Prion diseases: pathogenesis and public health concerns

Transmissible spongiform encephalopathy (TSE) agents or prions induce neurodegenerative fatal diseases in humans and in some mammalian species. Human TSEs include Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker syndrome, kuru and fatal familial insomnia. In animals, scrapie in sheep and goats, feline spongiform encephalopathy, transmissible mink encephalopathy, chronic wasting disease in wild ruminants, and bovine spongiform encephalopathy (BSE), which appeared in the UK in the mid-1980s [Wells, G.A.H. et al. (1987) Vet. Rec. 121, 419-420], belong to the TSE group. Prions have biological and physicochemical characteristics that differ significantly from those of other microorganisms; for example, they are resistant to inactivation processes that are effective against conventional viruses, including those that alter nucleic acid structure or function. Alternatively, infectivity is highly susceptible to procedures that modify protein conformation. Today, the exact nature of prions remains unknown even though it is likely that they consist of protein only. At the biochemical level, TSEs are characterised by the accumulation, within the central nervous system of the infected individual, of an abnormal isoform of a particular protein from the host, the prion protein [Prusiner, S.B. (1982) Science 216, 136-144]. TSEs are transmissible among their species of origin, but they can also cross the species barrier and induce chronic infection and/or disease in other species. Transmissibility has been proven in natural situations such as the outbreak of CJD among patients treated with pituitary-derived hormones and the appearance of BSE that affected UK cattle in the mid-1980s.     

PrPTSE distribution in a primate model of variant, sporadic, and iatrogenic Creutzfeldt-Jakob disease

Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), are neurodegenerative and fatal. Sporadic CJD (sCJD) can be transmitted between humans through medical procedures involving highly infected organs, such as the central nervous system. However, in variant CJD (vCJD), which is due to human contamination with the bovine spongiform encephalopathy (BSE) agent, lymphoreticular tissue also harbors the transmissible spongiform encephalopathy-associated prion protein (PrP(TSE)), which poses a particularly acute risk for iatrogenic transmission. Two blood transfusion-related cases are already documented. In addition, the recent observation of PrP(TSE) in spleen and muscle in sCJD raised the possibility that peripheral PrP(TSE) is not limited to vCJD cases. We aimed to clarify the peripheral pathogenesis of human TSEs by using a nonhuman primate model which mimics human diseases. A highly sensitive enzyme-linked immunosorbent assay was adapted to the detection of extraneural PrP(TSE). We show that affected organs can be divided into two groups. The first is peripheral organs accumulating large amounts of PrP(TSE), which represent a high risk of iatrogenic transmission. This category comprises only lymphoreticular organs in the vCJD/BSE model. The second is organs with small amounts of PrP(TSE) associated with nervous structures. These are the muscles, adrenal glands, and enteric nervous system in the sporadic, iatrogenic, and variant CJD models. In contrast to the first set of organs, this low level of tissue contamination is not strain restricted and seems to be linked to secondary centrifugal spread of the agent through nerves. It might represent a risk for iatrogenic transmission, formerly underestimated despite previous reports of low rates of transmission from peripheral organs of humans to nonhuman primates (5, 10). This study provides an additional experimental basis for the classification of human organs into different risk categories and a rational re-evaluation of current risk management measures.     

Prions: where do we stand 20 years after the appearance of bovine spongiform encephalopathy?

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) identified twenty years ago in the British cattle herds. Creutzfeldt-Jakob disease (CJD) is a TSE that occurs in humans. In 1996, scientists found a possible link between BSE and a new variant of CJD (vCJD). The fact that the non conventional infectious agent of TSE, named prions, could cross the species barrier from cattle to human through meat consumption, raised a tremendous concern for public safety in Europe. This led to the development in the following two decades of substantial and expensive measures to contain BSE and prevent its transmission to humans. In parallel, scientific programs have been funded to progress through the comprehension of the physiopathology of these fatal disorders. In Europe, the BSE epidemics is now ending and the number of cases is decreasing thanks to the strict control of animal foodstuff that was the main source of prion contamination. Only a small number of vCJD have been detected, however, additional concerns have been raised recently for public safety as secondary transmission of CJD through medical procedure and blood transfusion is possible. In addition, the possibility that the BSE was transmitted to other animals including small ruminants is also worrisome. Research efforts are now focussing on decontamination and ante mortem diagnosis of TSE to prevent animal and human transmission. However, needs for fundamental research are still important as many questions remain to be addressed to understand the mechanism of prion transmission, as well as its pathogenesis.     

Novel Aspects of Prions,Their Receptor Molecules,and Innovative Approaches for TSE Therapy

1. Prion diseases are a group of rare, fatal neurodegenerative diseases, also known as transmissible spongiform encephalopathies (TSEs), that affect both animals and humans and include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, chronic wasting disease (CWD) in deer and elk, and Creutzfeldt–Jakob disease (CJD) in humans. TSEs are usually rapidly progressive and clinical symptoms comprise dementia and loss of movement coordination due to the accumulation of an abnormal isoform (PrP^Sc) of the host-encoded prion protein (PrP^c). 2. This article reviews the current knowledge on PrP^c and PrP^Sc, prion replication mechanisms, interaction partners of prions, and their cell surface receptors. Several strategies, summarized in this article, have been investigated for an effective antiprion treatment including development of a vaccination therapy and screening for potent chemical compounds. Currently, no effective treatment for prion diseases is available. 3. The identification of the 37 kDa/67 kDa laminin receptor (LRP/LR) and heparan sulfate as cell surface receptors for prions, however, opens new avenues for the development of alternative TSE therapies.     

BSE case associated with prion protein gene mutation

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle and was first detected in 1986 in the United Kingdom. It is the most likely cause of variant Creutzfeldt-Jakob disease (CJD) in humans. The origin of BSE remains an enigma. Here we report an H-type BSE case associated with the novel mutation E211K within the prion protein gene (Prnp). Sequence analysis revealed that the animal with H-type BSE was heterozygous at Prnp nucleotides 631 through 633. An identical pathogenic mutation at the homologous codon position (E200K) in the human Prnp has been described as the most common cause of genetic CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. A recent epidemiological study revealed that the K211 allele was not detected in 6062 cattle from commercial beef processing plants and 42 cattle breeds, indicating an extremely low prevalence of the E211K variant (less than 1 in 2000) in cattle.     

Molecular aspects of disease pathogenesis in the transmissible spongiform encephalopathies

The transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of rare, fatal, and transmissible neurodegenerative diseases of mammals for which there are no known viral or bacterial etiological agents. The bovine form of these diseases, bovine spongiform encephalopathy (BSE), has crossed over into humans to cause variant Creutzfeldt-Jakob disease. As a result, BSE and the TSE diseases are now considered a significant threat to human health. Understanding the basic mechanisms of TSE pathogenesis is essential for the development of effective TSE diagnostic tests and anti-TSE therapeutic regimens. This review provides an overview of the molecular mechanisms that underlie this enigmatic group of diseases.     

Recombinant human prion protein fragment 90-231, a useful model to study prion neurotoxicity

Transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of fatal neurodegenerative disorders of animals and humans. Human diseases include Creutzfeldt-Jakob (CJD) and Gerstmann-Straussler-Scheinker (GSSD) diseases, fatal familial insomnia, and Kuru. Human and animal TSEs share a common histopathology with a pathognomonic triad: spongiform vacuolation of the grey matter, neuronal death, glial proliferation, and, more inconstantly, amyloid deposition. According to the "protein only" hypothesis, TSEs are caused by a unique post-translational conversion of normal, host-encoded, protease-sensitive prion protein (PrP(sen) or PrP(C)) to an abnormal disease-associated isoform (PrP(res) or PrP(Sc)). To investigate the molecular mechanism of neurotoxicity induced by PrP(Sc) we developed a protocol to obtain millimolar amounts of soluble recombinant polypeptide encompassing the amino acid sequence 90-231 of human PrP (hPrP90-231). This protein corresponds to the protease-resistant prion protein fragment that originates after amino-terminal truncation. Importantly, hPrP90-231 has a flexible backbone that, similar to PrP(C), can undergo to structural rearrangement. This peptide, structurally resembling PrP(C), can be converted in a PrP(Sc)-like conformation, and thus represents a valuable model to study prion neurotoxicity. In this article we summarized our experimental evidence on the molecular and structural mechanisms responsible of hPrP90-231 neurotoxicity on neuroectodermal cell line SHSY5Y and the effects of some PrP pathogen mutations identified in familial TSE.     

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.     

Prions and prion diseases: fundamentals and mechanistic details

Prion diseases, often called transmissible spongiform encephalopathies (TSEs), are infectious diseases that accompany neurological dysfunctions in many mammalian hosts. Prion diseases include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE, "mad cow disease") in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elks. The cause of these fatal diseases is a proteinaceous pathogen termed prion that lacks functional nucleic acids. As demonstrated in the BSE outbreak and its transmission to humans, the onset of disease is not limited to a certain species but can be transmissible from one host species to another. Such a striking nature ofprions has generated huge concerns in public health and attracted serious attention in the scientific communities. To date, the potential transmission ofprions to humans via foodbome infectiorn and iatrogenic routes has not been alleviated. Rather, the possible transmission of human to human or cervids to human aggravates the terrifying situation across the globe. In this review, basic features about prion diseases including clinical and pathological characteristics, etiology, and transmission of diseases are described. Based on recently accumulated evidences, the molecular and biochemical aspects of prions, with an emphasis on the molecular interactions involved in prion conversion that is critical during prion replication and pathogenesis, are also addressed.     

Production of cattle lacking prion protein

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP(C), such as PrP(BSE) in bovine spongiform encephalopathy (BSE) in cattle and PrP(CJD) in Creutzfeldt-Jakob disease (CJD) in humans. Disruption of PrP(C) expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities. However, the impact of ablating PrP(C) function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP(C)-deficient cattle produced by a sequential gene-targeting system. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification. PrP(C)-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.     

传染性海绵状脑病的诊断进展和存在的问题

传染性海绵状脑病是由朊病毒引起的人和多种哺乳动物以神经退行性变化为主要特征的一种慢性致死性传染病。引起这类疾病的病原因子是一种编码宿主蛋白的PrPC转变为异常的PrPSC沉积在大脑,导致传染性海绵状脑病的发生。本文从临床症状识别、组织病理学诊断、致病性朊蛋白检测、生物学测定以及毒株鉴定等几个方面作一回顾和总结,为揭示朊病毒疾病致病机理和诊断研究提供借鉴。     

A potential cerebrospinal fluid and plasmatic marker for the diagnosis of Creutzfeldt-Jakob disease

The recent occurrence of the new variant of Creutzfeldt-Jakob disease (CJD), probably transmitted to humans by cattle affected by the bovine form of spongiform encephalopathy, has generated renewed interest in the clinical issues related to human spongiform encephalopathies. Using the current set of diagnostic tools, these rare but devastating conditions may be difficult to diagnose with accuracy before death. The objective of the present communication is to describe the discovery of a potential cerebrospinal fluid (CSF) and plasmatic marker of human transmissible spongiform encephalopathies. A preliminary two-dimensional electrophoresis approach highlighted a potential neurodegenerative disorder marker called the fatty acid binding protein, FABP. Its heart form, H-FABP, was investigated in a small group of CJD affected patients (n = 8 ) by an immunoassay approach. The amount of FABP appeared to be significantly (p< or = 0.05) increased in all tested samples. H-FABP detection could therefore be helpful as a blood screening test for a pre-mortem diagnosis of the disease and also to prevent the risk of iatrogenic transmission of CJD through blood transfusion.     

Distinct Transmissibility Features of TSE Sources Derived from Ruminant Prion Diseases by the Oral Route in a Transgenic Mouse Model (TgOvPrP4) Overexpressing the Ovine Prion Protein

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases associated with a misfolded form of host-encoded prion protein (PrP). Some of them, such as classical bovine spongiform encephalopathy in cattle (BSE), transmissible mink encephalopathy (TME), kuru and variant Creutzfeldt–Jakob disease in humans, are acquired by the oral route exposure to infected tissues. We investigated the possible transmission by the oral route of a panel of strains derived from ruminant prion diseases in a transgenic mouse model (TgOvPrP4) overexpressing the ovine prion protein (A₁₃₆R₁₅₄Q₁₇₁) under the control of the neuron-specific enolase promoter. Sources derived from Nor98, CH1641 or 87V scrapie sources, as well as sources derived from L-type BSE or cattle-passaged TME, failed to transmit by the oral route, whereas those derived from classical BSE and classical scrapie were successfully transmitted. Apart from a possible effect of passage history of the TSE agent in the inocula, this implied the occurrence of subtle molecular changes in the protease-resistant prion protein (PrPres) following oral transmission that can raises concerns about our ability to correctly identify sheep that might be orally infected by the BSE agent in the field. Our results provide proof of principle that transgenic mouse models can be used to examine the transmissibility of TSE agents by the oral route, providing novel insights regarding the pathogenesis of prion diseases.     

Proteomic profiling of cerebrospinal fluid in Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare fatal neurodegenerative disease belonging to the group of transmissible spongiform encephalopathies or prion diseases. The agent responsible for the disease is the prion protein in an altered conformational form. Although there have been countless studies performed on the prion protein, the mechanisms that induce the structural change of the normal protein, and the harmful action the altered protein has on nervous cells, are still not fully understood. Furthermore, the final diagnosis for CJD can only occur with a postmortem histopathological analysis of the brain; the antemortem diagnosis is only possible for some specific CJD forms. Finally, there is no current treatment able to stop or delay the progression of the disease. Studies directed at resolving these issues are, therefore, extremely relevant. The proteomic approach is a very good strategy to be applied in such contexts because it allows easy identification of proteins and peptides possibly involved in the disease processes. In this article, the existing data regarding prion infection, biomarkers for CJD diagnosis and the use of several modern proteomic technologies for the identification of new cerebrospinal fluid polypeptides involved in CJD are reviewed.     

Proteomic profiling of cerebrospinal fluid in Creutzfeldt–Jakob disease

Creutzfeldt–Jakob disease (CJD) is a rare fatal neurodegenerative disease belonging to the group of transmissible spongiform encephalopathies or prion diseases. The agent responsible for the disease is the prion protein in an altered conformational form. Although there have been countless studies performed on the prion protein, the mechanisms that induce the structural change of the normal protein, and the harmful action the altered protein has on nervous cells, are still not fully understood. Furthermore, the final diagnosis for CJD can only occur with a postmortem histopathological analysis of the brain; the antemortem diagnosis is only possible for some specific CJD forms. Finally, there is no current treatment able to stop or delay the progression of the disease. Studies directed at resolving these issues are, therefore, extremely relevant. The proteomic approach is a very good strategy to be applied in such contexts because it allows easy identification of proteins and peptides possibly involved in the disease processes. In this article, the existing data regarding prion infection, biomarkers for CJD diagnosis and the use of several modern proteomic technologies for the identification of new cerebrospinal fluid polypeptides involved in CJD are reviewed.     

Nanoimaging for prion related diseases

Misfolding and aggregation of prion proteins is linked to a number of neurodegenerative disorders such as Creutzfeldt-Jacob disease (CJD) and its variants, kuru, Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. In prion diseases, infectious particles are proteins that propagate by transmitting a misfolded state of a protein, leading to the formation of aggregates and ultimately to neurodegeneration. Prion phenomenon is not restricted to humans. There is a number of prion-related diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as "mad cow disease") in cattle. All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), are untreatable and fatal. Prion proteins were also found in some fungi where they are responsible for heritable traits. Prion proteins in fungi are easily accessible and provide a powerful model for understanding the general principles of prion phenomenon and molecular mechanisms of mammalian prion diseases. Presently, several fundamental questions related to prions remain unanswered. For example, it is not clear how prions cause the disease. Other unknowns include the nature and structure of infectious agent and how prions replicate? Generally, the phenomenon of misfolding of prion protein into infectious conformations that have the ability to propagate their properties via aggregation is of significant interest. Despite the crucial importance of misfolding and aggregation, very little is currently known about the molecular mechanisms of these processes. While there is an apparent critical need to study molecular mechanisms underlying misfolding and aggregation, the detailed characterization of these single molecule processes is hindered by the limitation of conventional methods. Although some issues remain unresolved, much progress has been recently made primarily due to the application of nanoimaging tools. The use of nanoimaging methods shows great promise for understanding the molecular mechanisms of prion phenomenon, possibly leading toward early diagnosis and effective treatment of these devastating diseases. This review article summarizes recent reports which advanced our understanding of the prion phenomenon through the use of nanoimaging methods.     

The structural stability of wild-type horse prion protein

Prion diseases (e.g. Creutzfeldt-Jakob disease (CJD), variant CJD (vCJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal Familial Insomnia (FFI) and Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE or 'mad-cow' disease) and chronic wasting disease (CWD) in cattles) are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches or medications to treat all these prion diseases. Rabbits, dogs, and horses are the only mammalian species reported to be resistant to infection from prion diseases isolated from other species. Recently, the β2-α2 loop has been reported to contribute to their protein structural stabilities. The author has found that rabbit prion protein has a strong salt bridge ASP177-ARG163 (like a taut bow string) keeping this loop linked. This paper confirms that this salt bridge also contributes to the structural stability of horse prion protein. Thus, the region of β2-α2 loop might be a potential drug target region. Besides this very important salt bridge, other four important salt bridges GLU196-ARG156-HIS187, ARG156-ASP202 and GLU211-HIS177 are also found to greatly contribute to the structural stability of horse prion protein. Rich databases of salt bridges, hydrogen bonds and hydrophobic contacts for horse prion protein can be found in this paper.     

Seeded Fibrillation as Molecular Basis of the Species Barrier in Human Prion Diseases

Prion diseases are transmissible spongiform encephalopathies in humans and animals, including scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and Creutzfeldt-Jakob disease (CJD) in humans. The hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP^C) to its pathological isoform PrP^Sc, which is accompanied by PrP fibrillation. Transmission is not restricted within one species, but can also occur between species. In some cases a species barrier can be observed that results in limited or unsuccessful transmission. The mechanism behind interspecies transmissibility or species barriers is not completely understood. To analyse this process at a molecular level, we previously established an in vitro fibrillation assay, in which recombinant PrP (recPrP) as substrate can be specifically seeded by PrP^Sc as seed. Seeding with purified components, with no additional cellular components, is a direct consequence of the “prion-protein-only” hypothesis. We therefore hypothesise, that the species barrier is based on the interaction of PrP^C and PrP^Sc. Whereas in our earlier studies, the interspecies transmission in animal systems was analysed, the focus of this study lies on the transmission from animals to humans. We therefore combined seeds from species cattle, sheep and deer (BSE, scrapie, CWD) with human recPrP. Homologous seeding served as a control. Our results are consistent with epidemiology, other in vitro aggregation studies, and bioassays investigating the transmission between humans, cattle, sheep, and deer. In contrast to CJD and BSE seeds, which show a seeding activity we can demonstrate a species barrier for seeds from scrapie and CWD in vitro. We could show that the seeding activity and therewith the molecular interaction of PrP as substrate and PrP^Sc as seed is sufficient to explain the phenomenon of species barriers. Therefore our data supports the hypothesis that CWD is not transmissible to humans.