RESUMO
BACKGROUND: Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from classical bovine spongiform encephalopathy (C-BSE) may copropagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. METHODS: To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. RESULTS: No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, 1 isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. CONCLUSIONS: The results obtained suggest a low zoonotic potential for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.
Assuntos
Encéfalo , Síndrome de Creutzfeldt-Jakob , Cabras , Camundongos Transgênicos , Príons , Scrapie , Zoonoses , Animais , Síndrome de Creutzfeldt-Jakob/transmissão , Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Scrapie/transmissão , Scrapie/patologia , Camundongos , Zoonoses/transmissão , Encéfalo/patologia , Encéfalo/metabolismo , Ovinos , Bovinos , Príons/metabolismo , Fenótipo , Baço/patologia , Encefalopatia Espongiforme Bovina/transmissão , Encefalopatia Espongiforme Bovina/patologia , Encefalopatia Espongiforme Bovina/metabolismo , Doenças das Cabras/transmissão , Doenças das Cabras/patologia , Modelos Animais de DoençasRESUMO
It is commonly accepted that the prion replicative propensity and strain structural determinant (SSD) are encoded in the fold of PrPSc amyloid fibril assemblies. By exploring the quaternary structure dynamicity of several prion strains, we revealed that all mammalian prion assemblies exhibit the generic property of spontaneously generating two sets of discreet infectious tetrameric and dimeric species differing significantly by their specific infectivity. By using perturbation approaches such as dilution and ionic strength variation, we demonstrated that these two oligomeric species were highly dynamic and evolved differently in the presence of chaotropic agents. In general, our observations of seven different prion strains from three distinct species highlight the high dynamicity of PrPSc assemblies as a common and intrinsic property of mammalian prions. The existence of such small infectious PrPSc species harboring the SSD indicates that the prion infectivity and the SSD are not restricted only to the amyloid fold but can also be encoded in other alternative quaternary structures. Such diversity in the quaternary structure of prion assemblies tends to indicate that the structure of PrPSc can be divided into two independent folding domains: a domain encoding the strain structural determinant and a second domain whose fold determines the type of quaternary structure that could adopt PrPSc assemblies.
Assuntos
Doenças Priônicas , Proteínas Priônicas , Dobramento de Proteína , Animais , Amiloide/química , Amiloide/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Camundongos , Humanos , Ovinos , Conformação ProteicaRESUMO
Prions are neurotropic pathogens composed of misfolded assemblies of the host-encoded prion protein PrPC which replicate by recruitment and conversion of further PrPC by an autocatalytic seeding polymerization process. While it has long been shown that mouse-adapted prions cannot replicate and are rapidly cleared in transgenic PrP0/0 mice invalidated for PrPC, these experiments have not been done with other prions, including from natural resources, and more sensitive methods to detect prion biological activity. Using transgenic mice expressing human PrP to bioassay prion infectivity and RT-QuIC cell-free assay to measure prion seeding activity, we report that prions responsible for the most prevalent form of sporadic Creutzfeldt-Jakob disease in human (MM1-sCJD) can persist indefinitely in the brain of intra-cerebrally inoculated PrP0/0 mice. While low levels of seeding activity were measured by RT-QuIC in the brain of the challenged PrP0/0 mice, the bio-indicator humanized mice succumbed at a high attack rate, suggesting relatively high levels of persistent infectivity. Remarkably, these humanized mice succumbed with delayed kinetics as compared to MM1-sCJD prions directly inoculated at low doses, including the limiting one. Yet, the disease that did occur in the humanized mice on primary and subsequent back-passage from PrP0/0 mice shared the neuropathological and molecular characteristics of MM1-sCJD prions, suggesting no apparent strain evolution during lifelong dormancy in PrP0/0 brain. Thus, MM1-sCJD prions can persist for the entire life in PrP0/0 brain with potential disease potentiation on retrotransmission to susceptible hosts. These findings highlight the capacity of prions to persist and rejuvenate in non-replicative environments, interrogate on the type of prion assemblies at work and alert on the risk of indefinite prion persistence with PrP-lowering therapeutic strategies.
RESUMO
Prions are pathogenic infectious agents responsible for fatal, incurable neurodegenerative diseases in animals and humans. Prions are composed exclusively of an aggregated and misfolded form (PrP Sc ) of the cellular prion protein (PrPC). During the propagation of the disease, PrPSc recruits and misfolds PrPC into further PrPSc. In human, iatrogenic prion transmission has occurred with incompletely sterilized medical material because of the unusual resistance of prions to inactivation. Most commercial prion disinfectants validated against the historical, well-characterized laboratory strain of 263K hamster prions were recently shown to be ineffective against variant Creutzfeldt-Jakob disease human prions. These observations and previous reports support the view that any inactivation method must be validated against the prions for which they are intended to be used. Strain-specific variations in PrPSc physico-chemical properties and conformation are likely to explain the strain-specific efficacy of inactivation methods. Animal bioassays have long been used as gold standards to validate prion inactivation methods, by measuring reduction of prion infectivity. Cell-free assays such as the real-time quaking-induced conversion (RT-QuIC) assay and the protein misfolding cyclic amplification (PMCA) assay have emerged as attractive alternatives. They exploit the seeding capacities of PrPSc to exponentially amplify minute amounts of prions in biospecimens. European and certain national medicine agencies recently implemented their guidelines for prion inactivation of non-disposable medical material; they encourage or request the use of human prions and cell-free assays to improve the predictive value of the validation methods. In this review, we discuss the methodological and technical issues regarding the choice of (i) the cell-free assay, (ii) the human prion strain type, (iii) the prion-containing biological material. We also introduce a new optimized substrate for high-throughput PMCA amplification of human prions bound on steel wires, as translational model for prion-contaminated instruments.
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Prions are pathogens formed from abnormal conformers (PrPSc) of the host-encoded cellular prion protein (PrPC). PrPSc conformation to disease phenotype relationships extensively vary among prion strains. In particular, prions exhibit a strain-dependent tropism for lymphoid tissues. Prions can be composed of several substrain components. There is evidence that these substrains can propagate in distinct tissues (e.g. brain and spleen) of a single individual, providing an experimental paradigm to study the cause of prion tissue selectivity. Previously, we showed that PrPC expression levels feature in prion substrain selection in the brain. Transmission of sheep scrapie isolates (termed LAN) to multiple lines of transgenic mice expressing varying levels of ovine PrPC in their brains resulted in the phenotypic expression of the dominant sheep substrain in mice expressing near physiological PrPC levels, whereas a minor substrain replicated preferentially on high expresser mice. Considering that PrPC expression levels are markedly decreased in the spleen compared to the brain, we interrogate whether spleen PrPC dosage could drive prion selectivity. The outcome of the transmission of a large cohort of LAN isolates in the spleen from high expresser mice correlated with the replication rate dependency on PrPC amount. There was a prominent spleen colonization by the substrain preferentially replicating on low expresser mice and a relative incapacity of the substrain with higher-PrPC level need to propagate in the spleen. Early colonization of the spleen after intraperitoneal inoculation allowed neuropathological expression of the lymphoid substrain. In addition, a pair of substrain variants resulting from the adaptation of human prions to ovine high expresser mice, and exhibiting differing brain versus spleen tropism, showed different tropism on transmission to low expresser mice, with the lymphoid substrain colonizing the brain. Overall, these data suggest that PrPC expression levels are instrumental in prion lymphotropism.
Assuntos
Proteínas Priônicas/metabolismo , Baço/metabolismo , Animais , Encéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Doenças Priônicas/metabolismoRESUMO
Prion replication results from the autocatalytic templated assisted conversion of the host-encoded prion protein PrPC into misfolded, polydisperse PrPSc conformers. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Within and between prion strains, the biological activity (replicative efficacy and specific infectivity) of PrPSc assemblies is size dependent and thus reflects an intrinsic structural heterogeneity. The contribution of such PrPSc heterogeneity across species prion adaptation, which is believed to be based on fit adjustment between PrPSc template(s) and host PrPC, has not been explored. To define the structural-to-fitness PrPSc landscape, we measured the relative capacity of size-fractionated PrPSc assemblies from different prion strains to cross mounting species barriers in transgenic mice expressing foreign PrPC. In the absence of a transmission barrier, the relative efficacy of the isolated PrPSc assemblies to induce the disease is like the efficacy observed in the homotypic context. However, in the presence of a transmission barrier, size fractionation overtly delays and even abrogates prion pathogenesis in both the brain and spleen tissues, independently of the infectivity load of the isolated assemblies. Altering by serial dilution PrPSc assembly content of non-fractionated inocula aberrantly reduces their specific infectivity, solely in the presence of a transmission barrier. This suggests that synergy between structurally distinct PrPSc assemblies in the inoculum is requested for crossing the species barrier. Our data support a mechanism whereby overcoming prion species barrier requires complementation between structurally distinct PrPSc assemblies. This work provides key insight into the "quasispecies" concept applied to prions, which would not necessarily rely on prion substrains as constituent but on structural PrPSc heterogeneity within prion population.
Assuntos
Encéfalo/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Animais , Bovinos , Cricetinae , Camundongos , Camundongos Transgênicos , Proteínas PrPSc/metabolismo , OvinosRESUMO
To date, approximately 500 iatrogenic Creutzfeldt-Jakob disease cases have been reported worldwide, most of them resulting from cadaveric dura mater graft and from the administration of prion-contaminated human growth hormone. The unusual resistance of prions to decontamination processes, their large tissue distribution, and the uncertainty about the prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the general population lead to specific recommendations regarding identification of tissue at risk and reprocessing of reusable medical devices, including the use of dedicated treatment for prion inactivation. We previously described an in vitro assay, called Surf-PMCA, which allowed us to classify prion decontamination treatments according to their efficacy on vCJD prions by monitoring residual seeding activity (RSA). Here, we used a transgenic mouse line permissive to vCJD prions to study the correlation between the RSA measured in vitro and the in vivo infectivity. Implantation in mouse brains of prion-contaminated steel wires subjected to different decontamination procedures allows us to demonstrate a good concordance between RSA measured by Surf-PMCA (in vitro) and residual infectivity (in vivo). These experiments emphasize the strength of the Surf-PMCA method as a rapid and sensitive assay for the evaluation of prion decontamination procedures and also confirm the lack of efficacy of several marketed reagents on vCJD prion decontamination.IMPORTANCE Creutzfeldt-Jakob diseases are neurodegenerative disorders for which transmission linked to medical procedures have been reported in hundreds of patients. As prion diseases, they are characterized by an unusual resistance to conventional decontamination processes. Moreover, their large tissue distribution and the ability of prions to attach to many surfaces raised the risk of transmission in health care facilities. It is therefore of major importance that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated for prion inactivation. We previously described an in vitro assay, which allowed us to classify accurately prion decontamination treatments according to their efficacy on variant Creutzfeldt-Jakob disease. The significance of this study is in demonstrating the concordance between previous in vitro results and infectivity studies in transgenic mice. Furthermore, commercial reagents currently used in hospitals were tested by both protocols, and we observed that most of them were ineffective on human prions.
Assuntos
Síndrome de Creutzfeldt-Jakob/patologia , Descontaminação/métodos , Contaminação de Equipamentos , Proteínas Priônicas/química , Animais , Síndrome de Creutzfeldt-Jakob/etiologia , Feminino , Humanos , Doença Iatrogênica , Camundongos , Camundongos Transgênicos , Deficiências na Proteostase/patologiaRESUMO
In peripherally acquired prion diseases, prions move through several tissues of the infected host, notably in the lymphoid tissue, long before the occurrence of neuroinvasion. Accumulation can even be restricted to the lymphoid tissue without neuroinvasion and clinical disease. Several experimental observations indicated that the presence of differentiated follicular dendritic cells (FDCs) in the lymphoid structures and the strain type are critical determinants of prion extraneural replication. In this context, the report that granulomatous structures apparently devoid of FDCs could support prion replication raised the question of the requirements for prion lymphotropism. The report also raised the possibility that nonlymphoid tissue-tropic prions could actually target these inflammatory structures. To investigate these issues, we examined the capacity of closely related prions, albeit with opposite lymphotropism (or FDC dependency), for establishment in experimentally-induced granuloma in ovine PrP transgenic mice. We found a positive correlation between the prion capacity to accumulate in the lymphoid tissue and granuloma, regardless of the prion detection method used. Surprisingly, we also revealed that the accumulation of prions in granulomas involved lymphoid-like structures associated with the granulomas and containing cells that stain positive for PrP, Mfge-8 but not CD45 that strongly suggest FDCs. These results suggest that the FDC requirement for prion replication in lymphoid/inflammatory tissues may be strain-dependent.
Assuntos
Células Dendríticas Foliculares/metabolismo , Granuloma/patologia , Doenças Priônicas/patologia , Proteínas Priônicas/metabolismo , Animais , Antígenos de Superfície/metabolismo , Modelos Animais de Doenças , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Leite/metabolismo , Proteínas Priônicas/genética , Proteínas Priônicas/isolamento & purificação , Proteínas Priônicas/toxicidade , Dobramento de Proteína , Ovinos , Baço/citologia , TropismoRESUMO
The dynamics of aggregation and structural diversification of misfolded, host-encoded proteins in neurodegenerative diseases are poorly understood. In many of these disorders, including Alzheimer's, Parkinson's and prion diseases, the misfolded proteins are self-organized into conformationally distinct assemblies or strains. The existence of intrastrain structural heterogeneity is increasingly recognized. However, the underlying processes of emergence and coevolution of structurally distinct assemblies are not mechanistically understood. Here, we show that early prion replication generates two subsets of structurally different assemblies by two sequential processes of formation, regardless of the strain considered. The first process corresponds to a quaternary structural convergence, by reducing the parental strain polydispersity to generate small oligomers. The second process transforms these oligomers into larger ones, by a secondary autocatalytic templating pathway requiring the prion protein. This pathway provides mechanistic insights into prion structural diversification, a key determinant for prion adaptation and toxicity.
Assuntos
Proteínas Priônicas/química , Multimerização Proteica , Animais , Simulação por Computador , Humanos , Cinética , Camundongos , Modelos Moleculares , Proteínas Priônicas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica , OvinosRESUMO
Prions are proteinaceous infectious agents responsible for a range of neurodegenerative diseases in animals and humans. Prion particles are assemblies formed from a misfolded, ß-sheet rich, aggregation-prone isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). Prions replicate by recruiting and converting PrPC into PrPSc, by an autocatalytic process. PrPSc is a pleiomorphic protein as different conformations can dictate different disease phenotypes in the same host species. This is the basis of the strain phenomenon in prion diseases. Recent experimental evidence suggests further structural heterogeneity in PrPSc assemblies within specific prion populations and strains. Still, this diversity is rather seen as a size continuum of assemblies with the same core structure, while analysis of the available experimental data points to the existence of structurally distinct arrangements. The atomic structure of PrPSc has not been elucidated so far, making the prion replication process difficult to understand. All currently available models suggest that PrPSc assemblies exhibit a PrPSc subunit as core constituent, which was recently identified. This review summarizes our current knowledge on prion assembly heterogeneity down to the subunit level and will discuss its importance with regard to the current molecular principles of the prion replication process.
Assuntos
Doenças Neurodegenerativas/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Animais , Humanos , Doenças Neurodegenerativas/genética , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPSc/química , Proteínas PrPSc/genética , Dobramento de ProteínaRESUMO
Among the ever-growing number of self-replicating proteins involved in neurodegenerative diseases, the prion protein PrP remains the most infamous for its central role in transmissible spongiform encephalopathies (TSEs). In these diseases, pathogenic prions propagate through a seeding mechanism, where normal PrPC molecules are converted into abnormally folded scrapie isoforms termed PrPSc. Since its discovery over 30 years ago, much advance has contributed to define the host-encoded cellular prion protein PrPC as a critical relay of prion-induced neuronal cell demise. A current consensual view is that the conversion of PrPC into PrPSc in neuronal cells diverts the former from its normal function with subsequent molecular alterations affecting synaptic plasticity. Here, we report that prion infection is associated with reduced expression of key effectors of the Notch pathway in vitro and in vivo, recapitulating changes fostered by the absence of PrPC. We further show that both prion infection and PrPC depletion promote drastic alterations in the expression of a defined set of Eph receptors and their ephrin ligands, which represent important players in synaptic function. Our data indicate that defects in the Notch and Eph axes can be mitigated in response to histone deacetylase inhibition in PrPC-depleted as well as prion-infected cells. We thus conclude that infectious prions cause a loss-of-function phenotype with respect to Notch and Eph signaling and that these alterations are sustained by epigenetic mechanisms.
Assuntos
Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Receptores da Família Eph/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Epigênese Genética , Camundongos , Neurônios/metabolismo , Doenças Priônicas/genéticaRESUMO
Mammalian prions, the pathogens that cause transmissible spongiform encephalopathies, propagate by self-perpetuating the structural information stored in the abnormally folded, aggregated conformer (PrPSc) of the host-encoded prion protein (PrPC). To date, no structural model related to prion assembly organization satisfactorily describes how strain-specified structural information is encoded and by which mechanism this information is transferred to PrPC. To achieve progress on this issue, we correlated the PrPSc quaternary structural transition from three distinct prion strains during unfolding and refolding with their templating activity. We reveal the existence of a mesoscopic organization in PrPSc through the packing of a highly stable oligomeric elementary subunit (suPrP), in which the strain structural determinant (SSD) is encoded. Once kinetically trapped, this elementary subunit reversibly loses all replicative information. We demonstrate that acquisition of the templating interface and infectivity requires structural rearrangement of suPrP, in concert with its condensation. The existence of such an elementary brick scales down the SSD support to a small oligomer and provide a basis of reflexion for prion templating process and propagation.
Assuntos
Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Desdobramento de Proteína , Animais , Doenças Transmissíveis , Camundongos , Conformação ProteicaRESUMO
Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a ß-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrPC structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrPC nor for the formation of a stable prion structure.
Assuntos
Príons/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Camundongos , Modelos Moleculares , Príons/patogenicidade , Conformação Proteica , VirulênciaRESUMO
Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission.
Assuntos
Evolução Molecular , Regulação da Expressão Gênica/fisiologia , Proteínas PrPC/metabolismo , Animais , Genótipo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas PrPC/genética , OvinosRESUMO
Prions are proteinaceous pathogens responsible for subacute spongiform encephalopathies in animals and humans. The prions responsible for bovine spongiform encephalopathy (BSE) are zoonotic agents, causing variant Creutzfeldt-Jakob disease (CJD) in humans. The transfer of prions between species is limited by a species barrier, which is thought to reflect structural incompatibilities between the host cellular prion protein (PrPC) and the infecting pathological PrP assemblies (PrPSc) constituting the prion. A BSE strain variant, designated L-BSE and responsible for atypical, supposedly spontaneous forms of prion diseases in aged cattle, demonstrates zoonotic potential, as evidenced by its capacity to propagate more easily than classical BSE in transgenic mice expressing human PrPC and in nonhuman primates. In humanized mice, L-BSE propagates without any apparent species barrier and shares similar biochemical PrPSc signatures with the CJD subtype designated MM2-cortical, thus opening the possibility that certain CJD cases classified as sporadic may actually originate from L-type BSE cross-transmission. To address this issue, we compared the biological properties of L-BSE and those of a panel of CJD subtypes representative of the human prion strain diversity using standard strain-typing criteria in human PrP transgenic mice. We found no evidence that L-BSE causes a known form of sporadic CJD. IMPORTANCE: Since the quasi-extinction of classical BSE, atypical BSE forms are the sole BSE variants circulating in cattle worldwide. They are observed in rare cases of old cattle, making them difficult to detect. Extrapolation of our results suggests that L-BSE may propagate in humans as an unrecognized form of CJD, and we urge both the continued utilization of precautionary measures to eliminate these agents from the human food chain and active surveillance for CJD phenotypes in the general population.
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Síndrome de Creutzfeldt-Jakob/genética , Encefalopatia Espongiforme Bovina/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Bovinos , Síndrome de Creutzfeldt-Jakob/etiologia , Síndrome de Creutzfeldt-Jakob/transmissão , Modelos Animais de Doenças , Encefalopatia Espongiforme Bovina/etiologia , Encefalopatia Espongiforme Bovina/transmissão , Variação Genética , Especificidade de Hospedeiro , Humanos , Camundongos , Camundongos Transgênicos , Proteínas PrPC/genética , Proteínas PrPC/patogenicidade , Proteínas PrPSc/genética , Proteínas PrPSc/patogenicidadeRESUMO
Prions are formed of misfolded assemblies (PrP(Sc)) of the variably N-glycosylated cellular prion protein (PrP(C)). In infected species, prions replicate by seeding the conversion and polymerization of host PrP(C). Distinct prion strains can be recognized, exhibiting defined PrP(Sc) biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrP(Sc) assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrP(C) glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrP(C) species of interest as substrate. Applying the technique to PrP(C) glycosylation mutants expressing cells revealed that neither PrP(C) nor PrP(Sc) glycoform stoichiometry was instrumental to PrP(Sc) formation and strainness perpetuation. Our study supports the view that strain properties, including PrP(Sc) glycotype are enciphered within PrP(Sc) structural backbone, not in the attached glycans.
Assuntos
Bioquímica/métodos , Príons/metabolismo , Dobramento de Proteína , Animais , Encéfalo/metabolismo , Extratos Celulares , Linhagem Celular , Células Cultivadas , Eletroforese , Técnicas de Inativação de Genes , Glicosilação , Humanos , Camundongos Transgênicos , Microesferas , Miniaturização , Proteínas Mutantes/metabolismo , Fatores de TempoRESUMO
UNLABELLED: Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions. IMPORTANCE: Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.
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Células Epiteliais/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Scrapie/metabolismo , Deleção de Sequência , Sequência de Aminoácidos , Animais , Camundongos , Camundongos Transgênicos , Proteínas PrPC/química , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ovinos , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: Mammalian prions are proteinaceous pathogens responsible for a broad range of fatal neurodegenerative diseases in humans and animals. These diseases can occur spontaneously, such as Creutzfeldt-Jakob disease (CJD) in humans, or be acquired or inherited. Prions are primarily formed of macromolecular assemblies of the disease-associated prion protein PrP(Sc), a misfolded isoform of the host-encoded prion protein PrP(C). Within defined host-species, prions can exist as conformational variants or strains. Based on both the M/V polymorphism at codon 129 of PrP and the electrophoretic signature of PrP(Sc) in the brain, sporadic CJD is classified in different subtypes, which may encode different strains. A transmission barrier, the mechanism of which remains unknown, limits prion cross-species propagation. To adapt to the new host, prions have the capacity to 'mutate' conformationally, leading to the emergence of a variant with new biological properties. Here, we transmitted experimentally one rare subtype of human CJD, designated cortical MM2 (129 MM with type 2 PrP(Sc)), to transgenic mice overexpressing either human or the VRQ allele of ovine PrP(C). RESULTS: In marked contrast with the reported absence of transmission to knock-in mice expressing physiological levels of human PrP, this subtype transmitted faithfully to mice overexpressing human PrP, and exhibited unique strain features. Onto the ovine PrP sequence, the cortical MM2 subtype abruptly evolved on second passage, thereby allowing emergence of a pair of strain variants with distinct PrP(Sc) biochemical characteristics and differing tropism for the central and lymphoid tissues. These two strain components exhibited remarkably distinct replicative properties in cell-free amplification assay, allowing the 'physical' cloning of the minor, lymphotropic component, and subsequent isolation in ovine PrP mice and RK13 cells. CONCLUSIONS: Here, we provide in-depth assessment of the transmissibility and evolution of one rare subtype of sporadic CJD upon homologous and heterologous transmission. The notion that the environment or matrix where replication is occurring is key to the selection and preferential amplification of prion substrain components raises new questions on the determinants of prion replication within and between species. These data also further interrogate on the interplay between animal and human prions.
Assuntos
Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Síndrome de Creutzfeldt-Jakob , Polimorfismo Genético/genética , Príons/genética , Animais , Linhagem Celular Transformada , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/transmissão , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Príons/classificação , Dobramento de Proteína , Ovinos , Baço/metabolismo , Baço/patologia , Suínos , TransfecçãoRESUMO
UNLABELLED: Mammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the N-terminal polybasic region of residues 23 to 31 of PrP has been shown to be involved in its endocytic trafficking and interactions with glycosaminoglycans or putative ectodomains of membrane-associated proteins. Several recent reports also describe this PrP region as important for the toxicity of mutant prion proteins and the efficiency of prion propagation, both in vitro and in vivo. The question remains as to whether the latter observations made with mouse PrP and mouse prions would be relevant to other PrP species/prion strain combinations given the dramatic impact on prion susceptibility of minimal amino acid substitutions and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP with a deletion of residues 23 to 26 (KKRP) or mutated in this N-terminal region (KQHPH instead of KKRPK) exhibited a variable, strain-dependent susceptibility to prion infection with regard to the proportion of affected mice and disease tempo relative to findings in their wild-type counterparts. Deletion has no major effect on 127S scrapie prion pathogenesis, whereas mutation increased by almost 3-fold the survival time of the mice. Deletion marginally affected the incubation time of scrapie LA19K and ovine bovine spongiform encephalopathy (BSE) prions, whereas mutation caused apparent resistance to disease. IMPORTANCE: Recent reports suggested that the N-terminal polybasic region of the prion protein could be a therapeutic target to prevent prion propagation or toxic signaling associated with more common neurodegenerative diseases such as Alzheimer's disease. Mutating or deleting this region in ovine PrP completes the data previously obtained with the mouse protein by identifying the key amino acid residues involved.
Assuntos
Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Doenças Priônicas/patologia , Animais , Modelos Animais de Doenças , Camundongos Transgênicos , Mutação de Sentido Incorreto , Deleção de Sequência , OvinosRESUMO
Although Bovine Spongiform Encephalopathy (BSE) is the cause of variant Creutzfeldt Jakob disease (vCJD) in humans, the zoonotic potential of scrapie prions remains unknown. Mice genetically engineered to overexpress the human prion protein (tgHu) have emerged as highly relevant models for gauging the capacity of prions to transmit to humans. These models can propagate human prions without any apparent transmission barrier and have been used used to confirm the zoonotic ability of BSE. Here we show that a panel of sheep scrapie prions transmit to several tgHu mice models with an efficiency comparable to that of cattle BSE. The serial transmission of different scrapie isolates in these mice led to the propagation of prions that are phenotypically identical to those causing sporadic CJD (sCJD) in humans. These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.