Ovine serum biomarkers of early and late phase scrapie

Background  

Transmissible spongiform encephalopathies are fatal neurodegenerative disease occurring in animals and humans for which no ante-mortem diagnostic test in biological fluids is available. In such pathologies, detection of the pathological form of the prion protein (i.e., the causative factor) in blood is difficult and therefore identification of new biomarkers implicated in the pathway of prion infection is relevant.     

Human and animal spongiform encephalopathies are the result of chronic autoimmune attack in the CNS: a novel medical theory supported by overwhelming experimental evidence

Spongiform encephalopathies, also called "prion diseases", are fatal degenerative diseases of the central nervous system which can occur in animals (such as the "mad cow disease" in cattle) and also in humans. This paper presents a novel medical theory concerning the pathogenic mechanisms for various human and animal spongiform encephalopathies. It is hypothesized that various forms of prion diseases are essentially autoimmune diseases, resulting from chronic autoimmune attack of the central nervous system. A key step in the pathogenic process leading towards the development of spongiform encephalopathies involves the production of specific autoimmune antibodies against the disease-causing prion protein (PrPsc) and possibly other immunogenic macromolecules present in the brain. As precisely explained in this paper, the autoimmune antibodies produced against PrPsc are responsible for the conversion of the normal cellular prion protein (PrPc) to PrPsc, for the accumulation of PrPsc in the brain and other peripheral tissues, and also for the initiation of an antibody-mediated chronic autoimmune attack of the central nervous system neurons, which would contribute to the development of characteristic pathological changes and clinical symptoms associated with spongiform encephalopathies. The validity and correctness of the proposed theory is supported by an overwhelming body of experimental observations that are scattered in the biomedical literature. In addition, the theory also offers practical new strategies for early diagnosis, treatment, and prevention of various human and animal prion diseases.     

Detection of pathological molecular alterations in scrapie-infected hamster brain by Fourier transform infrared (FT-IR) spectroscopy

In this report a new approach for the identification of pathological changes in scrapie-infected Syrian hamster brains using Fourier transform infrared microspectroscopy is discussed. Using computer-based pattern recognition techniques and imaging, infrared maps with high structural contrast were obtained. This strategy permitted comparison of spectroscopic data from identical anatomical structures in scrapie-infected and control brains. Consistent alterations in membrane state-of-order, protein composition, carbohydrate and nucleic acid constituents were detected in scrapie-infected tissues. Cluster analysis performed on spectra of homogenized medulla oblongata and pons samples also reliably separated uninfected from infected specimens. This method provides a useful tool not only for the exploration of the disease process but also for the development of rapid diagnostic and screening techniques of transmissible spongiform encephalopathies.     

Peptidylarginine deiminase and protein citrullination in prion diseases

The post-translational citrullination (deimination) process is mediated by peptidylarginine deiminases (PADs), which convert peptidylarginine into peptidylcitrulline in the presence of high calcium concentrations. Over the past decade, PADs and protein citrullination have been commonly implicated as abnormal pathological features in neurodegeneration and inflammatory responses associated with diseases such as multiple sclerosis, Alzheimer disease and rheumatoid arthritis. Based on this evidence, we investigated the roles of PADs and citrullination in the pathogenesis of prion diseases. Prion diseases (also known as transmissible spongiform encephalopathies) are fatal neurodegenerative diseases that are pathologically well characterized as the accumulation of disease-associated misfolded prion proteins, spongiform changes, glial cell activation and neuronal loss. We previously demonstrated that the upregulation of PAD2, mainly found in reactive astrocytes of infected brains, leads to excessive citrullination, which is correlated with disease progression. Further, we demonstrated that various cytoskeletal and energy metabolism-associated proteins are particularly vulnerable to citrullination. Our recent in vivo and in vitro studies elicited altered functions of enolase as the result of citrullination; these altered functions included reduced enzyme activity, increased protease sensitivity and enhanced plasminogen-binding affinity. These findings suggest that PAD2 and citrullinated proteins may play a key role in the brain pathology of prion diseases. By extension, we believe that abnormal increases in protein citrullination may be strong evidence of neurodegeneration.     

The ubiquitin-proteasome system in spongiform degenerative disorders

Spongiform degeneration is characterized by vacuolation in nervous tissue accompanied by neuronal death and gliosis. Although spongiform degeneration is a hallmark of prion diseases, this pathology is also present in the brains of patients suffering from Alzheimer's disease, diffuse Lewy body disease, human immunodeficiency virus (HIV) infection, and Canavan's spongiform leukodystrophy. The shared outcome of spongiform degeneration in these diverse diseases suggests that common cellular mechanisms must underlie the processes of spongiform change and neurodegeneration in the central nervous system. Immunohistochemical analysis of brain tissues reveals increased ubiquitin immunoreactivity in and around areas of spongiform change, suggesting the involvement of ubiquitin-proteasome system dysfunction in the pathogenesis of spongiform neurodegeneration. The link between aberrant ubiquitination and spongiform neurodegeneration has been strengthened by the discovery that a null mutation in the E3 ubiquitin-protein ligase mahogunin ring finger-1 (Mgrn1) causes an autosomal recessively inherited form of spongiform neurodegeneration in animals. Recent studies have begun to suggest that abnormal ubiquitination may alter intracellular signaling and cell functions via proteasome-dependent and proteasome-independent mechanisms, leading to spongiform degeneration and neuronal cell death. Further elucidation of the pathogenic pathways involved in spongiform neurodegeneration should facilitate the development of novel rational therapies for treating prion diseases, HIV infection, and other spongiform degenerative disorders.     

Copper and prion diseases

Transmissible spongiform encephalopathies are diseases of animals and humans that are also termed prion diseases. These diseases are linked together because a normal brain glycoprotein termed the prion protein is converted to a readily detectable protease-resistant isoform. There is now strong evidence to suggest that apart from this difference in resistance a major difference between the isoforms is that the normal prion protein binds copper and has an anti-oxidant function. Brains from Creutzfeldt-Jakob disease patients and brains from mice with experimental mouse scrapie have been shown to have changes in the levels of both copper and manganese. There is growing evidence that links prion diseases to disturbances of metal metabolism.     

Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion

A characteristic feature of prion diseases such as bovine spongiform encephalopathy (BSE) is the accumulation of a pathological isoform of the host-encoded prion protein, PrP. In contrast to its cellular isoform PrP(C), the pathological isoform PrP(Sc) forms insoluble aggregates. All commercial BSE tests currently used for routine testing are based on the proteinase K (PK) resistance of PrP, but not all pathological PrP is PK-resistant. In the present study, single prion particles were counted by fluorescence correlation spectroscopy (FCS). The property of PK resistance is not required, i.e., both the PK-resistant and the PK-sensitive parts of the prion particles are detectable. PrP aggregates were prepared from the brains of BSE-infected cattle, as well as from scrapie-infected hamsters, by the NaPTA precipitation method without PK digestion. They were labeled using two different PrP-specific antibodies for FCS measurements in the dual-color mode (2D-FIDA). Within the limited number of samples tested, BSE-infected cattle and scrapie-infected hamsters in the clinical stage of the disease could be distinguished with 100% specificity from a control group. Thus, a diagnostic tool for BSE detection with complete avoidance of PK treatment is presented, which should have particular advantages for testing animals in the preclinical stage.     

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

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

Inoculation of Scrapie with the Self-Assembling RADA-Peptide Disrupts Prion Accumulation and Extends Hamster Survival

Intracerebral inoculation of 263K Scrapie brain homogenate (PrPsc) with a self-assembling RADA-peptide (RADA) significantly delayed disease onset and increased hamster survival. Time of survival was dependent on the dose of RADA and pre-incubation with PrPsc prior to inoculation. RADA treatment resulted in the absence of detectable PrPsc at 40 d followed by an increased rate of PrPsc accumulation at 75 d up to sacrifice. In all PrPsc inoculated animals, clinical symptoms were observed ∼10 d prior to sacrifice and brains showed spongiform degeneration with Congo red positive plaques. A time-dependent increase in reactive gliosis was observed in both groups with more GFAP detected in RADA treated animals at all time points. The PrP protein showed dose-dependent binding to RADA and this binding was competitively inhibited by Congo Red. We conclude that RADA disrupts the efficacy of prion transmission by altering the rate of PrPsc accumulation. This is the first demonstration that a self-assembling biomolecular peptide can interact with PrPsc, disrupt the course of Scrapie disease process, and extend survival.     

Metallic prions

Prion diseases, also referred to as transmissible spongiform encephalopathies, are characterized by the deposition of an abnormal isoform of the prion protein in the brain. However, this aggregated, fibrillar, amyloid protein, termed PrPSc, is an altered conformer of a normal brain glycoprotein, PrPc. Understanding the nature of the normal cellular isoform of the prion protein is considered essential to understanding the conversion process that generates PrPSc. To this end much work has focused on elucidation of the normal function and activity of PrPc. Substantial evidence supports the notion that PrPc is a copper-binding protein. In conversion to the abnormal isoform, this Cu-binding activity is lost. Instead, there are some suggestions that the protein might bind other metals such as Mn or Zn. PrPc functions currently under investigation include the possibility that the protein is involved in signal transduction, cell adhesion, Cu transport and resistance to oxidative stress. Of these possibilities, only a role in Cu transport and its action as an antioxidant take into consideration PrPc's Cu-binding capacity. There are also more published data supporting these two functions. There is strong evidence that during the course of prion disease, there is a loss of function of the prion protein. This manifests as a change in metal balance in the brain and other organs and substantial oxidative damage throughout the brain. Thus prions and metals have become tightly linked in the quest to understand the nature of transmissible spongiform encephalopathies.     

Evaluation of the human transmission risk of an atypical bovine spongiform encephalopathy prion strain

Bovine spongiform encephalopathy (BSE), the prion disease in cattle, was widely believed to be caused by only one strain, BSE-C. BSE-C causes the fatal prion disease named new variant Creutzfeldt-Jacob disease in humans. Two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H, have been discovered in several countries since 2004; their transmissibility and phenotypes in humans are unknown. We investigated the infectivity and human phenotype of BASE strains by inoculating transgenic (Tg) mice expressing the human prion protein with brain homogenates from two BASE strain-infected cattle. Sixty percent of the inoculated Tg mice became infected after 20 to 22 months of incubation, a transmission rate higher than those reported for BSE-C. A quarter of BASE strain-infected Tg mice, but none of the Tg mice infected with prions causing a sporadic human prion disease, showed the presence of pathogenic prion protein isoforms in the spleen, indicating that the BASE prion is intrinsically lymphotropic. The pathological prion protein isoforms in BASE strain-infected humanized Tg mouse brains are different from those from the original cattle BASE or sporadic human prion disease. Minimal brain spongiosis and long incubation times are observed for the BASE strain-infected Tg mice. These results suggest that in humans, the BASE strain is a more virulent BSE strain and likely lymphotropic.     

Abnormal tubulovesicular particles in brains of hamsters with scrapie

Abnormal tubulovesicular particles of an average diameter of 23 nm have been observed in brains of mice with scrapie as well as in other animals with spongiform encephalopathies, but they were thought to be absent from the brains of hamsters with scrapie in which the highest known concentrations of the infectious agent occur. We observed in neuronal processes of hamsters as well as mice clusters of those tubulovesicular structures, most often in postsynaptic terminals. Such particles have now been seen regularly in both experimental and natural scrapie in all species examined as well as in other spongiform encephalopathies.     

Biochemical and strain properties of CJD prions: complexity versus simplicity

Prions, the agents responsible for transmissible spongiform encephalopathies, are infectious proteins consisting primarily of scrapie prion protein (PrP(Sc)), a misfolded, β-sheet enriched and aggregated form of the host-encoded cellular prion protein (PrP(C)). Their propagation is based on an autocatalytic PrP conversion process. Despite the lack of a nucleic acid genome, different prion strains have been isolated from animal diseases. Increasing evidence supports the view that strain-specific properties may be enciphered within conformational variations of PrP(Sc). In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most frequent form of prion diseases and has demonstrated a wide phenotypic and molecular spectrum. In contrast, variant Creutzfeldt-Jakob disease (vCJD), which results from oral exposure to the agent of bovine spongiform encephalopathy, is a highly stereotyped disease, that, until now, has only occurred in patients who are methionine homozygous at codon 129 of the PrP gene. Recent research has provided consistent evidence of strain diversity in sCJD and also, unexpectedly enough, in vCJD. Here, we discuss the puzzling biochemical/pathological diversity of human prion disorders and the relationship of that diversity to the biological properties of the agent as demonstrated by strain typing in experimental models.     

The role of glycophosphatidylinositol anchor in the amplification of the scrapie isoform of prion protein in vitro

Transmissible spongiform encephalopathies are associated with an autocatalytic conversion of normal prion protein, PrP^C, to a protease-resistant form, PrPres. This autocatalytic reaction can be reproduced in vitro using a procedure called protein misfolding cyclic amplification (PMCA). Here we show that, unlike brain-derived PrP^C, bacterially-expressed recombinant prion protein (rPrP) is a poor substrate for PrPres amplification in a standard PMCA reaction. The differences between PrP^C and rPrP appear to be due to the lack of the glycophosphatidylinositol anchor in the recombinant protein. These findings shed a new light on prion protein conversion process and have important implications for the efforts to generate synthetic prions for structural and biophysical studies.     

Quantum dots and prion proteins

A diagnostics of infectious diseases can be done by the immunologic methods or by the amplification of nucleic acid specific to contagious agent using polymerase chain reaction. However, in transmissible spongiform encephalopathies, the infectious agent, prion protein (PrP^Sc), has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrP^Sc detection is that the pathological form of prion protein is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of prion protein caused diseases represent a sort of challenges as that hosts can incubate infectious prion proteins for many months or even years. Therefore, new in vivo assays for detection of prion proteins and for diagnosis of their relation to neurodegenerative diseases are summarized. Their applicability and future prospects in this field are discussed with particular aim at using quantum dots as fluorescent labels.     

Prion immunoreactivity in brain,tonsil, gastrointestinal epithelial cells,and blood and lymph vessels in lemurian zoo primates with spongiform encephalopathy

We report on two animals of a non-human primate species Eulemur fulvus mayottensis, housed in the local zoo and fed over a number of years with a food containing cattle meat, that developed serious neurological symptoms associated with prion immunoreactivity in brain and various viscera. Microscopy of the brains showed neuronal vacuolation with patchylperivacuolar immunolabelling with an abnormal isoform of prion protein (IR-PrP), an important characteristic of spongiform encephalopathy. For the first time, we report the presence in the same severely ill animals of IR-PrP in the gastrointestinal tract, detected by immunocytochemistry with mono- andpolyclonal antibodies directed against various parts of the PrP. Strong PrP labelling was observed in the epithelial cells lining the pharyngeal and gastrointestinal lumen. The tonsils and the walls of the lymph and blood vessels below the intestinal epithelium were also labelled. There were no such immunoreactions in healthy lemurians killed as controls, i.e. a younger congener of the same species housed under the same conditions, and others belonging to the smaller species Microcebus murinus, reared in the laboratory and never fed on commercial food products containing cattle meat. These results demonstrate a strong PrP accumulation in the brain, the gastrointestinal tract and underlying lymphoreticular structures in these primates living in a zoological park and suffering from a spongiform encephalopathy.     

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

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

Molecular behaviors of "CH1641-like" sheep scrapie isolates in ovine transgenic mice (TgOvPrP4)

Molecular analyses of the protease-resistant prion protein (PrP(res)) from a few natural scrapie isolates showed by Western blotting some partial similarities with those observed in experimental ovine bovine spongiform encephalopathy (BSE). They showed a low apparent molecular mass of unglycosylated PrP(res), although diglycosylated PrP(res) was less abundant than in ovine BSE. The prototype of such cases is the CH1641 experimental scrapie isolate. We analyzed PrP(res) molecular features from three French natural "CH1641-like" isolates, in comparison with CH1641 and BSE, after transmission of the disease in ovine transgenic mice (TgOvPrP4). One of these isolates (TR316211) behaved like the CH1641 isolate, with PrP(res) features in mice similar to those in the sheep brain. From two other isolates (O100 and O104), two distinct PrP(res) phenotypes were identified in mouse brains, with either high (h-type) or low (l-type) apparent molecular masses of unglycosylated PrP(res), the latter being similar to that observed with CH1641, TR316211, or BSE. Both phenotypes could be found in variable proportions in the brains of the individual mice. In contrast with BSE, l-type PrP(res) from "CH1641-like" isolates showed lower levels of diglycosylated PrP(res). From one of these cases (O104), a second passage in mice was performed for two mice with distinct PrP(res) profiles. This showed a partial selection of the l-type phenotype in mice infected with a mouse brain with predominant l-type PrP(res), and it was accompanied by a significant increase in the proportions of the diglycosylated band. These results are discussed in relation to the diversity of scrapie and BSE strains.     

A study of neurological diseases in farmed deer in Switzerland,with emphasis on chronic wasting disease

A study of neurological diseases in farmed deer, with emphasis on chronic wasting disease, was conducted during 2 years in Switzerland. Deer breeders were asked to submit the heads of all deer at least 2 years of age, found dead or slaughtered, for examination. A complete histological examination of the brain and immunohistochemical detection of the prion protein on selected regions of the brain and lymphoid tissues were performed on 120 apparently healthy and 40 diseased animals. In a number of cases, a full necropsy was performed. Significant inflammatory and/or degenerative changes were seen in 25% of the brains. No evidence for a transmissible spongiform encephalopathy was established.     

Bradykinin release and inactivation in brain of rats submitted to an experimental model of Alzheimer's disease

The kallikrein-kinin system is involved in a variety of physiological and pathological processes. Components of this system, identified in rat and human brains, can be altered in neurodegenerative processes such as Alzheimer's disease. Here, we studied kinin release and its inactivation in rats submitted to chronic cerebroventricular infusion of β-amyloid (Aβ) peptide. Neurodegeneration was confirmed by histological analysis of brain samples. In cerebrospinal fluid of animals infused with Aβ, bradykinin concentration was increased, as determined by radioimmunoassay. However, in the brain of Aβ group, we only detected the tripeptide Arg-Pro-Pro, purified by reversed-phase chromatography and characterized by liquid chromatography-electrospray ionization mass spectrometry. This fragment of bradykinin indicated the possible participation of kinin-processing enzymes in the brain such as a prolyl oligopeptidase.