Insights into prion strains and neurotoxicity

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that are caused by prions and affect humans and many animal species. It is now widely accepted that the infectious agent that causes TSEs is PrP(Sc), an aggregated moiety of the host-derived membrane glycolipoprotein PrP(C). Although PrP(C) is encoded by the host genome, prions themselves encipher many phenotypic TSE variants, known as prion strains. Prion strains are TSE isolates that, after inoculation into distinct hosts, cause disease with consistent characteristics, such as incubation period, distinct patterns of PrP(Sc) distribution and spongiosis and relative severity of the spongiform changes in the brain. The existence of such strains poses a fascinating challenge to prion research.     

Experimental approaches to TSE prevention via inhibition of prion formation

Transmissible spongiform encepahalopathies (TSEs) are fatal diseases that damage the central nervous system. TSEs are unique in that they may be inherited, infectious or spontaneous. The central pathogenic agent is thought to be a conformationally distinct form (PrP(Sc;)) of the endogenous prion protein(PrP(c)), which is high in beta-sheet content and is resistant to proteases; infectivity is thought to involve formation of PrP(Sc) via imprinting of abnormal conformation on the normal form of the protein (PrP(c)) by seeds of PrP(Sc). A number of compounds found to inhibit the conversion of PrP(c) to PrP(Sc) have been proposed as therapeutics to halt TSEs.     

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.     

Adaptation and selection of prion protein strain conformations following interspecies transmission of transmissible mink encephalopathy

Interspecies transmission of the transmissible spongiform encephalopathies (TSEs), or prion diseases, can result in the adaptation and selection of TSE strains with an expanded host range and increased virulence such as in the case of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease. To investigate TSE strain adaptation, we serially passaged a biological clone of transmissible mink encephalopathy (TME) into Syrian golden hamsters and examined the selection of distinct strain phenotypes and conformations of the disease-specific isoform of the prion protein (PrP(Sc)). The long-incubation-period drowsy (DY) TME strain was the predominate strain, based on the presence of its strain-specific PrP(Sc) following interspecies passage. Additional serial passages in hamsters resulted in the selection of the hyper (HY) TME PrP(Sc) strain-dependent conformation and its short incubation period phenotype unless the passages were performed with a low-dose inoculum (e.g., 10(-5) dilution), in which case the DY TME clinical phenotype continued to predominate. For both TME strains, the PrP(Sc) strain pattern preceded stabilization of the TME strain phenotype. These findings demonstrate that interspecies transmission of a single cloned TSE strain resulted in adaptation of at least two strain-associated PrP(Sc) conformations that underwent selection until one type of PrP(Sc) conformation and strain phenotype became predominant. To examine TME strain selection in the absence of host adaptation, hamsters were coinfected with hamster-adapted HY and DY TME. DY TME was able to interfere with the selection of the short-incubation HY TME phenotype. Coinfection could result in the DY TME phenotype and PrP(Sc) conformation on first passage, but on subsequent passages, the disease pattern converted to HY TME. These findings indicate that during TSE strain adaptation, there is selection of a strain-specific PrP(Sc) conformation that can determine the TSE strain phenotype.     

Amyloid formation by recombinant full-length prion proteins in phospholipid bicelle solutions

A soluble, oligomeric beta-sheet-rich conformational variant of recombinant full-length prion protein, PrP beta, was generated that aggregates into amyloid fibrils, PrP betaf. These fibrils have physico-chemical and structural properties closely similar to those of pathogenic PrP Sc in scrapie-associated fibrils and prion rods, including a closely similar proteinase K digestion pattern and Congo red birefringence. The conformational transition from PrP C to PrP beta occurs at pH 5.0 in bicellar solutions containing equimolar mixtures of dihexanoyl-phosphocholine and dimyristoyl-phospholipids, and a small percentage of negatively charged dimyristoyl-phosphoserine. The same protocol was applicable to human, cow, elk, pig, dog and mouse PrP. Comparison of full-length hPrP 23-230 with the N-terminally truncated human PrP fragments hPrP 90-230, hPrP 96-230, hPrP 105-230 and hPrP 121-230 showed that the flexible peptide segment 105-120 must be present for the generation of PrP beta. Dimerization of PrP C represents the rate-limiting step of the PrP C-to-PrP beta conformational transition, which is dependent on the amino acid sequence. The activation enthalpy of dimerization is about 130 kJ/mol for the recombinant full-length human and bovine prion proteins, and between 260 and 320 kJ/mol for the other species investigated. The in vitro conversion assay described here permits direct molecular characterization of processes that might be closely related to conformational transitions of the prion protein in transmissible spongiform encephalopathies.     

Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein

Transmissible spongiform encephalopathies (TSEs) are caused by a unique infectious agent which appears to be identical with PrPSc, an oligomeric, misfolded isoform of the cellular prion protein, PrPC. All inherited forms of human TSEs, i.e., familial Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker syndrome, and fatal familial insomnia, segregate with specific point mutations or insertions in the gene coding for human PrP. Here we have tested the hypothesis that these mutations destabilize PrPC and thus facilitate its conversion into PrPSc. Eight of the disease-specific amino acid replacements are located in the C-terminal domain of PrPC, PrP(121-231), which constitutes the only part of PrPC with a defined tertiary structure. Introduction of all these replacements into PrP(121-231) yielded variants with the same spectroscopic characteristics as wild-type PrP(121-231) and similar to full-length PrP(23-231), which excludes the possibility that the exchanges a priori induce a PrPSc-like conformation. The thermodynamic stabilities of the variants do not correlate with specific disease phenotypes. Five of the amino acid replacements destabilize PrP(121-231), but the other variants have the same stability as the wild-type protein. These data suggest that destabilization of PrPC is neither a general mechanism underlying the formation of PrPSc nor the basis of disease phenotypes in inherited human TSEs.     

The highways and byways of prion protein trafficking

Prions are defined as infectious agents that comprise only proteins and are responsible for transmissible spongiform encephalopathies (TSEs)--fatal neurodegenerative diseases that affect humans and other mammals and include Creutzfeldt-Jacob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. Prions have been proposed to arise from the conformational conversion of the cellular prion protein PrP(C) to a misfolded form termed PrP(Sc) that precipitates into aggregates and fibrils. The conversion process might be triggered by interaction of the infectious form with the cellular form or it might result from a mutation in the gene encoding PrP(C). Exactly how and where in the cell the interaction and the conversion of PrP(C) to PrP(Sc) occur, however, remain controversial. Recent studies have shed light on the intracellular trafficking of PrP(C), the role of protein mis-sorting and the cellular factors that are thought to be required for the conformational conversion of prion proteins.     

Relationships between PrP(Sc) Stability and Incubation Time for United States Scrapie Isolates in a Natural Host System

Transmissible spongiform encephalopathies (TSEs), including scrapie in sheep (Ovis aries), are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP(C)) into a a-rich conformer (PrP(Sc)) that accumulates into higher-order structures in the brain and other tissues. Distinct strains of TSEs exist, characterized by different pathologic profiles upon passage into rodents and representing distinct conformations of PrP(Sc). One biochemical method of distinguishing strains is the stability of PrP(Sc) as determined by unfolding in guanidine hydrochloride (GdnHCl), which is tightly and positively correlated with the incubation time of disease upon passage into mice. Here, we utilize a rapid, protease-free version of the stability assay to characterize naturally occurring scrapie samples, including a fast-acting scrapie inoculum for which incubation time is highly dependent on the amino acid at codon 136 of the prion protein. We utilize the stability methodology to identify the presence of two distinct isolates in the inoculum, and compare isolate properties to those of a host-stabilized reference scrapie isolate (NADC 13-7) in order to assess the stability/incubation time correlation in a natural host system. We demonstrate the utility of the stability methodology in characterizing TSE isolates throughout serial passage in livestock, which is applicable to a range of natural host systems, including strains of bovine spongiform encephalopathy and chronic wasting disease.     

Fish models in prion biology: Underwater issues

Transmissible spongiform encephalopathies (TSEs), otherwise known as prion disorders, are fatal diseases causing neurodegeneration in a wide range of mammalian hosts, including humans. The causative agents - prions - are thought to be composed of a rogue isoform of the endogenous prion protein (PrP). Beyond these and other basic concepts, fundamental questions in prion biology remain unanswered, such as the physiological function of PrP, the molecular mechanisms underlying prion pathogenesis, and the origin of prions. To date, the occurrence of TSEs in lower vertebrates like fish and birds has received only limited attention, despite the fact that these animals possess bona fide PrPs. Recent findings, however, have brought fish before the footlights of prion research. Fish models are beginning to provide useful insights into the roles of PrP in health and disease, as well as the potential risk of prion transmission between fish and mammals. Although still in its infancy, the use of fish models in TSE research could significantly improve our basic understanding of prion diseases, and also help anticipate risks to public health. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Kinetic intermediate in the folding of human prion protein

Transmissible spongiform encephalopathies are associated with the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). Here we have examined the kinetics of folding and unfolding reactions for the recombinant human prion protein C-terminal fragment 90-231 at pH 4.8 and 7.0. The stopped-flow data provide clear evidence for the population of an intermediate on the refolding pathway of the prion protein as indicated by a pronounced curvature in chevron plots and the presence of significant burst phase amplitude in the refolding kinetics. In addition to its role in the normal prion protein folding, this intermediate likely represents a crucial monomeric precursor of the pathogenic PrP(Sc) isoform.     

Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein

Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain.The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases.Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin.The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.     

Prion diseases of humans and farm animals: epidemiology, genetics, and pathogenesis

Neuronal vacuolation (spongiosis), neuronal death, and pronounced glial reactions are the hallmarks of transmissible spongiform encephalopathies (TSEs), or prion diseases. A wealth of physical, biochemical, and immunological evidence indicates that the TSE agent, termed prion, does not contain agent-specific nucleic acid encoding its own constituents, as is the case for all other infectious pathogens. Also, no adaptive immune responses are elicited upon infection. A defining feature of TSEs is the deposition, mainly in the brain and lymphoreticular tissues, of an aggregated and structurally abnormal protein, designated PrP(Sc) or PrP-res, which represents a conformational isomer of the ubiquitous surface protein PrP(C). Biochemical and genetic evidence link PrP and its gene to the disease. Although TSEs are by definition transmissible, a growing number of Prnp-associated non-infectious neurodegenerative proteinopathies are now being recognized.     

Aggregation of cellular prion protein is initiated by proximity-induced dimerization

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP(C)) into an altered disease-associated conformation generally designated PrP(Sc), abnormal deposition of PrP(Sc) aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP(C) aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP(C) molecules may trigger its aggregation in neuronal cells. A chimeric PrP(C) composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP(C) were created, and transfected in N2a cells. Similar to PrP(C), Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP(C), both dimerization and oligomerization of PrP(C) resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP(C) molecules may constitute a minimal and sufficient molecular event leading to PrP(C) aggregation and extracellular deposition.     

Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography-mass spectrometry

The conversion of the cellular prion protein (PrP(C)) into a misfolded isoform (PrP(TSE)) that accumulates in the brain of affected individuals is the key feature of transmissible spongiform encephalopaties (TSEs). Susceptibility to TSEs is influenced by polymorphisms of the prion gene suggesting that the presence of certain amino acid residues may facilitate the pathological conversion. In this work, we describe a quantitative, fast and reliable HPLC-MS method that allowed to demonstrate that in the brain of 109(Met/Ile) heterozygous bank voles infected with the mouse adapted scrapie strain 139A, there are comparable amounts of PrP(TSE) with methionine or isoleucine in position 109, suggesting that in this TSE model the two allotypes have similar rates of accumulation. This method can be easily adapted for the quantitative determination of PrP allotypes in the brain of other natural or experimental TSE models.     

Amidation and structure relaxation abolish the neurotoxicity of the prion peptide PrP106-126 in vivo and in vitro

One of the major pathological hallmarks of transmissible spongiform encephalopathies (TSEs) is the accumulation of a pathogenic (scrapie) isoform (PrP(Sc)) of the cellular prion protein (PrP(C)) primarily in the central nervous system. The synthetic prion peptide PrP106-126 shares many characteristics with PrP(Sc) in that it shows PrP(C)-dependent neurotoxicity both in vivo and in vitro. Moreover, PrP106-126 in vitro neurotoxicity has been closely associated with the ability to form fibrils. Here, we studied the in vivo neurotoxicity of molecular variants of PrP106-126 toward retinal neurons using electroretinographic recordings in mice after intraocular injections of the peptides. We found that amidation and structure relaxation of PrP106-126 significantly reduced the neurotoxicity in vivo. This was also found in vitro in primary neuronal cultures from mouse and rat brain. Thioflavin T binding studies showed that amidation and structure relaxation significantly reduced the ability of PrP106-126 to attain fibrillar structures in physiological salt solutions. This study hence supports the assumption that the neurotoxic potential of PrP106-126 is closely related to its ability to attain secondary structure.     

Prion protein and the transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that occur in a wide variety of mammals. In humans, TSE diseases include kuru, sporadic and iatrogenic Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). So far, TSE diseases occur only rarely in humans; however, scrapie is a widespread problem in sheep, and the recent epidemic of bovine spongiform encephalopathy (BSE or mad cow disease) has seriously affected the British cattle industry. Of special concern is the recent appearance of a new variant of CJD in humans that is suspected of being caused by infections from BSE-infected cattle products. In all these diseases, an abnormal form of a host protein, prion protein (PrP), is essential for the pathogenic process. The relationship of this protein to the transmissible agent is currently the subject of great interest and controversy and is the subject of this review.     

New inhibitors of scrapie-associated prion protein formation in a library of 2000 drugs and natural products

Transmissible spongiform encephalopathies (TSEs) are fatal, untreatable neurodegenerative diseases associated with the accumulation of a disease-specific form of prion protein (PrP) in the brain. One approach to TSE therapeutics is the inhibition of PrP accumulation. Indeed, many inhibitors of the accumulation of PrP associated with scrapie (PrP(Sc)) in scrapie-infected mouse neuroblastoma cells (ScN(2)a) also have antiscrapie activity in rodents. To expedite the search for potential TSE therapeutic agents, we have developed a high-throughput screening assay for PrP(Sc) inhibitors using ScN(2)a cells in a 96-well format. A library of 2000 drugs and natural products was screened in ScN(2)a cells infected with scrapie strain RML (Chandler) or 22L. Forty compounds were found to have concentrations causing 50% inhibition (IC(50)s) of PrP(Sc) accumulation of 相似文献   

Computerized morphometric analysis of pathological prion protein deposition in scrapie-infected hamster brain.

Transmissible spongiform encephalopathies (TSEs or prion diseases) are characterized by a constellation of typical though variable pathological changes in the brain. Deposition of disease-associated abnormal prion protein (PrP(Sc)) is the pathological feature of TSEs most consistent and accessible for quantification. However, the evaluation of PrP(Sc) deposits detected by immunohistochemical techniques has been traditionally based on arbitrarily assigned semiquantitative scores. This approach is limited by its subjectivity and bias, yielding considerable variability. In this study, we used MetaMorph 6.1 image analysis software for quantitative analysis of immunostained PrP(Sc) deposits in the CNS of hamsters infected with the 263K strain of scrapie agent. Computerized morphometric analysis (CMA) allowed unambiguous detection of even minimal amounts of immunostained PrP(Sc). CMA values for intensity of staining and area stained correlated well with semiquantitative scores, providing reproducible quantitative data and objective criteria for analyzing PrP(Sc) deposition. CMA provides a simple and reliable method for improved and consistent diagnosis of TSEs that may also be used to quantify other immunostained biomarkers.     

Dynamics of the nucleated polymerization model of prion replication

The disease process for transmissible spongiform encephalopathies (TSEs), in one way or another, involves the conversion of a predominantly alpha-helical normal host-coded prion protein (PrP(C)) to an abnormally folded (predominantly beta sheet) protease resistant isoform (PrP(Sc)). Several alternative mechanisms have been proposed for this auto-catalytic process. Here the dynamical behavior of one of these models, the nucleated polymerization model, is studied by Monte Carlo discrete-event simulation of the explicit conversion reactions. These simulations demonstrate the characteristic dynamical behavior of this model for prion replication. Using estimates for the reaction rates and concentrations, time courses are estimated for concentration of PrP(Sc), PrP(Sc) aggregates, and PrP(C) as well as size distributions for the aggregates. The implications of these dynamics on protein misfolding cyclic amplification (PMCA) is discussed.