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.

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

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.

Facilitated cross-species transmission of prions in extraneural tissue.

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

Atypical/Nor98 scrapie infectivity in sheep peripheral tissues.

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrP(Sc) negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed.

Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model.

Prion strains yield specific neuropathological features including spongiform degeneration and deposition patterns of pathological prion protein. Their invariant regional distribution, following variations in the infection route, has led to the proposal that prions replicate preferentially in defined neuro-anatomical areas. The molecular mechanisms underlying this apparent strain-specific neuronal tropism are currently unknown. However, a possible explanation may be that prion replication is relatively innocuous, resulting in long-term propagation, thus masking initial regional distribution variations linked to different infection routes. This "low neurotoxicity" may be imputable either to the rodent model used or the prion strain(s) inoculated. To investigate this possibility, we studied prion pathogenesis in a prototypal short-incubation disease model consisting of 127S scrapie strain propagated in tg338 transgenic mice expressing the VRQ allele of ovine PrP. This prion strain derives from a natural sheep scrapie isolate that was serially transmitted to tg338 mice without any obvious transmission barrier and biologically cloned by limiting dilution. We compared the pathology induced by the peripheral or intracerebral inoculation of 127S strain. Surprisingly, we found that the disease greatly differed in clinical signs, abnormal prion protein levels, and neuropathology among the routes of infection. Secondary transmission performed with brain material from mice inoculated either intracranially or intraperitoneally produced similar neuropathological features. These results therefore indicate that the route of infection can strongly influence the apparent phenotype of a scrapie strain.

Characterization of atypical scrapie cases from Great Britain in transgenic ovine PrP mice.

Twenty-four atypical scrapie cases from sheep with different prion protein genotypes from Great Britain were transmitted to transgenic tg338 and/or TgshpXI mice expressing sheep PrP alleles, but failed to transmit to wild-type mice. Mean incubation periods were 200-300 days in tg338 mice and 300-500 days in TgshpXI mice. Survival times in C57BL/6 and VM/Dk mice were >700 days. Western blot analysis of mouse brain samples revealed similar multi-band, protease-resistant prion protein (PrP(res)) profiles, including an unglycosylated band at approximately 8-11 kDa, which was shown by antibody mapping to correspond to the approximately 93-148 aa portion of the PrP molecule. In transgenic mice, the incubation periods, Western blot PrP(res) profiles, brain lesion profiles and abnormal PrP (PrP(Sc)) distribution patterns produced by the Great Britain atypical scrapie isolates were similar and compatible with the biological characteristics of other European atypical scrapie or Nor98 cases.

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

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

Glycosylation-related gene expression profiling in the brain and spleen of scrapie-affected mouse.

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). The molecular requirements for efficient PrP conversion remain unknown. Altered glycosylation has been linked to various pathologies and the N-glycans harbored by two prion protein isoforms are different. In order to search for glycosylation-related genes that could mark prion infection, we used a glycosylation-dedicated microarray that allowed the simultaneous analysis of the expression of 165 glycosylation-related genes encoding proteins of the glycosyltransferase, glycosidase, lectin, and sulfotransferase families to compare the gene expression profiles of normal and scrapie-infected mouse brain and spleen. Eight genes were found upregulated in "scrapie brain" at the final state of the disease. In the spleen, five genes presented a modified expression. Three genes were also upregulated in the spleen of infected mice, and two (Pigq and St3gal5) downregulated. All changes were confirmed by qPCR and biochemical analyses applied to Pigq and St3gal5 proteins.

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

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

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

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

Prnp knockdown in transgenic mice using RNA interference.

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

Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD.

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 neuropathological 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.

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

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.

Isolation from cattle of a prion strain distinct from that causing bovine spongiform encephalopathy.

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 (PrP(res)) isolated from brains of affected animals or humans. However, alternate PrP(res) 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 PrP(res) 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.

A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes.

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.

Cultured peripheral neuroglial cells are highly permissive to sheep prion infection.

Transmissible spongiform encephalopathies arise as a consequence of infection of the central nervous system (CNS) by prions. Spreading of the infectious agent through the peripheral nervous system (PNS) may represent a crucial step toward CNS neuroinvasion, but the modalities of this process have yet to be clarified. Here we provide further evidence that PNS glial cells are likely targets for infection by prions. Glial cell clones originating from dorsal root ganglia of transgenic mice expressing ovine PrP (tgOv) and simian virus 40 T antigen were found to be readily infectible by sheep scrapie agent. This led us to establish two stable cell lines that exhibited features of Schwann cells. These cells were shown to sustain an efficient and stable replication of sheep prion based on the high level of accumulation of abnormal PrP and infectivity in exposed cultures. We also provide evidence for abnormal PrP deposition in peripheral neuroglial cells from scrapie-infected tgOv mice and sheep. These findings have potential implications in terms of designing new cell systems permissive to prions and of peripheral pathobiology of prion infections.

PrP polymorphisms tightly control sheep prion replication in cultured cells.

Prion diseases are fatal neurodegenerative disorders of animals and humans that are characterized by the conversion of the host-encoded prion protein (PrP) to an abnormal isoform. In several species, including humans, polymorphisms in the gene encoding the PrP protein tightly control susceptibility of individuals toward this disease. In the present study we show that Rov cells expressing an ovine PrP allele ((VRQ)PrP) associated with high susceptibility of sheep to scrapie were very sensitive to sheep prion transmission and replicated the agent to high titers. In contrast, we did not find any evidence of infection when Rov cells expressed similar levels of a PrP variant ((ARR)PrP) linked to resistance. Our data provide the first direct evidence that natural PrP polymorphisms may affect prion susceptibility by controlling prion replication at the cell level. The study of how PrP polymorphisms influence the genetic control of prion propagation in cultured Rov cells may help elucidate basic mechanisms of prion replication.

New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives.

Sheep scrapie is a prototypical transmissible spongiform encephalopathy (TSE), and the most widespread of these diseases. Experimental study of TSE infectious agents from sheep and other species essentially depends on bioassays in rodents. Transmission of natural sheep scrapie to conventional mice commonly requires one or two years. In an effort to develop laboratory models in which investigations on the sheep TSE agent would be facilitated, we have established mice and cell lines that were genetically engineered to express ovine PrP protein and examined their susceptibility to the infection. A series of transgenic mice lines (tgOv) expressing the high susceptibility allele (VRQ) of the ovine PrP gene from different constructs was expanded. Following intracerebral inoculation with natural scrapie isolates, all animals developed typical TSE neurological signs and accumulated abnormal PrP in their brain. The survival time in the highest expressing tgOv lines ranged from 2 to 7 months, depending on the isolate. It was inversely related to the brain PrP content, and essentially unchanged on further passaging. Ovine PrP transgene expression thus enhanced scrapie disease transmission from sheep to mice. Such tgOv mice may bring new opportunities for analysing the natural variation of scrapie strains and measuring infectivity. As no relevant cell culture models for agents of naturally-occurring TSE exist, we have explored various strategies in order to obtain stable cell lines that would propagate the sheep agent ex vivo without prior adaptation to rodent. In one otherwise refractory rabbit epithelial cell line, a regulable expression of ovine PrP was achieved and found to enable an efficient replication of the scrapie agent in inoculated cultures. Cells derived from sheep embryos or from tgOv mice were also used in an attempt to establish permissive cell lines derived from the nervous system. Cells engineered to express PrP proteins of a specified sequence may thus represent a promising strategy to further explore, at the cellular level, various aspects of TSE diseases.