Prions

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.     

Evaluation of the antineoplastic activity of guduchi (Tinospora cordifolia) in cultured HeLa cells

The transmissible spongiform encephalopathies (TSEs) (in both humans and animals) have been reviewed with the principal focus on bovine spongiform encephalopathy (BSE), its recent outbreak amongst cattle in the UK ('mad cow disease') and its sequelae. The possible reasons for this outbreak are discussed as well as a number of hypotheses reviewed for TSEs (e.g. prions, organophosphates, etc.) and current measures attendant with food safety and surveillance taken in the European Union. It is generally accepted that the combined weight of all the evidence to date supports the conclusion that the new rare but lethal variant Creutzfeldt-Jacob Disease (vCJD) is the human counterpart of the aetiological agent BSE and that eating meat from the infected animals is probably to blame for 24 deaths (23 in the UK and one in France) to date from vCJD. Considerable evidence from biochemical, immunologic, pathologic and genetic studies strongly suggests that PrpSC is the major component of the transmissible prion particle responsible for the rare fatal brain TSE diseases.     

BSE: can we predict the future?

Prion diseases are transmissible neurodegenerative disorders of humans and animals. The prion protein (PrPc) gene is expressed to some extent in many cell types but principally in neurons. Normal PrPc may contribute in the protection of neurons and are protease sensitive. Abnormal prions consist of a post-translationally modified form of PrP, PrPsc, which is partly protease resistant. PrPsc is a protein with high resistance to inactivation by irradiation, heat and harsh chemical treatments. It is currently proposed that PrPsc is an infectious protein that propagates by inducing the normal PrPc to become the abnormal PrPsc. PrPsc cause transmissible spongiform encephalopathies (TSE), an unusual group of degenerative brain diseases that can be transmitted by inoculation or ingestion of diseased brain or other tissues. The human diseases occur in an inherited, acquired and sporadic forms. Transmission of prion diseases between species is limited by a species barrier, determined in part by the degree of sequence homology between the host PrP and inoculated PrPsc. The epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom is a new disease that has affected over 160,000 cattle and has presumably arisen from dietary exposure to PrPsc from sheep with scrapie. Until shown otherwise we must assume that oral consumption of infectious BSE protein is a new factor for Creutzfeldt-Jakob (CJD) disease in man. This disease is a new variant of CJD (nvCJD) and has a different clinical picture. Early diagnostic markers to facilitate the diagnosis and screen blood and organ donors are not available. The control of the disease relies on the measures to eradicate the spread of BSE by banning the use of ruminant tissues in ruminant feed and slaughter and rendering procedures to ensure inactivation of prions of all infected animals. The control of nvCJD is based on reduction of exposure to BSE by banning a variety of tissues for consumption. A surveillance worldwide is increased for both BSE and nv-CJD and the WHO update regularly recommendations to limit the risk of transmitting the disease. The extent and size of the nvCJD will depend on different factors among others the dose of infectious material, the route of infection, the genetic susceptibility of the person. Therefore is not possible to predict how important the disease will be.     

Diagnosis of Creutzfeldt-Jakob disease and related human spongiform encephalopathies

Spongiform encephalopathies are transmissible diseases (TSE) of animals and humans. With the appearance of bovine spongiform encephalopathy (BSE) in 1986 and in 1996 with the identification of an apparently new variant of the human spongiform encephalopathy Creutzfeldt-Jakob disease (CJD), great concerns of a potential transmission of BSE to humans have been voiced. The agent known to transmit CJD and other human and animal spongiform encephalopathies is designated as prion, i.e., proteinaceous infectious agent, due to the absence of evidence for the involvement of a nucleic acid in disease transmission. In humans the clinical diagnosis of typical CJD cases can now be supported by paraclinical parameters. Electroencephalographic changes, so called periodic sharp wave complexes, are pathognomonic for CJD but by no means specific. The detection of neuronal enzymes in the cerebrospinal fluid (CSF) such as neuron specific enolase (NSE) or glial proteins such as S-100 aids greatly in the diagnosis of a human spongiform encephalopathy. By far the most specific marker in CSF are a group of proteins designated 14-3-3. Current evidence suggests that by including elevated levels of NSE (> or = 35 ng/mL), S-100 (> or = 8 ng/mL) and tau protein in the CSF and the presence of 14-3-3, a laboratory supported diagnosis of CJD can be achieved which in the appropriate clinical setting has a better diagnostic accuracy than the currently used clinical and paraclinical diagnostic criteria alone.     

Abnormalities in stress proteins in prion diseases

1. Prion diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker disease (GSS), and fatal familia insomnia (FFI) of humans, as well as scrapie and bovine spongiform encephalopathy (BSE) of animals. 2. All these disorders involve conversion of the normal, cellular prion protein (PrPC) into the corresponding scrapie isoform (PrPSc). PrPC adopts a structure rich in alpha-helices and devoid of beta-sheet, in contrast to PrPSc, which has a high beta-sheet content and is resistant to limited digestion by proteases. That a conformational transition features in the conversion of PrPC into PrPSc implies that prion diseases are disorders of protein conformation. 3. This concept has been extended by our studies with heat shock proteins (Hsp), many of which are thought to function as molecular chaperones. We found that the induction of some Hsps but not others was profoundly altered in scrapie-infected cells and that the distribution of Hsp73 is unusual in these cells. 4. Whether the conversion of PrPC into PrPSc is assisted by molecular chaperones, or if the accumulation of the abnormally folded PrPSc is complexed with Hsps remains to be established.     

The use of transgenic mice in the investigation of transmissible spongiform encephalopathies

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and to be identical to PrPSc (prion protein: scrapie form), a modified form of the normal host protein PrPC (prion protein: cellular form) which is encoded by the single copy gene Prnp. The 'protein only' hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal devoid of PrPC should be resistant to prion diseases. The authors generated homozygous Prnp(o/o) ('PrP knockout') mice and showed that, after inoculation with prions, these mice remained free from scrapie for at least two years while wild-type controls all died within six months. There was no propagation of prions in the Prnp(o/o) animals. Surprisingly, heterozygous Prnp(o/+) mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie despite high levels of infectious agent and PrPSc in the brain at an early stage. After introduction of murine PrP transgenes, Prnp(o/o) mice became highly susceptible to mouse--but not to hamster--prions, while the insertion of Syrian hamster PrP transgenes rendered the mice susceptible to hamster prions but much less susceptible to mouse prions. These complementation experiments enabled the application of reverse genetics. The authors prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and found that PrP that lacks 48 amino proximal amino acids (which comprise four of the five octa repeats of PrP) is still biologically active.     

Sequencing analysis of prion genes from red deer and camel

An abnormal isoform of the prion protein (PrP) appears to be the agent responsible for transmissible spongiform encephalopathies (TSE). The normal isoform of PrP is host-encoded and expressed in the central nervous system. The recent bovine spongiform encephalopathy (BSE) epidemic in the UK and the incidence of prion-related diseases in other animals could indicate that ruminants are highly susceptible to infection via ingestion of prion-contaminated food. Sequence analysis of PrP gene open reading frames from red deer and camel was carried out to investigate sequence variability of these genes among ruminants.     

The prion diseases

The human prion diseases are fatal neurodegenerative maladies that may present as sporadic, genetic, or infectious illnesses. The sporadic form is called Creutzfeldt-Jakob disease (CJD) while the inherited disorders are called familial (f) CJD, Gerstmann-Straussler-Scheinker (GSS) disease and fatal familial insomnia (FFI). Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. In fCJD, GSS, and FFI, mutations in the PrP gene located on the short arm of chromosome 20 are the cause of disease. Considerable evidence argues that the prion diseases are disorders of protein conformation.     

Transmissible spongiform encephalopathies in animals

Transmissible spongiform encephalopathies in animals are known for centuries. In particular scrapie in sheep and goats occurs worldwide; it spreads as a natural disease and is genetically controlled. Chronic wasting disease (CWD) in the United States (Wyoming and Colorado) also spreads as natural disease among free ranging and captive elk and mule deer. In contrast, transmissible mink encephalopathy (TME) of mink in fur producing farms is caused by contaminated feed; the source of this food contamination is still controversial. The only occurrence of a TSE in an avian species was reported from a flock of ostriches in a German zoo. The origin of the outbreak of bovine spongiform encephalopathy (BSE) in Great Britain could be traced back to feeding concentrates containing animal proteins contaminated with the agent of sheep scrapie. BSE was introduced into other countries, e.g. Switzerland and France, through the import of contaminated feeds from Great Britain. In addition, sporadic cases of TSE occurred in 10 other species, in particular domestic cats and zoo animals, e.g. antelopes and large cats. The diagnosis is based on the neuropathological examination of the brain as well as the demonstration of the disease specific protease resistant prion protein (PrPres). The first measures against the disease aim at eliminating the risk factors. The most important is the prohibition of feeding animal protein concentrates to ruminants. Thanks to this measure the incidence of BSE diminished remarkably. To protect consumers of beef products, in countries with BSE the potentially infectious organs of all cattle are confiscated at slaughter. Yet, in Great Britain this measure was introduced only at a point of time when BSE had already spread all over the country. Therefore, there is a strong probability of an exposition of consumers of beef with the BSE agent, which might have caused the new variant of Creutzfeldt-Jakob disease (nvCJD).     

The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells

Prions are thought to consist of infectious proteins that cause transmissible spongiform encephalopathies. According to overwhelming evidence, the pathogenic prion protein PrPSc converts its host encoded isoform PrPC into insoluble aggregates of PrPSc, concomitant with pathological modifications (for review, see refs. 1-3). Although the physiological role of PrPC is poorly understood, studies with PrP knockout mice demonstrated that PrPC is required for the development of prion diseases. Using the yeast two-hybrid technology in Saccharomyces cerevisiae, we identified the 37-kDa laminin receptor precursor (LRP) as interacting with the cellular prion protein PrPC. Mapping analysis of the LRP-PrP interaction site in S. cerevisiae revealed that PrP and laminin share the same binding domain (amino acids 161 to 180) on LRP. The LRP-PrP interaction was confirmed in vivo in insect (Sf9) and mammalian cells (COS-7). The LRP level was increased in scrapie-infected murine N2a cells and in brain and spleen of scrapie-infected mice. In contrast, the LRP concentration was not significantly altered in these organs from mice infected with the bovine spongiform encephalopathic agent (BSE), which have a lower PrPSc accumulation. LRP levels, however, were dramatically increased in brain and pancreas, slightly increased in the spleen and not altered in the liver of crapie-infected hamsters. These data show that enhanced LRP concentrations are correlated with PrPSc accumulation in organs from mice and hamsters. The laminin receptor precursor, which is highly conserved among mammals and is located on the cell surface, may act as a receptor or co-receptor for the prion protein on mammalian cells.     

A review of the current research on prions. The evidence suggests the possibility of transmission of the mad cow disease to humans

Further evidence of the transmissibility of bovine spongiform encephalopathy (BSE) across the species barrier from cow to man has been derived from epidemiological analysis and the characterisation of prion strains. Recent research has shown the persistence of prions after experimental transmission to resistant murine species, and subclinical persistence in cows. The accumulation of pathological prion proteins in tonsils and appendix has been demonstrated prior to clinical confirmation of the presence of the new variant of Creutzfeldt-Jakob disease. Current prion research is focused on the involvement of B lymphocytes as carriers, on the species barrier and cellular receptors, and on macromolecules involved in the conformational change from normal to pathological prion proteins.     

Abnormal properties of prion protein with insertional mutations in different cell types

Inherited forms of the human transmissible spongiform encephalopathy Creutzfeldt-Jakob disease (CJD) have been associated with mutations in the normal soluble, protease-sensitive form of the host prion protein (PrP-sen). Normal PrP protein contains five copies of a repeating eight-amino acid region, and PrP molecules with six or more copies of this region are associated with disease in familial CJD. It has been hypothesized that these mutations might facilitate spontaneous formation of the abnormal, aggregated protease-resistant PrP isoform, PrP-res, associated with clinical CJD and other transmissible spongiform encephalopathies (TSE). In the present experiments, hamster PrP molecules with 5 (wild-type), 7, 9, or 11 copies of this repeat region were generated and expressed in mouse fibroblast cells or mouse neuroblastoma cells. In mouse fibroblast cells, mutant hamster PrP molecules expressing 7, 9, and 11 copies of the octapeptide repeat sequence showed altered cell surface expression, but both mutant and wild-type hamster PrP-sen molecules demonstrated abnormal properties of aggregation and increased protease resistance. By contrast in mouse neuroblastoma cells, hamster PrP-sen with 5, 9, and 11 octapeptide repeats were expressed normally on the cell surface, but only PrP-sen molecules with 9 or 11 copies of the repeat motif had abnormal properties of aggregation and increased protease resistance. Overall, regardless of cell type, hamster PrP molecules with greater than 7 octapeptide repeats were more aggregated and more protease-resistant than molecules with 7 repeats or less. However, these abnormal molecules were at least 1000-fold less protease-resistant than bona fide PrP-res derived from TSE-infected brain tissue, and they showed no increased ability to form PrP-res in a cell-free system.     

A trend of molecular genetics on prion diseases and prion protein

Infectious amyloid filaments designated as prion rods or scrapie associated fibrils (SAF) present in brain tissues affected by transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker disease (GSS) and kuru of humans, and scrapie of sheep. A hydrophobic glycoprotein, PrPSc is a major component of SAF, and is known to be associated with the infectivity of these diseases. Both PrPSc and the normal isoform of this glycoprotein, PrPC are encoded by a single host gene, PrP gene, and the conversion of PrPC to PrPSc is a posttranslational event. Several mutations on the PrP gene are associated with variations of the phenotype and the occurrence in familial CJD and GSS.     

Creutzfeldt-Jakob disease(CJD) and Gerstmann-Str?ussler-Scheinker syndrome(GSS)

Prion diseases are now a focus of attention since the diseases are known to disseminate beyond expectation. They are various types of Gerstmann-Str?ussler-Scheinker syndrome(GSS), fatal familial insomnia, iatrogenic Creutzfeldt-Jakob disease(CJD), and variant CJD which suspected to have disseminated from bovine spongiform encephalopathy. Abnormal prion protein deposits as amyloid structure in the brain of the patients with these diseases. Mutation of the prion protein gene exists in a part of these diseases. Depending on their kind, patients represent a variety of clinical and neuropathological manifestations. They can be clinically divided into two general groups of the CJD type and the GSS type. For clinical diagnosis of the GSS type prion diseases, analysis of prion protein gene is useful.     

Significance of prion protein in transmission of prions and in pathogenesis of spongiform encephalopathies

Prion disease or transmissible spongiform encephalopathies are caused by novel pathogens termed prions. Unlike classical infectious agents such as viruses or bacteria, prions lack an independent genome and consist largely if not entirely of an abnormal form of the host-encoded prion protein. How prions multiply is not known. A wealth of experimental evidence supports an essential role for the host-encoded prion protein in susceptibility and pathogenesis of prion diseases and in the propagation and spread of prions. In addition, B lymphocytes have been found to play a crucial role in the neuroinvasiveness of prions.     

Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (prion diseases)

Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human transmissible spongiform encephalopathies (prion diseases) are proposed for the following disease entities: CJD--sporadic, iatrogenic (recognised risk) or familial (same disease in 1st degree relative): spongiform encephalopathy in cerebral and/or cerebellar cortex and/or subcortical grey matter; or encephalopathy with prion protein (PrP) immunoreactivity (plaque and/or diffuse synaptic and/or patchy/perivacuolar types). Gerstmann-Str?ussler-Scheinker disease (GSS) (in family with dominantly inherited progressive ataxia and/or dementia): encephalo(myelo)pathy with multicentric PrP plaques. Familial fatal insomnia (FFI) (in member of a family with PRNP178 mutation): thalamic degeneration, variable spongiform change in cerebrum. Kuru (in the Fore population). Without PrP data, the crucial feature is the spongiform change accompanied by neuronal loss and gliosis. This spongiform change is characterised by diffuse or focally clustered small round or oval vacuoles in the neuropil of the deep cortical layers, cerebellar cortex or subcortical grey matter, which might become confluent. Spongiform change should not be confused with non-specific spongiosis. This includes status spongiosus ("spongiform state"), comprising irregular cavities in gliotic neuropil following extensive neuronal loss (including also lesions of "burnt-out" CJD), "spongy" changes in brain oedema and metabolic encephalopathies, and artefacts such as superficial cortical, perineuronal, or perivascular vacuolation; focal changes indistinguishable from spongiform change may occur in some cases of Alzheimer's and diffuse Lewy body diseases. Very rare cases might not be diagnosed by these criteria. Then confirmation must be sought by additional techniques such as PrP immunoblotting, preparations for electron microscopic examination of scrapie associated fibrils (SAF), molecular biologic studies, or experimental transmission.     

Biochemical properties of protease resistant prion protein PrPsc in natural sheep scrapie

Prions are infectious agents involved in neurodegenerative diseases, such as scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cows and Creutzfeldt-Jakob disease (CJD) in humans. These pathogens are characterized by unusual properties, and, in particular, by their strong resistance to common procedures of disinfection used against conventional microorganisms. A major component of highly infectious fractions is a proteinase K-resistant prion protein PrPsc (PrP-res), the normal host prion protein PrPc being sensitive to PK (PrP-sen). We used a biochemical approach to further characterize PrPsc protein in natural sheep scrapie. Western blot analyses using rabbit antiserum that recognized both normal and pathologic sheep prion proteins, were undertaken to study the biochemical behaviour of PrPsc extracted from brains of sheep naturally infected with scrapie after protease digestion and under denaturing conditions. Increasing concentrations of urea (1-7 M) or GdnSCN (0.25-3 M) and different pH from 2 to 11 were tested for their effects on protease resistance of PrPsc. Alkaline pH (pH = 10) and high concentrations of urea (> 3 M) and GdnSCN (> 0.75 M) greatly decreased the protease resistance of the prion protein. Identical experiments carried out on three different sheep from the same flock gave similar results. The biochemical behaviour of PrPsc under denaturing conditions and in the presence of proteinase K could thus provide a biochemical means for further characterization of different natural scrapie isolates.     

Making sense of bovine spongiform encephalopathy

The possibility of animals infected with bovine spongiform encephalopathy (BSE) causing Creutzfeldt-Jakob disease (CJD) in humans has caused public alarm resulting in the banning of the export of British beef. This paper explores the current understanding of BSE and its relationship to CJD. Although there are similarities between the diseases, there is no clear evidence of a causal link between the two.