RESUMO
While animal prion diseases are a threat to human health, their zoonotic potential is generally inefficient because of interspecies prion transmission barriers. New animal models are required to provide an understanding of these prion transmission barriers and to assess the zoonotic potential of animal prion diseases. To address this goal, we generated Drosophila transgenic for human or non-human primate PrP and determined their susceptibility to known pathogenic prion diseases, namely vCJD and classical BSE, and that with unknown pathogenic potential, namely CWD. Adult Drosophila transgenic for M129 or V129 human PrP, or non-human primate PrP developed a neurotoxic phenotype and showed an accelerated loss of survival after exposure to vCJD, classical BSE, or CWD prions at the larval stage. vCJD prion strain identity was retained after passage in both M129 and V129 human PrP Drosophila. All of the primate PrP fly lines accumulated prion seeding activity and concomitantly developed a neurotoxic phenotype, generally including accelerated loss of survival, after exposure to CWD prions derived from different cervid species, including North American white-tailed deer and muntjac, and European reindeer and moose. These novel studies show that primate PrP transgenic Drosophila lack known prion transmission barriers since, in mammalian hosts, V129 human PrP is associated with severe resistance to classical BSE prions, while both human and cynomolgus macaque PrP are associated with resistance to CWD prions. Significantly, our data suggest that interspecies differences in the amino acid sequence of PrP may not be a principal determinant of the prion transmission barrier.
RESUMO
The presence of prion infectivity in the blood of patients affected by variant Creutzfeldt-Jakob disease (v-CJD), the human prion disease linked to the bovine spongiform encephalopathy (BSE), poses the risk of inter-human transmission of this fatal prion disease through transfusion. In the frame of various experiments, we have previously described that several cynomolgus macaques experimentally exposed to prion-contaminated blood products developed c-BSE/v-CJD, but the vast majority of them developed an unexpected, fatal disease phenotype focused on spinal cord involvement, which does not fulfill the classical diagnostic criteria of v-CJD. Here, we show that extensive analyses with current conventional techniques failed to detect any accumulation of abnormal prion protein (PrPv-CJD) in the CNS of these myelopathic animals, i.e., the biomarker considered responsible for neuronal death and subsequent clinical signs in prion diseases. Conversely, in the spinal cord of these myelopathic primates, we observed an alteration of their physiological cellular PrP pattern: PrP was not detectable under its full-length classical expression but mainly under its physiological terminal-truncated C1 fragment. This observed disappearance of the N-terminal fragment of cellular PrP at the level of the lesions may provide the first experimental evidence of a link between loss of function of the cellular prion protein and disease onset. This original prion-induced myelopathic syndrome suggests an unexpected wide extension in the field of prion diseases that is so far limited to pathologies associated with abnormal changes of the cellular PrP to highly structured conformations.
RESUMO
The fascinating history of prion diseases is intimately linked to the use of nonhuman primates as experimental models, which brought so fundamental and founding information about transmissibility, pathogenesis, and resistance of prions. These models are still of crucial need for risk assessment of human health and may contribute to pave a new way towards the moving field of prion-like entities which now includes the main human neurodegenerative diseases (especially Alzheimer's and Parkinson's diseases).
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Doenças Priônicas , Príons , Animais , Humanos , PrimatasRESUMO
Treatment with human pituitary-derived growth hormone (hGH) was responsible for a significant proportion of iatrogenic Creutzfeldt-Jakob disease (iCJD) cases. France and the UK experienced the largest case numbers of hGH-iCJD, with 122 and 81 cases respectively. Differences in the frequency of the three PRNP codon 129 polymorphisms (MM, MV and VV) and the estimated incubation periods associated with each of these genotypes in the French and the UK hGH-iCJD cohorts led to the suggestion that the prion strains responsible for these two hGH-iCJD cohorts were different. In this study, we characterized the prion strains responsible for hGH-iCJD cases originating from UK (n = 11) and France (n = 11) using human PrP expressing mouse models. The cases included PRNP MM, MV and VV genotypes from both countries. UK and French sporadic CJD (sCJD) cases were included as controls. The prion strains identified following inoculation with hGH-iCJD homogenates corresponded to the two most frequently observed sCJD prion strains (M1CJD and V2CJD). However, in clear contradiction to the initial hypothesis, the prion strains that were identified in the UK and the French hGH-iCJD cases were not radically different. In the vast majority of the cases originating from both countries, the V2CJD strain or a mixture of M1CJD + V2CJD strains were identified. These data strongly support the contention that the differences in the epidemiological and genetic profiles observed in the UK and France hGH-iCJD cohorts cannot be attributed only to the transmission of different prion strains.
Assuntos
Síndrome de Creutzfeldt-Jakob/epidemiologia , Síndrome de Creutzfeldt-Jakob/patologia , Encefalopatia Espongiforme Bovina/epidemiologia , Encefalopatia Espongiforme Bovina/patologia , Hormônio do Crescimento Humano/efeitos adversos , Proteínas PrPSc/efeitos adversos , Adulto , Animais , Estudos de Coortes , Síndrome de Creutzfeldt-Jakob/transmissão , Encefalopatia Espongiforme Bovina/transmissão , Feminino , França/epidemiologia , Hormônio do Crescimento Humano/administração & dosagem , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas PrPSc/administração & dosagem , Proteínas PrPSc/isolamento & purificação , Reino Unido/epidemiologiaAssuntos
Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/transmissão , Modelos Animais de Doenças , Dedos , Condução Nervosa , Príons , Animais , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Imuno-Histoquímica , Macaca , Nervo Mediano/fisiopatologia , Neurônios Motores , Proteínas Priônicas/metabolismo , SensaçãoRESUMO
Cynomolgus macaque has been used for the evaluation of the zoonotic potential of prion diseases, especially for classical-Bovine Spongiform Encephalopathy (classical-BSE) infectious agent. PrP amino acid sequence is considered to play a key role in the susceptibility to prion strains and only one amino acid change may alter this susceptibility. Macaque and human-PrP sequences have only nine amino acid differences, but the effect of these amino acid changes in the susceptibility to dissimilar prion strains is unknown. In this work, the transmissibility of a panel of different prions from several species was compared in transgenic mice expressing either macaque-PrPC (TgMac) or human-PrPC (Hu-Tg340). Similarities in the transmissibility of most prion strains were observed suggesting that macaque is an adequate model for the evaluation of human susceptibility to most of the prion strains tested. Interestingly, TgMac were more susceptible to classical-BSE strain infection than Hu-Tg340. This differential susceptibility to classical-BSE transmission should be taken into account for the interpretation of the results obtained in macaques. It could notably explain why the macaque model turned out to be so efficient (worst case model) until now to model human situation towards classical-BSE despite the limited number of animals inoculated in the laboratory experiments.
Assuntos
Síndrome de Creutzfeldt-Jakob/genética , Encefalopatia Espongiforme Bovina/genética , Doenças Priônicas/genética , Proteínas Priônicas/genética , Sequência de Aminoácidos/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Bovinos , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Modelos Animais de Doenças , Encefalopatia Espongiforme Bovina/metabolismo , Encefalopatia Espongiforme Bovina/patologia , Predisposição Genética para Doença , Humanos , Macaca , Macaca fascicularis/genética , Camundongos , Camundongos Transgênicos , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Proteínas Priônicas/metabolismoRESUMO
Alzheimer's disease is characterized by cognitive alterations, cerebral atrophy and neuropathological lesions including neuronal loss, accumulation of misfolded and aggregated ß-amyloid peptides (Aß) and tau proteins. Iatrogenic induction of Aß is suspected in patients exposed to pituitary-derived hormones, dural grafts, or surgical instruments, presumably contaminated with Aß. Induction of Aß and tau lesions has been demonstrated in transgenic mice after contamination with Alzheimer's disease brain homogenates, with very limited functional consequences. Unlike rodents, primates naturally express Aß or tau under normal conditions and attempts to transmit Alzheimer pathology to primates have been made for decades. However, none of earlier studies performed any detailed functional assessments. For the first time we demonstrate long term memory and learning impairments in a non-human primate (Microcebus murinus) following intracerebral injections with Alzheimer human brain extracts. Animals inoculated with Alzheimer brain homogenates displayed progressive cognitive impairments (clinical tests assessing cognitive and motor functions), modifications of neuronal activity (detected by electroencephalography), widespread and progressive cerebral atrophy (in vivo MRI assessing cerebral volume loss using automated voxel-based analysis), neuronal loss in the hippocampus and entorhinal cortex (post mortem stereology). They displayed parenchymal and vascular Aß depositions and tau lesions for some of them, in regions close to the inoculation sites. Although these lesions were sparse, they were never detected in control animals. Tau-positive animals had the lowest performances in a memory task and displayed the greatest neuronal loss. Our study is timely and important as it is the first one to highlight neuronal and clinical dysfunction following inoculation of Alzheimer's disease brain homogenates in a primate. Clinical signs in a chronic disease such as Alzheimer take a long time to be detectable. Documentation of clinical deterioration and/or dysfunction following intracerebral inoculations with Alzheimer human brain extracts could lead to important new insights about Alzheimer initiation processes.
Assuntos
Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/genética , Encefalopatias/diagnóstico por imagem , Encefalopatias/genética , Encéfalo/diagnóstico por imagem , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Encefalopatias/patologia , Cheirogaleidae , Eletroencefalografia/métodos , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Transgênicos , Primatas , Especificidade da EspécieRESUMO
The European Commission asked EFSA for a Scientific Opinion: to revise the state of knowledge about the differences between the chronic wasting disease (CWD) strains found in North America (NA) and Europe and within Europe; to review new scientific evidence on the zoonotic potential of CWD and to provide recommendations to address the potential risks and to identify risk factors for the spread of CWD in the European Union. Full characterisation of European isolates is being pursued, whereas most NA CWD isolates have not been characterised in this way. The differing surveillance programmes in these continents result in biases in the types of cases that can be detected. Preliminary data support the contention that the CWD strains identified in Europe and NA are different and suggest the presence of strain diversity in European cervids. Current data do not allow any conclusion on the implications of strain diversity on transmissibility, pathogenesis or prevalence. Available data do not allow any conclusion on the zoonotic potential of NA or European CWD isolates. The risk of CWD to humans through consumption of meat cannot be directly assessed. At individual level, consumers of meat, meat products and offal derived from CWD-infected cervids will be exposed to the CWD agent(s). Measures to reduce human dietary exposure could be applied, but exclusion from the food chain of whole carcasses of infected animals would be required to eliminate exposure. Based on NA experiences, all the risk factors identified for the spread of CWD may be associated with animals accumulating infectivity in both the peripheral tissues and the central nervous system. A subset of risk factors is relevant for infected animals without involvement of peripheral tissues. All the risk factors should be taken into account due to the potential co-localisation of animals presenting with different disease phenotypes.
RESUMO
The recently reevaluated high prevalence of healthy carriers (1/2,000 in UK) of variant Creutzfeldt-Jakob Disease (v-CJD), whose blood might be infectious, suggests that the evolution of this prion disease might not be under full control as expected. After experimental transfusion of macaques and conventional mice with blood derived from v-CJD exposed (human and animal) individuals, we confirmed in these both models the transmissibility of v-CJD, but we also observed unexpected neurological syndromes transmissible by transfusion: despite their prion etiology confirmed through transmission experiments, these original cases would escape classical prion diagnosis, notably in the absence of detectable abnormal PrP with current techniques. It is noteworthy that macaques developed an original, yet undescribed myelopathic syndrome associating demyelination and pseudo-necrotic lesions of spinal cord, brainstem and optical tract without affecting encephalon, which is rather evocative of spinal cord disease than prion disease in human medicine. These observations strongly suggest that the spectrum of human prion diseases may extend the current field restricted to the phenotypes associated to protease-resistant PrP, and may notably include spinal cord diseases.
RESUMO
Abeta deposits and tau pathology were investigated in 24 French patients that died from iatrogenic Creutzfeldt-Jakob disease after exposure to cadaver-derived human growth hormone (c-hGH) in the 1980s. Abeta deposits were found only in one case that had experienced one of the longest incubation periods. Three cases had also intracellular tau accumulation. The analysis of 24 batches of c-hGH, produced between 1974 and 1988, demonstrated for the first time the presence of Abeta and tau contaminants in c-hGH (in 17 and 6 batches, respectively). The incubation of prion disease was shorter in the French patients than the incubation times reported in two previously published British series. We interpreted the low incidence of Abeta in this French series as a consequence of the shorter incubation period observed in France, as compared to that observed in the United Kingdom. This concept suggested that a mean incubation period for the development of detectable Abeta deposits would be longer than 18 years after the first exposure. Moreover, we hypothesized that tau pathology might also be transmissible in humans.
Assuntos
Encéfalo/patologia , Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/transmissão , Contaminação de Medicamentos , Hormônio do Crescimento Humano , Adulto , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Cadáver , Estudos de Coortes , Síndrome de Creutzfeldt-Jakob/metabolismo , França , Hormônio do Crescimento Humano/administração & dosagem , Humanos , Doença Iatrogênica , Imunoensaio , Período de Incubação de Doenças Infecciosas , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Adulto Jovem , Proteínas tau/metabolismoRESUMO
Exposure of human populations to bovine spongiform encephalopathy through contaminated food has resulted in <250 cases of variant Creutzfeldt-Jakob disease (vCJD). However, more than 99% of vCJD infections could have remained silent suggesting a long-term risk of secondary transmission particularly through blood. Here, we present experimental evidence that transfusion in mice and non-human primates of blood products from symptomatic and non-symptomatic infected donors induces not only vCJD, but also a different class of neurological impairments. These impairments can all be retransmitted to mice with a pathognomonic accumulation of abnormal prion protein, thus expanding the spectrum of known prion diseases. Our findings suggest that the intravenous route promotes propagation of masked prion variants according to different mechanisms involved in peripheral replication.
Assuntos
Transfusão de Sangue , Síndrome de Creutzfeldt-Jakob/transmissão , Reação Transfusional , Animais , Doenças Assintomáticas , Doadores de Sangue , Bovinos , Síndrome de Creutzfeldt-Jakob/metabolismo , Encefalopatia Espongiforme Bovina/transmissão , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Doenças Priônicas/classificação , Doenças Priônicas/metabolismo , Doenças Priônicas/transmissão , Proteínas Priônicas/metabolismoRESUMO
Gadolinium (Gd)-stained MRI is based on Gd contrast agent (CA) administration into the brain parenchyma. The strong signal increase induced by Gd CA can be converted into resolution enhancement to record microscopic MR images. Moreover, inhomogeneous distribution of the Gd CA in the brain improves the contrast between different tissues and provides new contrasts in MR images. Gd-stained MRI detects amyloid plaques, one of the microscopic lesions of Alzheimer's disease (AD), in APPSL/PS1M146L mice or in primates. Numerous transgenic mice with various plaque typologies have been developed to mimic cerebral amyloidosis and comparison of plaque detection between animal models and humans with new imaging methods is a recurrent concern. Here, we investigated detection of amyloid plaques by Gd-stained MRI in five mouse models of amyloidosis (APPSL/PS1M146L, APP/PS1dE9, APP23, APPSwDI, and 3xTg) presenting with compact, diffuse and intracellular plaques as well as in post mortem human-AD brains. The brains were then evaluated by histology to investigate the impact of size, compactness, and iron load of amyloid plaques on their detection by MRI. We show that Gd-stained MRI allows detection of compact amyloid plaques as small as 25 µm, independently of their iron load, in mice as well as in human-AD brains.
Assuntos
Doença de Alzheimer/diagnóstico por imagem , Amiloidose/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Placa Amiloide/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Amiloidose/metabolismo , Animais , Autopsia , Meios de Contraste/administração & dosagem , Modelos Animais de Doenças , Gadolínio/administração & dosagem , Humanos , Ferro/metabolismo , Camundongos , Camundongos Transgênicos , Placa Amiloide/metabolismoRESUMO
The transmission of classical bovine spongiform encephalopathy (C-BSE) through contaminated meat product consumption is responsible for variant Creutzfeldt-Jakob disease (vCJD) in humans. More recent and atypical forms of BSE (L-BSE and H-BSE) have been identified in cattle since the C-BSE epidemic. Their low incidence and advanced age of onset are compatible with a sporadic origin, as are most cases of Creutzfeldt-Jakob disease (CJD) in humans. Transmissions studies in primates and transgenic mice expressing a human prion protein (PrP) indicated that atypical forms of BSE may be associated with a higher zoonotic potential than classical BSE, and require particular attention for public health. Recently, methods designed to amplify misfolded forms of PrP have emerged as promising tools to detect prion strains and to study their diversity. Here, we validated real-time quaking-induced conversion assay for the discrimination of atypical and classical BSE strains using a large series of bovine samples encompassing all the atypical BSE cases detected by the French Centre of Reference during 10 years of exhaustive active surveillance. We obtained a 100% sensitivity and specificity for atypical BSE detection. In addition, the assay was able to discriminate atypical and classical BSE in non-human primates, and also sporadic CJD and vCJD in humans. The RT-QuIC assay appears as a practical means for a reliable detection of atypical BSE strains in a homologous or heterologous PrP context.
Assuntos
Síndrome de Creutzfeldt-Jakob/veterinária , Encefalopatia Espongiforme Bovina/diagnóstico , Encefalopatia Espongiforme Bovina/patologia , Doenças dos Primatas/diagnóstico , Proteínas Priônicas/análise , Animais , Encéfalo/patologia , Química Encefálica , Bovinos , Síndrome de Creutzfeldt-Jakob/diagnóstico , Síndrome de Creutzfeldt-Jakob/patologia , Humanos , Proteínas Recombinantes/análise , Sensibilidade e EspecificidadeRESUMO
Classical bovine spongiform encephalopathy (c-BSE) is the only animal prion disease reputed to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans and having guided protective measures for animal and human health against animal prion diseases. Recently, partial transmissions to humanized mice showed that the zoonotic potential of scrapie might be similar to c-BSE. We here report the direct transmission of a natural classical scrapie isolate to cynomolgus macaque, a highly relevant model for human prion diseases, after a 10-year silent incubation period, with features similar to those reported for human cases of sporadic CJD. Scrapie is thus actually transmissible to primates with incubation periods compatible with their life expectancy, although fourfold longer than BSE. Long-term experimental transmission studies are necessary to better assess the zoonotic potential of other prion diseases with high prevalence, notably Chronic Wasting Disease of deer and elk and atypical/Nor98 scrapie.
Assuntos
Scrapie/patologia , Animais , Bovinos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Humanos , Macaca fascicularis , Masculino , Camundongos , Príons/metabolismo , Scrapie/transmissão , Lobo Temporal/patologia , Fatores de TempoRESUMO
In most forms of prion diseases, blood is infectious, but detection by immunochemistry techniques of the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the finding that PrPTSE is associated with plasma exosomes. However, further purification of the exosomes on a sucrose gradient was necessary to remove plasma immunoglobulins, which interfere with PrPTSE, masking its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with exosomes.
Assuntos
Exossomos/química , Príons/análise , Animais , Análise Química do Sangue , Feminino , Imunoquímica , Mesocricetus , Doenças Priônicas/diagnóstico , Manejo de Espécimes/métodosRESUMO
BACKGROUND: Analysis of archived appendix samples reveals that one in 2000 individuals in the United Kingdom may carry the infectious prion protein associated with variant Creutzfeldt-Jakob disease (vCJD), raising questions about the risk of transfusion transmission from apparently healthy carriers. Blood leukoreduction shows limited efficiency against prions. Therefore, in absence of antemortem diagnostic tests, prion removal filters, including the P-Capt filter were designed to improve blood transfusion safety. STUDY DESIGN AND METHODS: We evaluated the performances of two filters, the P-Capt and one prototype (PMC#005), with blood-borne infectivity in two independent experiments. Blood was drawn twice from prion-infected macaques. Corresponding RBCCs were prepared according to two different procedures: in Study A, the leukoreduction step was followed by the filtration through the P-Capt. In Study B, the leukoreduction and prion removal were performed simultaneously through the PMC#005. For each study, two groups of three animals were transfused twice with samples before or after filtration. RESULTS: Among the six macaques transfused with nonfiltered samples, five developed neurologic signs but only four exhibited peripheral detectable protease-resistant prion protein (PrPres) accumulation. In Study A, the three animals transfused with P-Capt-filtered samples remain asymptomatic and devoid of PrPres in lymph node biopsies 6 years after the transfusion. In Study B, one animal transfused with PMC#005-filtered samples developed vCJD. CONCLUSION: After 5 to 6 years of progress, this ongoing study provides encouraging results on the prion blood removal performances of the P-Capt filter in macaques, an utmost relevant model for human prion diseases.
Assuntos
Transfusão de Componentes Sanguíneos/efeitos adversos , Segurança do Sangue/instrumentação , Patógenos Transmitidos pelo Sangue/isolamento & purificação , Síndrome de Creutzfeldt-Jakob/prevenção & controle , Encefalopatia Espongiforme Bovina/prevenção & controle , Procedimentos de Redução de Leucócitos/instrumentação , Príons/isolamento & purificação , Ultrafiltração/instrumentação , Adsorção , Animais , Segurança do Sangue/métodos , Química Encefálica , Bovinos , Síndrome de Creutzfeldt-Jakob/sangue , Síndrome de Creutzfeldt-Jakob/transmissão , Encefalopatia Espongiforme Bovina/sangue , Encefalopatia Espongiforme Bovina/transmissão , Macaca fascicularis , Masculino , Filtros Microporos , Microesferas , Príons/análise , Príons/toxicidade , Resinas Sintéticas , Medula Espinal/química , Baço/químicaRESUMO
The key molecular event in human cerebral proteinopathies, which include Alzheimer's, Parkinson's and Huntington's diseases, is the structural conversion of a specific host protein into a ß-sheet-rich conformer. With regards to this common mechanism, it appears difficult to explain the outstanding infectious properties attributed to PrP(Sc), the hallmark of another intriguing family of cerebral proteinopathies known as transmissible spongiform encephalopathies (TSE) or prion diseases. The infectious PrP(Sc) or "prion" is thought to be composed solely of a misfolded form of the otherwise harmless cellular prion protein (PrP(c)). To gain insight into this unique situation, we used the 263K scrapie hamster model to search for a putative PrP(Sc)-associated factor that contributes to the infectivity of PrP(Sc) amyloid. In a rigorously controlled set of experiments that included several bioassays, we showed that originally innocuous recombinant prion protein (recPrP) equivalent to PrP(c) is capable of initiating prion disease in hamsters when it is converted to a prion-like conformation (ß-sheet-rich) in the presence of RNA purified from scrapie-associated fibril (SAF) preparations. Analysis of the recPrP-RNA infectious mixture reveals the presence of 2 populations of small RNAs of approximately 27 and 55 nucleotides. These unprecedented findings are discussed in light of the distinct relationship that may exist between this RNA material and the 2 biological properties, infectivity and strain features, attributed to prion amyloid.
Assuntos
Amiloide/análise , Química Encefálica , Encéfalo/patologia , Proteínas PrPSc/patogenicidade , RNA/metabolismo , Scrapie/etiologia , Animais , Encéfalo/ultraestrutura , Cricetinae , Microscopia Eletrônica , Proteínas PrPSc/análise , Proteínas PrPSc/química , Proteínas PrPSc/genética , Estrutura Secundária de Proteína , RNA/análise , RNA/química , RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The prion protein (PrP) is highly conserved and ubiquitously expressed, suggesting that it plays an important physiological function. However, despite decades of investigation, this role remains elusive. Here, by using animal and cellular models, we unveil a key role of PrP in the DNA damage response. Exposure of neurons to a genotoxic stress activates PRNP transcription leading to an increased amount of PrP in the nucleus where it interacts with APE1, the major mammalian endonuclease essential for base excision repair, and stimulates its activity. Preventing the induction of PRNP results in accumulation of abasic sites in DNA and impairs cell survival after genotoxic treatment. Brains from Prnp(-/-) mice display a reduced APE1 activity and a defect in the repair of induced DNA damage in vivo. Thus, PrP is required to maintain genomic stability in response to genotoxic stresses.
Assuntos
Reparo do DNA , Príons/metabolismo , Animais , Encéfalo/enzimologia , Linhagem Celular , Núcleo Celular/química , Sobrevivência Celular , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Humanos , Metanossulfonato de Metila/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Mutagênicos/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Priônicas , Príons/análise , Príons/biossíntese , Príons/genética , Ativação TranscricionalRESUMO
BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative infection that can be transmitted by blood and blood products from donors in the latent phase of the disease. Currently, there is no validated antemortem vCJD blood screening test. Several blood tests are under development. Any useful test must be validated with disease-relevant blood reference panels. STUDY DESIGN AND METHODS: To generate blood reference materials, we infected four cynomolgus macaques with macaque-adapted vCJD brain homogenates. Blood was collected throughout the preclinical and clinical phases of infection. In parallel, equivalent blood was collected from one uninfected macaque. For each blood collection, an aliquot was stored as whole blood and the remainder was separated into components. Aliquots of plasma from terminally ill macaques were assayed for the presence of PrP(TSE) with the protein misfolding cyclic amplification (PMCA) method. Infectivity of the macaque brain homogenate used to infect macaques was titrated in C57BL/6 and RIII J/S inbred wild-type mice. RESULTS: We sampled blood 19 times from the inoculated monkeys at various stages of the disease over a period of 29 months, generating liters of vCJD-infected macaque blood. vCJD was confirmed in all inoculated macaques. After PMCA, PrP(TSE) was detected in plasma from infected monkeys, but not from uninfected animals. Both mouse models were more sensitive to infection with macaque-adapted vCJD agent than to primary human vCJD agent. CONCLUSION: The macaque vCJD blood panels generated in this study provide a unique resource to support vCJD assay development and to characterize vCJD infectivity in blood.
Assuntos
Síndrome de Creutzfeldt-Jakob/sangue , Príons/sangue , Sequência de Aminoácidos , Animais , Síndrome de Creutzfeldt-Jakob/transmissão , Modelos Animais de Doenças , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Dados de Sequência Molecular , Padrões de ReferênciaRESUMO
The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of 3 or more copies of the specific protein. We have developed an SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10(7)-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at 100-fold higher concentration. Using either of the 2 monoclonal anti-PrP antibodies, 3F4 and 6H4, we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases.