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1.
Vet Res ; 55(1): 62, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38750594

RESUMEN

The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrPSc immunohistochemistry staining in the brainstem and the absence of detectable PrPSc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.


Asunto(s)
Ciervos , Encefalopatía Espongiforme Bovina , Enfermedad Debilitante Crónica , Animales , Noruega , Western Blotting/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Priones/metabolismo , Bovinos , Inmunohistoquímica/veterinaria , Proteínas PrPSc/metabolismo
2.
PLoS Pathog ; 15(10): e1008117, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31644574

RESUMEN

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.


Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Priónicas/metabolismo , Priones/metabolismo , Animales , Arvicolinae , Sistema Nervioso Central/patología , Sulfato de Dextran/farmacología , Modelos Animales de Enfermedad , Ratones Transgénicos , Enfermedades por Prión/patología , Estructura Terciaria de Proteína , Deficiencias en la Proteostasis/patología
3.
FASEB J ; 34(3): 3969-3982, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31944411

RESUMEN

Unlike other species, prion disease has never been described in dogs even though they were similarly exposed to the bovine spongiform encephalopathy (BSE) agent. This resistance prompted a thorough analysis of the canine PRNP gene and the presence of a negatively charged amino acid residue in position 163 was readily identified as potentially fundamental as it differed from all known susceptible species. In the present study, the first transgenic mouse model expressing dog prion protein (PrP) was generated and challenged intracerebrally with a panel of prion isolates, none of which could infect them. The brains of these mice were subjected to in vitro prion amplification and failed to find even minimal amounts of misfolded prions providing definitive experimental evidence that dogs are resistant to prion disease. Subsequently, a second transgenic model was generated in which aspartic acid in position 163 was substituted for asparagine (the most common in prion susceptible species) resulting in susceptibility to BSE-derived isolates. These findings strongly support the hypothesis that the amino acid residue at position 163 of canine cellular prion protein (PrPC ) is a major determinant of the exceptional resistance of the canidae family to prion infection and establish this as a promising therapeutic target for prion diseases.


Asunto(s)
Ácido Aspártico/química , Ácido Glutámico/química , Priones/química , Priones/patogenicidad , Animales , Bioensayo , Encéfalo/patología , Perros , Ratones , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo
4.
Vet Res ; 52(1): 57, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33858518

RESUMEN

The diversity of goat scrapie strains in Europe has recently been studied using bioassays in a wide collection of rodent models, resulting in the classification of classical scrapie into four different categories. However, the sole use of the first passage does not lead to isolate adaptation and identification of the strains involved and might therefore lead to misclassification of some scrapie isolates. Therefore, this work reports the complete transmission study of a wide collection of goat transmissible spongiform encephalopathy (TSE) isolates by intracranial inoculation in two transgenic mouse lines overexpressing either small ruminant (TgGoat-ARQ) or bovine (TgBov) PrPC. To compare scrapie strains in sheep and goats, sheep scrapie isolates from different European countries were also included in the study. Once the species barrier phenomenon was overcome, an accurate classification of the isolates was attained. Thus, the use of just two rodent models allowed us to fully differentiate at least four different classical scrapie strains in small ruminants and to identify isolates containing mixtures of strains. This work reinforces the idea that classical scrapie in small ruminants is a prion disease caused by multiple different prion strains and not by a single strain, as is the case for epidemic classical bovine spongiform encephalopathy (BSE-C). In addition, the clear dissimilarity between the different scrapie strains and BSE-C does not support the idea that classical scrapie is the origin of epidemic BSE-C.


Asunto(s)
Enfermedades de las Cabras/etiología , Priones/efectos adversos , Scrapie/etiología , Enfermedades de las Ovejas/etiología , Animales , Europa (Continente) , Cabras , Ovinos , Oveja Doméstica
5.
Int J Mol Sci ; 22(1)2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-33466523

RESUMEN

Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrPC to its abnormal and misfolded isoform PrPSc is the main event in the pathogenesis of prion diseases of all origins. In spontaneous prion diseases, the mechanisms that trigger the formation of PrPSc in the central nervous system remain unknown. Several reports have demonstrated that the accumulation of PrPSc can induce endoplasmic reticulum (ER) stress and proteasome impairment from the early stages of the prion disease. Both mechanisms lead to an increment of PrP aggregates in the secretory pathway, which could explain the pathogenesis of spontaneous prion diseases. Here, we investigate the role of ER stress and proteasome impairment during prion disorders in a murine model of spontaneous prion disease (TgVole) co-expressing the UbG76V-GFP reporter, which allows measuring the proteasome activity in vivo. Spontaneously prion-affected mice showed a significantly higher accumulation of the PKR-like ER kinase (PERK), the ER chaperone binding immunoglobulin protein (BiP/Grp78), the ER protein disulfide isomerase (PDI) and the UbG76V-GFP reporter than age-matched controls in certain brain areas. The upregulation of PERK, BiP, PDI and ubiquitin was detected from the preclinical stage of the disease, indicating that ER stress and proteasome impairment begin at early stages of the spontaneous disease. Strong correlations were found between the deposition of these markers and neuropathological markers of prion disease in both preclinical and clinical mice. Our results suggest that both ER stress and proteasome impairment occur during the pathogenesis of spontaneous prion diseases.


Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas Priónicas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Femenino , Masculino , Ratones , Enfermedades por Prión/metabolismo , Transporte de Proteínas/fisiología , Ubiquitina/metabolismo
6.
PLoS Pathog ; 14(1): e1006802, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29357384

RESUMEN

Increasing evidence indicates that microRNAs (miRNAs) are contributing factors to neurodegeneration. Alterations in miRNA signatures have been reported in several neurodegenerative dementias, but data in prion diseases are restricted to ex vivo and animal models. The present study identified significant miRNA expression pattern alterations in the frontal cortex and cerebellum of sporadic Creutzfeldt-Jakob disease (sCJD) patients. These changes display a highly regional and disease subtype-dependent regulation that correlates with brain pathology. We demonstrate that selected miRNAs are enriched in sCJD isolated Argonaute(Ago)-binding complexes in disease, indicating their incorporation into RNA-induced silencing complexes, and further suggesting their contribution to disease-associated gene expression changes. Alterations in the miRNA-mRNA regulatory machinery and perturbed levels of miRNA biogenesis key components in sCJD brain samples reported here further implicate miRNAs in sCJD gene expression (de)regulation. We also show that a subset of sCJD-altered miRNAs are commonly changed in Alzheimer's disease, dementia with Lewy bodies and fatal familial insomnia, suggesting potential common mechanisms underlying these neurodegenerative processes. Additionally, we report no correlation between brain and cerebrospinal fluid (CSF) miRNA-profiles in sCJD, indicating that CSF-miRNA profiles do not faithfully mirror miRNA alterations detected in brain tissue of human prion diseases. Finally, utilizing a sCJD MM1 mouse model, we analyzed the miRNA deregulation patterns observed in sCJD in a temporal manner. While fourteen sCJD-related miRNAs were validated at clinical stages, only two of those were changed at early symptomatic phase, suggesting that the miRNAs altered in sCJD may contribute to later pathogenic processes. Altogether, the present work identifies alterations in the miRNA network, biogenesis and miRNA-mRNA silencing machinery in sCJD, whereby contributions to disease mechanisms deserve further investigation.


Asunto(s)
Síndrome de Creutzfeldt-Jakob/clasificación , Síndrome de Creutzfeldt-Jakob/genética , MicroARNs/genética , Interferencia de ARN , Transcriptoma , Adulto , Anciano , Anciano de 80 o más Años , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Síndrome de Creutzfeldt-Jakob/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , MicroARNs/biosíntesis , Persona de Mediana Edad
7.
PLoS Pathog ; 14(1): e1006797, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29385212

RESUMEN

Very solid evidence suggests that the core of full length PrPSc is a 4-rung ß-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the ß-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of ß-strands, helping us to predict the threading of the ß-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.


Asunto(s)
Encéfalo/metabolismo , Proteínas PrPSc/química , Priones/química , Proteolisis , Proteínas Recombinantes/química , Animales , Arvicolinae , Femenino , Ratones , Ratones Transgénicos , Proteínas PrPSc/metabolismo , Priones/metabolismo , Estructura Secundaria de Proteína
8.
Proc Natl Acad Sci U S A ; 114(5): 1141-1146, 2017 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-28096357

RESUMEN

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.


Asunto(s)
Especificidad del Huésped/fisiología , Proteínas PrPC/fisiología , Enfermedades por Prión/transmisión , Enfermedades por Prión/veterinaria , Priones/fisiología , Animales , Ciervos , Guanidina/farmacología , Caballos , Ratones , Ratones Endogámicos C57BL , Proteínas PrPC/química , Proteínas PrPC/genética , Priones/química , Conformación Proteica , Desnaturalización Proteica , Conejos , Ovinos , Especificidad de la Especie , Relación Estructura-Actividad
9.
PLoS Pathog ; 13(11): e1006716, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29131852

RESUMEN

One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different in vitro and in vivo models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the Leporidae and Equidae families. Here we undertake in vitro and in vivo approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis in silico, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel transgenic mouse model carrying this substitution and these mice showed complete resistance to disease despite intracerebral challenge with three different mouse prion strains (RML, 22L and 301C) known to cause disease in wild-type mice. These findings suggest that dog D163 amino acid is primarily, if not totally, responsible for the prion resistance of canids.


Asunto(s)
Canidae/inmunología , Proteínas PrPC/química , Enfermedades por Prión/veterinaria , Secuencia de Aminoácidos , Animales , Antílopes , Encéfalo/patología , Gatos , Bovinos , Quirópteros , Ciervos , Resistencia a la Enfermedad , Perros , Encefalopatía Espongiforme Bovina/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas PrPC/ultraestructura , Enfermedades por Prión/inmunología , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Conejos , Alineación de Secuencia , Ovinos , Electricidad Estática , Xenarthra
10.
J Virol ; 91(24)2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-28978705

RESUMEN

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of rare progressive neurodegenerative disorders caused by an abnormally folded prion protein (PrPSc). This is capable of transforming the normal cellular prion protein (PrPC) into new infectious PrPSc Interspecies prion transmissibility studies performed by experimental challenge and the outbreak of bovine spongiform encephalopathy that occurred in the late 1980s and 1990s showed that while some species (sheep, mice, and cats) are readily susceptible to TSEs, others are apparently resistant (rabbits, dogs, and horses) to the same agent. To study the mechanisms of low susceptibility to TSEs of certain species, the mouse-rabbit transmission barrier was used as a model. To identify which specific amino acid residues determine high or low susceptibility to PrPSc propagation, protein misfolding cyclic amplification (PMCA), which mimics PrPC-to-PrPSc conversion with accelerated kinetics, was used. This allowed amino acid substitutions in rabbit PrP and accurate analysis of misfolding propensities. Wild-type rabbit recombinant PrP could not be misfolded into a protease-resistant self-propagating isoform in vitro despite seeding with at least 12 different infectious prions from diverse origins. Therefore, rabbit recombinant PrP mutants were designed to contain every single amino acid substitution that distinguishes rabbit recombinant PrP from mouse recombinant PrP. Key amino acid residue substitutions were identified that make rabbit recombinant PrP susceptible to misfolding, and using these, protease-resistant misfolded recombinant rabbit PrP was generated. Additional studies characterized the mechanisms by which these critical amino acid residue substitutions increased the misfolding susceptibility of rabbit PrP.IMPORTANCE Prion disorders are invariably fatal, untreatable diseases typically associated with long incubation periods and characteristic spongiform changes associated with neuronal loss in the brain. Development of any treatment or preventative measure is dependent upon a detailed understanding of the pathogenesis of these diseases, and understanding the mechanism by which certain species appear to be resistant to TSEs is critical. Rabbits are highly resistant to naturally acquired TSEs, and even under experimental conditions, induction of clinical disease is not easy. Using recombinant rabbit PrP as a model, this study describes critical molecular determinants that confer this high resistance to transmissible spongiform encephalopathies.


Asunto(s)
Aminoácidos/química , Proteínas Priónicas/química , Pliegue de Proteína , Sustitución de Aminoácidos , Aminoácidos/aislamiento & purificación , Animales , Bovinos , Susceptibilidad a Enfermedades , Ratones , Mutación , Enfermedades por Prión/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
11.
Acta Neuropathol ; 135(2): 179-199, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29094186

RESUMEN

Prion diseases are caused by a misfolding of the cellular prion protein (PrP) to a pathogenic isoform named PrPSc. Prions exist as strains, which are characterized by specific pathological and biochemical properties likely encoded in the three-dimensional structure of PrPSc. However, whether cofactors determine these different PrPSc conformations and how this relates to their specific biological properties is largely unknown. To understand how different cofactors modulate prion strain generation and selection, Protein Misfolding Cyclic Amplification was used to create a diversity of infectious recombinant prion strains by propagation in the presence of brain homogenate. Brain homogenate is known to contain these mentioned cofactors, whose identity is only partially known, and which facilitate conversion of PrPC to PrPSc. We thus obtained a mix of distinguishable infectious prion strains. Subsequently, we replaced brain homogenate, by different polyanionic cofactors that were able to drive the evolution of mixed prion populations toward specific strains. Thus, our results show that a variety of infectious recombinant prions can be generated in vitro and that their specific type of conformation, i.e., the strain, is dependent on the cofactors available during the propagation process. These observations have significant implications for understanding the pathogenesis of prion diseases and their ability to replicate in different tissues and hosts. Importantly, these considerations might apply to other neurodegenerative diseases for which different conformations of misfolded proteins have been described.


Asunto(s)
Encéfalo/metabolismo , Enfermedades por Prión/metabolismo , Proteínas Priónicas/metabolismo , Animales , Arvicolinae , Encéfalo/patología , Escherichia coli , Ratones Transgénicos , Polimorfismo Genético , Proteínas Priónicas/genética , Pliegue de Proteína , Proteínas Recombinantes/metabolismo
12.
Emerg Infect Dis ; 23(9): 1522-1530, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28820136

RESUMEN

Bovine spongiform encephalopathy (BSE) is the only known zoonotic prion that causes variant Creutzfeldt-Jakob disease (vCJD) in humans. The major risk determinant for this disease is the polymorphic codon 129 of the human prion protein (Hu-PrP), where either methionine (Met129) or valine (Val129) can be encoded. To date, all clinical and neuropathologically confirmed vCJD cases have been Met129 homozygous, with the exception of 1 recently reported Met/Val heterozygous case. Here, we found that transgenic mice homozygous for Val129 Hu-PrP show severely restricted propagation of the BSE prion strain, but this constraint can be partially overcome by adaptation of the BSE agent to the Met129 Hu-PrP. In addition, the transmission of vCJD to transgenic mice homozygous for Val129 Hu-PrP resulted in a prion with distinct strain features. These observations may indicate increased risk for vCJD secondary transmission in Val129 Hu-PrP-positive humans with the emergence of new strain features.


Asunto(s)
Síndrome de Creutzfeldt-Jakob/patología , Resistencia a la Enfermedad/genética , Encefalopatía Espongiforme Bovina/inmunología , Proteínas Priónicas/inmunología , Valina/inmunología , Sustitución de Aminoácidos , Animales , Encéfalo/patología , Bovinos , Codón , Síndrome de Creutzfeldt-Jakob/transmisión , Encefalopatía Espongiforme Bovina/patología , Encefalopatía Espongiforme Bovina/transmisión , Expresión Génica , Humanos , Inyecciones Intraventriculares , Metionina/genética , Metionina/inmunología , Ratones , Ratones Transgénicos , Péptido Hidrolasas/química , Proteínas Priónicas/química , Proteínas Priónicas/genética , Valina/genética
13.
PLoS Pathog ; 11(8): e1004977, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26247589

RESUMEN

Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrPC was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrPd) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrPd originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits.


Asunto(s)
Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Enfermedades por Prión/transmisión , Priones , Animales , Transmisión de Enfermedad Infecciosa , Ratones , Ratones Transgénicos , Conejos
14.
PLoS Pathog ; 11(4): e1004796, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25880443

RESUMEN

Fatal familial insomnia (FFI) and a genetic form of Creutzfeldt-Jakob disease (CJD178) are clinically different prion disorders linked to the D178N prion protein (PrP) mutation. The disease phenotype is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg) mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD) mice modeling CJD178. No prion infectivity was detectable in Tg(FFI) and Tg(CJD) brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI) and Tg(CJD) neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype.


Asunto(s)
Insomnio Familiar Fatal/genética , Insomnio Familiar Fatal/fisiopatología , Priones/genética , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Electroencefalografía , Imagen por Resonancia Magnética , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mutación , Fenotipo , Proteínas Priónicas
15.
Environ Res ; 151: 587-594, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27591838

RESUMEN

The environment plays a key role in horizontal transmission of prion diseases, since prions are extremely resistant to classical inactivation procedures. In prior work, we observed the high stability of bovine spongiform encephalopathy (BSE) infectivity when these prions were incubated in aqueous media such as phosphate-buffered saline (PBS) or wastewater for nearly nine months. As a continuation of this experiment, the same samples were maintained in PBS or wastewater for five additional years and residual BSE infectivity was assessed in bovine PrPC transgenic mice. Over this long time period (more than six years), BSE infectivity was reduced by three and one orders of magnitude in wastewater and PBS respectively. To rule out a possible agent specific effect, sheep scrapie prions were subjected to the same experimental protocol, using eight years as the experimental end-point. No significant reduction in scrapie infectivity was observed over the first nine months of wastewater incubation while PBS incubation for eight years only produced a two logarithmic unit reduction in infectivity. By contrast, the dynamics of PrPRes persistence was different, disappearing progressively over the first year. The long persistence of prion infectivity observed in this study for two different agents provides supporting evidence of the assumed high stability of these agents in aquatic environments and that environmental processes or conventional wastewater treatments with low retention times would have little impact on prion infectivity. These results could have great repercussions in terms of risk assessment and safety for animals and human populations.


Asunto(s)
Encefalopatía Espongiforme Bovina/transmisión , Fosfatos/química , Priones/patogenicidad , Scrapie/transmisión , Aguas Residuales/química , Contaminantes Químicos del Agua/análisis , Animales , Bioensayo , Bovinos , Ratones Transgénicos , Priones/análisis , Priones/genética , Ovinos , Factores de Tiempo , Purificación del Agua/métodos
16.
J Neurosci ; 34(3): 1022-7, 2014 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-24431459

RESUMEN

Zoonotic prion transmission was reported after the bovine spongiform encephalopathy (BSE) epidemic, when >200 cases of prion disease in humans were diagnosed as variant Creutzfeldt-Jakob disease. Assessing the risk of cross-species prion transmission remains challenging. We and others have studied how specific amino acid residue differences between species impact prion conversion and have found that the ß2-α2 loop region of the mouse prion protein (residues 165-175) markedly influences infection by sheep scrapie, BSE, mouse-adapted scrapie, deer chronic wasting disease, and hamster-adapted scrapie prions. The tyrosine residue at position 169 is strictly conserved among mammals and an aromatic side chain in this position is essential to maintain a 310-helical turn in the ß2-α2 loop. Here we examined the impact of the Y169G substitution together with the previously described S170N, N174T "rigid loop" substitutions on cross-species prion transmission in vivo and in vitro. We found that transgenic mice expressing mouse PrP containing the triple-amino acid substitution completely resisted infection with two strains of mouse prions and with deer chronic wasting disease prions. These studies indicate that Y169 is important for prion formation, and they provide a strong indication that variation of the ß2-α2 loop structure can modulate interspecies prion transmission.


Asunto(s)
Sustitución de Aminoácidos/genética , Enfermedades por Prión/genética , Enfermedades por Prión/transmisión , Priones/genética , Animales , Bovinos , Cricetinae , Ciervos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedades por Prión/prevención & control , Proteínas Priónicas , Estructura Secundaria de Proteína , Ovinos , Especificidad de la Especie
17.
J Gen Virol ; 96(12): 3715-3726, 2015 12.
Artículo en Inglés | MEDLINE | ID: mdl-26431976

RESUMEN

Mesenchymal stem cells (MSCs) can be infected with prions and have been proposed as in vitro cell-based models for prion replication. In addition, autologous MSCs are of interest for cell therapy in neurodegenerative diseases. To the best of our knowledge, the effect of prion diseases on the characteristics of these cells has never been investigated. Here, we analysed the properties of MSCs obtained from bone marrow (BM-MSCs) and peripheral blood (PB-MSCs) of sheep naturally infected with scrapie ­ a large mammal model for the study of prion diseases. After three passages of expansion, MSCs derived from scrapie animals displayed similar adipogenic, chondrogenic and osteogenic differentiation ability as cells from healthy controls, although a subtle decrease in the proliferation potential was observed. Exceptionally, mesenchymal markers such as CD29 were significantly upregulated at the transcript level compared with controls. Scrapie MSCs were able to transdifferentiate into neuron-like cells, but displayed lower levels of neurogenic markers at basal conditions, which could limit this potential .The expression levels of cellular prion protein (PrPC) were highly variable between cultures, and no significant differences were observed between control and scrapie-derived MSCs. However, during neurogenic differentiation the expression of PrPC was upregulated in MSCs. This characteristic could be useful for developing in vitro models for prion replication. Despite the infectivity reported for MSCs obtained from scrapie-infected mice and Creutzfeldt­Jakob disease patients, protein misfolding cyclic amplification did not detect PrPSc in BM- or PB-MSCs from scrapie-infected sheep, which limits their use for in vivo diagnosis for scrapie.


Asunto(s)
Células Madre Mesenquimatosas/fisiología , Scrapie/patología , Animales , Diferenciación Celular , Extensiones de la Superficie Celular/genética , Extensiones de la Superficie Celular/metabolismo , Regulación de la Expresión Génica , Ovinos
18.
Proc Natl Acad Sci U S A ; 109(13): 5080-5, 2012 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-22416127

RESUMEN

The ability of prions to infect some species and not others is determined by the transmission barrier. This unexplained phenomenon has led to the belief that certain species were not susceptible to transmissible spongiform encephalopathies (TSEs) and therefore represented negligible risk to human health if consumed. Using the protein misfolding cyclic amplification (PMCA) technique, we were able to overcome the species barrier in rabbits, which have been classified as TSE resistant for four decades. Rabbit brain homogenate, either unseeded or seeded in vitro with disease-related prions obtained from different species, was subjected to serial rounds of PMCA. De novo rabbit prions produced in vitro from unseeded material were tested for infectivity in rabbits, with one of three intracerebrally challenged animals succumbing to disease at 766 d and displaying all of the characteristics of a TSE, thereby demonstrating that leporids are not resistant to prion infection. Material from the brain of the clinically affected rabbit containing abnormal prion protein resulted in a 100% attack rate after its inoculation in transgenic mice overexpressing rabbit PrP. Transmissibility to rabbits (>470 d) has been confirmed in 2 of 10 rabbits after intracerebral challenge. Despite rabbits no longer being able to be classified as resistant to TSEs, an outbreak of "mad rabbit disease" is unlikely.


Asunto(s)
Enfermedades por Prión/patología , Enfermedades por Prión/transmisión , Priones/metabolismo , Priones/patogenicidad , Animales , Encéfalo/metabolismo , Encéfalo/patología , Resistencia a la Enfermedad , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Enfermedades por Prión/metabolismo , Priones/química , Desnaturalización Proteica , Pliegue de Proteína , Conejos , Especificidad de la Especie
19.
J Neurosci ; 33(18): 7778-86, 2013 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-23637170

RESUMEN

Bovine spongiform encephalopathy (BSE) prions were responsible for an unforeseen epizootic in cattle which had a vast social, economic, and public health impact. This was primarily because BSE prions were found to be transmissible to humans. Other species were also susceptible to BSE either by natural infection (e.g., felids, caprids) or in experimental settings (e.g., sheep, mice). However, certain species closely related to humans, such as canids and leporids, were apparently resistant to BSE. In vitro prion amplification techniques (saPMCA) were used to successfully misfold the cellular prion protein (PrP(c)) of these allegedly resistant species into a BSE-type prion protein. The biochemical and biological properties of the new prions generated in vitro after seeding rabbit and dog brain homogenates with classical BSE were studied. Pathobiological features of the resultant prion strains were determined after their inoculation into transgenic mice expressing bovine and human PrP(C). Strain characteristics of the in vitro-adapted rabbit and dog BSE agent remained invariable with respect to the original cattle BSE prion, suggesting that the naturally low susceptibility of rabbits and dogs to prion infections should not alter their zoonotic potential if these animals became infected with BSE. This study provides a sound basis for risk assessment regarding prion diseases in purportedly resistant species.


Asunto(s)
Susceptibilidad a Enfermedades , Encefalopatía Espongiforme Bovina/metabolismo , Encefalopatía Espongiforme Bovina/transmisión , Priones/metabolismo , Deficiencias en la Proteostasis/etiología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Bovinos , Modelos Animales de Enfermedad , Perros , Encefalopatía Espongiforme Bovina/mortalidad , Humanos , Ratones , Ratones Transgénicos , Técnicas de Amplificación de Ácido Nucleico/métodos , Deficiencias en la Proteostasis/mortalidad , Deficiencias en la Proteostasis/patología , Conejos , Especificidad de la Especie , Análisis de Supervivencia
20.
J Virol ; 86(4): 2056-66, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22156536

RESUMEN

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.


Asunto(s)
Plaquetas/virología , Modelos Animales de Enfermedad , Leucocitos Mononucleares/virología , Ratones , Enfermedades por Prión/veterinaria , Enfermedades por Prión/virología , Scrapie/virología , Animales , Humanos , Ratones Transgénicos , Enfermedades por Prión/transmisión , Scrapie/transmisión , Ovinos
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