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Prions are neurotropic pathogens composed of misfolded assemblies of the host-encoded prion protein PrPC which replicate by recruitment and conversion of further PrPC by an autocatalytic seeding polymerization process. While it has long been shown that mouse-adapted prions cannot replicate and are rapidly cleared in transgenic PrP0/0 mice invalidated for PrPC, these experiments have not been done with other prions, including from natural resources, and more sensitive methods to detect prion biological activity. Using transgenic mice expressing human PrP to bioassay prion infectivity and RT-QuIC cell-free assay to measure prion seeding activity, we report that prions responsible for the most prevalent form of sporadic Creutzfeldt–Jakob disease in human (MM1-sCJD) can persist indefinitely in the brain of intra-cerebrally inoculated PrP0/0 mice. While low levels of seeding activity were measured by RT-QuIC in the brain of the challenged PrP0/0 mice, the bio-indicator humanized mice succumbed at a high attack rate, suggesting relatively high levels of persistent infectivity. Remarkably, these humanized mice succumbed with delayed kinetics as compared to MM1-sCJD prions directly inoculated at low doses, including the limiting one. Yet, the disease that did occur in the humanized mice on primary and subsequent back-passage from PrP0/0 mice shared the neuropathological and molecular characteristics of MM1-sCJD prions, suggesting no apparent strain evolution during lifelong dormancy in PrP0/0 brain. Thus, MM1-sCJD prions can persist for the entire life in PrP0/0 brain with potential disease potentiation on retrotransmission to susceptible hosts. These findings highlight the capacity of prions to persist and rejuvenate in non-replicative environments, interrogate on the type of prion assemblies at work and alert on the risk of indefinite prion persistence with PrP-lowering therapeutic strategies., Abbreviated summary Martin et al. report lifelong persistence of sporadic Creutzfeldt–Jakob disease prions in the brain of mice lacking the endogenous prion protein gene and disease potentiation—without strain evolution—on retrotransmission to humanized mice. These findings highlight the risk of indefinite prion persistence with therapeutic strategies lowering the prion protein., Graphical Abstract Graphical Abstract

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 (PrPSc) of the cellular prion protein (PrPC). During the propagation of the disease, PrPScrecruits and misfolds PrPCinto further PrPSc. 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 PrPScphysico-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 PrPScto 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.

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 in vitro 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 in vitro 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., 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 in vitro 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 in vitro and the in vivo 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 (in vitro) and residual infectivity (in vivo). 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. IMPORTANCE 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 in vitro 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 in vitro 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.

International audience; Prion replication results from the autocatalytic templated assisted conversion of the host-encoded prion protein PrPC into misfolded, polydisperse PrPSc conformers. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Within and between prion strains, the biological activity (replicative efficacy and specific infectivity) of PrPSc assemblies is size dependent and thus reflects an intrinsic structural heterogeneity. The contribution of such PrPSc heterogeneity across species prion adaptation, which is believed to be based on fit adjustment between PrPSc template(s) and host PrPC, has not been explored. To define the structural-to-fitness PrPSc landscape, we measured the relative capacity of size-fractionated PrPSc assemblies from different prion strains to cross mounting species barriers in transgenic mice expressing foreign PrPC. In the absence of a transmission barrier, the relative efficacy of the isolated PrPSc 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 PrPSc 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 PrPSc 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 PrPSc 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 PrPSc heterogeneity within prion population.

Backgroundprion replication results from the autocatalytic templated assisted conversion of the host-encoded prion protein PrPCinto misfolded, polydisperse PrPSc conformers. Structurally distinct PrPScconformers can give rise to multiple prion strains. Within and between prion strains, the biological activity (replicative efficacy and specific infectivity) of PrPScassemblies is size-dependent and thus reflects an intrinsic structural heterogeneity. The contribution of such PrPScheterogeneity across species prion adaptation, - which is believed to be based on fit-adjustment between PrPSctemplate(s) and host PrPC-, has not been explored.Methodsto define the structural-to-fitness PrPSclandscape, we measured the relative capacity of size-fractionated PrPScassemblies from different prion strains to cross mounting species barriers in transgenic mice expressing foreign PrPc.Resultsin the absence of a transmission barrier, the relative efficacy of the isolated PrPScassemblies to induce the disease is superimposable to 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 neural and extraneural, prion-permissive tissues, for reason independent of the infectivity load of the isolated assemblies. This suggests that a synergy between structurally distinct PrPScassemblies in the inoculum is requested for crossing the species barrier. We further strengthen this hypothesis by showing that altering, by serial dilution, PrPScassemblies content of unfractionated inocula reduce their specific infectivity in an aberrant manner, solely in the presence of a transmission barrier.Conclusionsour data support a mechanism whereby overcoming prion species barrier requires complementation between structurally distinct PrPScassemblies. This work provides key insight into the “quasi-species” concept applied to prions, which would not necessarily rely on prion sub-strains as constituent but on structural PrPScheterogeneity within prion population.

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., Through sedimentation velocity-based methods, Igel-Egalon et al. characterize the mechanism of prion protein misfolding during early prion replication. They identify two subsets of structurally different assemblies, Ai and Bi, and two sequential processes, the first generating Ai and the second transforming Ai into Bi.

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 (PrPSc) of the host-encoded cellular prion protein (PrPC). Prions replicate by recruiting and converting PrPC into PrPSc, by an autocatalytic process. PrPSc 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 PrPSc 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 PrPSc has not been elucidated so far, making the prion replication process difficult to understand. All currently available models suggest that PrPSc assemblies exhibit a PrPSc 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.

Aggregation of misfolded forms from host-encoded proteins is key to the pathogenesis of a number of neurodegenerative disorders, including prion diseases, Alzheimer’s disease and Parkinson’s disease. In prion diseases, the cellular prion protein PrPCcan misfold into PrPScand auto-organize into conformationally distinct assemblies or strains. A plethora of observations reports the existence of PrPScstructural heterogeneity within prion strains, suggesting the emergence and coevolution of structurally distinct PrPScassemblies during prion replication in controlled environment. Such PrPScdiversification processes remain poorly understood. Although central to prion host-adaptation, structural diversification of PrPScassemblies is also a key issue for the formation of PrP conformers involved in neuronal injury. Here, we characterized the evolution of the PrPScquaternary structure during prion replicationin vivoand inbona fidecell-free amplification assays. Regardless of the strain studied, the early replication stage conduced to the preferential formation of small PrPScoligomers, thus highlighting a quaternary structural convergence phenomenon. Their evolutionary kinetics revealed the existence of a PrPC-dependent secondary templating pathway in concert with a structural rearrangement. This secondary templating pathway provides, for the first time, a mechanistic explanation for prion structural diversification during replication, a key determinant for prion adaptation on further transmission, including to other host species. The uncovered processes are also key for a better understanding of the accumulation mechanisms of other misfolded assemblies believed to propagate by a prion-like process.

International audience; 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 PrPC molecules are converted into abnormally folded scrapie isoforms termed PrPSc. Since its discovery over 30 years ago, much advance has contributed to define the host-encoded cellular prion protein PrPC as a critical relay of prion-induced neuronal cell demise. A current consensual view is that the conversion of PrPC into PrPSc 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 PrPC. We further show that both prion infection and PrPC 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 PrPC-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.

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., Author summary The cause of prion phenotype variation among prion strains remains poorly understood. In particular, prions replicate in a strain-dependent manner in the spleen. This can result in prion asymptomatic carriers. Based on our previous observations that dosage of the prion precursor (PrP) determined prion substrain selection in the brain, we examine whether PrP levels in the spleen could drive prion replication in this tissue, due to the low levels of the protein. We observe that the prion substrain with higher PrP need for replication does barely replicate in the spleen, while the component with low PrP need replicates efficiently. In addition, other human co-propagating prions with differing spleen and brain tropism showed different tropism on transmission to mice expressing low PrP levels, with the lymphoid substrain colonizing the brain. PrPC expression levels may thus be instrumental in prion tropism for the lymphoid tissue. From a diagnostic point of view, given the apparent complexity of prion diseases with respect to prion substrain composition, these data advocate to type extraneural tissues or fluids for a comprehensive identification of the circulating prions in susceptible mammals.

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.

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., Author summary Prions are self-propagating assemblies with all necessary and sufficient replicative information stored in the 3D structure of the misfolded form of PrP called PrPSc. Since the emergence of the prion theory in the 80s, many attempts have been done to identify prion replicative information at molecular scale. Different models have been constructed based on a broad panel of experimental observations and some of them predict the existence of periodic elements constituting prion assemblies. Here, by using partial unfolding approaches, we trapped an oligomeric conformer that we called suPrP, which could constitute the elementary brick of prion assemblies. Once isolated, this elementary brick is devoid of infectivity. However, it becomes infectious once condensated into larger assemblies. The identification of the elementary PrP building block provides a new structural basis for understanding prion replicative information storage and spreading.

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 PrPC 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 PrPC nor for the formation of a stable prion structure.

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (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 PrPC. 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 PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission.

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.

International audience; 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 Met with fi eld 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.

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

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 (PrPC) and the infecting pathological PrP assemblies (PrPSc) 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 PrPCand in nonhuman primates. In humanized mice, L-BSE propagates without any apparent species barrier and shares similar biochemical PrPScsignatures 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.IMPORTANCESince 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.

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.

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 PrPSc, a misfolded isoform of the host-encoded prion protein PrPC. 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 PrPSc 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 PrPSc), to transgenic mice overexpressing either human or the VRQ allele of ovine PrPC. 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 PrPSc 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. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0284-9) contains supplementary material, which is available to authorized users.

Prion Problem Prion disease, like “mad cow disease,” has shown a frightening ability to cross the species transmission barrier, but, mercifully, with low efficiency. However, the role of different tissues in prion cross-species transmission is unclear. Béringue et al. (p. 472 ; see the cover; see the Perspective by Collinge ) compared the ability of brain and lymphoid tissues from “ovinized” (sheeplike) and “humanized” transgenic mouse models to replicate prion transmission across a robust transmission barrier. Lymphoid tissue of these mice was consistently more permissive than brain tissue to prions such as those causing chronic wasting disease and bovine spongiform encephalopathy. Because previous measures of the transmission barrier have focused on the brain, this heightened susceptibility of lymphoid tissues could strongly impact estimates of the number of silent carriers of prion disease.

Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access; International audience; Natural mutations in the LIPH gene were shown to be responsible for hair growth defects in humans and for the rex short hair phenotype in rabbits. In this species, we identified a single nucleotide deletion in LIPH (1362delA) introducing a stop codon in the C-terminal region of the protein. We investigated the expression of LIPH between normal coat and rex rabbits during critical fetal stages of hair follicle genesis, in adults and during hair follicle cycles. Transcripts were three times less expressed in both fetal and adult stages of the rex rabbits than in normal rabbits. In addition, the hair growth cycle phases affected the regulation of the transcription level in the normal and mutant phenotypes differently. LIPH mRNA and protein levels were higher in the outer root sheath (ORS) than in the inner root sheath (IRS), with a very weak signal in the IRS of rex rabbits. In vitro transfection shows that the mutant protein has a reduced lipase activity compared to the wild type form. Our results contribute to the characterization of the LIPH mode of action and confirm the crucial role of LIPH in hair production.

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., Author Summary Prion diseases, also referred as transmissible spongiform encephalopathies, are fatal neurodegenerative diseases caused by proteinaceous infectious particles denominated “prions.” Prion diseases acquired their first real public relevance with the outbreak of bovine spongiform encephalopathy (BSE) (“mad cow disease”) in the United Kingdom in the 80s and its link with the appearance of a new, variant form of Creutzfeldt-Jakob disease in humans. Recycling of ruminant tissues in meat and bone meal has been proposed as origin of the BSE epidemic. During this episode, sheep and goats have also been exposed to BSE-contaminated meal, so transmission to this species may have occurred. We analyzed the human susceptibility to sheep and goat passaged-BSE prions by using transgenic mice expressing human prion protein (PrP). When different sheep and goat BSE isolates were inoculated in these transgenic mice, higher susceptibility than that observed for cattle BSE was detected and the disease manifestation was similar to that observed in mice inoculated with the new variant of Creutzfeldt-Jakob disease. Our findings suggest that humans are at least equally, and might be even more, susceptible to a sheep or goat BSE agent compared to a cattle BSE one.

International audience; Background. The evolution of the variant Creutzfeldt-Jakob disease (vCJD) epidemic is hazardous to predict due to uncertainty in ascertaining the prevalence of infection and because the disease might remain asymptomatic or produce an alternate, sporadic-like phenotype. Methodology/Principal Findings. Transgenic mice were produced that overexpress human prion protein with methionine at codon 129, the only allele found so far in vCJD-affected patients. These mice were infected with prions derived from variant and sporadic CJD (sCJD) cases by intracerebral or intraperitoneal route, and transmission efficiency and strain phenotype were analyzed in brain and spleen. We showed that i) the main features of vCJD infection in humans, including a prominent involvement of the lymphoid tissues compared to that in sCJD infection were faithfully reproduced in such mice; ii) transmission of vCJD agent by intracerebral route could lead to the propagation of either vCJD or sCJD-like prion in the brain, whereas vCJD prion was invariably propagated in the spleen, iii) after peripheral exposure, inefficient neuroinvasion was observed, resulting in an asymptomatic infection with life-long persistence of vCJD prion in the spleen at stable and elevated levels. Conclusion/Significance. Our findings emphasize the possibility that human-to-human transmission of vCJD might produce alternative neuropathogical phenotypes and that lymphoid tissue examination of CJD cases classified as sporadic might reveal an infection by vCJD-type prions. They also provide evidence for the strong propensity of this agent to establish long-lasting, subclinical vCJD infection of lymphoreticular tissues, thus amplifying the risk for iatrogenic transmission.

International audience; 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.

To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical profile of abnormal protease-resistant prion protein (PrPres) isolated from brains of affected animals or humans. However, alternate PrPres signatures in cattle have recently been discovered through large-scale screening. To test whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrPres of higher apparent molecular mass—called H-type—into transgenic mice expressing bovine or ovine PrP. All mice developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals., Synopsis Prions are unconventional agents of proteic nature that are formed of abnormal conformations of the host-encoded prion protein (PrP). They cause fatal neurodegenerative diseases in both animals and humans, and can be transmitted between species as exemplified in humans by the emergence of variant Creutzfeldt-Jakob disease following the epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom. Since diagnosis of prion infection is only possible once the central nervous system has been invaded, brains of slaughtered or fallen cattle are routinely screened in Europe to protect the consumers from BSE. This has unexpectedly led to the discovery of unprecedented PrP conformations that were distinct from the single one associated so far with BSE or BSE-related diseases. To precisely determine their etiology, the authors have studied the transmissibility of these new conformations, termed H-type, to transgenic mice expressing either bovine or ovine PrP. They show that these cases are highly pathogenic for these mice. The authors also demonstrate that they are not directly related to the agent involved in the BSE epidemic, supporting the view for isolation of a new prion strain from cattle, whose prevalence and associated zoonotic risk should be carefully monitored in the future.

Scrapie in small ruminants belongs to transmissible spongiform encephalopathies (TSEs), or prion diseases, a family of fatal neurodegenerative disorders that affect humans and animals and can transmit within and between species by ingestion or inoculation. Conversion of the host-encoded prion protein (PrP), normal cellular PrP (PrP c ), into a misfolded form, abnormal PrP (PrP Sc ), plays a key role in TSE transmission and pathogenesis. The intensified surveillance of scrapie in the European Union, together with the improvement of PrP Sc detection techniques, has led to the discovery of a growing number of so-called atypical scrapie cases. These include clinical Nor98 cases first identified in Norwegian sheep on the basis of unusual pathological and PrP Sc molecular features and “cases” that produced discordant responses in the rapid tests currently applied to the large-scale random screening of slaughtered or fallen animals. Worryingly, a substantial proportion of such cases involved sheep with PrP genotypes known until now to confer natural resistance to conventional scrapie. Here we report that both Nor98 and discordant cases, including three sheep homozygous for the resistant PrP ARR allele (A 136 R 154 R 171 ), efficiently transmitted the disease to transgenic mice expressing ovine PrP, and that they shared unique biological and biochemical features upon propagation in mice. These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health.

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrPSc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). Stable variations in PrPSc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrPSc 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 PrPSc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrPSc. 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 PrPSc 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 PrPSc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrPSc 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., Author Summary Prions are infectious agents causing irremediably fatal neurodegenerative diseases in human and in farmed or wild animals. They are thought to be formed from abnormally folded assemblies (PrPSc) of the host-encoded prion protein (PrPC). Different PrPSc conformational variants associated with distinct biological phenotypes, or ‘strains,’ can propagate in the same host. To gain some structural information on the physical relationship between packing order (i.e. quaternary structure) and the strain-specific biological information, we previously subjected PrPSc assemblies from prion strains classified as fast or slow (according to their survival time in susceptible laboratory animals) to sedimentation velocity ultracentrifugation experiments. For the fast strains specifically, the most infectious assemblies sedimented slowly and partitioned from the bulk of PrPSc macromolecular complexes. By changing the solubilization and sedimentation conditions, we established here that a small PrPSc aggregation size and not a low density accounts for these hydrodynamic properties. We further showed that these small assemblies resist proteolytic digestion and outcompete by several orders of magnitude the larger-size assemblies in cell-free prion conversion assays. Thus PrPSc quaternary structure appears to be a determining factor of prion replication dynamics. For certain strains, a discrete subset of PrPSc assemblies appears to be the best template for prion replication.

Prions are unconventional infectious agents thought to be primarily composed of PrPSc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrPSc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrPSc 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 PrPSc aggregates from PrPC. The distribution of PrPSc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrPSc 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 PrPSc 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., Author Summary Prions are unconventional transmissible agents causing fatal neurodegenerative diseases in human and animals. They are thought to be formed from polymers of abnormal conformations of the host-encoded prion protein (PrP), but little is known about the physical organization of the infectious particles and any relationship between packing order and infectivity. As an additional layer of complexity, different PrP conformational variants associated with distinct biological phenotypes, or ‘strains’, can propagate in the same host. We subjected PrP polymers from eight different ovine and hamster prion strains to sedimentation velocity centrifugation, which allows separation of macromolecular complexes according to their size, density or shape. We showed that, whereas the PrP sedimentation profiles share common features, the infectivity profiles exhibit striking differences amongst the strains. For four of them, the infectious component was predominantly associated with slowly sedimenting particles, suggestive of small size oligomers and/or low density PrP aggregates. Such particles appeared to be a feature of strains able to induce a rapidly lethal disease in the recipient host. 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.