and chronic wasting disease has been recently achieved. These models have improved our knowledge on the molecular and cellular events controlling the conversion of the PrP(C) protein into abnormal isoforms and on the cell-to-cell spreading of prions. Infected cultured cells will also facilitate investigations on the molecular basis of strain identity and on the mechanisms that lead to neurodegeneration. The ongoing development of new cell models with improved characteristics will certainly be useful for a number of unanswered critical issues in the prion field.     

Immunisation strategies against prion diseases: prime-boost immunisation with a PrP DNA vaccine containing foreign helper T-cell epitopes does not prevent mouse scrapie

Vaccination against prion diseases constitutes a promising approach for the treatment and prevention of the disease. Passive immunisation with antibodies binding to the cellular prion protein (PrP(C)) can protect against prion disease. However, immunotherapeutic strategies with active immunisation are limited due to the immune tolerance against the self-antigen. In order to develop an anti-prion vaccine, we designed a novel DNA fusion vaccine composed of mouse PrP and immune stimulatory helper T-cell epitopes of the tetanus toxin that have previously been reported to break tolerance to other self-antigens. This approach provoked a strong PrP(C)-specific humoral and cellular immune response in PrP null mice, but only low antibody titres were found in vaccinated wild-type mice. Furthermore, prime-boost immunisation with the DNA vaccine and recombinant PrP protein increased antibody titres in PrP null mice, but failed to protect wild-type mice from mouse scrapie.     

Recent developments in prion disease research: diagnostic tools and in vitro cell culture models

After prion infection, an abnormal isoform of prion protein (PrP(Sc)) converts the cellular isoform of prion protein (PrP(C)) into PrP(Sc). PrP(C)-to-PrP(Sc) conversion leads to PrP(Sc) accumulation and PrP(C) deficiency, contributing etiologically to induction of prion diseases. Presently, most of the diagnostic methods for prion diseases are dependent on PrP(Sc) detection. Highly sensitive/accurate specific detection of PrP(Sc) in many different samples is a prerequisite for attempts to develop reliable detection methods. Towards this goal, several methods have recently been developed to facilitate sensitive and precise detection of PrP(Sc), namely, protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, capillary gel electrophoresis, fluorescence correlation spectroscopy, flow microbead immunoassay, etc. Additionally, functionally relevant prion-susceptible cell culture models that recognize the complexity of the mechanisms of prion infection have also been pursued, not only in relation to diagnosis, but also in relation to prion biology. Prion protein (PrP) gene-deficient neuronal cell lines that can clearly elucidate PrP(C) functions would contribute to understanding of the prion infection mechanism. In this review, we describe the trend in recent development of diagnostic methods and cell culture models for prion diseases and their potential applications in prion biology.     

A prion disease of cervids: chronic wasting disease

Chronic wasting disease (CWD) is a prion disease of deer, elk, and moose, initially recognized in Colorado mule deer. The discovery of CWD beyond the borders of Colorado and Wyoming, in Canada and as far east as New York, has led to its emergence as a prion disease of international importance. Epidemiological studies indicate that CWD is horizontally transmitted among free-ranging animals, potentially indirectly by prion-containing secreta or excreta contaminating the environment. Experimental CWD transmission attempts to other wild and domestic mammals and to transgenic mice expressing the prion protein of cattle, sheep, and humans have shed light on CWD species barriers. Transgenic mice expressing the cervid prion protein have proven useful for assessing the genetic influences of Prnp polymorphisms on CWD susceptibility. Accumulating evidence of CWD pathogenesis indicates that the misfolded prion protein or prion infectivity seems to be widely disseminated in many nonneural organs and in blood. This review highlights contemporary research findings in this prion disease of free-ranging wildlife.     

Expression of alpha-hemoglobin stabilizing protein and cellular prion protein in a subclone of murine erythroleukemia cell line MEL

Alpha-Hemoglobin stabilizing protein (AHSP) functions as the erythroid-specific molecular chaperon for alpha-globin. AHSP gene expression has been reported to be downregulated in hematopoietic tissues of animals suffering from prion diseases though the mechanism remains to be clarified. Herein, we demonstrate that MELhipod8 cells, a subclone of murine erythroleukemia (MEL) cells, have prion protein (PrPc) on the cell surface and have highly inducible expression of the AHSP and alpha- and beta-globin genes, resembling the expression pattern of the PrP and AHSP genes in bipotential erythroid- and megakaryocyte-lineage cells followed by erythroid differentiation in normal erythropoiesis. Moreover, MELhipod8 cells exhibit greater effective erythroid differentiation with a population of hemoglobinized normoblast-like cells than that observed for the parental MEL cells. These findings suggest that MELhipod8 cells could provide a mechanism for downregulation of the AHSP gene in prion diseases.     

Elevated manganese levels in blood and central nervous system occur before onset of clinical signs in scrapie and bovine spongiform encephalopathy

Prion diseases, or transmissible spongiform encephalopathies, are neurodegenerative diseases that can only be accurately diagnosed by analysis of central nervous system tissue for the presence of an abnormal isoform of the prion protein known as PrP(Sc). Furthermore, these diseases have long incubation periods during which there are no clear symptoms but where the infectious agent could still be present in the tissues. Therefore, the development of diagnostic assays to detect a surrogate marker for the presence of prion disease is essential. Previous studies on mice experimentally infected with scrapie, an ovine spongiform encephalopathy, suggested that changes in the levels of Mn occur in the blood and brain before the onset of symptoms of the disease. To assess whether these findings have relevance to the animal diseases scrapie and bovine spongiform encephalopathy, tissues from bovine spongiform encephalopathy- and scrapie-infected cattle and sheep were analyzed for their metal content and compared with values for noninfected animals. In field cases and experimentally infected animals, elevated Mn was associated with prion infection. Although some central nervous system regions showed elevated Mn, other regions did not. The most consistent finding was an elevation of Mn in blood. This change was present in experimentally infected animals before the onset of symptoms. In scrapie-infected sheep, elevated Mn levels occurred regardless of the genotype of the sheep and were even detected in scrapie-resistant sheep in which no symptoms of disease were detected. These findings suggest that elevated blood Mn could be a potential diagnostic marker for prion infection even in the absence of apparent clinical disease.     

Prion protein in sheep urine.

The misfolded form of cellular prion protein (PrP(C)) is the main component of the infectious agent of transmissible spongiform encephalopathies and the validated biomarker for these diseases. The expression of PrP(C) is highest in the central nervous system and has been found in peripheral tissues. Soluble PrP(C) has been detected in cerebrospinal fluid, urine, serum, milk, and seminal plasma. In this study, attempts were made to characterize prion protein in urine samples from normal and scrapie-infected sheep. Urine samples from scrapie-infected sheep and age-matched healthy sheep were collected and analyzed by Western blot following concentration. A protease K-sensitive protein band with a molecular weight of approximately 27-30 kDa was visualized after immunoblotting with anti-PrP monoclonal antibodies to a C-terminal part of PrP(C), but not after immunoblotting with monoclonal antibodies to an N-terminal epitope of PrP(C) or with secondary antibodies only. The amount of PrP(C) in the urine of 49 animals (control group: n = 16; naturally scrapie-infected group: n = 33) was estimated by comparison with known amounts of ovine recombinant PrP in the immunoblot. Background concentration of PrP(C) in urine was found to be 0-0.16 ng/ml (adjusted to the initial nonconcentrated volume of the urine samples). Seven out of 33 naturally scrapie-infected animals had an elevated level (0.3-4.7 ng/ml) of PrP(C) in urine. The origin of PrP(C) in urine and the reason for the increased level of PrP(C) in scrapie-infected sheep urine has yet to be explored.     

First and second cattle passage of transmissible mink encephalopathy by intracerebral inoculation

To compare clinicopathologic findings of transmissible mink encephalopathy (TME) with other transmissible spongiform encephalopathies (TSE, prion diseases) that have been shown to be experimentally transmissible to cattle (sheep scrapie and chronic wasting disease [CWD]), two groups of calves (n = 4 each) were intracerebrally inoculated with TME agents from two different sources (mink with TME and a steer with TME). Two uninoculated calves served as controls. Within 15.3 months postinoculation, all animals from both inoculated groups developed clinical signs of central nervous system (CNS) abnormality; their CNS tissues had microscopic spongiform encephalopathy (SE); and abnormal prion protein (PrP(res)) as detected in their CNS tissues by immunohistochemistry (IHC) and Western blot (WB) techniques. These findings demonstrate that intracerebrally inoculated cattle not only amplify TME PrP(res) but also develop clinical CNS signs and extensive lesions of SE. The latter has not been shown with other TSE agents (scrapie and CWD) similarly inoculated into cattle. The findings also suggest that the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) would detect TME in cattle should it occur naturally. However, it would be a diagnostic challenge to differentiate TME in cattle from BSE by clinical signs, neuropathology, or the presence of PrP(res) by IHC and WB.     

Western blot detection of PrP Sc in archived paraffin-embedded brainstem from scrapie-affected sheep

Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies (TSE) or prion diseases. Immunoassays that identify disease-associated prion protein (PrP Sc) are integral to the diagnosis of scrapie and other prion diseases. Results obtained by either immunohistochemistry (IHC) or Western blot (WB) assay are generally adequate for the definitive diagnosis. Approved or accepted methods for WB diagnosis of TSEs requires the use of fresh or frozen nonfixed tissue samples, whereas formalin-fixed, paraffin-embedded tissue is required for the localization of PrP Sc by IHC. Because disparate processing methods are used for these accepted diagnostic techniques, separate tissue samples are collected from the same animal. Occasions arise in which there is either insufficient quantity of tissue available to complete analysis by both techniques or initial tissue processing is incompatible with one of the assays. Also, results between the assays may differ because of the vagaries of sampling, especially in case material that contains moderate-to-low levels of PrP Sc. The present article describes a method to conduct a WB assay from the same paraffin-embedded brainstem sample used for the IHC diagnosis of experimentally induced sheep scrapie.     

The first Canadian indigenous case of bovine spongiform encephalopathy (BSE) has molecular characteristics for prion protein that are similar to those of BSE in the United Kingdom but differ from those of chronic wasting disease in captive elk and deer

Brain tissue from a case of bovine spongiform encephalopathy (BSE) from Alberta was subjected to a Western immunoblotting technique to ascertain the molecular profile of any disease-specific, abnormal prion protein, that is, prion protein that is protease-resistant (PrP(res)). This technique can discriminate between isolates from BSE, ovine scrapie, and sheep experimentally infected with BSE. Isolates of brain tissue from the BSE case in Alberta, 3 farmed elk with chronic wasting disease (CWD) from different parts of Saskatchewan, and 1 farmed white-tailed deer with CWD from Edmonton, Alberta, were examined alongside isolates of brain tissue from BSE, ovine scrapie, and sheep experimentally infected with BSE from the United Kingdom (UK). The molecular weights of PrP(res) and the cross reactions to 2 specific monoclonal antibodies (mAbs) were determined for each sample. The BSE isolates from Canada and the UK had very similar PrP(res) molecular weights and reacted with only 1 of the 2 mAbs. The PrP(res) isolated from both elk and white-tailed deer with CWD had a higher molecular weight profile than did the corresponding PrP(res) from the scrapie and BSE isolates. The PrP(res) from CWD cases cross reacted with both mAbs, a property shared with PrP(res) in isolates from scrapie but not with PrP(res) isolates from BSE or sheep experimentally infected with BSE. The results from this study seem to confirm that the PrP(res) isolated from the BSE case in Alberta has similar molecular properties to the PrP(res) isolated from a BSE case in the UK, and that it differs in its molecular and immunological characteristics from the CWD and scrapie cases studied.     

Detergents modify proteinase K resistance of PrP Sc in different transmissible spongiform encephalopathies (TSEs)

Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrP(Sc)). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrP(Sc) against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorption assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrP(Sc) after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrP(Sc) can be very different. The results obtained here may be helpful during the development or improvement of a PrP(Sc) detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrP(Sc) that may stabilize the aggregates.     

Emerging therapeutic agents for transmissible spongiform encephalopathies: a review

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative disorders associated with misfolding of prion protein, from PrPC to PrPSc. Different types of experimental studies have resulted in a better understanding of the pathogenesis of the prion diseases. Genetic and molecular properties of PrP isoforms have been explained but the conformational conversion of the PrPC isoform to the PrPSc isoform has not yet been entirely elucidated. However, a number of possible therapeutic agents have been tried and some have proven to be effective against TSEs but most have limitations in terms of toxicity and pharmacokinetics. Congo red (CR), anthracyclines, and polyanionic dextran sulfate have limited ability to cross the blood-brain barrier and may be toxic. The efficacy of polyene antibiotics seems to be restricted to certain scrapie strains. Tetrapyrroles and tetracyclines with low toxicities and favorable pharmacokinetics could be useful in preventing PrPSc accumulation. Compounds like branched polyamines, Cp-60, analogs of CR, quinacrine and chlorpromazine, beta-sheet breaker peptides and inhibitory peptides, active immunization using recombinant PrP and passive immunization with anti-PrP antibodies, have potential use as therapeutic agents but would need further research and clinical trials.     

Enrichment of prion protein in exosomes derived from ovine cerebral spinal fluid

Prion diseases are transmissible neurodegenerative disorders affecting humans and a wide variety of animal species including sheep and cattle. The transmissible agent, the prion, is an abnormally folded form (PrP(Sc)) of the host encoded cellular prion protein (PrP(C)). Distribution of the prion protein in the fluids of species susceptible to these diseases is of importance to human health and the iatrogenic spread of prion disease. Aside from blood which is confirmed to be a source of prion infectivity, it is currently unclear which other body fluids harbor a significant transmission risk. In the current study we examined two ovine fluids; pseudo-afferent lymph and cerebral spinal fluid (CSF), for the presence of exosomes and concurrent enrichment of the normal, cellular form of the prion protein (PrP(C)). Here we demonstrate the existence of exosomes in both pseudo-afferent lymph and CSF isolated from sheep. In the CSF derived exosomes we were able to show an enrichment of PrP(C) over unfractionated CSF. This experimental approach suggests that CSF derived exosomes could be used as a novel means of detecting abnormal forms of the prion protein and provide an in vivo link between these vesicles and prion disease pathogenesis.     

PrP genetics in ruminant transmissible spongiform encephalopathies

Scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) are prion diseases in ruminants with considerable impact on animal health and welfare. They can also pose a risk to human health and control is therefore an important issue. Prion protein (PrP) genetics may be used to control and eventually eradicate animal prion diseases. The PrP gene in sheep and other representatives of the order Artiodactyles has many polymorphisms of which several are crucial determinants of susceptibility to prion diseases, also known as transmissible spongiform encephalopathies (TSE). This review will present the current understanding of PrP genetics in ruminants highlighting similarity and difference between the species in the context of TSE.