Publications by authors named "Laetitia Herzog"

30 Publications

  • Page 1 of 1

Improving the Predictive Value of Prion Inactivation Validation Methods to Minimize the Risks of Iatrogenic Transmission With Medical Instruments.

Front Bioeng Biotechnol 2020 1;8:591024. Epub 2020 Dec 1.

Université Paris Saclay, INRAE, UVSQ, VIM, Jouy-en-Josas, France.

Prions are pathogenic infectious agents responsible for fatal, incurable neurodegenerative diseases in animals and humans. Prions are composed exclusively of an aggregated and misfolded form (PrP ) of the cellular prion protein (PrP). During the propagation of the disease, PrP recruits and misfolds PrP into further PrP. In human, iatrogenic prion transmission has occurred with incompletely sterilized medical material because of the unusual resistance of prions to inactivation. Most commercial prion disinfectants validated against the historical, well-characterized laboratory strain of 263K hamster prions were recently shown to be ineffective against variant Creutzfeldt-Jakob disease human prions. These observations and previous reports support the view that any inactivation method must be validated against the prions for which they are intended to be used. Strain-specific variations in PrP physico-chemical properties and conformation are likely to explain the strain-specific efficacy of inactivation methods. Animal bioassays have long been used as gold standards to validate prion inactivation methods, by measuring reduction of prion infectivity. Cell-free assays such as the real-time quaking-induced conversion (RT-QuIC) assay and the protein misfolding cyclic amplification (PMCA) assay have emerged as attractive alternatives. They exploit the seeding capacities of PrP to exponentially amplify minute amounts of prions in biospecimens. European and certain national medicine agencies recently implemented their guidelines for prion inactivation of non-disposable medical material; they encourage or request the use of human prions and cell-free assays to improve the predictive value of the validation methods. In this review, we discuss the methodological and technical issues regarding the choice of (i) the cell-free assay, (ii) the human prion strain type, (iii) the prion-containing biological material. We also introduce a new optimized substrate for high-throughput PMCA amplification of human prions bound on steel wires, as translational model for prion-contaminated instruments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fbioe.2020.591024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736614PMC
December 2020

Host prion protein expression levels impact prion tropism for the spleen.

PLoS Pathog 2020 07 23;16(7):e1008283. Epub 2020 Jul 23.

Université Paris-Saclay, INRAE, UVSQ, VIM Jouy-en-Josas, France.

Prions are pathogens formed from abnormal conformers (PrPSc) of the host-encoded cellular prion protein (PrPC). PrPSc conformation to disease phenotype relationships extensively vary among prion strains. In particular, prions exhibit a strain-dependent tropism for lymphoid tissues. Prions can be composed of several substrain components. There is evidence that these substrains can propagate in distinct tissues (e.g. brain and spleen) of a single individual, providing an experimental paradigm to study the cause of prion tissue selectivity. Previously, we showed that PrPC expression levels feature in prion substrain selection in the brain. Transmission of sheep scrapie isolates (termed LAN) to multiple lines of transgenic mice expressing varying levels of ovine PrPC in their brains resulted in the phenotypic expression of the dominant sheep substrain in mice expressing near physiological PrPC levels, whereas a minor substrain replicated preferentially on high expresser mice. Considering that PrPC expression levels are markedly decreased in the spleen compared to the brain, we interrogate whether spleen PrPC dosage could drive prion selectivity. The outcome of the transmission of a large cohort of LAN isolates in the spleen from high expresser mice correlated with the replication rate dependency on PrPC amount. There was a prominent spleen colonization by the substrain preferentially replicating on low expresser mice and a relative incapacity of the substrain with higher-PrPC level need to propagate in the spleen. Early colonization of the spleen after intraperitoneal inoculation allowed neuropathological expression of the lymphoid substrain. In addition, a pair of substrain variants resulting from the adaptation of human prions to ovine high expresser mice, and exhibiting differing brain versus spleen tropism, showed different tropism on transmission to low expresser mice, with the lymphoid substrain colonizing the brain. Overall, these data suggest that PrPC expression levels are instrumental in prion lymphotropism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1008283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402522PMC
July 2020

Crossing Species Barriers Relies on Structurally Distinct Prion Assemblies and Their Complementation.

Mol Neurobiol 2020 Jun 1;57(6):2572-2587. Epub 2020 Apr 1.

Université Paris-Saclay, INRAE, UVSQ, VIM, 78350, Jouy-en-Josas, France.

Prion replication results from the autocatalytic templated assisted conversion of the host-encoded prion protein PrP into misfolded, polydisperse PrP conformers. Structurally distinct PrP conformers can give rise to multiple prion strains. Within and between prion strains, the biological activity (replicative efficacy and specific infectivity) of PrP assemblies is size dependent and thus reflects an intrinsic structural heterogeneity. The contribution of such PrP heterogeneity across species prion adaptation, which is believed to be based on fit adjustment between PrP template(s) and host PrP, has not been explored. To define the structural-to-fitness PrP landscape, we measured the relative capacity of size-fractionated PrP assemblies from different prion strains to cross mounting species barriers in transgenic mice expressing foreign PrP. In the absence of a transmission barrier, the relative efficacy of the isolated PrP assemblies to induce the disease is like the efficacy observed in the homotypic context. However, in the presence of a transmission barrier, size fractionation overtly delays and even abrogates prion pathogenesis in both the brain and spleen tissues, independently of the infectivity load of the isolated assemblies. Altering by serial dilution PrP assembly content of non-fractionated inocula aberrantly reduces their specific infectivity, solely in the presence of a transmission barrier. This suggests that synergy between structurally distinct PrP assemblies in the inoculum is requested for crossing the species barrier. Our data support a mechanism whereby overcoming prion species barrier requires complementation between structurally distinct PrP assemblies. This work provides key insight into the "quasispecies" concept applied to prions, which would not necessarily rely on prion substrains as constituent but on structural PrP heterogeneity within prion population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-020-01897-3DOI Listing
June 2020

Correlation between Bioassay and Protein Misfolding Cyclic Amplification for Variant Creutzfeldt-Jakob Disease Decontamination Studies.

mSphere 2020 Jan 29;5(1). Epub 2020 Jan 29.

Pathogenesis and Control of Chronic Infections, Etablissement Français du Sang, INSERM, Université de Montpellier, Montpellier, France

To date, approximately 500 iatrogenic Creutzfeldt-Jakob disease cases have been reported worldwide, most of them resulting from cadaveric dura mater graft and from the administration of prion-contaminated human growth hormone. The unusual resistance of prions to decontamination processes, their large tissue distribution, and the uncertainty about the prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the general population lead to specific recommendations regarding identification of tissue at risk and reprocessing of reusable medical devices, including the use of dedicated treatment for prion inactivation. We previously described an assay, called Surf-PMCA, which allowed us to classify prion decontamination treatments according to their efficacy on vCJD prions by monitoring residual seeding activity (RSA). Here, we used a transgenic mouse line permissive to vCJD prions to study the correlation between the RSA measured and the infectivity. Implantation in mouse brains of prion-contaminated steel wires subjected to different decontamination procedures allows us to demonstrate a good concordance between RSA measured by Surf-PMCA () and residual infectivity (). These experiments emphasize the strength of the Surf-PMCA method as a rapid and sensitive assay for the evaluation of prion decontamination procedures and also confirm the lack of efficacy of several marketed reagents on vCJD prion decontamination. Creutzfeldt-Jakob diseases are neurodegenerative disorders for which transmission linked to medical procedures have been reported in hundreds of patients. As prion diseases, they are characterized by an unusual resistance to conventional decontamination processes. Moreover, their large tissue distribution and the ability of prions to attach to many surfaces raised the risk of transmission in health care facilities. It is therefore of major importance that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated for prion inactivation. We previously described an assay, which allowed us to classify accurately prion decontamination treatments according to their efficacy on variant Creutzfeldt-Jakob disease. The significance of this study is in demonstrating the concordance between previous results and infectivity studies in transgenic mice. Furthermore, commercial reagents currently used in hospitals were tested by both protocols, and we observed that most of them were ineffective on human prions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mSphere.00649-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992370PMC
January 2020

Early stage prion assembly involves two subpopulations with different quaternary structures and a secondary templating pathway.

Commun Biol 2019 4;2:363. Epub 2019 Oct 4.

1VIM, INRA, Université Paris-Saclay, 78350 Jouy-en-Josas, France.

The dynamics of aggregation and structural diversification of misfolded, host-encoded proteins in neurodegenerative diseases are poorly understood. In many of these disorders, including Alzheimer's, Parkinson's and prion diseases, the misfolded proteins are self-organized into conformationally distinct assemblies or strains. The existence of intrastrain structural heterogeneity is increasingly recognized. However, the underlying processes of emergence and coevolution of structurally distinct assemblies are not mechanistically understood. Here, we show that early prion replication generates two subsets of structurally different assemblies by two sequential processes of formation, regardless of the strain considered. The first process corresponds to a quaternary structural convergence, by reducing the parental strain polydispersity to generate small oligomers. The second process transforms these oligomers into larger ones, by a secondary autocatalytic templating pathway requiring the prion protein. This pathway provides mechanistic insights into prion structural diversification, a key determinant for prion adaptation and toxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-019-0608-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778151PMC
May 2020

Prion strain-dependent tropism is maintained between spleen and granuloma and relies on lymphofollicular structures.

Sci Rep 2019 10 10;9(1):14656. Epub 2019 Oct 10.

VIM, INRA, Université Paris-Saclay, 78350, Jouy-en-Josas, France.

In peripherally acquired prion diseases, prions move through several tissues of the infected host, notably in the lymphoid tissue, long before the occurrence of neuroinvasion. Accumulation can even be restricted to the lymphoid tissue without neuroinvasion and clinical disease. Several experimental observations indicated that the presence of differentiated follicular dendritic cells (FDCs) in the lymphoid structures and the strain type are critical determinants of prion extraneural replication. In this context, the report that granulomatous structures apparently devoid of FDCs could support prion replication raised the question of the requirements for prion lymphotropism. The report also raised the possibility that nonlymphoid tissue-tropic prions could actually target these inflammatory structures. To investigate these issues, we examined the capacity of closely related prions, albeit with opposite lymphotropism (or FDC dependency), for establishment in experimentally-induced granuloma in ovine PrP transgenic mice. We found a positive correlation between the prion capacity to accumulate in the lymphoid tissue and granuloma, regardless of the prion detection method used. Surprisingly, we also revealed that the accumulation of prions in granulomas involved lymphoid-like structures associated with the granulomas and containing cells that stain positive for PrP, Mfge-8 but not CD45 that strongly suggest FDCs. These results suggest that the FDC requirement for prion replication in lymphoid/inflammatory tissues may be strain-dependent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-51084-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787085PMC
October 2019

Heterogeneity and Architecture of Pathological Prion Protein Assemblies: Time to Revisit the Molecular Basis of the Prion Replication Process?

Viruses 2019 05 10;11(5). Epub 2019 May 10.

Molecular Virology and Immunology Unit (VIM), INRA, Université Paris-Saclay, 78350 Jouy-en-Josas, France.

Prions are proteinaceous infectious agents responsible for a range of neurodegenerative diseases in animals and humans. Prion particles are assemblies formed from a misfolded, β-sheet rich, aggregation-prone isoform (PrP) of the host-encoded cellular prion protein (PrP). Prions replicate by recruiting and converting PrP into PrP, by an autocatalytic process. PrP is a pleiomorphic protein as different conformations can dictate different disease phenotypes in the same host species. This is the basis of the strain phenomenon in prion diseases. Recent experimental evidence suggests further structural heterogeneity in PrP assemblies within specific prion populations and strains. Still, this diversity is rather seen as a size continuum of assemblies with the same core structure, while analysis of the available experimental data points to the existence of structurally distinct arrangements. The atomic structure of PrP has not been elucidated so far, making the prion replication process difficult to understand. All currently available models suggest that PrP assemblies exhibit a PrP subunit as core constituent, which was recently identified. This review summarizes our current knowledge on prion assembly heterogeneity down to the subunit level and will discuss its importance with regard to the current molecular principles of the prion replication process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v11050429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563208PMC
May 2019

Epigenetic Control of the Notch and Eph Signaling Pathways by the Prion Protein: Implications for Prion Diseases.

Mol Neurobiol 2019 Mar 11;56(3):2159-2173. Epub 2018 Jul 11.

INSERM UMR 1124, 75006, Paris, France.

Among the ever-growing number of self-replicating proteins involved in neurodegenerative diseases, the prion protein PrP remains the most infamous for its central role in transmissible spongiform encephalopathies (TSEs). In these diseases, pathogenic prions propagate through a seeding mechanism, where normal PrP molecules are converted into abnormally folded scrapie isoforms termed PrP. Since its discovery over 30 years ago, much advance has contributed to define the host-encoded cellular prion protein PrP as a critical relay of prion-induced neuronal cell demise. A current consensual view is that the conversion of PrP into PrP in neuronal cells diverts the former from its normal function with subsequent molecular alterations affecting synaptic plasticity. Here, we report that prion infection is associated with reduced expression of key effectors of the Notch pathway in vitro and in vivo, recapitulating changes fostered by the absence of PrP. We further show that both prion infection and PrP depletion promote drastic alterations in the expression of a defined set of Eph receptors and their ephrin ligands, which represent important players in synaptic function. Our data indicate that defects in the Notch and Eph axes can be mitigated in response to histone deacetylase inhibition in PrP-depleted as well as prion-infected cells. We thus conclude that infectious prions cause a loss-of-function phenotype with respect to Notch and Eph signaling and that these alterations are sustained by epigenetic mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-018-1193-7DOI Listing
March 2019

Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick.

PLoS Pathog 2017 Sep 7;13(9):e1006557. Epub 2017 Sep 7.

INRA, Université Paris-Saclay, UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France.

Mammalian prions, the pathogens that cause transmissible spongiform encephalopathies, propagate by self-perpetuating the structural information stored in the abnormally folded, aggregated conformer (PrPSc) of the host-encoded prion protein (PrPC). To date, no structural model related to prion assembly organization satisfactorily describes how strain-specified structural information is encoded and by which mechanism this information is transferred to PrPC. To achieve progress on this issue, we correlated the PrPSc quaternary structural transition from three distinct prion strains during unfolding and refolding with their templating activity. We reveal the existence of a mesoscopic organization in PrPSc through the packing of a highly stable oligomeric elementary subunit (suPrP), in which the strain structural determinant (SSD) is encoded. Once kinetically trapped, this elementary subunit reversibly loses all replicative information. We demonstrate that acquisition of the templating interface and infectivity requires structural rearrangement of suPrP, in concert with its condensation. The existence of such an elementary brick scales down the SSD support to a small oligomer and provide a basis of reflexion for prion templating process and propagation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1006557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589264PMC
September 2017

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity.

Prion 2017 01 8;11(1):25-30. Epub 2017 Feb 8.

a VIM, INRA, Université Paris-Saclay , Jouy-en-Josas , France.

Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrP structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrP nor for the formation of a stable prion structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/19336896.2016.1274851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360129PMC
January 2017

Divergent prion strain evolution driven by PrP expression level in transgenic mice.

Nat Commun 2017 01 23;8:14170. Epub 2017 Jan 23.

Virologie Immunologie Moléculaires (VIM), INRA, Université Paris-Saclay, 78350 Jouy-en-Josas, France.

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrP into PrP. Structurally distinct PrP conformers can give rise to multiple prion strains. Constrained interactions between PrP and different PrP strains can in turn lead to certain PrP (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrP. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrP. While breeding true in mice expressing PrP at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrP overexpression. Our results support the view that PrP gene dosage can influence prion evolution on homotypic transmission.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms14170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264111PMC
January 2017

Absence of Evidence for a Causal Link between Bovine Spongiform Encephalopathy Strain Variant L-BSE and Known Forms of Sporadic Creutzfeldt-Jakob Disease in Human PrP Transgenic Mice.

J Virol 2016 Dec 14;90(23):10867-10874. Epub 2016 Nov 14.

VIM, INRA, Université Paris-Saclay, Jouy-en-Josas, France

Prions are proteinaceous pathogens responsible for subacute spongiform encephalopathies in animals and humans. The prions responsible for bovine spongiform encephalopathy (BSE) are zoonotic agents, causing variant Creutzfeldt-Jakob disease (CJD) in humans. The transfer of prions between species is limited by a species barrier, which is thought to reflect structural incompatibilities between the host cellular prion protein (PrP) and the infecting pathological PrP assemblies (PrP) constituting the prion. A BSE strain variant, designated L-BSE and responsible for atypical, supposedly spontaneous forms of prion diseases in aged cattle, demonstrates zoonotic potential, as evidenced by its capacity to propagate more easily than classical BSE in transgenic mice expressing human PrP and in nonhuman primates. In humanized mice, L-BSE propagates without any apparent species barrier and shares similar biochemical PrP signatures with the CJD subtype designated MM2-cortical, thus opening the possibility that certain CJD cases classified as sporadic may actually originate from L-type BSE cross-transmission. To address this issue, we compared the biological properties of L-BSE and those of a panel of CJD subtypes representative of the human prion strain diversity using standard strain-typing criteria in human PrP transgenic mice. We found no evidence that L-BSE causes a known form of sporadic CJD.

Importance: Since the quasi-extinction of classical BSE, atypical BSE forms are the sole BSE variants circulating in cattle worldwide. They are observed in rare cases of old cattle, making them difficult to detect. Extrapolation of our results suggests that L-BSE may propagate in humans as an unrecognized form of CJD, and we urge both the continued utilization of precautionary measures to eliminate these agents from the human food chain and active surveillance for CJD phenotypes in the general population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01383-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110181PMC
December 2016

Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification.

Sci Rep 2016 07 7;6:29116. Epub 2016 Jul 7.

VIM, INRA, Université Paris-Saclay, 78350, Jouy-en-Josas, France.

Prions are formed of misfolded assemblies (PrP(Sc)) of the variably N-glycosylated cellular prion protein (PrP(C)). In infected species, prions replicate by seeding the conversion and polymerization of host PrP(C). Distinct prion strains can be recognized, exhibiting defined PrP(Sc) biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrP(Sc) assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrP(C) glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrP(C) species of interest as substrate. Applying the technique to PrP(C) glycosylation mutants expressing cells revealed that neither PrP(C) nor PrP(Sc) glycoform stoichiometry was instrumental to PrP(Sc) formation and strainness perpetuation. Our study supports the view that strain properties, including PrP(Sc) glycotype are enciphered within PrP(Sc) structural backbone, not in the attached glycans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep29116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935985PMC
July 2016

Generating Bona Fide Mammalian Prions with Internal Deletions.

J Virol 2016 08 11;90(15):6963-6975. Epub 2016 Jul 11.

INRA U892, Virologie et Immunologie Moléculaires, Jouy-en-Josas, France

Unlabelled: Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions.

Importance: Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00555-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4944278PMC
August 2016

Emergence of two prion subtypes in ovine PrP transgenic mice infected with human MM2-cortical Creutzfeldt-Jakob disease prions.

Acta Neuropathol Commun 2016 Feb 5;4:10. Epub 2016 Feb 5.

INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, F-78350, Jouy-en-Josas, France.

Introduction: 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).

Results: 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.

Conclusions: 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40478-016-0284-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743415PMC
February 2016

Mutated but Not Deleted Ovine PrP(C) N-Terminal Polybasic Region Strongly Interferes with Prion Propagation in Transgenic Mice.

J Virol 2016 02 25;90(3):1638-46. Epub 2015 Nov 25.

UMR1313 Génétique Animale et Biologie Intégrative, Institut National de la Recherche Agronomique, Jouy-en-Josas, France

Unlabelled: Mammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the N-terminal polybasic region of residues 23 to 31 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 in vitro and in vivo. 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 substitutions and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP with a deletion of residues 23 to 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 findings in 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 bovine spongiform encephalopathy (BSE) prions, whereas mutation caused apparent resistance to disease.

Importance: 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02805-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719590PMC
February 2016

Interaction of prion protein with acetylcholinesterase: potential pathobiological implications in prion diseases.

Acta Neuropathol Commun 2015 Apr 3;3:18. Epub 2015 Apr 3.

Introduction: The prion protein (PrP) binds to various molecular partners, but little is known about their potential impact on the pathogenesis of prion diseases

Results: 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.

Conclusion: Our results indicate that AChE deserves consideration as a new actor in expanding pathologically relevant PrP morphotypes and as a therapeutic target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40478-015-0188-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383067PMC
April 2015

Evidence for zoonotic potential of ovine scrapie prions.

Nat Commun 2014 Dec 16;5:5821. Epub 2014 Dec 16.

UMR INRA ENVT 1225, Interactions Hôtes Agents Pathogènes, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31076 Toulouse, France.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms6821DOI Listing
December 2014

Accelerated, spleen-based titration of variant Creutzfeldt-Jakob disease infectivity in transgenic mice expressing human prion protein with sensitivity comparable to that of survival time bioassay.

J Virol 2014 Aug 21;88(15):8678-86. Epub 2014 May 21.

INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France

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.

Importance: 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01118-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135959PMC
August 2014

Highly infectious prions generated by a single round of microplate-based protein misfolding cyclic amplification.

mBio 2013 Dec 31;5(1):e00829-13. Epub 2013 Dec 31.

Measurements of the presence of prions in biological tissues or fluids rely more and more on cell-free assays. Although protein misfolding cyclic amplification (PMCA) has emerged as a valuable, sensitive tool, it is currently hampered by its lack of robustness and rapidity for high-throughput purposes. Here, we made a number of improvements making it possible to amplify the maximum levels of scrapie prions in a single 48-h round and in a microplate format. The amplification rates and the infectious titer of the PMCA-formed prions appeared similar to those derived from the in vivo laboratory bioassays. This enhanced technique also amplified efficiently prions from different species, including those responsible for human variant Creutzfeldt-Jakob disease. This new format should help in developing ultrasensitive, high-throughput prion assays for cognitive, diagnostic, and therapeutic applications. IMPORTANCE The method developed here allows large-scale, fast, and reliable cell-free amplification of subinfectious levels of prions from different species. The sensitivity and rapidity achieved approach or equal those of other recently developed prion-seeded conversion assays. Our simplified assay may be amenable to high-throughput, automated purposes and serve in a complementary manner with other recently developed assays for urgently needed antemortem diagnostic tests, by using bodily fluids containing small amounts of prion infectivity. Such a combination of assays is of paramount importance to reduce the transfusion risk in the human population and to identify asymptomatic carriers of variant Creutzfeldt-Jakob disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00829-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3884057PMC
December 2013

Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics.

PLoS Pathog 2013 10;9(10):e1003702. Epub 2013 Oct 10.

INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France.

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrP(Sc), an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP(C)). Stable variations in PrP(Sc) conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrP(Sc) quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrP(Sc) quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrP(Sc). To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrP(Sc) tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrP(Sc) aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrP(Sc) quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1003702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795044PMC
May 2014

Integrity of helix 2-helix 3 domain of the PrP protein is not mandatory for prion replication.

J Biol Chem 2012 Jun 16;287(23):18953-64. Epub 2012 Apr 16.

INRA, UR892 Virologie Immunologie Moléculaires, F-78350 Jouy-en-Josas, France.

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.341677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365929PMC
June 2012

Facilitated cross-species transmission of prions in extraneural tissue.

Science 2012 Jan;335(6067):472-5

Institut National de la Recherche Agronomique UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France.

Prions are infectious pathogens essentially composed of PrP(Sc), an abnormally folded form of the host-encoded prion protein PrP(C). Constrained steric interactions between PrP(Sc) and PrP(C) are thought to provide prions with species specificity and to control cross-species transmission into other host populations, including humans. We compared the ability of brain and lymphoid tissues from ovine and human PrP transgenic mice to replicate foreign, inefficiently transmitted prions. Lymphoid tissue was consistently more permissive than the brain to prions such as those causing chronic wasting disease and bovine spongiform encephalopathy. Furthermore, when the transmission barrier was overcome through strain shifting in the brain, a distinct agent propagated in the spleen, which retained the ability to infect the original host. Thus, prion cross-species transmission efficacy can exhibit a marked tissue dependence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1215659DOI Listing
January 2012

Sheep and goat BSE propagate more efficiently than cattle BSE in human PrP transgenic mice.

PLoS Pathog 2011 Mar 17;7(3):e1001319. Epub 2011 Mar 17.

Centro de Investigación en Sanidad Animal, Madrid, Spain.

A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met) allele of human PrP at codon 129 (tg650 and tg340) with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1001319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060172PMC
March 2011

Three-dimensional fluorescence in situ hybridization in mouse embryos using repetitive probe sequences.

Methods Mol Biol 2010 ;659:401-8

UMR 1198 Biologie du Développement et Reproduction, INRA, Jouy en Josas, France.

A common problem in research laboratories that study the mammalian embryo is the limited supply of live material. For this reason, new methods are constantly being developed and existing methods for in vitro models using cells in culture are being adapted to represent embryogenesis. Three-dimensional fluorescence in situ hybridization (3D-FISH) is an important tool to study where genomic sequences are positioned within nuclei without interfering with this 3D organization. When used in the embryo, this technique provides vital information about the distribution of specific sequences in relation to embryonic nuclear substructures such as nucleolar precursor bodies and chromocenters. In this chapter, we will present a detailed description of FISH in order to perform 3D-FISH in the early preimplantation murine embryos.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-60761-789-1_31DOI Listing
December 2010

The physical relationship between infectivity and prion protein aggregates is strain-dependent.

PLoS Pathog 2010 Apr 15;6(4):e1000859. Epub 2010 Apr 15.

INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France.

Prions are unconventional infectious agents thought to be primarily composed of PrP(Sc), a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP(C)). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP(Sc) conformation could encode this 'strain' diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP(Sc) aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrP(Sc) aggregates from PrP(C). The distribution of PrP(Sc) and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP(Sc) peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12-30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrP(Sc) aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1000859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855332PMC
April 2010

Transmission of atypical bovine prions to mice transgenic for human prion protein.

Emerg Infect Dis 2008 Dec;14(12):1898-901

Institut National de la Recherche Agronomique, Jouy-en-Josas, France.

To assess risk for cattle-to-human transmission of prions that cause uncommon forms of bovine spongiform encephalopathy (BSE), we inoculated mice expressing human PrP Met129 with field isolates. Unlike classical BSE agent, L-type prions appeared to propagate in these mice with no obvious transmission barrier. H-type prions failed to infect the mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634647PMC
http://dx.doi.org/10.3201/eid1412.080941DOI Listing
December 2008

Spatial and temporal down-regulation of transgene expression using the TRSID-silencer in mice: application to Prnp.

FEBS Lett 2008 Jun 22;582(15):2219-24. Epub 2008 May 22.

Laboratoire de Génétique Biochimique et de Cytogénétique, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France.

Spatial and temporal control of ovine prion protein (Prnp) gene expression was achieved in mice using two transgenes: a Prnp minigene with tet-operator sequences inserted 5' to exon 1 and a mouse neurofilament genomic clone carrying the chimeric-repressor TRSID cDNA. In bi-transgenic mice, ovine PrP(C) expression could be reversibly controlled in neuronal cells by doxycycline treatment whereas it remains constant in other cell types. Overall, this model opens opportunities to assess the involvement of cell types in prion diseases and PrP physiological function. It demonstrates the potentiality of the TRSID-silencer to precisely control temporal and spatial gene expression in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2008.05.014DOI Listing
June 2008

Prnp knockdown in transgenic mice using RNA interference.

Transgenic Res 2008 Oct 19;17(5):783-91. Epub 2008 Mar 19.

UR 339, Laboratoire de Génétique Biochimique et de Cytogénétique, Institut National de la Recherche Agronomique, 78350, Jouy-en-Josas, France.

RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11248-008-9179-2DOI Listing
October 2008

A bovine prion acquires an epidemic bovine spongiform encephalopathy strain-like phenotype on interspecies transmission.

J Neurosci 2007 Jun;27(26):6965-71

Institut National de la Recherche Agronomique, Unité de Recherche 892, Virologie Immunologie Moléculaires, F-78350 Jouy-en-Josas, France.

Implementation in Europe of large-scale testing to detect bovine spongiform encephalopathy (BSE)-infected cattle and prevent the transmission of this prion disease to humans has recently led to the discovery of novel types of bovine prions. We characterized atypical isolates called BSE L-type by analyzing their molecular and neuropathological properties during transmission to several mouse lines transgenic for the prion protein (PrP). Unexpectedly, such isolates acquired strain features closely similar to those of BSE-type agents when propagated in mice expressing ovine PrP, although they retained phenotypic traits distinct from BSE in other lines, including bovine PrP mice. These findings further underline the relationship between the crossing of species barrier and prion strain diversification, and, although the origin of the epidemic BSE agent has only been speculative until now, they provide new insight into the nature of the events that could have led to the appearance of this agent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.0693-07.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6672218PMC
June 2007