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1.
J Biol Chem ; 298(4): 101834, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35304100

RESUMO

Chronic wasting disease (CWD) is an invariably fatal prion disease affecting cervid species worldwide. Prions can manifest as distinct strains that can influence disease pathology and transmission. CWD is profoundly lymphotropic, and most infected cervids likely shed peripheral prions replicated in lymphoid organs. However, CWD is a neurodegenerative disease, and most research on prion strains has focused on neurogenic prions. Thus, a knowledge gap exists comparing neurogenic prions to lymphogenic prions. In this study, we compared prions from the obex and lymph nodes of naturally exposed white-tailed deer to identify potential biochemical strain differences. Here, we report biochemical evidence of strain differences between the brain and lymph node from these animals. Conformational stability assays, glycoform ratio analyses, and immunoreactivity scanning across the structured domain of the prion protein that refolds into the amyloid aggregate of the infectious prion reveal significantly more structural and glycoform variation in lymphogenic prions than neurogenic prions. Surprisingly, we observed greater biochemical differences among neurogenic prions than lymphogenic prions across individuals. We propose that the lymphoreticular system propagates a diverse array of prions from which the brain selects a more restricted pool of prions that may be quite different than those from another individual of the same species. Future work should examine the biological and zoonotic impact of these biochemical differences and examine more cervids from multiple locations to determine if these differences are conserved across species and locations.


Assuntos
Cervos , Príons , Doença de Emaciação Crônica , Animais , Príons/química , Príons/metabolismo , Doença de Emaciação Crônica/fisiopatologia
2.
Emerg Infect Dis ; 29(2): 323-332, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36692340

RESUMO

Our previous studies using gene-targeted mouse models of chronic wasting disease (CWD) demonstrated that Norway and North America cervids are infected with distinct prion strains that respond differently to naturally occurring amino acid variation at residue 226 of the prion protein. Here we performed transmissions in gene-targeted mice to investigate the properties of prions causing newly emergent CWD in moose in Finland. Although CWD prions from Finland and Norway moose had comparable responses to primary structural differences at residue 226, other distinctive criteria, including transmission kinetics, patterns of neuronal degeneration, and conformational features of prions generated in the brains of diseased mice, demonstrated that the strain properties of Finland moose CWD prions are different from those previously characterized in Norway CWD. Our findings add to a growing body of evidence for a diverse portfolio of emergent strains in Nordic countries that are etiologically distinct from the comparatively consistent strain profile of North America CWD.


Assuntos
Cervos , Príons , Doença de Emaciação Crônica , Animais , Camundongos , Príons/genética , Doença de Emaciação Crônica/epidemiologia , Finlândia/epidemiologia , Proteínas Priônicas/genética
3.
PLoS Pathog ; 17(7): e1009748, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34310663

RESUMO

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.


Assuntos
Proteínas PrPSc/genética , Proteínas Priônicas/genética , Doença de Emaciação Crônica/genética , Doença de Emaciação Crônica/transmissão , Animais , Animais Geneticamente Modificados , Cervos , Camundongos , América do Norte , Noruega
4.
Cell Tissue Res ; 392(1): 33-46, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36929219

RESUMO

While much of what we know about the general principles of protein-based information transfer derives from studies of experimentally adapted rodent prions, these laboratory strains are limited in their ability to recapitulate features of human and animal prions and the diseases they produce. Here, we review how recent approaches using genetically modified mice have informed our understanding of naturally occurring prion diseases, their strain properties, and the factors controlling their transmission and evolution. In light of the increasing importance of chronic wasting disease, the application of mouse transgenesis to study this burgeoning and highly contagious prion disorder, in particular recent insights derived from gene-targeting approaches, will be a major focus of this review.


Assuntos
Cervos , Doenças Priônicas , Príons , Doença de Emaciação Crônica , Camundongos , Animais , Humanos , Camundongos Transgênicos , Doenças Priônicas/genética , Príons/genética , Marcação de Genes , Modelos Animais de Doenças
5.
Proc Natl Acad Sci U S A ; 117(49): 31417-31426, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33229531

RESUMO

Chronic wasting disease (CWD) is a relentless epidemic disorder caused by infectious prions that threatens the survival of cervid populations and raises increasing public health concerns in North America. In Europe, CWD was detected for the first time in wild Norwegian reindeer (Rangifer tarandus) and moose (Alces alces) in 2016. In this study, we aimed at comparing the strain properties of CWD prions derived from different cervid species in Norway and North America. Using a classical strain typing approach involving transmission and adaptation to bank voles (Myodes glareolus), we found that prions causing CWD in Norway induced incubation times, neuropathology, regional deposition of misfolded prion protein aggregates in the brain, and size of their protease-resistant core, different from those that characterize North American CWD. These findings show that CWD prion strains affecting Norwegian cervids are distinct from those found in North America, implying that the highly contagious North American CWD prions are not the proximate cause of the newly discovered Norwegian CWD cases. In addition, Norwegian CWD isolates showed an unexpected strain variability, with reindeer and moose being caused by different CWD strains. Our findings shed light on the origin of emergent European CWD, have significant implications for understanding the nature and the ecology of CWD in Europe, and highlight the need to assess the zoonotic potential of the new CWD strains detected in Europe.


Assuntos
Arvicolinae/fisiologia , Príons/metabolismo , Doença de Emaciação Crônica/epidemiologia , Adaptação Fisiológica , Animais , Encéfalo/patologia , Degeneração Neural/complicações , Degeneração Neural/patologia , América do Norte/epidemiologia , Noruega/epidemiologia , Fenótipo , Especificidade da Espécie , Doença de Emaciação Crônica/complicações , Doença de Emaciação Crônica/transmissão
6.
J Infect Dis ; 225(3): 542-551, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34302479

RESUMO

BACKGROUND: Chronic wasting disease (CWD) is a rapidly spreading prion disorder affecting various species of wild and captive cervids. The risk that CWD poses to cohabiting animals or more importantly to humans is largely unknown. METHODS: In this study, we investigated differences in the capacity of CWD isolates obtained from 6 different cervid species to induce prion conversion in vitro by protein misfolding cyclic amplification. We define and quantify spillover and zoonotic potential indices as the efficiency by which CWD prions sustain prion generation in vitro at expenses of normal prion proteins from various mammals and human, respectively. RESULTS: Our data suggest that reindeer and red deer from Norway could be the most transmissible CWD prions to other mammals, whereas North American CWD prions were more prone to generate human prions in vitro. CONCLUSIONS: Our results suggest that Norway and North American CWD prions correspond to different strains with distinct spillover and zoonotic potentials.


Assuntos
Cervos , Príons , Doença de Emaciação Crônica , Animais , Cervos/metabolismo , Humanos , América do Norte/epidemiologia , Noruega , Príons/metabolismo , Doença de Emaciação Crônica/metabolismo
7.
Proc Natl Acad Sci U S A ; 116(25): 12478-12487, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31147460

RESUMO

Although the unifying hallmark of prion diseases is CNS neurodegeneration caused by conformational corruption of host prion protein (PrP) to its infective counterpart, contagious transmission of chronic wasting disease (CWD) results from shedding of prions produced at high titers in the periphery of diseased cervids. While deer and elk PrP primary structures are equivalent except at residue 226, which is glutamate in elk and glutamine in deer, the effect of this difference on CWD pathogenesis is largely unknown. Using a gene-targeting approach where the mouse PrP coding sequence was replaced with elk or deer PrP, we show that the resulting GtE226 and GtQ226 mice had distinct kinetics of disease onset, prion conformations, and distributions of prions in the brains of diseased mice following intracerebral CWD challenge. These findings indicate that amino acid differences at PrP residue 226 dictate the selection and propagation of divergent strains in deer and elk with CWD. Because prion strain properties largely dictate host-range potential, our findings suggest that prion strains from elk and deer pose distinct risks to sympatric species or humans exposed to CWD. GtE226 and GtQ226 mice were also highly susceptible to CWD prions following intraperitoneal and oral exposures, a characteristic that stood in stark contrast to previously produced transgenic models. Remarkably, disease transmission was effective when infected mice were cohoused with naïve cagemates. Our findings indicate that gene-targeted mice provide unprecedented opportunities to accurately investigate CWD peripheral pathogenesis, CWD strains, and mechanisms of horizontal CWD transmission.


Assuntos
Marcação de Genes , Proteínas Priônicas/química , Proteínas Priônicas/genética , Doença de Emaciação Crônica/genética , Substituição de Aminoácidos , Animais , Cervos , Redes Reguladoras de Genes , Camundongos , Camundongos Transgênicos , Conformação Proteica , Especificidade da Espécie , Doença de Emaciação Crônica/transmissão
8.
J Biol Chem ; 295(30): 10420-10433, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32513872

RESUMO

The causative factors underlying conformational conversion of cellular prion protein (PrPC) into its infectious counterpart (PrPSc) during prion infection remain undetermined, in part because of a lack of monoclonal antibodies (mAbs) that can distinguish these conformational isoforms. Here we show that the anti-PrP mAb PRC7 recognizes an epitope that is shielded from detection when glycans are attached to Asn-196. We observed that whereas PrPC is predisposed to full glycosylation and is therefore refractory to PRC7 detection, prion infection leads to diminished PrPSc glycosylation at Asn-196, resulting in an unshielded PRC7 epitope that is amenable to mAb recognition upon renaturation. Detection of PRC7-reactive PrPSc in experimental and natural infections with various mouse-adapted scrapie strains and with prions causing deer and elk chronic wasting disease and transmissible mink encephalopathy uncovered that incomplete PrPSc glycosylation is a consistent feature of prion pathogenesis. We also show that interrogating the conformational properties of the PRC7 epitope affords a direct means of distinguishing different prion strains. Because the specificity of our approach for prion detection and strain discrimination relies on the extent to which N-linked glycosylation shields or unshields PrP epitopes from antibody recognition, it dispenses with the requirement for additional standard manipulations to distinguish PrPSc from PrPC, including evaluation of protease resistance. Our findings not only highlight an innovative and facile strategy for prion detection and strain differentiation, but are also consistent with a mechanism of prion replication in which structural instability of incompletely glycosylated PrP contributes to the conformational conversion of PrPC to PrPSc.


Assuntos
Anticorpos Monoclonais Murinos/química , Epitopos/química , Proteínas PrPC/química , Animais , Linhagem Celular , Epitopos/metabolismo , Glicosilação , Proteínas PrPC/metabolismo , Coelhos
9.
PLoS Pathog ; 15(10): e1008117, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31644574

RESUMO

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Priônicas/metabolismo , Príons/metabolismo , Animais , Arvicolinae , Sistema Nervoso Central/patologia , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Camundongos Transgênicos , Doenças Priônicas/patologia , Estrutura Terciária de Proteína , Deficiências na Proteostase/patologia
10.
J Biol Chem ; 294(37): 13619-13628, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31320473

RESUMO

Prion diseases are a group of incurable neurodegenerative disorders that affect humans and animals via infection with proteinaceous particles called prions. Prions are composed of PrPSc, a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc As PrPSc accumulates, cellular stress mechanisms are activated to maintain cellular proteostasis, including increased protein chaperone levels. However, the exact roles of several of these chaperones remain unclear. Here, using various methodologies to monitor prion replication (i.e. protein misfolding cyclic amplification and cellular and animal infectivity bioassays), we studied the potential role of the molecular chaperone heat shock protein 70 (HSP70) in prion replication in vitro and in vivo Our results indicated that pharmacological induction of the heat shock response in cells chronically infected with prions significantly decreased PrPSc accumulation. We also found that HSP70 alters prion replication in vitro More importantly, prion infection of mice lacking the genes encoding stress-induced HSP70 exhibited accelerated prion disease progression compared with WT mice. In parallel with HSP70 being known to respond to endogenous and exogenous stressors such as heat, infection, toxicants, and ischemia, our results indicate that HSP70 may also play an important role in suppressing or delaying prion disease progression, opening opportunities for therapeutic intervention.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Doenças Priônicas/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Progressão da Doença , Retículo Endoplasmático/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Feminino , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Priônicas/metabolismo , Príons/metabolismo , Dobramento de Proteína
11.
Proc Natl Acad Sci U S A ; 114(5): 1141-1146, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28096357

RESUMO

Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrPC) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrPC Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrPC conversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrPC was similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrPC from the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrPC also failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders.


Assuntos
Especificidade de Hospedeiro/fisiologia , Proteínas PrPC/fisiologia , Doenças Priônicas/transmissão , Doenças Priônicas/veterinária , Príons/fisiologia , Animais , Cervos , Guanidina/farmacologia , Cavalos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPC/química , Proteínas PrPC/genética , Príons/química , Conformação Proteica , Desnaturação Proteica , Coelhos , Ovinos , Especificidade da Espécie , Relação Estrutura-Atividade
12.
Dev Biol ; 438(1): 23-32, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29577883

RESUMO

The cellular prion protein (PrPC) has been associated with diverse biological processes including cell signaling, neurogenesis, and neuroprotection, but its physiological function(s) remain ambiguous. Here we determine the role of PrPC in adult neurogenesis using the olfactory system model in transgenic mice. Olfactory sensory neurons (OSNs) within the olfactory sensory epithelium (OSE) undergo neurogenesis, integration, and turnover even into adulthood. The neurogenic processes of proliferation, differentiation/maturation, and axon targeting were evaluated in wild type, PrP-overexpressing, and PrP-null transgenic mice. Our results indicate that PrPC plays a role in maintaining mature OSNs within the epithelium: overexpression of PrPC resulted in greater survival of mitotically active cells within the OSE, whereas absence of prion protein resulted in fewer cells being maintained over time. These results are supported by both quantitative PCR analysis of gene expression and protein analysis characteristic of OSN differentiation. Finally, evaluation of axon migration determined that OSN axon targeting in the olfactory bulb is PrPC dose-dependent. Together, these findings provide new mechanistic insight into the neuroprotective role for PrPC in adult OSE neurogenesis, whereby more mature neurons are stably maintained in animals expressing PrPC.


Assuntos
Axônios/fisiologia , Neurogênese/genética , Neurônios Receptores Olfatórios/metabolismo , Proteínas PrPC/genética , Animais , Axônios/metabolismo , Western Blotting , Diferenciação Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Neurogênese/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Proteínas PrPC/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
13.
J Immunol ; 199(11): 3821-3827, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29070671

RESUMO

Several complement proteins exacerbate prion disease, including C3, C1q, and CD21/35. These proteins of the complement cascade likely increase uptake, trafficking, and retention of prions in the lymphoreticular system, hallmark sites of early prion propagation. Complement regulatory protein factor H (fH) binds modified host proteins and lipids to prevent C3b deposition and, thus, autoimmune cell lysis. Previous reports show that fH binds various conformations of the cellular prion protein, leading us to question the role of fH in prion disease. In this article, we report that transgenic mice lacking Cfh alleles exhibit delayed peripheral prion accumulation, replication, and pathogenesis and onset of terminal disease in a gene-dose manner. We also report a biophysical interaction between purified fH and prion rods enriched from prion-diseased brain. fH also influences prion deposition in brains of infected mice. We conclude from these data and previous findings that the interplay between complement and prions likely involves a complex balance of prion sequestration and destruction via local tissue macrophages, prion trafficking by B and dendritic cells within the lymphoreticular system, intranodal prion replication by B and follicular dendritic cells, and potential prion strain selection by CD21/35 and fH. These findings reveal a novel role for complement-regulatory proteins in prion disease.


Assuntos
Linfócitos B/imunologia , Encéfalo/metabolismo , Fator H do Complemento/metabolismo , Células Dendríticas/imunologia , Macrófagos/imunologia , Doenças Priônicas/imunologia , Príons/imunologia , Animais , Encéfalo/patologia , Células Cultivadas , Fator H do Complemento/genética , Inativadores do Complemento , Via Alternativa do Complemento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Priônicas/genética , Ligação Proteica
14.
J Gen Virol ; 99(5): 753-758, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29580373

RESUMO

The prevalence, host range and geographical bounds of chronic wasting disease (CWD), the prion disease of cervids, are expanding. Horizontal transmission likely contributes the majority of new CWD cases, but the mechanism by which prions are transmitted among CWD-affected cervids remains unclear. To address the extent to which prion amplification in peripheral tissues contributes to contagious transmission, we assessed the prion levels in central nervous and lymphoreticular system tissues in white-tailed deer (Odocoileus virginianus), red deer (Cervus elaphus elaphus) and elk (Cervus canadensis). Using real-time quaking-induced conversion, cervid prion cell assay and transgenic mouse bioassay, we found that the retropharyngeal lymph nodes of red deer, white-tailed deer and elk contained similar prion titres to brain from the same individuals. We propose that marked lymphotropism is essential for the horizontal transmission of prion diseases and postulate that shed CWD prions are produced in the periphery.


Assuntos
Transmissão de Doença Infecciosa/veterinária , Príons/patogenicidade , Doença de Emaciação Crônica/patologia , Animais , Encéfalo/patologia , Cervos , Tecido Linfoide/patologia , Camundongos , Camundongos Transgênicos , Príons/isolamento & purificação , Doença de Emaciação Crônica/transmissão
15.
16.
Proc Natl Acad Sci U S A ; 111(16): 6028-33, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24711410

RESUMO

Quinacrine's ability to reduce levels of pathogenic prion protein (PrP(Sc)) in mouse cells infected with experimentally adapted prions led to several unsuccessful clinical studies in patients with prion diseases, a 10-y investment to understand its mechanism of action, and the production of related compounds with expectations of greater efficacy. We show here, in stark contrast to this reported inhibitory effect, that quinacrine enhances deer and elk PrP(Sc) accumulation and promotes propagation of prions causing chronic wasting disease (CWD), a fatal, transmissible, neurodegenerative disorder of cervids of uncertain zoonotic potential. Surprisingly, despite increased prion titers in quinacrine-treated cells, transmission of the resulting prions produced prolonged incubation times and altered PrP(Sc) deposition patterns in the brains of diseased transgenic mice. This unexpected outcome is consistent with quinacrine affecting the intrinsic properties of the CWD prion. Accordingly, quinacrine-treated CWD prions were comprised of an altered PrP(Sc) conformation. Our findings provide convincing evidence for drug-induced conformational mutation of prions without the prerequisite of generating drug-resistant variants of the original strain. More specifically, they show that a drug capable of restraining prions in one species/strain setting, and consequently used to treat human prion diseases, improves replicative ability in another and therefore force reconsideration of current strategies to screen antiprion compounds.


Assuntos
Mutação/genética , Príons/química , Príons/genética , Quinacrina/farmacologia , Doença de Emaciação Crônica/patologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cervos , Humanos , Camundongos , Proteínas PrPSc/metabolismo , Doenças Priônicas/patologia , Doenças Priônicas/transmissão , Conformação Proteica
17.
Proc Natl Acad Sci U S A ; 111(30): 11169-74, 2014 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-25034251

RESUMO

Understanding the molecular parameters governing prion propagation is crucial for controlling these lethal, proteinaceous, and infectious neurodegenerative diseases. To explore the effects of prion protein (PrP) sequence and structural variations on intra- and interspecies transmission, we integrated studies in deer, a species naturally susceptible to chronic wasting disease (CWD), a burgeoning, contagious epidemic of uncertain origin and zoonotic potential, with structural and transgenic (Tg) mouse modeling and cell-free prion amplification. CWD properties were faithfully maintained in deer following passage through Tg mice expressing cognate PrP, and the influences of naturally occurring PrP polymorphisms on CWD susceptibility were accurately reproduced in Tg mice or cell-free systems. Although Tg mice also recapitulated susceptibility of deer to sheep prions, polymorphisms that provided protection against CWD had distinct and varied influences. Whereas substitutions at residues 95 and 96 in the unstructured region affected CWD propagation, their protective effects were overridden during replication of sheep prions in Tg mice and, in the case of residue 96, deer. The inhibitory effects on sheep prions of glutamate at residue 226 in elk PrP, compared with glutamine in deer PrP, and the protective effects of the phenylalanine for serine substitution at the adjacent residue 225, coincided with structural rearrangements in the globular domain affecting interaction between α-helix 3 and the loop between ß2 and α-helix 2. These structure-function analyses are consistent with previous structural investigations and confirm a role for plasticity of this tertiary structural epitope in the control of PrP conversion and strain propagation.


Assuntos
Polimorfismo Genético , Proteínas PrPSc/genética , Substituição de Aminoácidos , Animais , Cervos , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Proteínas PrPSc/metabolismo , Estrutura Secundária de Proteína , Ovinos , Doenças dos Ovinos/genética , Doenças dos Ovinos/metabolismo , Doença de Emaciação Crônica/genética , Doença de Emaciação Crônica/metabolismo
18.
J Virol ; 89(18): 9524-31, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26157118

RESUMO

UNLABELLED: The propensity for transspecies prion transmission is related to the structural characteristics of the enciphering and new host PrP, although the exact mechanism remains incompletely understood. The effects of variability in prion protein on cross-species prion transmission have been studied with animal bioassays, but the influence of prion protein structure versus that of host cofactors (e.g., cellular constituents, trafficking, and innate immune interactions) remains difficult to dissect. To isolate the effects of protein-protein interactions on transspecies conversion, we used recombinant PrP(C) and real-time quaking-induced conversion (RT-QuIC) and compared chronic wasting disease (CWD) and classical bovine spongiform encephalopathy (cBSE) prions. To assess the impact of transmission to a new species, we studied feline CWD (fCWD) and feline BSE (i.e., feline spongiform encephalopathy [FSE]). We cross-seeded fCWD and FSE into each species' full-length, recombinant PrP(C) and measured the time required for conversion to the amyloid (PrP(Res)) form, which we describe here as the rate of amyloid conversion. These studies revealed the following: (i) CWD and BSE seeded their homologous species' PrP best; (ii) fCWD was a more efficient seed for feline rPrP than for white-tailed deer rPrP; (iii) conversely, FSE more efficiently converted bovine than feline rPrP; (iv) and CWD, fCWD, BSE, and FSE all converted human rPrP, although not as efficiently as homologous sCJD prions. These results suggest that (i) at the level of protein-protein interactions, CWD adapts to a new species more readily than does BSE and (ii) the barrier preventing transmission of CWD to humans may be less robust than estimated. IMPORTANCE: We demonstrate that bovine spongiform encephalopathy prions maintain their transspecies conversion characteristics upon passage to cats but that chronic wasting disease prions adapt to the cat and are distinguishable from the original prion. Additionally, we showed that chronic wasting disease prions are effective at seeding the conversion of normal human prion protein to an amyloid conformation, perhaps the first step in crossing the species barrier.


Assuntos
Síndrome de Creutzfeldt-Jakob , Encefalopatia Espongiforme Bovina , Proteínas PrPC , Doença de Emaciação Crônica , Animais , Arvicolinae , Gatos , Bovinos , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/transmissão , Encefalopatia Espongiforme Bovina/metabolismo , Encefalopatia Espongiforme Bovina/transmissão , Humanos , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPC/patogenicidade , Especificidade da Espécie , Doença de Emaciação Crônica/metabolismo , Doença de Emaciação Crônica/transmissão
19.
J Virol ; 88(3): 1830-3, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24257620

RESUMO

Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536(+/-), to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536(+/-) mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer.


Assuntos
Cervos/metabolismo , Modelos Animais de Doenças , Encefalopatia Espongiforme Bovina/metabolismo , Camundongos , Príons/metabolismo , Doença de Emaciação Crônica/metabolismo , Animais , Bovinos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Suscetibilidade a Doenças , Encefalopatia Espongiforme Bovina/patologia , Encefalopatia Espongiforme Bovina/transmissão , Feminino , Masculino , Camundongos Transgênicos , Especificidade da Espécie , Doença de Emaciação Crônica/patologia , Doença de Emaciação Crônica/transmissão
20.
PLoS Pathog ; 9(10): e1003692, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24204258

RESUMO

Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer's and Parkinson's diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg) mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A) at (OvPrP-A136) infected with SSBP/1 scrapie prions propagated a relatively stable (S) prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V) at 136 (OvPrP-V136) infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U), diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb) PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to the otherwise unfavorable U conformer. This epigenetic mechanism thus expands the range of selectable conformations that can be adopted by PrP, and therefore the variety of options for strain propagation.


Assuntos
Epigênese Genética , Mutação de Sentido Incorreto , Proteínas PrPSc/biossíntese , Doenças Priônicas/metabolismo , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/farmacologia , Bovinos , Humanos , Camundongos , Camundongos Transgênicos , Proteínas PrPSc/genética , Doenças Priônicas/genética , Doenças Priônicas/patologia , Ovinos
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