RESUMO
Prions can exist as different strains that consist of conformational variants of the misfolded, pathogenic prion protein isoform PrPSc. Defined by stably transmissible biological and biochemical properties, strains have been identified in a spectrum of prion diseases, including chronic wasting disease (CWD) of wild and farmed cervids. CWD is highly contagious and spreads via direct and indirect transmission involving extraneural sites of infection, peripheral replication and neuroinvasion of prions. Here, we investigated the impact of infection route on CWD prion conformational selection and propagation. We used gene-targeted mouse models expressing deer PrP for intracerebral or intraperitoneal inoculation with fractionated or unfractionated brain homogenates from white-tailed deer, harboring CWD strains Wisc-1 or 116AG. Upon intracerebral inoculation, Wisc-1 and 116AG-inoculated mice differed in conformational stability of PrPSc. In brains of mice infected intraperitoneally with either inoculum, PrPSc propagated with identical conformational stability and fewer PrPSc deposits in most brain regions than intracerebrally inoculated animals. For either inoculum, PrPSc conformational stability in brain and spinal cord was similar upon intracerebral infection but significantly higher in spinal cords of intraperitoneally infected animals. Inoculation with fractionated brain homogenates resulted in lower variance of survival times upon intraperitoneal compared to intracerebral infection. In summary, we demonstrate that extraneural infection mitigates the impact of PrPSc quaternary structure on infection and reduces conformational variability of PrPSc propagated in the brain. These findings provide new insights into the evolution of stable CWD strains in natural, extraneural transmissions.
Assuntos
Encéfalo , Cervos , Proteínas PrPSc , Doença de Emaciação Crônica , Animais , Camundongos , Doença de Emaciação Crônica/transmissão , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas PrPSc/metabolismo , Conformação Proteica , Príons/metabolismo , Príons/patogenicidade , Doenças Priônicas/transmissão , Doenças Priônicas/patologia , Doenças Priônicas/metabolismo , Camundongos TransgênicosRESUMO
Chronic wasting disease (CWD) is a prion disease affecting deer, elk and moose in North America and reindeer, moose and red deer in Northern Europe. Pathogenesis is driven by the accumulation of PrPSc, a pathological form of the host's cellular prion protein (PrPC), in the brain. CWD is contagious among North American cervids and Norwegian reindeer, with prions commonly found in lymphatic tissue. In Nordic moose and red deer CWD appears exclusively in older animals, and prions are confined to the CNS and undetectable in lymphatic tissues, indicating a sporadic origin. We aimed to determine transmissibility, neuroinvasion and lymphotropism of Nordic CWD isolates using gene-targeted mice expressing either wild-type (138SS/226QQ) or S138N (138NN/226QQ) deer PrP. When challenged with North American CWD strains, mice expressing S138N PrP did not develop clinical disease but harbored prion seeding activity in brain and spleen. Here, we infected these models intracerebrally or intraperitoneally with Norwegian moose, red deer and reindeer CWD isolates. The moose isolate was the first CWD type to cause full-blown disease in the 138NN/226QQ model in the first passage, with 100% attack rate and shortened survival times upon second passage. Furthermore, we detected prion seeding activity or PrPSc in brains and spinal cords, but not spleens, of 138NN/226QQ mice inoculated intraperitoneally with the moose isolate, providing evidence of prion neuroinvasion. We also demonstrate, for the first time, that transmissibility of the red deer CWD isolate was restricted to transgenic mice overexpressing elk PrPC (138SS/226EE), identical to the PrP primary structure of the inoculum. Our findings highlight that susceptibility to clinical disease is determined by the conformational compatibility between prion inoculum and host PrP primary structure. Our study indicates that neuroinvasion of Norwegian moose prions can occur without, or only very limited, replication in the spleen, an unprecedented finding for CWD.
Assuntos
Cervos , Doença de Emaciação Crônica , Animais , Doença de Emaciação Crônica/transmissão , Doença de Emaciação Crônica/metabolismo , Camundongos , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas Priônicas/metabolismo , Proteínas Priônicas/genética , Camundongos Transgênicos , Noruega , Marcação de Genes , Príons/metabolismo , Príons/genética , Príons/patogenicidadeRESUMO
The state of open science needs to be monitored to track changes over time and identify areas to create interventions to drive improvements. In order to monitor open science practices, they first need to be well defined and operationalized. To reach consensus on what open science practices to monitor at biomedical research institutions, we conducted a modified 3-round Delphi study. Participants were research administrators, researchers, specialists in dedicated open science roles, and librarians. In rounds 1 and 2, participants completed an online survey evaluating a set of potential open science practices, and for round 3, we hosted two half-day virtual meetings to discuss and vote on items that had not reached consensus. Ultimately, participants reached consensus on 19 open science practices. This core set of open science practices will form the foundation for institutional dashboards and may also be of value for the development of policy, education, and interventions.
Assuntos
Pesquisa Biomédica , Humanos , Consenso , Técnica Delphi , Inquéritos e Questionários , Projetos de PesquisaRESUMO
Prion diseases are fatal and infectious neurodegenerative diseases that occur in humans and animals. They are caused by the misfolding of the cellular prion protein PrPc into the infectious isoform PrPSc. PrPSc accumulates mostly in endolysosomal vesicles of prion-infected cells, eventually causing neurodegeneration. In response to prion infection, elevated cholesterol levels and a reduction in membrane-attached small GTPase Rab7 have been observed in neuronal cells. Here, we investigated the molecular events causing an impaired Rab7 membrane attachment and the potential mechanistic link with elevated cholesterol levels in prion infection. We demonstrate that prion infection is associated with reduced levels of active Rab7 (Rab7.GTP) in persistently prion-infected neuronal cell lines, primary cerebellar granular neurons, and neurons in the brain of mice with terminal prion disease. In primary cerebellar granular neurons, levels of active Rab7 were increased during the very early stages of the prion infection prior to a significant decrease concomitant with PrPSc accumulation. The reduced activation of Rab7 in prion-infected neuronal cell lines is also associated with its reduced ubiquitination status, decreased interaction with its effector RILP, and altered lysosomal positioning. Consequently, the Rab7-mediated trafficking of low-density lipoprotein to lysosomes is delayed. This results in an impaired feedback regulation of cholesterol synthesis leading to an increase in cholesterol levels. Notably, transient overexpression of the constitutively active mutant of Rab7 rescues the delay in the low-density lipoprotein trafficking, hence reducing cholesterol levels and attenuating PrPSc propagation, demonstrating a mechanistic link between the loss of Rab7.GTP and elevated cholesterol levels.
Assuntos
Hipercolesterolemia , Proteínas Monoméricas de Ligação ao GTP , Doenças Priônicas , Animais , Camundongos , Colesterol/metabolismo , Ativação Enzimática , Retroalimentação , Hipercolesterolemia/etiologia , Hipercolesterolemia/fisiopatologia , Lipoproteínas LDL/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Doenças Priônicas/metabolismo , Príons/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismoRESUMO
Alzheimer's disease (AD) is an incurable, progressive and devastating neurodegenerative disease. Pathogenesis of AD is associated with the aggregation and accumulation of amyloid beta (Aß), a major neurotoxic mediator that triggers neuroinflammation and memory impairment. Recently, we found that cellulose ether compounds (CEs) have beneficial effects against prion diseases by inhibiting protein misfolding and replication of prions, which share their replication mechanism with Aß. CEs are FDA-approved safe additives in foods and pharmaceuticals. Herein, for the first time we determined the therapeutic effects of the representative CE (TC-5RW) in AD using in vitro and in vivo models. Our in vitro studies showed that TC-5RW inhibits Aß aggregation, as well as neurotoxicity and immunoreactivity in Aß-exposed human and murine neuroblastoma cells. In in vivo studies, for the first time we observed that single and weekly TC-5RW administration, respectively, improved memory functions of transgenic 5XFAD mouse model of AD. We further demonstrate that TC-5RW treatment of 5XFAD mice significantly inhibited Aß oligomer and plaque burden and its associated neuroinflammation via regulating astrogliosis, microgliosis and proinflammatory mediator glial maturation factor beta (GMFß). Additionally, we determined that TC-5RW reduced lipopolysaccharide-induced activated gliosis and GMFß in vitro. In conclusion, our results demonstrate that CEs have therapeutic effects against Aß pathologies and cognitive impairments, and direct, potent anti-inflammatory activity to rescue neuroinflammation. Therefore, these FDA-approved compounds are effective candidates for developing therapeutics for AD and related neurodegenerative diseases associated with protein misfolding.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Doenças Neurodegenerativas , Camundongos , Animais , Humanos , Doença de Alzheimer/complicações , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Camundongos Transgênicos , Doenças Neuroinflamatórias , Éter , Fator de Maturação da Glia , Disfunção Cognitiva/tratamento farmacológico , Etil-Éteres/uso terapêutico , Éteres/uso terapêutico , Gliose/complicações , Cognição , Modelos Animais de DoençasRESUMO
Prion diseases are fatal infectious neurodegenerative disorders and prototypic conformational diseases, caused by the conformational conversion of the normal cellular prion protein (PrPC) into the pathological PrPSc isoform. Examples are scrapie in sheep and goat, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and Creutzfeldt-Jacob disease (CJD) in humans. There are no therapies available, and animal prion diseases like BSE and CWD can negatively affect the economy, ecology, animal health, and possibly human health. BSE is a confirmed threat to human health, and mounting evidence supports the zoonotic potential of CWD. CWD is continuously expanding in North America in numbers and distribution and was recently identified in Scandinavian countries. CWD is the only prion disease occurring both in wild and farmed animals, which, together with extensive shedding of infectivity into the environment, impedes containment strategies. There is currently a strong push to develop vaccines against CWD, including ones that can be used in wildlife. The immune system does not develop a bona fide immune response against prion infection, as PrPC and PrPSc share an identical protein primary structure, and prions seem not to represent a trigger for immune responses. This asks for alternative vaccine strategies, which focus on PrPC-directed self-antibodies or exposure of disease-specific structures and epitopes. Several groups have established a proof-of-concept that such vaccine candidates can induce some levels of protective immunity in cervid and rodent models without inducing unwanted side effects. This review will highlight the most recent developments and discuss progress and challenges remaining.
Assuntos
Cervos , Encefalopatia Espongiforme Bovina , Doenças Priônicas , Príons , Vacinas , Doença de Emaciação Crônica , Animais , Bovinos , Humanos , Ovinos , Objetivos , Doenças Priônicas/prevenção & controle , Doenças Priônicas/metabolismo , Príons/metabolismo , Encefalopatia Espongiforme Bovina/metabolismo , Doença de Emaciação Crônica/prevenção & controle , Doença de Emaciação Crônica/metabolismo , Cervos/metabolismo , CabrasRESUMO
Prions cause infectious and fatal neurodegenerative diseases in mammals. Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we provide evidence for a zoonotic potential of CWD prions, and its probable signature using mice expressing human prion protein (PrP) as an infection model. Inoculation of these mice with deer CWD isolates resulted in atypical clinical manifestation with prion seeding activity and efficient transmissible infectivity in the brain and, remarkably, in feces, but without classical neuropathological or Western blot appearances of prion diseases. Intriguingly, the protease-resistant PrP in the brain resembled that found in a familial human prion disease and was transmissible upon second passage. Our results suggest that CWD might infect humans, although the transmission barrier is likely higher compared to zoonotic transmission of cattle prions. Notably, our data suggest a different clinical presentation, prion signature, and tissue tropism, which causes challenges for detection by current diagnostic assays. Furthermore, the presence of infectious prions in feces is concerning because if this occurs in humans, it is a source for human-to-human transmission. These findings have strong implications for public health and CWD management.
Assuntos
Cervos , Príons , Doença de Emaciação Crônica , Animais , Western Blotting , Bovinos , Cervos/metabolismo , Humanos , Camundongos , Proteínas Priônicas/metabolismo , Príons/metabolismo , Doença de Emaciação Crônica/metabolismo , Doença de Emaciação Crônica/patologiaRESUMO
Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc Elucidating the molecular and cellular mechanisms underlying prion propagation may help to develop disease interventions. Cell culture systems for prion propagation have greatly advanced molecular insights into prion biology, but translation of in vitro to in vivo findings is often disappointing. A wider range of cell culture systems might help overcome these shortcomings. Here, we describe an immortalized mouse neuronal astrocyte cell line (C8D1A) that can be infected with murine prions. Both PrPC protein and mRNA levels in astrocytes were comparable with those in neuronal and non-neuronal cell lines permitting persistent prion infection. We challenged astrocytes with three mouse-adapted prion strains (22L, RML, and ME7) and cultured them for six passages. Immunoblotting results revealed that the astrocytes propagated 22L prions well over all six passages, whereas ME7 prions did not replicate, and RML prions replicated only very weakly after five passages. Immunofluorescence analysis indicated similar results for PrPSc Interestingly, when we used prion conversion activity as a readout in real-time quaking-induced conversion assays with RML-infected cell lysates, we observed a strong signal over all six passages, comparable with that for 22L-infected cells. These data indicate that the C8D1A cell line is permissive to prion infection. Moreover, the propagated prions differed in conversion and proteinase K-resistance levels in these astrocytes. We propose that the C8D1A cell line could be used to decipher prion strain biology.
Assuntos
Astrócitos/patologia , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/patologia , Agregação Patológica de Proteínas/patologia , Animais , Astrócitos/metabolismo , Linhagem Celular , Expressão Gênica , Humanos , Camundongos , Proteínas PrPC/análise , Proteínas PrPSc/análise , Doenças Priônicas/metabolismo , Agregação Patológica de Proteínas/metabolismoRESUMO
Misfolding of the prion protein (PrP) and templating of its pathological conformation onto cognate proteins causes a number of lethal disorders of central nervous system in humans and animals, such as Creutzfeldt-Jacob disease, chronic wasting disease and bovine spongiform encephalopathy. Structural rearrangement of PrPC into PrPSc promotes aggregation of misfolded proteins into ß-sheet-rich fibrils, which can be visualized by conformationally sensitive fluorescent probes. Early detection of prion misfolding and deposition might provide useful insights into its pathophysiology. Pentameric formyl thiophene acetic acid (pFTAA) is a novel amyloid probe that was shown to sensitively detect various misfolded proteins, including PrP. Here, we compared sensitivity of pFTAA staining and spectral microscopy with conventional methods of prion detection in mouse brains infected with mouse-adapted 22L prions. pFTAA bound to prion deposits in mouse brain sections exhibited a red-shifted fluorescence emission spectrum, which quantitatively increased with disease progression. Small prion deposits were detected as early as 50 days post-inoculation, well before appearance of clinical signs. Moreover, we detected significant spectral shifts in the greater brain parenchyma as early as 25 days post-inoculation, rivaling the most sensitive conventional method (real-time quaking-induced conversion). These results showcase the potential of pFTAA staining combined with spectral imaging for screening of prion-infected tissue. Not only does this method have comparable sensitivity to established techniques, it is faster and technically simpler. Finally, this readout provides valuable information about the spatial distribution of prion aggregates across tissue in the earliest stages of infection, potentially providing valuable pathophysiological insight into prion transmission.
Assuntos
Proteínas Priônicas/química , Acetatos , Animais , Química Encefálica , Corantes , Feminino , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia Confocal , Proteínas PrPSc/química , Doenças Priônicas/patologia , Agregados Proteicos , Deficiências na Proteostase/patologia , Proteínas Recombinantes/química , Espectrometria de Fluorescência , TiofenosRESUMO
Chronic wasting disease (CWD) is a prion disease found in both free-ranging and farmed cervids. Susceptibility of these animals to CWD is governed by various exogenous and endogenous factors. Past studies have demonstrated that polymorphisms within the prion protein (PrP) sequence itself affect an animal's susceptibility to CWD. PrP polymorphisms can modulate CWD pathogenesis in two ways: the ability of the endogenous prion protein (PrPC) to convert into infectious prions (PrPSc) or it can give rise to novel prion strains. In vivo studies in susceptible cervids, complemented by studies in transgenic mice expressing the corresponding cervid PrP sequence, show that each polymorphism has distinct effects on both PrPC and PrPSc. It is not entirely clear how these polymorphisms are responsible for these effects, but in vitro studies suggest they play a role in modifying PrP epitopes crucial for PrPC to PrPSc conversion and determining PrPC stability. PrP polymorphisms are unique to one or two cervid species and most confer a certain degree of reduced susceptibility to CWD. However, to date, there are no reports of polymorphic cervid PrP alleles providing absolute resistance to CWD. Studies on polymorphisms have focused on those found in CWD-endemic areas, with the hope that understanding the role of an animal's genetics in CWD can help to predict, contain, or prevent transmission of CWD.
Assuntos
Cervos/genética , Polimorfismo Genético , Proteínas Priônicas/genética , Doença de Emaciação Crônica/patologia , Sequência de Aminoácidos , Animais , Proteínas Priônicas/química , Zoonoses/patologia , Zoonoses/transmissãoRESUMO
Chronic wasting disease (CWD) is a prion disease of free-ranging and farmed cervids that is highly contagious because of extensive prion shedding and prion persistence in the environment. Previously, cellulose ether compounds (CEs) have been shown to significantly extend the survival of mice inoculated with mouse-adapted prion strains. In this study, we used CEs, TC-5RW, and 60SH-50, in vitro and in vivo to assess their efficacy to interfere with CWD prion propagation. In vitro, CEs inhibited CWD prion amplification in a dose-dependent manner. Transgenic mice over-expressing elk PrPC (tgElk) were injected subcutaneously with a single dose of either of the CEs, followed by intracerebral inoculation with different CWD isolates from white tailed deer, mule deer, or elk. All treated groups showed a prolonged survival of up to more than 30 % when compared to the control group regardless of the CWD isolate used for infection. The extended survival in the treated groups correlated with reduced proteinase K resistance of prions. Remarkably, passage of brain homogenates from treated or untreated animals in tgElk mice resulted in a prolonged life span of mice inoculated with homogenates from CE-treated mice (of + 17%) even in the absence of further treatment. Besides the delayed disease onset upon passage in TgElk mice, the reduced proteinase K resistance was maintained but less pronounced. Therefore, these compounds can be very useful in limiting the spread of CWD in captive and wild-ranging cervids.
Assuntos
Celulose/administração & dosagem , Éter/administração & dosagem , Peptídeo Hidrolases/metabolismo , Príons/metabolismo , Doença de Emaciação Crônica/metabolismo , Doença de Emaciação Crônica/prevenção & controle , Animais , Química Encefálica , Cervos , Expressão Gênica , Camundongos , Camundongos Transgênicos , Proteínas PrPSc/química , Proteínas Priônicas/química , Proteínas Priônicas/genética , Príons/administração & dosagem , Príons/efeitos dos fármacos , Conformação Proteica , Proteínas RecombinantesRESUMO
Prion diseases are fatal infectious neurodegenerative disorders in human and animals that are caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform PrPSc. No effective treatment is available for prion diseases. Metformin is a first-line medication for treatment of type 2 diabetes which is known to activate AMPK and induce autophagy through the inhibition of mammalian target of rapamycin (mTOR1) signaling. Metformin was reported to be beneficial in various protein misfolding and neurodegenerative diseases like Alzheimer's and Huntington's diseases. In this study we investigated the anti-prion effect of metformin in persistently prion-infected neuronal cells. Our data showed that metformin significantly decreased the PrPSc load in the treated cells, as shown by less PK resistant PrP in Western blots and reduced prion conversion activity in Real-Time Quaking-Induced Conversion (RT-QuIC) assay in both 22L-ScN2a and RML-ScCAD5 cells. Additionally, metformin induced autophagy as shown by higher levels of LC3-II in treated cells compared with control cells. On the other hand, our mouse bioassay showed that oral metformin at a dose of 2 mg/ml in drinking water had no effect on the survival of prion-infected mice. In conclusion, our findings describe the anti-prion effect of metformin in two persistently prion-infected neuronal cell lines. This effect can be explained at least partially by the autophagy inducing activity of metformin. This study sheds light on metformin as an anti-prion candidate for the combination therapy of prion diseases.
Assuntos
Autofagia/efeitos dos fármacos , Metformina/farmacologia , Doenças Priônicas/tratamento farmacológico , Animais , Linhagem Celular , Feminino , Camundongos Endogâmicos , Doenças Priônicas/mortalidade , Doenças Priônicas/patologia , Príons/metabolismoRESUMO
Prions are protein-based infectious agents that autocatalytically convert the cellular prion protein PrPC to its pathological isoform PrPSc Subsequent aggregation and accumulation of PrPSc in nervous tissues causes several invariably fatal neurodegenerative diseases in humans and animals. Prions can infect recipient cells when packaged into endosome-derived nanoparticles called exosomes, which are present in biological fluids such as blood, urine, and saliva. Autophagy is a basic cellular degradation and recycling machinery that also affects exosomal processing, but whether autophagy controls release of prions in exosomes is unclear. Our work investigated the effect of autophagy modulation on exosomal release of prions and how this interplay affects cellular prion infection. Exosomes isolated from cultured murine central neuronal cells (CAD5) and peripheral neuronal cells (N2a) contained prions as shown by immunoblotting for PrPSc, prion-conversion activity, and cell culture infection. We observed that autophagy stimulation with the mTOR inhibitor rapamycin strongly inhibited exosomal prion release. In contrast, inhibition of autophagy by wortmannin or CRISPR/Cas9-mediated knockout of the autophagy protein Atg5 (autophagy-related 5) greatly increased the release of exosomes and exosome-associated prions. We also show that a difference in exosomal prion release between CAD5 and N2a cells is related to differences at the level of basal autophagy. Taken together, our results indicate that autophagy modulation can control lateral transfer of prions by interfering with their exosomal release. We describe a novel role of autophagy in the prion life cycle, an understanding that may provide useful targets for containing prion diseases.
Assuntos
Autofagia , Exossomos/metabolismo , Neurônios/metabolismo , Proteínas Priônicas/metabolismo , Animais , Linhagem Celular , Exossomos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/patologiaRESUMO
Prions are composed solely of the pathological isoform (PrPSc) of the normal cellular prion protein (PrPC). Identification of different PrPSc structures is crucially important for understanding prion biology because the pathogenic properties of prions are hypothesized to be encoded in the structures of PrPSc However, these structures remain yet to be identified, because of the incompatibility of PrPSc with conventional high-resolution structural analysis methods. Previously, we reported that the region between the first and the second α-helix (H1â¼H2) of PrPC might cooperate with the more C-terminal side region for efficient interactions with PrPSc From this starting point, we created a series of PrP variants with two cysteine substitutions (C;C-PrP) forming a disulfide-crosslink between H1â¼H2 and the distal region of the third helix (Ctrm). We then assessed the conversion capabilities of the C;C-PrP variants in N2a cells infected with mouse-adapted scrapie prions (22L-ScN2a). Specifically, Cys substitutions at residues 165, 166, or 168 in H1â¼H2 were combined with cysteine scanning along Ctrm residues 220-229. We found that C;C-PrPs are expressed normally with glycosylation patterns and subcellular localization similar to WT PrP, albeit differing in expression levels. Interestingly, some C;C-PrPs converted to protease-resistant isoforms in the 22L-ScN2a cells, but not in Fukuoka1 prion-infected cells. Crosslink patterns of convertible C;C-PrPs indicated a positional change of H1â¼H2 toward Ctrm in PrPSc-induced conformational conversion. Given the properties of the C;C-PrPs reported here, we propose that these PrP variants may be useful tools for investigating prion strain-specific structures and structure-phenotype relationships of PrPSc.
Assuntos
Reagentes de Ligações Cruzadas/química , Dissulfetos/química , Neuroblastoma/patologia , Proteínas PrPSc/metabolismo , Príons/patogenicidade , Conformação Proteica , Scrapie/patologia , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Neuroblastoma/metabolismo , Proteínas PrPSc/química , Príons/química , Príons/genética , Scrapie/metabolismo , Homologia de Sequência , Células Tumorais CultivadasRESUMO
Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrPSc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type- or prion strain-specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrPSc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy.
Assuntos
Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Feminino , Expressão Gênica , Humanos , Lectinas de Ligação a Manose/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Isomerases de Dissulfetos de Proteínas/genéticaRESUMO
Chronic wasting disease (CWD) is a fatal neurodegenerative disease that affects cervids in North America and now Europe. No effective measures are available to control CWD. We hypothesized that active vaccination with homologous and aggregation-prone recombinant prion protein (PrP) could overcome self-tolerance and induce autoantibody production against the cellular isoform of PrP (PrPC), which would be protective against CWD infection from peripheral routes. Five groups of transgenic mice expressing elk PrP (TgElk) were vaccinated with either the adjuvant CpG alone or one of four recombinant PrP immunogens: deer dimer (Ddi); deer monomer (Dmo); mouse dimer (Mdi); and mouse monomer (Mmo). Mice were then challenged intraperitoneally with elk CWD prions. All vaccinated mice developed ELISA-detectable antibody titers against PrP. Importantly, all four vaccinated groups survived longer than the control group, with the Mmo-immunized group exhibiting 60% prolongation of mean survival time compared with the control group (183 versus 114 days post-inoculation). We tested for prion infection in brain and spleen of all clinically sick mice. Notably, the attack rate was 100% as revealed by positive CWD signals in all tested tissues when assessed with Western blotting, real-time quaking-induced conversion, and immunohistochemistry. Our pilot study in reindeer indicated appreciable humoral immune responses to Mdi and Ddi immunogens, and the post-immune sera from the Ddi-vaccinated reindeer mitigated CWD propagation in a cell culture model (CWD-RK13). Taken together, our study provides very promising vaccine candidates against CWD, but further studies in cervids are required to investigate vaccine efficacy in the natural CWD hosts.
Assuntos
Proteínas Priônicas/imunologia , Proteínas Recombinantes/imunologia , Rena/imunologia , Vacinação , Doença de Emaciação Crônica/prevenção & controle , Animais , Autoanticorpos/imunologia , Feminino , Camundongos , Camundongos Transgênicos , Doença de Emaciação Crônica/imunologiaRESUMO
A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.
Assuntos
Neuroblastoma/prevenção & controle , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Multimerização Proteica , Scrapie/prevenção & controle , Animais , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Neuroblastoma/patologia , Transporte Proteico , Scrapie/patologia , Células Tumorais CultivadasRESUMO
Prions are self-templating protein conformers that replicate by recruitment and conversion of homotypic proteins into growing protein aggregates. Originally identified as causative agents of transmissible spongiform encephalopathies, increasing evidence now suggests that prion-like phenomena are more common in nature than previously anticipated. In contrast to fungal prions that replicate in the cytoplasm, propagation of mammalian prions derived from the precursor protein PrP is confined to the cell membrane or endocytic vesicles. Here we demonstrate that cytosolic protein aggregates can also behave as infectious entities in mammalian cells. When expressed in the mammalian cytosol, protein aggregates derived from the prion domain NM of yeast translation termination factor Sup35 persistently propagate and invade neighboring cells, thereby inducing a self-perpetuating aggregation state of NM. Cell contact is required for efficient infection. Aggregates can also be induced in primary astrocytes, neurons, and organotypic cultures, demonstrating that this phenomenon is not specific to immortalized cells. Our data have important implications for understanding prion-like phenomena of protein aggregates associated with human diseases and for the growing number of amyloidogenic proteins discovered in mammals.
Assuntos
Comunicação Celular , Citosol/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Astrócitos/citologia , Técnicas de Cocultura , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Camundongos , Microscopia Confocal , Doenças Priônicas , Conformação Proteica , Saccharomyces cerevisiae/metabolismoRESUMO
Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrP(Sc) of the host-encoded prion protein (PrP(c)). A profound conformational change of PrP(c) underlies formation of PrP(Sc) and prion propagation involves conversion of PrP(c) substrate by direct interaction with PrP(Sc) template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI) effects of conversion-incompetent, internally-deleted PrP (ΔPrP) on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1â¼H2) which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1â¼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrP(C) into PrP(Sc). To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale of prion conversion and PrP(Sc) recycling and can exert DNI there. This shows that the intracellular trafficking of PrPs is more complex than previously anticipated.