[Show abstract][Hide abstract] ABSTRACT: Mammalian prions are proteinaceous pathogens responsible for a broad range of fatal neurodegenerative diseases in humans and animals. These diseases can occur spontaneously, such as Creutzfeldt-Jakob disease (CJD) in humans, or be acquired or inherited. Prions are primarily formed of macromolecular assemblies of the disease-associated prion protein PrP Sc , a misfolded isoform of the host-encoded prion protein PrP C . Within defined host-species, prions can exist as conformational variants or strains. Based on both the M/V polymorphism at codon 129 of PrP and the electrophoretic signature of PrP Sc in the brain, sporadic CJD is classified in different subtypes, which may encode different strains. A transmission barrier, the mechanism of which remains unknown, limits prion cross-species propagation. To adapt to the new host, prions have the capacity to ‘mutate’ conformationally, leading to the emergence of a variant with new biological properties. Here, we transmitted experimentally one rare subtype of human CJD, designated cortical MM2 (129 MM with type 2 PrP Sc ), to transgenic mice overexpressing either human or the VRQ allele of ovine PrP C .
In marked contrast with the reported absence of transmission to knock-in mice expressing physiological levels of human PrP, this subtype transmitted faithfully to mice overexpressing human PrP, and exhibited unique strain features. Onto the ovine PrP sequence, the cortical MM2 subtype abruptly evolved on second passage, thereby allowing emergence of a pair of strain variants with distinct PrP Sc biochemical characteristics and differing tropism for the central and lymphoid tissues. These two strain components exhibited remarkably distinct replicative properties in cell-free amplification assay, allowing the ‘physical’ cloning of the minor, lymphotropic component, and subsequent isolation in ovine PrP mice and RK13 cells.
Here, we provide in-depth assessment of the transmissibility and evolution of one rare subtype of sporadic CJD upon homologous and heterologous transmission. The notion that the environment or matrix where replication is occurring is key to the selection and preferential amplification of prion substrain components raises new questions on the determinants of prion replication within and between species. These data also further interrogate on the interplay between animal and human prions.
[Show abstract][Hide abstract] ABSTRACT: The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10-8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.
[Show abstract][Hide abstract] ABSTRACT: Although conversion of the cellular form of the prion protein (PrPC) into a misfolded isoform is the underlying cause of prion diseases, understanding PrPC physiological functions has remained challenging. PrPC depletion or overexpression alters the proliferation and differentiation properties of various types of stem and progenitor cells in vitro by unknown mechanisms. Such involvement remains uncertain in vivo in the absence of any drastic phenotype of mice lacking PrPC. Here, we report PrPC enrichment at the base of the primary cilium in stem and progenitor cells from the central nervous system and cardiovascular system of developing mouse embryos. PrPC depletion in a neuroepithelial cell line dramatically altered key cilium-dependent processes, such as Sonic hedgehog signalling and alpha-tubulin post-translational modifications. These processes were also affected over a limited time window in PrPC-ablated embryos. Thus, our study reveals PrPC as a potential actor in the developmental regulation of microtubule dynamics and ciliary functions.
Full-text · Article · Jan 2016 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: The phenomenon of protein superstructural polymorphism has become the subject of increased research activity. Besides the relevance to explain the existence of multiple prion strains, such activity is partly driven by the recent finding that in many age-related neurodegenerative diseases highly ordered self-associated forms of peptides and proteins might be the structural basis of prion-like processes and strains giving rise to different disease phenotypes. Biophysical studies of prion strains have been hindered by a lack of tools to characterize inherently noncrystalline, heterogeneous and insoluble proteins. A description of the pressure response of prion quaternary structures might change this picture. This is because applying pressure induces quaternary structural changes of PrP, such as misfolding and self-assembly. From the thermodynamics of these processes structural features in terms of associated volume changes can then be deduced. We suggest that conformation-enciphered prion strains can be distinguished in terms of voids in the interfaces of the constituting PrP protomers and thus in their volumetric properties.
[Show abstract][Hide abstract] ABSTRACT: Mammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the 23-31 N-terminal polybasic region of PrP has been shown to be involved in its endocytic trafficking and interactions with glycosaminoglycans or putative ectodomains of membrane-associated proteins. Several recent reports also describe this PrP region as important for the toxicity of mutant prion proteins and the efficiency of prion propagation, both
. The question remains as to whether the latter observations made with mouse PrP and mouse prions would be relevant to other PrP species/prion strain combinations given the dramatic impact on prion susceptibility of minimal amino acid substitution and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP deleted for residues 23-26 (KKRP) or mutated in this N-terminal region (KQHPH instead of KKRPK) exhibited a variable, strain-dependent susceptibility to prion infection with regard to the proportion of affected mice and disease tempo relative to their wild-type counterparts. Deletion has no major effect on 127S scrapie prion pathogenesis, whereas mutation increased by almost 3-fold the survival time of the mice. Deletion marginally affected the incubation time of scrapie LA19K and ovine BSE prions, whereas mutation caused apparent resistance to disease.
Recent reports suggested that the N-terminal polybasic region of the prion protein could be a therapeutic target to prevent prion propagation or toxic signaling associated with more common neurodegenerative diseases such as Alzheimer's disease. Mutating or deleting this region in ovine PrP completes the data previously obtained with the mouse protein, by identifying the key amino acid residues involved.
Full-text · Article · Nov 2015 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide range of mammalian species. They are caused by prions, a proteinaceous pathogen essentially composed of PrPSc, an abnormal isoform of the host encoded cellular prion protein PrPC. Constrained steric interactions between PrPSc and PrPC are thought to provide prions with species specificity, and to control cross-species transmission into other host populations, including humans. Transgenetic expression of foreign PrP genes has been successfully and widely used to overcome the recognized resistance of mouse to foreign TSE sources. Rabbit is one of the species that exhibit a pronounced resistance to TSEs. Most attempts to infect experimentally rabbit have failed, except after inoculation with cell-free generated rabbit prions. To gain insights on the molecular determinants of the relative resistance of rabbits to prions, we generated transgenic rabbits expressing the susceptible V136R154Q171 allele of the ovine PRNP gene on a rabbit wild type PRNP New Zealand background and assessed their experimental susceptibility to scrapie prions. All transgenic animals developed a typical TSE 6-8 months after intracerebral inoculation, whereas wild type rabbits remained healthy more than 700 days after inoculation. Despite the endogenous presence of rabbit PrPC, only ovine PrPSc was detectable in the brains of diseased animals. Collectively these data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background.
[Show abstract][Hide abstract] ABSTRACT: Prion diseases are characterised by conformational changes of a cellular prion protein (PrPC) into a β-sheet-enriched and aggregated conformer (PrPSc). Shadoo (Sho), a member of the prion protein family, is expressed in the central nervous system (CNS) and is highly conserved among vertebrates. Based on histo-anatomical co-localisation and sequence similarities, it is suspected that Sho and PrP may be functionally related. The down-regulation of Sho expression during prion pathology and the direct interaction between Sho and PrP, as revealed by the two-hybrid analysis, suggest a relationship between Sho and prion replication. Using biochemical and biophysical approaches, we demonstrate that Sho forms a 1:1 complex with full-length PrP, with a Kd in the micromolar range, and this interaction consequently modifies the PrP folding pathway. Using a truncated PrP that mimics the C1 fragment, an allosteric binding behaviour with a Hill number of 4 was observed, suggesting that at least a tetramerisation state occurs. Cell-based prion titration assay performed with different concentrations of Sho revealed an increase in the PrPSc conversion rate in the presence of Sho. Collectively, our observations suggest that Sho can affect the prion replication process by 1) acting as a holdase and 2) interfering with the dominant-negative inhibitor effect of the C1 fragment.
Since the inception of the prion theory, the search for a cofactor involved in the conversion process has been an active field of research. Although the PrP interactome presents a broad landscape, candidates corresponding to specific criteria for cofactors are currently missing. Here, we describe for the first time that Sho can affect PrP structural dynamics and therefore increase the prion conversion rate. A biochemical characterisation of Sho:PrP indicates that Sho acts as an ATP-independent holdase.
Full-text · Article · Apr 2015 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The prion protein (PrP) binds to various molecular partners, but little is known about their potential impact on the pathogenesis of prion diseases
Here, we show that PrP can interact in vitro with acetylcholinesterase (AChE), a key protein of the cholinergic system in neural and non-neural tissues. This heterologous association induced aggregation of monomeric PrP and modified the structural properties of PrP amyloid fibrils. Following its recruitment into PrP fibrils, AChE loses its enzymatic activity and enhances PrP-mediated cytotoxicity. Using several truncated PrP variants and specific tight-binding AChE inhibitors (AChEis), we then demonstrate that the PrP-AChE interaction requires two mutually exclusive sub-sites in PrP N-terminal domain and an aromatic-rich region at the entrance of AChE active center gorge. We show that AChEis that target this site impair PrP-AChE complex formation and also limit the accumulation of pathological prion protein (PrPSc) in prion-infected cell cultures. Furthermore, reduction of AChE levels in prion-infected heterozygous AChE knock-out mice leads to slightly but significantly prolonged incubation time. Finally, we found that AChE levels were altered in prion-infected cells and tissues, suggesting that AChE might be directly associated with abnormal PrP.
Our results indicate that AChE deserves consideration as a new actor in expanding pathologically relevant PrP morphotypes and as a therapeutic target.
[Show abstract][Hide abstract] ABSTRACT: Although Bovine Spongiform Encephalopathy (BSE) is the cause of variant Creutzfeldt Jakob disease (vCJD) in humans, the zoonotic potential of scrapie prions remains unknown. Mice genetically engineered to overexpress the human prion protein (tgHu) have emerged as highly relevant models for gauging the capacity of prions to transmit to humans. These models can propagate human prions without any apparent transmission barrier and have been used used to confirm the zoonotic ability of BSE. Here we show that a panel of sheep scrapie prions transmit to several tgHu mice models with an efficiency comparable to that of cattle BSE. The serial transmission of different scrapie isolates in these mice led to the propagation of prions that are phenotypically identical to those causing sporadic CJD (sCJD) in humans. These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.
Full-text · Article · Dec 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: The deletion of the cellular form of the prion protein (PrP(C)) in mouse, goat, and cattle has no drastic phenotypic consequence. This stands in apparent contradiction with PrP(C) quasi-ubiquitous expression and conserved primary and tertiary structures in mammals, and its pivotal role in neurodegenerative diseases such as prion and Alzheimer's diseases. In zebrafish embryos, depletion of PrP ortholog leads to a severe loss-of-function phenotype. This raises the question of a potential role of PrP(C) in the development of all vertebrates. This view is further supported by the early expression of the PrP(C) encoding gene (Prnp) in many tissues of the mouse embryo, the transient disruption of a broad number of cellular pathways in early Prnp(-/-) mouse embryos, and a growing body of evidence for PrP(C) involvement in the regulation of cell proliferation and differentiation in various types of mammalian stem cells and progenitors. Finally, several studies in both zebrafish embryos and in mammalian cells and tissues in formation support a role for PrP(C) in cell adhesion, extra-cellular matrix interactions and cytoskeleton. In this review, we summarize and compare the different models used to decipher PrP(C) functions at early developmental stages during embryo- and organo-genesis and discuss their relevance.
Full-text · Article · Oct 2014 · Frontiers in Cell and Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: Guanabenz (GA) is an orally active α2-adrenergic agonist that has been used for many years for the treatment of hypertension. We recently described that GA is also active against both yeast and mammalian prions in an α2-adrenergic receptor-independent manner. These data suggest that this side-activity of GA could be explored for the treatment of prion-based diseases and other amyloid-based disorders. In this perspective, the potent antihypertensive activity of GA happens to be an annoying side-effect that could limit its use. In order to get rid of GA agonist activity at α2-adrenergic receptors, we performed a structure-activity relationship study around GA based on changes of the chlorine positions on the benzene moiety and then on the modifications of the guanidine group. Hence, we identified the two derivatives 6 and 7 that still possess a potent antiprion activity but were totally devoid of any agonist activity at α2-adrenergic receptors. Similarly to GA, 6 and 7 were also able to inhibit the protein folding activity of the ribosome (PFAR) which has been suggested to be involved in prion appearance/maintenance. Therefore, these two GA derivatives are worth being considered as drug candidates.
No preview · Article · Sep 2014 · ACS Chemical Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods - in vitro, ex vivo and in vivo assays - to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA) and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages). However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.
[Show abstract][Hide abstract] ABSTRACT: Based on its developmental pattern of expression, early studies suggested the implication of the mammalian Prion protein PrP, a glycosylphosphatidylinositol-anchored ubiquitously expressed and evolutionary conserved glycoprotein encoded by the Prnp gene, in early embryogenesis. However, gene invalidation in several species did not result in obvious developmental abnormalities and it was only recently that it was associated in mice with intra-uterine growth retardation and placental dysfunction. A proposed explanation for this lack of easily detectable developmental-related phenotype is the existence in the genome of one or more gene (s) able to compensate for the absence of PrP. Indeed, two other members of the Prnp gene family have been recently described, Doppel and Shadoo, and the consequences of their invalidation alongside that of PrP tested in mice. No embryonic defect was observed in mice depleted for Doppel and PrP. Interestingly, the co-invalidation of PrP and Shadoo in two independent studies led to apparently conflicting observations, with no apparent consequences in one report and the observation of a developmental defect of the ectoplacental cone that leads to early embryonic lethality in the other. This short review aims at summarizing these recent, apparently conflicting data highlighting the related biological questions and associated implications in terms of animal and human health.
Full-text · Article · Aug 2014 · Frontiers in Cell and Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context, the risk of vCJD blood borne transmission is considered as a serious concern by health authorities. In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Amplification (PMCA) were first identified. This showed that whatever the origin (species) of the vCJD/BSE agent, the ovine Q171 PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent propagation in vitro. The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, sample panels that included buffy coat from vCJD affected patients and healthy controls were tested blind. The assay identified three out of the four tested vCJD affected patients and no false positive was observed in 141 healthy controls. The negative results observed in one of the tested vCJD cases concurs with results reported by others using a different vCJD agent blood detection assay and raises the question of the potential absence of prionemia in certain patients.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
The dietary exposure of the human population to the prions responsible for the bovine spongiform encephalopathy (BSE) epizooty has led to the emergence of variant Creutzfeldt-Jakob disease (vCJD). This fatal, untreatable neurodegenerative disorder is a growing public health concern because the prevalence of the infection seems much greater than the disease incidence and because secondary transmission of vCJD by blood transfusion or use of blood products has occurred. A current limitation in variant CJD risk assessment is the lack of quantitative information on the infectivity of contaminated tissues. To address this limitation, we tested the potential of a transgenic mouse line overexpressing human prion protein (PrP), which was previously reported to propagate vCJD prions. Endpoint titration of vCJD infectivity in different tissues was evaluated by two different methods: (i) the "classical" bioassay, based on the appearance of clinical symptoms and the detection of pathological prion protein in tissues of the inoculated mouse, and (ii) a shortened bioassay based on the detection of the protein in the mouse spleen at defined time points. The two methods proved equally sensitive in quantifying infectivity, even after very-low-dose inoculation of infected material, but the time schedule was shortened from ~2.5 years to ~1 year with the spleen bioassay. Compared to the "gold-standard" RIII model routinely used for endpoint titration of vCJD/BSE prions, either method improved the sensitivity by >2 orders of magnitude and allowed reevaluating the infectious titer of spleen from a vCJD individual at disease end stage to >1,000-fold-higher values.
Here, we provide key reevaluation of the infectious titer of variant CJD brain and spleen tissues. The highly sensitive, accelerated spleen-based assay should thus constitute a key advance for variant CJD epidemiological and risk assessment purposes and should greatly facilitate future titration studies, including, for example, those aimed at validating decontamination procedures. The overlooked notion that the lymphoid tissue exhibits a higher capacity than the brain to replicate prions even after low-dose infection raises new questions about the molecular and/or cellular determinant(s) involved, a key issue regarding potent silent carriers of variant CJD in the lymphoid tissue.
Full-text · Article · May 2014 · Journal of Virology