Scrapie strains maintain biological phenotypes on propagation in a cell line in culture
2001; Springer Nature; Volume: 20; Issue: 13 Linguagem: Inglês
10.1093/emboj/20.13.3351
ISSN1460-2075
Autores Tópico(s)Trace Elements in Health
ResumoArticle2 July 2001free access Scrapie strains maintain biological phenotypes on propagation in a cell line in culture Christopher R. Birkett Corresponding Author Christopher R. Birkett Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Ruth M. Hennion Ruth M. Hennion Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Dawn A. Bembridge Dawn A. Bembridge Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Michael C. Clarke Michael C. Clarke Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Aileen Chree Aileen Chree Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Moira E. Bruce Moira E. Bruce Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Christopher J. Bostock Christopher J. Bostock Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Christopher R. Birkett Corresponding Author Christopher R. Birkett Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Ruth M. Hennion Ruth M. Hennion Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Dawn A. Bembridge Dawn A. Bembridge Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Michael C. Clarke Michael C. Clarke Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Aileen Chree Aileen Chree Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Moira E. Bruce Moira E. Bruce Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Christopher J. Bostock Christopher J. Bostock Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK Search for more papers by this author Author Information Christopher R. Birkett 1, Ruth M. Hennion1, Dawn A. Bembridge1, Michael C. Clarke1, Aileen Chree2, Moira E. Bruce2 and Christopher J. Bostock1 1Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN UK 2Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:3351-3358https://doi.org/10.1093/emboj/20.13.3351 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Bovine spongiform encephalopathy (BSE) and its human equivalent, variant Creutzfeldt–Jakob disease (vCJD), are caused by the same strain of infectious agent, which is similar to, but distinct from, >20 strains of their sheep scrapie homologue. A better understanding of the molecular strain determinants could be obtained from cells in monoculture than from whole animal studies where different cell targeting is commonly a strain-related feature. Although a few cell types can be infected with different strains, the phenotypes of the emergent strains have not been studied. We have cured the scrapie-infected, clonal SMB cell line with pentosan sulfate, stably re-infected it with a different strain of scrapie and shown that biological properties and prion protein profiles characteristic of each original strain are propagated faithfully in this single non-neuronal cell type. These findings attest to the fact that scrapie strain determinants are stable and host-independent in isolated cells. Introduction The prion concept holds that a phenotype can be transmitted across generations using a protein as the carrier of information rather than a nucleic acid (Prusiner, 1982). It originated in the field of the transmissible spongiform encephalopathies (TSEs) which are progressive neurodegenerative diseases such as scrapie of sheep and goats, Creutzfeldt–Jakob disease (CJD) of humans and bovine spongiform encephalopathy (BSE), and has been extended recently to explain non-Mendelian inheritance of certain traits in yeast and fungi (Masison, 2000). The existence of several distinct strains of mammalian agent has been recognized for several years and these strains were first defined by two principal criteria, their incubation times in Sincs7 and Sincp7 inbred mice and their heterozygote cross, and the types and patterns of brain lesions they induce at the terminal stage of disease in these mice (Dickinson et al., 1986). The prion protein (PrP) is encoded at the Sinc (or Prn-i) locus, and one (or both) of two amino acid differences in the mouse protein encoded by the s7 or p7 alleles is the molecular basis of these host differences in survival time (Moore et al., 1998). Although genetic elements other than the protein-coding element within the Sinc locus can influence the brain pathology following infection with a particular strain (Moore et al., 1998), distinct features can be seen which are obviously determined by the strain of inoculated agent independently of the host species (Fraser, 1976; Bruce, 1993). The conversion of normal PrP (PrPC) to a protease-resistant isoform, PrPSc, is a key event in the pathogenesis of scrapie and all other prion diseases, and PrPSc, according to prion theory, is the sole component of the infectious particle (Prusiner, 1998) so molecular differences in PrPSc must form the basis of the different phenotypes of CJD and scrapie. It has been speculated that variation in protease-resistant fragment size or conformation and the degree of its asparagine-linked glycosylation (Bessen and Marsh, 1994; Collinge et al., 1996; Telling et al., 1996; Caughey et al., 1998; Parchi et al., 1998) form the basis of agent strain differences. Such physicochemical differences could 'translate' into differences in the survival times and pathology seen with various strains as a consequence of differing rates of production, accumulation and clearance of the prion protein by different cell types within the brain (Safar et al., 1998). It follows that cell-specific variation in conformation and/or degree of glycosylation of PrP would be the effectors of this strain propagation (Weissmann, 1991). Separating cause of infection from its pathological by-products has been hindered by the need to study strain effects in the whole organism: for example, it has been impossible to determine if a single PrPSc glycoform—one molecular signature of a particular strain (Aguzzi and Weissmann, 1997)—can target only one specific subset of nerve cells in the brain capable of producing only that glycoform (DeArmond et al., 1997, 1999). To investigate these processes requires the stable infection of a single cell type with more than one strain of scrapie. The best defined scrapie strains have been isolated and passaged in mice (Dickinson et al., 1986); therefore, to avoid interpretational difficulties arising from inter- species transmission, a mouse cell line would be optimal. However, persistent scrapie infection can be established in only a very few cell lines in vitro. Scrapie-infected mouse C1300 neuroblastoma-derived cell lines ScMNB (Race et al., 1987) or ScN2a (Butler et al., 1988) have been valuable in defining events in PrPSc production (Race et al., 1988; Taraboulos et al., 1990, 1992; Caughey et al., 1991; McKinley et al., 1991; Vey et al., 1996; Naslavsky et al., 1997) but the wild-type cells have only a low susceptibility to in vitro challenge with scrapie. The cell line we chose for these experiments was SMB (Clarke and Haig, 1970) which was established originally by culture from a brain taken from a mouse clinically affected by the Chandler scrapie isolate and shown to be a cell line of mesodermal origin (Haig and Clarke, 1971). We reasoned that since SMB cells possessed a high degree of competence in propagating the original infection, a non-infected SMB variant might exhibit equal competence at hosting a newly introduced, different scrapie strain. Results Curing SMB with pentosan sulfate In preliminary experiments, when we examined clonally derived colonies of SMB for PrPSc, a surrogate marker for infectivity, no PrPSc-negative colonies were identified. We therefore investigated making an uninfected variant by treating the SMB cell line with pentosan sulfate (PS) to cure it of the infection with Chandler scrapie. PS is one of several polyanionic compounds capable of altering the scrapie incubation period in vivo (Diringer and Ehlers, 1991) and has been shown to curtail PrPSc production and replication of infectivity in ScMNB cells (Caughey and Raymond, 1993; Priola and Caughey, 1994). When grown for a single passage in the presence of PS at a range of concentrations up to 1 × 10−5 g/ml, the growth of SMB cells was unaffected, but PrPSc accumulation was inhibited in a dose-dependent manner (Figure 1A). The concentration required to inhibit PrPSc accumulation by 50% (IC50) was 1.5 × 10−7 g/ml. Even at the highest concentrations of PS tested, there was a residual PrPSc content of ∼12–14% of the control value, which is in accord with previous work showing that PS effectively inhibits de novo accumulation of PrPSc but has little effect on reducing the amount of pre-existing PrPSc in cells (Caughey and Raymond, 1993). Figure 1.PrPSc expression in SMB cells was cured by pentosan sulfate. (A) The inhibition by PS of PrPSc accumulation in SMB cells is dose dependent. Cultures of SMB cells were grown for 7 days in the continuous presence of the indicated concentrations of PS, after which time PrPSc levels were measured by quantitative dot-blotting. The points are the means (±SEM) from four experiments in which each data point was triplicated. (B) Western blot analysis of the PrP in SMB and the pentosan sulfate-treated SMB-PS cells. Proteins in post-nuclear cell extracts prepared either with (+) or without (−) proteinase K (PK) treatment, were precipitated with methanol/chloroform prior to electrophoresis. Successive lanes in each triplet of lanes represents a 5-fold dilution series of the previous sample. Molecular weight standards (kDa) are indicated on the left and the position of PK-treated PrPSc isoforms on the right: non-glycosylated (circle), monoglycosylated (single vertical bar) and diglycosylated (double vertical bar). Download figure Download PowerPoint To effect a permanent cure, SMB cells were grown in the continuous presence of 1 × 10−6 g/ml PS for seven passages so that all pre-formed accumulated PrPSc was effectively removed through dilution. With an ∼8-fold increase in cell number at each pass, PrPSc quickly became undetectable, the seven passages representing a dilution of some 2 × 107 in the initial amount of PrPSc. PrPSc subsequently has remained undetectable in this PS-treated cell line, designated SMB-PS, maintained in the absence of PS for >50 passages. Western blot analysis of total protein extracts of SMB and SMB-PS cells showed a complex pattern of PrP isoforms (Figure 1B). In SMB cells, the pattern was typical of a mixture of PrPSc and PrPC. In SMB-PS cells, a much simpler pattern of PrP isoforms existed composed predominantly of 35–37 kDa isoforms, most probably full-length diglycosylated PrPC, 30–33 kDa isoforms which were probably a mixture of full-length monoglycosylated and truncated diglycosylated PrPC (Chen et al., 1995), with very little evidence of non-glycosylated PrPC (26 kDa). Treatment with proteinase K (PK) produced a subset of proteinase-resistant bands from SMB cells typical of PrPSc, but resulted in the degradation of all the PrP in SMB-PS samples. To confirm that the PrPSc-cured cells had also been cured of infectious Chandler agent, the SMB-PS cell line was tested for scrapie infectivity. Of 18 C57BL mice inoculated intracerebrally with SMB-PS cells, 11 survived to the end of the experiment and were culled without clinical signs at 796 days post-inoculation; the other seven mice were killed as a result of contracting inter-current diseases (on days 370, 377, 473, 604, 628, 673 and 790). In the entire group of 18 (both inter-current and survivors), there were no symptomatic cases of scrapie, no post-mortem histopathological signs of scrapie and no western blot-detectable PrPSc in any of the brains. The survival curve for SMB-PS-inoculated mice (not shown) is not significantly different from that obtained for sham-inoculated controls but it is significantly longer than for mice inoculated with an equivalent number of SMB cells (see below), and is consistent with SMB-PS being cured of infectivity. Re-infection of the cured SMB-PS cell Two sources of scrapie have been particularly important in the derivation of individual scrapie strains, the 'drowsy goat' source and the Cheviot sheep-passaged SSBP/1 source (Dickinson et al., 1986). It was the drowsy goat source that first gave clues to the existence of different scrapie strains (Pattison and Millson, 1961; Pattison, 1972), was the first to be transmitted to mice (Chandler, 1961) and which was also the source of the SMB cell infection. Strains isolated from the drowsy goat source are 'Chandler', widely distributed and, after derivation and cloning, known as 139A or RML, and 79A which are all unique to this source (Dickinson et al., 1986). Among the several strains derived from SSBP/1 is 22F, and its very distinctive biological phenotype makes it ideal for comparison with the original infection of the SMB cell (Dickinson et al., 1986; Bruce, 1993). To survey the susceptibility of SMB-PS cells to in vitro infection using the 22F strain, we employed a colony-lift blotting procedure. The SMB cell is phagocytic (Haig and Clarke, 1971) and this gross screening method allowed us to introduce very large dilution factors post-challenge, favouring the subsequent detection of PrPSc synthesized de novo and discriminating it from any residual PrPSc of the inoculum taken up by the cells. Challenge of SMB-PS cells with 22F-infected brain homogenate resulted in consistently high stimulation of PrPSc (24.8% of the colonies, n = 4 experiments) while in SMB-PS exposed to normal mouse brain homogenate, PrPSc remained undetectable ( 1 year (>50 passages). The ratio diglycosyl/monoglycosyl PrPSc (D/M) from SMB cells was 0.46 ± 0.19 (mean ± SD; n = 11) compared with that from SMB[22F]F4 of 0.98 ± 0.31 (n = 14), marking the significance of these observations (ANOVA, P 150 passages (Clarke, 1979), with a constant infectious titre, was indicative of a high degree of competence as a host cell for scrapie. To exploit this competence in a study of scrapie strain determinants, we would first need to isolate a revertant, scrapie-free subline of SMB, but our search for subclones spontaneously 'cured' of PrPSc was unsuccessful. PS enabled us to redress this as it proved to be effective at inhibiting PrPSc accumulation, though when compared with the IC50 of 1 × 10−9 g/ml obtained with ScMNB cells (Priola and Caughey, 1994) SMB cells were far less sensitive to PS than the neuroblastoma line. There is no clear reason for this difference in sensitivity, but we can speculate that fundamental differences in cell type might be responsible. The mode of action of PS is uncertain although it is known to bind directly to isolated PrPC (Caughey et al., 1994) and recombinant PrP (Brimacombe et al., 1999) and to cause the subcellular re-distribution of PrPC expressed in mouse neuroblastoma cells (Shyng et al., 1995), so a direct interaction of PS with PrP during the curing process is implicated. Since polyanionic glycosaminoglycans such as PS can bind to numerous cell surface ligands, in particular to components of the extracellular matrix, differences in levels of expression of such ligands between SMB and neuroblastoma cells might serve to sequester PS to a greater extent and thereby modify the amount of polyanion available for binding PrP. It is probable that the susceptibility of a cell to infection with a TSE agent and its ability to sustain the replication of that infection are governed by separat
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