A simple, versatile and sensitive cell-based assay for prions from various species

Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.     

Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level.     

Mouse-adapted sporadic human Creutzfeldt-Jakob disease prions propagate in cell culture

Cell based models used for the study of prion diseases have traditionally employed mouse-adapted strains of sheep scrapie prions. To date, attempts to generate human prion propagation in cell culture have been unsuccessful. Rabbit kidney epithelial cells (RK13) are permissive to infection with prions from a variety of species upon expression of cognate PrP transgenes. We explored RK13 cells expressing human PrP for their utility as a cell line capable of sustaining infection with human prions. RK13 cells processed exogenously expressed human PrP similarly to exogenously expressed mouse PrP but were not permissive to infection when exposed to sporadic Creutzfeldt-Jakob disease prions. Transmission of the same sporadic Creutzfeldt Jakob disease prions to wild-type mice generated a strain of mouse-adapted human prions, which efficiently propagated in RK13 cells expressing mouse PrP, demonstrating these cells are permissive to infection by mouse-adapted human prions. Our observations underscore the likelihood that, in contrast to prions derived from non-human mammals, additional unidentified cofactors or subcellular environment are critical for the generation of human prions.     

Transmission barriers for bovine, ovine, and human prions in transgenic mice

Transgenic (Tg) mice expressing full-length bovine prion protein (BoPrP) serially propagate bovine spongiform encephalopathy (BSE) prions without posing a transmission barrier. These mice also posed no transmission barrier for Suffolk sheep scrapie prions, suggesting that cattle may be highly susceptible to some sheep scrapie strains. Tg(BoPrP) mice were also found to be susceptible to prions from humans with variant Creutzfeldt-Jakob disease (CJD); on second passage in Tg(BoPrP) mice, the incubation times shortened by 30 to 40 days. In contrast, Tg(BoPrP) mice were not susceptible to sporadic, familial, or iatrogenic CJD prions. While the conformational stabilities of bovine-derived and Tg(BoPrP)-passaged BSE prions were similar, the stability of sheep scrapie prions was higher than that found for the BSE prions but lower if the scrapie prions were passaged in Tg(BoPrP) mice. Our findings suggest that BSE prions did not arise from a sheep scrapie strain like the one described here; rather, BSE prions may have arisen spontaneously in a cow or by passage of a scrapie strain that maintains its stability upon passage in cattle. It may be possible to distinguish BSE prions from scrapie strains in sheep by combining conformational stability studies with studies using novel Tg mice expressing a chimeric mouse-BoPrP gene. Single-amino-acid substitutions in chimeric PrP transgenes produced profound changes in incubation times that allowed us to distinguish prions causing BSE from those causing scrapie.     

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP^Sc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP^res isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP^Sc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.     

Scrapie strain transmission studies in ovine PrP transgenic mice reveal dissimilar susceptibility

The Tg(OvPrP4) mouse line, expressing the sheep prion protein, is a sensitive model crucial for the identification of the bovine spongiform encephalopathy agent possibly present in natural sheep spongiform encephalopathies. It was also previously demonstrated as susceptible to infection with natural scrapie isolates from sheep harbouring various genotypes. The performance of this new transgenic mouse line in scrapie strain characterization was further assessed by intracranial inoculation of five groups of Tg(OvPrP4) mice with brain homogenate of the wild type mouse-adapted scrapie strains, C506M3, 22A, 79A, 87V, or Chandler. The Tg(OvPrP4) mice were susceptible to the scrapie agent transmitted using mouse-adapted scrapie strains but not equivalently. Strains 87V and Chandler were most readily transmissible followed by 79A and C506M3. Strain 22A was the least transmissible. Clinical signs, survival data, spongiosis, and PrP^sc distribution were also reported. These various data demonstrate the possibility of distinguishing between scrapie strains. Our findings are discussed with regard to agent strain and host factors and already demonstrate the dissimilar susceptibilities of Tg(OvPrP4) mice to the different murine strains studied, thus, reinforcing their potential use in strain typing studies.     

Microglial cell line established from prion protein-overexpressing mice is susceptible to various murine prion strains

Several lines of evidence suggest that microglia have important roles in the pathogenesis of prion diseases. Here, we establish a novel microglial cell line (MG20) from neonatal tga20 mice that overexpress murine prion protein. After exposure to Chandler scrapie, we observed the replication and accumulation of disease-associated forms of the prion protein in MG20 cells up to the 15th passage. Furthermore, MG20 cells were susceptible to ME7, Obihiro scrapie, and bovine spongiform encephalopathy agents. Thus, MG20 cell lines persistently infected with various murine prion strains provide a useful model for the study of the pathogenesis of prion diseases.     

Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication

Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.     

Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions

The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.     

Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques

Three transgenic mouse lines designated Tg 69, 71, and 81 were produced harboring a Syrian hamster (Ha) prion protein (PrP) gene; all expressed the cellular HaPrP isoform in their brains. Inoculation of Tg 81 mice or hamsters with Ha prions caused scrapie in integral of 75 days; nontransgenic control mice failed to develop scrapie after greater than 500 days. Tg 71 mice inoculated with Ha prions developed scrapie in integral of 170 days. Both Tg 71 and Tg 81 mice exhibited spongiform degeneration and reactive astrocytic gliosis, and they produced the scrapie HaPrP isoform in their brains. Tg 81 brains also showed HaPrP amyloid plaques characteristic of Ha scrapie and contained integral of 10(9) ID50 units of Ha prions based on Ha bioassays. Our findings argue that the PrP gene modulates scrapie susceptibility, incubation times, and neuropathology; furthermore, they demonstrate synthesis of infectious scrapie prions programmed by a recombinant DNA molecule.     

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.     

Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins.

Scrapie and Creutzfeldt-Jakob disease are transmissible, degenerative neurological diseases caused by prions. Considerable evidence argues that prions contain protease-resistant sialoglycoproteins, designated PrPSc, encoded by a cellular gene. The prion protein (PrP) gene also encodes a normal cellular protein designated PrPC. We established clonal cell lines which support the replication of mouse scrapie or Creutzfeldt-Jakob disease prions. Mouse neuroblastoma N2a cells were exposed to mouse scrapie prions and subsequently cloned. After limited proteinase K digestion, three PrP-immunoreactive proteins with apparent molecular masses ranging between 20 and 30 kilodaltons were detected in extracts of scrapie-infected N2a cells by Western (immuno-) blotting. The authenticity of these PrPSc molecules was established by using monospecific antiserum raised against a synthetic peptide corresponding to a portion of the prion protein. Those clones synthesizing PrPSc molecules possessed scrapie prion infectivity as measured by bioassay; clones without PrPSc failed to demonstrate infectivity. Detection of PrPSc molecules in scrapie-infected N2a cells supports the contention that PrPSc is a component of the infectious scrapie particle and opens new approaches to the study of prion diseases.     

Scrapie infection of prion protein-deficient cell line upon ectopic expression of mutant prion proteins

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.     

Prion pathogenesis in the absence of Toll-like receptor signalling

To reach the brain from peripheral sites, prions must colonize various cell types within the lymphoreticular compartment. However, no prion entry receptors are yet known. Toll-like receptors (TLRs) are pattern-recognition receptors that bind a multitude of pathogens and are therefore candidates as effectors of prion entry. Moreover, injection of unmethylated CpG oligodinucleotides, which stimulate TLR9, has been reported to delay peripherally initiated scrapie. We therefore studied prion infection in MyD88^−/− mice, which are defective in TLR signalling. Despite subtle defects in splenic microarchitecture, MyD88^−/− mice challenged intraperitoneally or intracerebrally were fully susceptible to disease and died of scrapie after similar incubation times to those of wild-type mice. Splenic infectivity titres rose to similar levels with the same kinetics, and brains showed similar histopathological changes. TLR signalling therefore does not have any major role in prion pathogenesis, and the protective effect of TLR stimulation is unlikely to result from direct interactions with prions.     

Transfer of scrapie prion infectivity by cell contact in culture

BACKGROUND: When a cell is infected with scrapie prions, newly synthesized molecules of the prion protein PrP(C) are expressed at the cell surface and may subsequently be converted to the abnormal form PrP(Sc). In an experimental scrapie infection of an animal, the initial innoculum of PrP(Sc) is cleared relatively rapidly, and the subsequent propagation of the infection depends on the ability of infected cells to convert uninfected target cells to stable production of PrP(Sc). The mechanism of such cell-based infection is not understood. RESULTS: We have established a system in dissociated cell culture in which scrapie-infected mouse SMB cells are able to stably convert genetically marked target cells by coculture. After coculture and rigorous removal of SMB cells, the target cells express PrP(Sc) and also incorporate [35S]methionine into PrP(Sc). The extent of conversion was sensitive to the ratio of the two cell types, and conversion by live SMB required 2500-fold less PrP(Sc) than conversion by a cell-free prion preparation. The conversion activity of SMB cells is not detectable in conditioned medium and apparently depends on close proximity or contact, as evidenced by culturing the SMB and target cells on neighboring but separate surfaces. SMB cells were killed by fixation in aldehydes, followed by washing, and were found to retain significant activity at conversion of target cells. CONCLUSIONS: Cell-mediated infection of target cells in this culture system is effective and requires significantly less PrP(Sc) than infection by a prion preparation. Several lines of evidence indicate that it depends on cell contact, in particular, the activity of aldehyde-fixed infected cells.     

Cultured cell sublines highly susceptible to prion infection

Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrP(Sc)). In order to derive cell lines producing sufficient quantities of PrP(Sc) for most studies, it has been necessary to subclone infected cultures and select the subclones producing the largest amounts of PrP(Sc). Since postinfection cloning can introduce differences between infected and uninfected cell lines, we sought an approach to generate prion-infected cell lines that would avoid clonal artifacts. Using an improved cell blot technique, which permits sensitive and rapid comparison of PrP(Sc) levels in multiple independent cell cultures, we discovered marked heterogeneity with regard to prion susceptibility in tumor cell sublines. We exploited this heterogeneity to derive sublines which are highly susceptible to prion infection and used these cells to generate prion-infected lines without further subcloning. These infected sublines can be compared to the cognate uninfected cultures without interference from cloning artifacts. We also used susceptible cell lines and our modified cell blot procedure to develop a sensitive and reproducible quantitative cell culture bioassay for prions. We found that the sublines were at least 100-fold more susceptible to strain RML prions than to strain ME7 prions. Comparisons between scrapie-susceptible and -resistant cell lines may reveal factors that modulate prion propagation.     

Sheep-passaged bovine spongiform encephalopathy agent exhibits altered pathobiological properties in bovine-PrP transgenic mice

Sheep can be experimentally infected with bovine spongiform encephalopathy (BSE), and the ensuing disease is similar to scrapie in terms of pathogenesis and clinical signs. BSE infection in sheep is an animal and human health concern. In this study, the transmission in BoPrP-Tg110 mice of prions from BSE-infected sheep was examined and compared to the transmission of original cattle BSE in cattle and sheep scrapie prions. Our results indicate no transmission barrier for sheep BSE prions to infect BoPrP-Tg110 mice, but the course of the disease is accelerated compared to the effects of the original BSE isolate. The shortened incubation period of sheep BSE in the model was conserved in subsequent passage in BoPrP-Tg110 mice, indicating that it is not related to infectious titer differences. Biochemical signature, lesion profile, and PrP(Sc) deposition pattern of both cattle and sheep BSE were similar. In contrast, all three sheep scrapie isolates tested showed an evident transmission barrier and further adaptation in subsequent passage. Taken together, those data indicate that BSE agent can be altered by crossing a species barrier, raising concerns about the virulence of this new prion towards other species, including humans. The BoPrP-Tg110 mouse bioassay should be considered as a valuable tool for discriminating scrapie and BSE in sheep.     

Aerosols transmit prions to immunocompetent and immunodeficient mice

Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrP(C), efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrP(C) selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrP(Sc) and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.     

Generation of a Persistently Infected MDBK Cell Line with Natural Bovine Spongiform Encephalopathy (BSE)

Bovine spongiform encephalopathy (BSE) is a zoonotic transmissible spongiform encephalopathy (TSE) thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD). Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrP^BSE) and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK) cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrP^BSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.     

Transgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates

Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrP^C was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrP^d) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrP^d originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits.