MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College, London WC1N 3BG, UK
Correspondence
John Collinge
j.collinge{at}prion.ucl.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Multiple prion strains have been described that are distinguished by their incubation periods and patterns of neuropathology when passaged in inbred lines of mice, and these distinctive phenotypes are preserved on multiple passage in the same host (for review, see Bruce et al., 1992). The existence of prion strains challenges the protein-only hypothesis of prion propagation. However, it is clear that prion strains are associated with biochemical differences in PrPSc itself including differences in conformation (Hill et al., 1997
, 2003
; Bessen & Marsh, 1992
, 1994
; Collinge et al., 1996
; Telling et al., 1996
; Wadsworth et al., 1999
), glycosylation (Hill et al., 1997
, 2003
; Collinge et al., 1996
) and overall protease resistance (Kuczius & Groschup, 1999
). That these strain-associated biochemical differences in PrPSc fragment sizes and glycoform ratios following proteolysis can be transmitted to PrP in an experimental host argues that they may be responsible for encoding strain diversity. While the precise nature of the molecular basis of prion strain diversity is unclear, that prion strains may be distinguished by the differing molecular mass of fragments following partial proteinase K digestion and by differing ratios of di-, mono- and unglycosylated PrPSc is clear. Using this approach, we described four common PrPSc types in humans (Collinge et al., 1996
; Wadsworth et al., 1999
; Hill et al., 2003
). PrPSc types 13 are seen in sporadic and iatrogenic CJD, while type 4 PrPSc is exclusively associated with variant CJD (vCJD) and is associated with a di-glycosylated-dominant PrPSc pattern on a Western blot (Collinge et al., 1996
; Hill et al., 2003
). This characteristic glycoform ratio is also seen in BSE-infected cattle brain and these observations, together with bioassay data from wild-type and transgenic mice and non-human primates, have proved critical in establishing a link between BSE and vCJD (Hill et al., 1997
; Bruce et al., 1997
; Lasmézas et al., 1996
; Collinge et al., 1996
; Asante et al., 2002
). This characteristic molecular signature of BSE-derived prion isolates is seen in all UK cattle BSE cases examined and, together with biological strain typing studies in inbred and transgenic mice, suggests that BSE is caused by a single strain of agent (Hill et al., 1997
; Bruce et al., 1994
, 2002
; Collinge et al., 1996
; Kuczius & Groschup, 1999
; and our unpublished data). This molecular pattern, in additional to biological characteristics, is also maintained on transmission to other hosts such as domestic cat, sheep, macaque and other exotic animals, either by natural exposure or by experimental transmission (Bruce et al., 1994
; Fraser et al., 1994
; Collinge et al., 1996
; Hill et al., 1998
; Lasmezas et al., 2001
). Recent data from French and Italian screening programmes suggest that more than one strain of BSE may exist in cattle (Biacabe et al., 2004
; Casalone et al., 2004
). We have also shown that on BSE transmission to a line of transgenic mice expressing only human PrP Met-129, Tg(HuPrP129M+/+Prnp0/0)-35, two distinct molecular phenotypes can be produced: one that mirrors the vCJD phenotype with type 4 PrPSc and an additional molecular phenotype that is indistinguishable from that of sporadic CJD with PrPSc type 2 (Asante et al., 2002
). These transgenic mice were generated on a mixed genetic background, and one possibility was that the different patterns were determined by background effects in individual mice (Asante et al., 2002
). This interpretation was supported by the demonstration that vCJD and BSE prions on primary passage to a series of inbred lines of mice were also able to produce two distinct molecular phenotypes on Western blotting, dependent only on the genetic background of the mice (Asante et al., 2002
). These data argued that two distinct strains had been propagated from cattle BSE. However, since the parameters that distinguish prion strains (incubation time, neuropathology and PrPSc type) are also known to be modulated by host genetic background, these cannot be adequately compared after primary passage in different lines of inbred mice (Bruce, 1993
; Moore et al., 1998
; Somerville, 1999
). We therefore subpassaged these mouse-adapted BSE prions in the same inbred mouse line to determine whether distinctive biological characteristics resulted, indicative of different strains, and whether these correlated with the different PrPSc types propagated in the animals.
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METHODS |
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For second passage to SJL mice, a single brain from a terminally sick mouse from the primary passage to SJL group (I783) was used to produce inoculum I1590 [cattle BSE (I783)SJL mice (I1590)
SJL mice]. For second passage to C57BL/6 mice, a single brain from a subclinically infected mouse from the primary passage to C57BL/6 group (I038) was used to produce inoculum I656 [cattle BSE (I038)
C57BL/6 mice (I656)
C57BL/6 mice].
For third passage, the SJL-passaged inoculum was generated from a single mouse brain from the SJL second passage group (I1891) and was used to inoculate a group of SJL mice [cattle BSE (I783)SJL (I1590)
SJL (I1891)
SJL mice]. To generate the C57BL/6-passaged BSE inoculum, 11 mouse brains from the second passage were pooled to generate a larger volume of homogenate (I874). The inoculum was generated in this way as it was originally intended for use in a survey of incubation times in inbred lines and for use in a large mapping study to identify genes that influence prion disease incubation time (Lloyd et al., 2002
). This pool (I874) was used to inoculate SJL mice [cattle BSE (I038)
C57BL/6 (I656)
C57BL/6 (I874)
SJL mice].
All inocula were prepared by homogenizing brain samples (1 % w/v in PBS) using disposable equipment for each inoculum in a microbiological containment level 3 laboratory and inoculations were performed within a class I microbiological safety cabinet. All mice were uniquely identified with a subcutaneous transponder tag. Disposable cages were used throughout and lids and water bottles were also uniquely tagged.
Mice were anaesthetized with halothane/O2 and inoculated intracerebrally into the right parietal lobe with 30 µl inoculum. Incubation time was defined as the number of days from inoculation to the onset of clinical signs. This was assessed by daily examination for neurological signs of disease. Criteria for clinical diagnosis of prion disease were as described (Carlson et al., 1986). Animals were killed as soon as clinical scrapie was confirmed or if showing signs of distress.
Western blotting.
Brain homogenates (10 % w/v in PBS) were prepared, proteinase K-digested (100 µg proteinase K ml1 for 1 h at 37 °C) and Western-blotted as described previously (Wadsworth et al., 2001). Blots were probed with a biotinylated anti-PrP monoclonal antibody ICSM-35 (Asante et al., 2002
) in conjunction with an avidinbiotinalkaline phosphatase conjugate (Dako) and developed in chemiluminescent substrate (CDP-Star; Tropix Inc.). For quantification and analysis of PrP glycoforms, blots were developed in chemifluorescent substrate (AttoPhos; Promega) and visualized on a Storm 840 PhosphorImager (Molecular Dynamics). Quantification of PrPSc glycoforms was performed using ImageQuaNT software (Molecular Dynamics). Sodium phosphotungstic acid pre-concentration of PrPSc was performed as described previously (Wadsworth et al., 2001
).
Neuropathology and immunohistochemistry.
Mouse brains were fixed in 10 % buffered formol-saline, immersed in 98 % formic acid for 1 h, formalin post-fixed and paraffin wax-embedded. Serial sections of 4 µm nominal thickness were pre-treated with Tris/citrate/EDTA buffer (1·3 mM EDTA, 2·1 mM Tris, 1·1 mM citrate, pH 7·8) for antigen retrieval. PrP deposition was visualized using ICSM-35 as the primary antibody (diluted 1 : 3000) and gliosis was detected with anti-glial fibrillary acidic protein rabbit polyclonal antiserum (diluted 1 : 1000; Dako), using an automated immunostaining system (www.ventanamed.com). Sections of brains were examined by the same person, who was blind to the identity of the animal and genotype. Sections were scored for spongiosis, neuronal loss, gliosis and PrPSc deposition. Photographs were taken on an ImageView digital camera (www.soft-imaging.de) and composed with Adobe Photoshop.
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RESULTS |
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Second passage of BSE to SJL and C57BL/6 mice
On second passage to either SJL or C57BL/6 mice, incubation times for both groups were substantially reduced, with a 100 % attack rate (Table 1), showing the expected adaptation on second passage to the mouse. It was not appropriate to compare the incubation times between inocula in this passage because of host genetic background effects. On Western blotting, the PrPSc type bred true, maintaining the pattern seen on primary passage, with the SJL-derived strain giving a mono-glycosylated-dominant pattern and the C57BL/6-derived strain showing a di-glycosylated-dominant pattern (Fig. 1c
).
Third passage of BSE to SJL and C57BL/6 mice
All third passages were carried out in SJL mice and were therefore appropriate for comparison of all criteria of prion strains: incubation time, PrPSc type and neuropathology. The incubation time on third passage was reduced for both the SJL- and C57BL/6-derived BSE, suggesting further adaptation to mouse (Table 1). The incubation times of 110±3 days for SJL-derived BSE and 155±1 days for C57BL/6-derived BSE were highly significantly different (P<0·0001, MannWhitney test). As inoculation was intracerebral with brain homogenate from terminally affected animals, there should be no titre effects, also evidenced by the remarkably consistent incubation periods, and there are no genetic background effects since all transmissions were to inbred SJL mice. These data are therefore consistent with propagation of two distinct prion strains in these SJL mice.
Brains from each of these groups were examined by Western blotting. The PrPSc type observed on primary and secondary passage was faithfully maintained on third passage. The SJL-passaged BSE showed a mono-glycosylated-dominant pattern and the C57BL/6-passaged BSE showed a di-glycosylated-dominant pattern (Fig. 1 and Fig. 2
) on further subpassage in SJL.
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DISCUSSION |
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SJL mice were able to support both the MRC1 and MRC2 strain patterns. However, it will be important to establish whether these strains are stable on further passage in SJL and other strains of mice. Both C57BL/6 and SJL mice share the same PrP amino acid sequence and therefore the strain selection must be a feature of other genetic loci. Within the limits of resolution available from Western blotting, the fragment sizes following proteinase K digestion were the same for the PrPSc associated with both strains, raising the possibility that the differences related to glycosylation and not to gross conformational differences, at least as differentiated by proteinase K digestion. Additional studies will be required to characterize precisely the conformation and physico-chemical properties of PrPSc associated with these prion strains.
Although recent reports suggest that alternative strains of BSE can be found in cattle, previous studies have suggested that BSE is caused by a single strain of agent (Bruce et al., 1994; Biacabe et al., 2004
; Casalone et al., 2004
). 301V and 301C represent previously reported independent BSE strains; however, these were propagated in different strains of mice, which not only had very different genetic backgrounds (VM/Dk and C57) but also had two amino acid coding differences in PrP and therefore their strain characteristics cannot be directly compared. It is possible that MRC2 represents the same strain as 301C; however, formal comparisons will be required to investigate this.
The strain characteristics associated with MRC1 resemble those seen with passage of Chandler/RML scrapie in these strains of mice with respect to incubation time (122±1 days), histology and PrPSc type on Western blotting (unpublished data). We are confident that our findings are not the result of contamination with mouse scrapie as these transmissions were performed in accordance with rigorous biosafety protocols for preparation of inocula, inoculations and care of mice. Disposable equipment was used for each inoculum and each mouse was identified with a unique transponder. In addition, the MRC1 strain was also seen on primary passage to RIIIS mice and also on primary passage of vCJD to SJL mice (Asante et al., 2002). vCJD is caused by a BSE-like prion strain (Hill et al., 1997
; Bruce et al., 1997
; Collinge et al., 1996
); therefore, it is not surprising that the same phenomenon is seen with both vCJD and BSE prions in the same strains of mice. Further transmission studies are under way to characterize these vCJD-derived mouse strains. A similar bifurcation of the BSE strain characteristics was also observed with independent BSE inocula in transgenic mice expressing human PrP with methionine at codon 129, Tg(HuPrP129M+/+Prnp0/0)-35 (Asante et al., 2002
).
MRC2 and MRC1 were derived from different sources of BSE prions, I038 and I783, respectively. I038 originated from a pool of five infected cow brainstems. However, our extensive transmission studies with these isolates excluded the pooling of material from five cows as the source of the strain variation that we observe in these transmissions. I038 gave the classical BSE signature and the novel phenotype was derived from I783, which originated from a single cow brain. I783 has been used for transmissions in several inbred lines of mice in our laboratory and consistently produces the MRC2 strain (as determined by Western blotting) in C57BL/6JOlaHsd, FVB/NHsd, NZW/OlaHsd, SM/J and SWR/OlaHsd mice. However, it also consistently produces the MRC1 strain (as determined by Western blotting) in SJL/OlaHsd and RIIIS/J mice (Fig. 1b, lane 3 and Asante et al., 2002). DNA sequence analysis of the bovine Prnp gene in I783 confirmed that the animal was homozygous for PrP amino acids, thereby excluding PrP heterozygosity as the source of the strain bifurcation. I038 and other independent BSE inocula have also been shown to produce two strains' in Tg(HuPrP129M+/+Prnp0/0)-35 transgenic mice, which are on a mixed genetic background. We therefore believe that the prion strain selection or mutation is a feature of the genetic background of the mice and studies are under way to identify the genes involved. Although recent data suggest that more than one strain of BSE may exist in cattle (Biacabe et al., 2004
; Casalone et al., 2004
), in this study, both I038 and I783 appeared to be the same single strain. However, Western blotting was unable to determine whether a mixed population of strains already existed in either of these brains or whether the alternative strains were generated de novo in the mice. The inbred lines studied to date represent only a small proportion of the allelic variation that exists in the mouse genome; therefore, the study of other strains of mice, particularly those derived from unrelated strains such as trapped wild mice, may reveal additional BSE-derived strains.
The human population exposed to BSE has a much more diverse genetic background than can be observed using laboratory strains of inbred mice. Therefore, it is possible that BSE infection may be revealed not only as the type 4 PrPSc and associated vCJD clinicopathological phenotype seen to date (Collinge et al., 1996; Hill et al., 2003
), but in future, may also present with alternative PrPSc types, incubation times and neuropathology that may not be distinguishable from sporadic CJD (Hill et al., 1997
; Asante et al., 2002
) or represent additional novel phenotypes (Collinge, 1999
). BSE may also have infected sheep flocks in the UK and, given the genetic diversity of sheep breeds, it is also possible that in some breeds BSE propagates as a strain type that is indistinguishable from natural sheep scrapie.
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ACKNOWLEDGEMENTS |
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Received 9 December 2003;
accepted 27 April 2004.