RESUMEN
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.
Asunto(s)
Encéfalo , Ciervos , Proteínas PrPSc , Enfermedad Debilitante Crónica , Animales , Ratones , Enfermedad Debilitante Crónica/transmisión , Encéfalo/metabolismo , Encéfalo/patología , Proteínas PrPSc/metabolismo , Conformación Proteica , Priones/metabolismo , Priones/patogenicidad , Enfermedades por Prión/transmisión , Enfermedades por Prión/patología , Enfermedades por Prión/metabolismo , Ratones TransgénicosRESUMEN
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.
Asunto(s)
Ciervos , Enfermedad Debilitante Crónica , Animales , Enfermedad Debilitante Crónica/transmisión , Enfermedad Debilitante Crónica/metabolismo , Ratones , Encéfalo/metabolismo , Encéfalo/patología , Proteínas Priónicas/metabolismo , Proteínas Priónicas/genética , Ratones Transgénicos , Noruega , Marcación de Gen , Priones/metabolismo , Priones/genética , Priones/patogenicidadRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Disfunción Cognitiva , Enfermedades Neurodegenerativas , Ratones , Animales , Humanos , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Péptidos beta-Amiloides/metabolismo , Ratones Transgénicos , Enfermedades Neuroinflamatorias , Éter , Factor de Maduración de la Glia , Disfunción Cognitiva/tratamiento farmacológico , Éteres de Etila/uso terapéutico , Éteres/uso terapéutico , Gliosis/complicaciones , Cognición , Modelos Animales de EnfermedadRESUMEN
Prion diseases are a novel class of infectious disease based in the misfolding of the cellular prion protein (PrPC) into a pathological, self-propagating isoform (PrPSc). These fatal, untreatable neurodegenerative disorders affect a variety of species causing scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and Creutzfeldt-Jacob disease (CJD) in humans. Of the animal prion diseases, CWD is currently regarded as the most significant threat due its ongoing geographical spread, environmental persistence, uptake into plants, unpredictable evolution, and emerging evidence of zoonotic potential. The extensive efforts to manage CWD have been largely ineffective, highlighting the need for new disease management tools, including vaccines. Development of an effective CWD vaccine is challenged by the unique biology of these diseases, including the necessity, and associated dangers, of overcoming immune tolerance, as well the logistical challenges of vaccinating wild animals. Despite these obstacles, there has been encouraging progress towards the identification of safe, protective antigens as well as effective strategies of formulation and delivery that would enable oral delivery to wild cervids. In this review we highlight recent strategies for antigen selection and optimization, as well as considerations of various platforms for oral delivery, that will enable researchers to accelerate the rate at which candidate CWD vaccines are developed and evaluated.
Asunto(s)
Antígenos , Ciervos , Proteínas PrPC , Vacunas de Subunidades Proteicas , Desarrollo de Vacunas , Enfermedad Debilitante Crónica , Zoonosis , Animales , Humanos , Administración Oral , Antígenos/administración & dosificación , Antígenos/inmunología , Vectores Genéticos , Inmunoterapia , Vacunas de Subunidades Proteicas/administración & dosificación , Vacunas de Subunidades Proteicas/inmunología , Proteínas PrPC/inmunología , Proteínas PrPC/uso terapéutico , Vacunación , Enfermedad Debilitante Crónica/prevención & control , Enfermedad Debilitante Crónica/transmisión , Zoonosis/prevención & control , Zoonosis/transmisiónRESUMEN
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.
Asunto(s)
Ciervos , Encefalopatía Espongiforme Bovina , Enfermedades por Prión , Priones , Vacunas , Enfermedad Debilitante Crónica , Animales , Bovinos , Humanos , Ovinos , Objetivos , Enfermedades por Prión/prevención & control , Enfermedades por Prión/metabolismo , Priones/metabolismo , Encefalopatía Espongiforme Bovina/metabolismo , Enfermedad Debilitante Crónica/prevención & control , Enfermedad Debilitante Crónica/metabolismo , Ciervos/metabolismo , CabrasRESUMEN
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.
Asunto(s)
Hipercolesterolemia , Proteínas de Unión al GTP Monoméricas , Enfermedades por Prión , Animales , Ratones , Colesterol/metabolismo , Activación Enzimática , Retroalimentación , Hipercolesterolemia/etiología , Hipercolesterolemia/fisiopatología , Lipoproteínas LDL/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuronas/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismoRESUMEN
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.
Asunto(s)
Investigación Biomédica , Humanos , Consenso , Técnica Delphi , Encuestas y Cuestionarios , Proyectos de InvestigaciónRESUMEN
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.
Asunto(s)
Ciervos , Priones , Enfermedad Debilitante Crónica , Animales , Western Blotting , Bovinos , Ciervos/metabolismo , Humanos , Ratones , Proteínas Priónicas/metabolismo , Priones/metabolismo , Enfermedad Debilitante Crónica/metabolismo , Enfermedad Debilitante Crónica/patologíaRESUMEN
Prion diseases are fatal infectious neurodegenerative disorders affecting both humans and animals. They are caused by the misfolded isoform of the cellular prion protein (PrPC), PrPSc, and currently no options exist to prevent or cure prion diseases. Chronic wasting disease (CWD) in deer, elk and other cervids is considered the most contagious prion disease, with extensive shedding of infectivity into the environment. Cell culture models provide a versatile platform for convenient quantification of prions, for studying the molecular and cellular biology of prions, and for performing high-throughput screening of potential therapeutic compounds. Unfortunately, only a very limited number of cell lines are available that facilitate robust and persistent propagation of CWD prions. Gene-editing using programmable nucleases (e.g., CRISPR-Cas9 (CC9)) has proven to be a valuable tool for high precision site-specific gene modification, including gene deletion, insertion, and replacement. CC9-based gene editing was used recently for replacing the PrP gene in mouse and cell culture models, as efficient prion propagation usually requires matching sequence homology between infecting prions and prion protein in the recipient host. As expected, such gene-editing proved to be useful for developing CWD models. Several transgenic mouse models were available that propagate CWD prions effectively, however, mostly fail to reproduce CWD pathogenesis as found in the cervid host, including CWD prion shedding. This is different for the few currently available knock-in mouse models that seem to do so. In this review, we discuss the available in vitro and in vivo models of CWD, and the impact of gene-editing strategies.
Asunto(s)
Ciervos , Enfermedades por Prión , Priones , Enfermedad Debilitante Crónica , Animales , Edición Génica , Ratones , Proteínas Priónicas/genética , Enfermedad Debilitante Crónica/genéticaRESUMEN
Prion diseases are infectious protein misfolding disorders of the central nervous system that result from misfolding of the cellular prion protein (PrPC) into the pathologic isoform PrPSc. Pathologic hallmarks of prion disease are depositions of pathological prion protein PrPSc, neuronal loss, spongiform degeneration and astrogliosis in the brain. Prion diseases affect human and animals, there is no effective therapy, and they invariably remain fatal. For a long time, neuronal loss was considered the sole reason for neurodegeneration in prion pathogenesis, and the contribution of non-neuronal cells like microglia and astrocytes was considered less important. Recent evidence suggests that neurodegeneration during prion pathogenesis is a consequence of a complex interplay between neuronal and non-neuronal cells in the brain, but the exact role of these non-neuronal cells during prion pathology is still elusive. Astrocytes are non-neuronal cells that regulate brain homeostasis under physiological conditions. However, astrocytes can deposit PrPSc aggregates and propagate prions in prion-infected brains. Additionally, sub-populations of reactive astrocytes that include neurotrophic and neurotoxic species have been identified, differentially expressed in the brain during prion infection. Revealing the exact role of astrocytes in prion disease is hampered by the lack of in vitro models of prion-infected astrocytes. Recently, we established a murine astrocyte cell line persistently infected with mouse-adapted prions, and showed how such astrocytes differentially process various prion strains. Considering the complexity of the role of astrocytes in prion pathogenesis, we need more in vitro and in vivo models for exploring the contribution of sub-populations of reactive astrocytes, their differential regulation of signaling cascades, and the interaction with neurons and microglia during prion pathogenesis. This will help to establish novel in vivo models and define new therapeutic targets against prion diseases. In this review, we will discuss the complex role of astrocytes in prion disease, the existing experimental resources, the challenges to analyze the contribution of astrocytes in prion disease pathogenesis, and future strategies to improve the understanding of their role in prion disease.
RESUMEN
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.
Asunto(s)
Ciervos/genética , Polimorfismo Genético , Proteínas Priónicas/genética , Enfermedad Debilitante Crónica/patología , Secuencia de Aminoácidos , Animales , Proteínas Priónicas/química , Zoonosis/patología , Zoonosis/transmisiónRESUMEN
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.
Asunto(s)
Proteínas Priónicas/química , Acetatos , Animales , Química Encefálica , Colorantes , Femenino , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía Confocal , Proteínas PrPSc/química , Enfermedades por Prión/patología , Agregado de Proteínas , Deficiencias en la Proteostasis/patología , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , TiofenosRESUMEN
Many devastating neurodegenerative diseases are driven by the misfolding of normal proteins into a pathogenic abnormal conformation. Examples of such protein misfolding diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases. The misfolded proteins involved in these diseases form self-templating oligomeric assemblies that recruit further correctly folded protein and induce their conversion. Over time, this leads to the formation of high molecular and mostly fibrillar aggregates that are increasingly inefficient at converting normal protein. Evidence from a multitude of in vitro models suggests that fibrils are fragmented to form new seeds, which can convert further normal protein and also spread to neighboring cells as observed in vivo. While fragmentation and seed generation were suggested as crucial steps in aggregate formation decades ago, the biological pathways involved remain largely unknown. Here, we show that mechanisms of aggregate clearance-namely the mammalian Hsp70-Hsp40-Hsp110 tri-chaperone system, macro-autophagy, and the proteasome system-may not only be protective, but also play a role in fragmentation. We further review the challenges that exist in determining the precise contribution of these mechanisms to protein misfolding diseases and suggest future directions to resolve these issues.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Proteínas Amiloidogénicas/química , Esclerosis Amiotrófica Lateral/metabolismo , Enfermedad de Huntington/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedades por Prión/metabolismo , Proteínas Priónicas/química , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Autofagia/genética , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP110/genética , Proteínas del Choque Térmico HSP110/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Enfermedades por Prión/genética , Enfermedades por Prión/patología , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas , Pliegue de ProteínaRESUMEN
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.
Asunto(s)
Astrocitos/patología , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/patología , Agregación Patológica de Proteínas/patología , Animales , Astrocitos/metabolismo , Línea Celular , Expresión Génica , Humanos , Ratones , Proteínas PrPC/análisis , Proteínas PrPSc/análisis , Enfermedades por Prión/metabolismo , Agregación Patológica de Proteínas/metabolismoRESUMEN
Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform PrPSc. These diseases have the potential to transmit within or between species, and no cure is available to date. Targeting the unfolded protein response (UPR) as an anti-prion therapeutic approach has been widely reported for prion diseases. Here, we describe the anti-prion effect of the chemical compound Sephin1 which has been shown to protect in mouse models of protein misfolding diseases including amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) by selectively inhibiting the stress-induced regulatory subunit of protein phosphatase 1, thus prolonging eIF2α phosphorylation. We show here that Sephin1 dose and time dependently reduced PrPSc in different neuronal cell lines which were persistently infected with various prion strains. In addition, prion seeding activity was reduced in Sephin1-treated cells. Importantly, we found that Sephin1 significantly overcame the endoplasmic reticulum (ER) stress induced in treated cells, as measured by lower expression of stress-induced aberrant prion protein. In a mouse model of prion infection, intraperitoneal treatment with Sephin1 significantly prolonged survival of prion-infected mice. When combining Sephin1 with the neuroprotective drug metformin, the survival of prion-infected mice was also prolonged. These results suggest that Sephin1 could be a potential anti-prion drug selectively targeting one component of the UPR pathway.
Asunto(s)
Guanabenzo/análogos & derivados , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Priones/efectos de los fármacos , Scrapie/tratamiento farmacológico , Respuesta de Proteína Desplegada/efectos de los fármacos , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Guanabenzo/administración & dosificación , Guanabenzo/farmacología , Guanabenzo/uso terapéutico , Metformina/administración & dosificación , Metformina/farmacología , Metformina/uso terapéutico , Ratones , Neuroblastoma/patología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 1/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Scrapie/patologíaRESUMEN
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.
Asunto(s)
Autofagia/efectos de los fármacos , Metformina/farmacología , Enfermedades por Prión/tratamiento farmacológico , Animales , Línea Celular , Femenino , Ratones Endogámicos , Enfermedades por Prión/mortalidad , Enfermedades por Prión/patología , Priones/metabolismoRESUMEN
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.
Asunto(s)
Celulosa/administración & dosificación , Éter/administración & dosificación , Péptido Hidrolasas/metabolismo , Priones/metabolismo , Enfermedad Debilitante Crónica/metabolismo , Enfermedad Debilitante Crónica/prevención & control , Animales , Química Encefálica , Ciervos , Expresión Génica , Ratones , Ratones Transgénicos , Proteínas PrPSc/química , Proteínas Priónicas/química , Proteínas Priónicas/genética , Priones/administración & dosificación , Priones/efectos de los fármacos , Conformación Proteica , Proteínas RecombinantesRESUMEN
Prion diseases are fatal transmissible neurodegenerative disorders that affect animals and humans. Prions are proteinaceous infectious particles consisting of a misfolded isoform of the cellular prion protein PrPC, termed PrPSc. PrPSc accumulates in infected neurons due to partial resistance to proteolytic digestion. Using compounds that interfere with the production of PrPSc or enhance its degradation cure prion infection in vitro, but most drugs failed when used to treat prion-infected rodents. In order to synergize the effect of anti-prion drugs, we combined drugs interfering with the generation of PrPSc with compounds inducing PrPSc degradation. Here, we tested autophagy stimulators (rapamycin or AR12) and cellulose ether compounds (TC-5RW or 60SH-50) either as single or combination treatment of mice infected with RML prions. Single drug treatments significantly extended the survival compared to the untreated group. As anticipated, also all the combination therapy groups showed extended survival compared to the untreated group, but no combination treatment showed superior effects to 60SH-50 or TC-5RW treatment alone. Unexpectedly, we later found that combining autophagy stimulator and cellulose ether treatment in cultured neuronal cells mitigated the pro-autophagic activity of AR12 and rapamycin, which can in part explain the in vivo results. Overall, we show that it is critical to exclude antagonizing drug effects when attempting combination therapy. In addition, we identified AR-12 as a pro-autophagic drug that significantly extends survival of prion-infected mice, has no adverse side effects on the animals used in this study, and can be useful in future studies.
Asunto(s)
Autofagia/efectos de los fármacos , Celulosa/uso terapéutico , Proteínas PrPSc/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Sirolimus/uso terapéutico , Animales , Celulosa/análogos & derivados , Sinergismo Farmacológico , Éteres/química , Éteres/uso terapéutico , Femenino , Ratones , Proteínas PrPSc/antagonistas & inhibidores , Enfermedades por Prión/metabolismo , Proteolisis/efectos de los fármacosRESUMEN
Prions cause fatal infectious neurodegenerative diseases in humans and animals. Cell culture models are essential for studying the molecular biology of prion propagation. Defining such culture models is mostly a random process, includes extensive subcloning, and for many prion diseases few or no models exist. One example is chronic wasting disease (CWD), a highly contagious prion disease of cervids. To extend the range of cell models propagating CWD prions, we gene-edited mouse cell lines known to efficiently propagate murine prions. Endogenous prion protein (PrP) was ablated in CAD5 and MEF cells, using CRISPR-Cas9 editing. PrP knock-out cells were reconstituted with mouse, bank vole and cervid PrP genes by lentiviral transduction. Reconstituted cells expressing mouse PrP provided proof-of-concept for re-established prion infection. Bank voles are considered universal receptors for prions from a variety of species. Bank vole PrP reconstituted cells propagated mouse prions and cervid prions, even without subcloning for highly susceptible cells. Cells reconstituted with cervid PrP and infected with CWD prions tested positive in prion conversion assay, whereas non-reconstituted cells were negative. This novel cell culture platform which is easily adjustable and allows testing of polymorphic alleles will provide important new insights into the biology of CWD prions.