Assessment of the zoonotic potential of atypical scrapie prions in humanized mice reveals rare phenotypic convergence but not identity with sporadic CJD prions.

BACKGROUND: Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from bovine spongiform encephalopathy (C-BSE) may co-propagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. METHODS: To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. RESULTS: No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, one isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. CONCLUSIONS: The results obtained suggest a low the zoonotic for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.

Characterization of the 263K-derived microsomal fraction: a source of prions for nanofiltration validation studies.

BACKGROUND: The manufacturing processes of plasma products include steps that can remove prions. The efficacy of these steps is measured in validation studies using animal brain-derived prion materials called spikes. Because the nature of the prion agent in blood is not known, the relevance of these spikes, particularly with steps that are based on retention mechanisms such as nanofiltration, is important to investigate. STUDY DESIGN AND METHODS: The aggregation and sizes of PrPres assemblies of microsomal fractions (MFs) extracted from 263K-infected hamster brains were analyzed using velocity gradients. The separated gradient fractions were either inoculated to Tg7 mice expressing hamster-PrPc to measure infectivity or used in Protein Misfolding Cyclic Amplification for measuring seeding activity. The collected data allowed for reanalyzing results from previous nanofiltration validation studies. RESULTS: A significant portion of MFs was found to be composed of small PrPres assemblies, estimated to have a size ≤24 mers (~22-528 kDa), and to contain a minimum of 20% of total prion infectivity. With this data we could calculate reductions of 4.10 log (15 N), 2.53 log (35 N), and 1.77 log (35 N) from validation studies specifically for these small PrPres objects. CONCLUSION: Our gradient data provided evidence that nanofilters can remove the majority of the smallest PrPres entities within microsomes spikes, estimated to be in a size below 24 mers, giving insight about the fact that, in our conditions, size exclusion may not be the only mechanism for retention nanofiltration.

Prion assemblies: structural heterogeneity, mechanisms of formation, and role in species barrier.

Prions are proteinaceous pathogens responsible for a wide range of neurodegenerative diseases in animal and human. Prions are formed from misfolded, ß-sheet rich, and aggregated conformers (PrPSc) of the host-encoded prion protein (PrPC). Prion replication stems from the capacity of PrPSc to self-replicate by templating PrPC conversion and polymerization. The question then arises about the molecular mechanisms of prion replication, host invasion, and capacity to contaminate other species. Studying these mechanisms has gained in recent years further complexity with evidence that PrPSc is a pleiomorphic protein. There is indeed compelling evidence for PrPSc structural heterogeneity at different scales: (i) within prion susceptible host populations with the existence of different strains with specific biological features due to different PrPSc conformers, (ii) within a single infected host with the co-propagation of different strains, and (iii) within a single strain with evidence for co-propagation of PrPSc assemblies differing in their secondary to quaternary structure. This review summarizes current knowledge of prion assembly heterogeneity, potential mechanisms of formation during the replication process, and importance when crossing the species barrier.

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

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.

A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP.

Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of ß-sheet-rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)-resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190-196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.

Probing amyloid fibril secondary structures by infrared nanospectroscopy: experimental and theoretical considerations.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillary structure with a higher ß-sheet content than their native structure. Attenuated total reflection Fourier transform infrared spectroscopy only provides bulk analysis of a sample therefore it is impossible to discriminate between different aggregated structures. To overcome this limitation, near-field techniques like AFM-IR have emerged in the last twenty years to allow infrared nanospectroscopy. This technique obtains IR spectra with a spatial resolution of ten nanometres, the size of isolated fibrils. Here, we present essential practical considerations to avoid misinterpretations and artefacts during these analyses. Effects of polarization of the incident IR laser, illumination configuration and coating of the AFM probes are discussed, including the advantages and drawbacks of their use. This approach will improve interpretation of AFM-IR spectra especially for the determination of secondary structures of species not accessible using classical ATR-FTIR.

Conformation-dependent membrane permeabilization by neurotoxic PrP oligomers: The role of the H2H3 oligomerization domain.

The relationship between prion propagation and the generation of neurotoxic species and clinical onset remains unclear. Several converging lines of evidence suggest that interactions with lipids promote various precursors to form aggregation-prone states that are involved in amyloid fibrils. Here, we compared the cytotoxicities of different soluble isolated oligomeric constructs from murine full-length PrP and from the restricted helical H2H3 domain with their effects on lipid vesicles. The helical H2H3 domain is suggested to be the minimal region of PrP involved in the oligomerization process. The discrete PrP oligomers of both the full-length sequence and the H2H3 domain have de novo ß-sheeted structure when interacting with the membrane. They were shown to permeabilize synthetic negatively charged vesicles in a dose-dependent manner. Restricting the polymerization domain of the full-length PrP to the H2H3 helices strongly diminished the ability of the corresponding oligomers to associate with the lipid vesicles. Furthermore, the membrane impairment mechanism occurs differently for the full-length PrP oligomers and the H2H3 helices, as shown by dye-release and black lipid membrane experiments. The membrane damage caused by the full-length PrP oligomers is correlated to their neuronal toxicity at submicromolar concentrations, as shown by cell culture assays. Although oligomers of synthetic H2H3 could compromise in vitro cell homeostasis, they followed a membrane-disruptive pattern that was different from the full-length oligomers, as revealed by the role of PrPC in cell viability assays.

Evidence of conformational landscape alteration and macromolecular complex formation in the early stages of in vitro human prion protein oxidation.

Oxidative stress is proposed to be one of the major causes of neurodegenerative diseases. Cellular prion protein (PrP) oxidation has been widely studied using chemical reagents such as hydrogen peroxide. However, the experimental conditions used do not faithfully reflect the physiological environment of the cell. With the goal to explore the conformational landscape of PrP under oxidative stress, we conducted a set of experiments combining the careful control of the nature and the amount of ROS produced by a60Co γ-irradiation source. Characterization of the resulting protein species was achieved using a set of analytical techniques. Under our experimental condition hydroxyl radical are the main reactive species produced. The most important findings are i) the formation of molecular assemblies under oxidative stress, ii) the detection of a majority of unmodified monomer mixed with oxidized monomers in these molecular assemblies at low hydroxyl radical concentration, iii) the absence of significant oxidation on the monomer fraction after irradiation. Molecular assemblies are produced in small amounts and were shown to be an octamer. These results suggest either i) an active recruitment of intact monomers by molecular assemblies' oxidized monomers then inducing a structural change of their intact counterparts or ii) an intrinsic capability of intact monomer conformers to spontaneously associate to form stable molecular assemblies when oxidized monomers are present. Finally, abundances of the intact monomer conformers after irradiation were modified. This suggests that monomers of the molecular assemblies exchange structural information with intact irradiated monomer. All these results shed a new light on structural exchange information between PrP monomers under oxidative stress.

Symmetry-breaking transitions in the early steps of protein self-assembly.

Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer's disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly ß-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aß(1-40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.

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

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.

A bi-monomeric, nonlinear Becker-Döring-type system to capture oscillatory aggregation kinetics in prion dynamics.

In this article, in order to understand the appearance of oscillations observed in protein aggregation experiments, we propose, motivate and analyse mathematically the differential system describing the kinetics of the following reactions: [Formula: see text] with n finite or infinite. This system may be viewed as a variant of the seminal Becker-Döring system, and is capable of displaying sustained though damped oscillations.

Stability analysis of a steady state of a model describing Alzheimer's disease and interactions with prion proteins.

Alzheimer's disease (AD) is a neuro-degenerative disease affecting more than 46 million people worldwide in 2015. AD is in part caused by the accumulation of A[Formula: see text] peptides inside the brain. These can aggregate to form insoluble oligomers or fibrils. Oligomers have the capacity to interact with neurons via membrane receptors such as prion proteins ([Formula: see text]). This interaction leads [Formula: see text] to be misfolded in oligomeric prion proteins ([Formula: see text]), transmitting a death signal to neurons. In the present work, we aim to describe the dynamics of A[Formula: see text] assemblies and the accumulation of toxic oligomeric species in the brain, by bringing together the fibrillation pathway of A[Formula: see text] peptides in one hand, and in the other hand A[Formula: see text] oligomerization process and their interaction with cellular prions, which has been reported to be involved in a cell-death signal transduction. The model is based on Becker-Döring equations for the polymerization process, with delayed differential equations accounting for structural rearrangement of the different reactants. We analyse the well-posedness of the model and show existence, uniqueness and non-negativity of solutions. Moreover, we demonstrate that this model admits a non-trivial steady state, which is found to be globally stable thanks to a Lyapunov function. We finally present numerical simulations and discuss the impact of model parameters on the whole dynamics, which could constitute the main targets for pharmaceutical industry.

Causative Links between Protein Aggregation and Oxidative Stress: A Review.

Compelling evidence supports a tight link between oxidative stress and protein aggregation processes, which are noticeably involved in the development of proteinopathies, such as Alzheimer's disease, Parkinson's disease, and prion disease. The literature is tremendously rich in studies that establish a functional link between both processes, revealing that oxidative stress can be either causative, or consecutive, to protein aggregation. Because oxidative stress monitoring is highly challenging and may often lead to artefactual results, cutting-edge technical tools have been developed recently in the redox field, improving the ability to measure oxidative perturbations in biological systems. This review aims at providing an update of the previously known functional links between oxidative stress and protein aggregation, thereby revisiting the long-established relationship between both processes.

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.

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 PrPC and in nonhuman primates. In humanized mice, L-BSE propagates without any apparent species barrier and shares similar biochemical PrPSc 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.

Generating Bona Fide Mammalian Prions with Internal Deletions.

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.

The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation.

Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct ß-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains.

Interaction between Shadoo and PrP Affects the PrP-Folding Pathway.

UNLABELLED: Prion diseases are characterized by conformational changes of a cellular prion protein (PrP(C)) into a ß-sheet-enriched and aggregated conformer (PrP(Sc)). Shadoo (Sho), a member of the prion protein family, is expressed in the central nervous system (CNS) and is highly conserved among vertebrates. On the basis of histoanatomical colocalization and sequence similarities, it is suspected that Sho and PrP may be functionally related. The downregulation of Sho expression during prion pathology and the direct interaction between Sho and PrP, as revealed by two-hybrid analysis, suggest a relationship between Sho and prion replication. Using biochemical and biophysical approaches, we demonstrate that Sho forms a 1:1 complex with full-length PrP with a dissociation constant in the micromolar range, and this interaction consequently modifies the PrP-folding pathway. Using a truncated PrP that mimics the C-terminal C1 fragment, an allosteric binding behavior with a Hill number of 4 was observed, suggesting that at least a tetramerization state occurs. A cell-based prion titration assay performed with different concentrations of Sho revealed an increase in the PrP(Sc) conversion rate in the presence of Sho. Collectively, our observations suggest that Sho can affect the prion replication process by (i) acting as a holdase and (ii) interfering with the dominant-negative inhibitor effect of the C1 fragment. IMPORTANCE: Since the inception of the prion theory, the search for a cofactor involved in the conversion process has been an active field of research. Although the PrP interactome presents a broad landscape, candidates corresponding to specific criteria for cofactors are currently missing. Here, we describe for the first time that Sho can affect PrP structural dynamics and therefore increase the prion conversion rate. A biochemical characterization of Sho-PrP indicates that Sho acts as an ATP-independent holdase.

The role of interruptions in polyQ in the pathology of SCA1.

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.

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.

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.

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

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.