Veterinary Laboratories Agency (VLA-Lasswade), Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
Correspondence
Martin Jeffrey
m.jeffrey{at}vla.defra.gsi.gov.uk
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ABSTRACT |
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INTRODUCTION |
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TSEs are widely believed to be caused by a structurally modified isomer of PrPc designated PrPsc (Prusiner, 1982, 1999a
, b
). This infectious prion would acquire pathogenic and infectious properties following its conversion from the normal isoform, but its precise nature still eludes definition. The structural and biochemical properties of PrPsc include increased
-pleated sheet content, decreased solubility and increased resistance to degradation by proteolytic enzymes. This latter property (PrPres) is widely used for diagnostic purposes and PrPres is often used as the operational definition of PrPsc. In many instances there is close correlation between PrPsc and infectivity, but sometimes infectivity and disease are associated with a PrP fraction lacking the biochemical properties of PrPsc (Lasmezas et al., 1997
; Somerville et al., 1991
). This has led to a modification of the prion hypothesis according to which only a subfraction of PrPsc molecules would be infectious. Nevertheless, the term prion continues to be used to describe simultaneously the causal agent and the abnormal PrP protein. Some biochemical variations of abnormal forms of PrP correlate with infections by particular TSE agent strains, but the way in which an abnormally modified host protein may code for the wide diversity of clinico-pathological phenotypes of different TSE strains remains a puzzle (Chesebro, 1998
; Farquhar et al., 1998
).
Strain diversity has long been recognized in experimental murine scrapie, but the extent to which strains occur in natural sheep scrapie is uncertain. Murine scrapie strains are isolated after cloning by limiting dilution in different strains of inbred mice and are principally characterized by their consistent incubation periods, patterns of vacuolation (Dickinson, 1976; Bruce & Fraser, 1991
) and PrPd deposition in the brain (Bruce et al., 1976
, 1989
). More recently, strains or sources of other TSE agents, including those of CJD and vCJD, have been characterized by N-terminal sequencing of the PrPres fragment (Bessen & Marsh, 1994
; Parchi et al., 2000
) or on the biochemical profile of the PrPres fraction. These biochemical changes consist of variation in the size, electrophoretic mobility, degree of protease resistance and glycosylation ratio of PrPres (Hill et al., 1997
; Kuczius & Groschup, 1999
; Somerville, 1999
; Somerville et al., 1997
). Only limited differences in biochemical features of PrPres (Hope et al., 1999
) and vacuolation profiles (Begara-McGorum et al., 2002
; Ligios et al., 2002
) between different sheep scrapie sources have so far been described. The biochemical properties of abnormal PrP extracted from infections by different scrapie strains generally overlap, preventing their definitive usage in strain characterization. However, in two separate studies, we have shown that IHC detection of PrPd may reveal differences between different scrapie sources. Different cellular patterns of PrPd deposition are found in the brain of sheep affected with different TSE sources, including scrapie and BSE (González et al., 2002
). Moreover, differences in intracellular PrPd labelling of phagocytic cells in the LRS and CNS are found when scrapie and ovine BSE are compared (Jeffrey et al., 2001b
).
Electron microscopy studies have shown that the majority of PrPd accumulation occurs at the plasmalemma of infected cells and in the adjacent extracellular space. Extracellular PrPd accumulations, including amyloid fibrils and plaques, found in rodent and sheep scrapie react with antibodies that recognize the extreme N terminus of the PrP molecule and are therefore assumed to be full-length proteins (Giaccone et al., 1992; Jeffrey et al., 1996
, 1998
). Some forms of PrPd accumulation are, however, intracellular and are located within lysosomes in CNS and LRS phagocytic cells of murine scrapie-infected tissues (Jeffrey et al., 1994
, 2000
). In sheep, these intracellular PrPd accumulations appear to lack the downstream segments closest to the signal peptide of the PrP molecule (Jeffrey et al., 2001a
).
In this study, we have further compared the effects of the TSE source or agent, PrP genotype, tissue, cell type and extracellular and intracellular locations of PrPd on antibody affinity. In so doing, we have defined more accurately differences in the IHC labelling patterns of the BSE agent and experimental scrapie sources. We conclude that there are consistent differences in the truncation patterns of intracellular PrPd between ovine BSE and infections by other UK scrapie sources. These differences are not, however, constant but change with the tissue and cellular site of infection, suggesting that the processing and truncation of PrPd depends on both the infecting agent and the tissues and cells where PrPd accumulation takes place.
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METHODS |
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Tissues were fixed in 10 % neutral phosphate-buffered formalin, trimmed, post-fixed and embedded according to standard procedures. Tissue sections (5 µm) were cut on a microtome, mounted on treated glass slides (Superfrost Plus; Menzel-Glaser) and dried overnight at 60 °C. Sections were dewaxed and hydrated by routine methods and then subjected to a retrieval procedure of formic acid and autoclaving in citrate buffer antigen and to an IHC protocol for the detection of PrPd, as described previously (González et al., 2002). Briefly, endogenous peroxidase activity was quenched using 1 % hydrogen peroxide in methanol. Blocking serum raised against the same species as the secondary antibody was applied for 1 h prior to overnight (16 h) application of optimally diluted primary antibody. Antigen/antibody interaction was visualized using biotinylated secondary antibody (Vector Laboratories) and ABC complex. The chromagen used was DAB. All dilutions were determined using standard chequerboard titration methods against a range of sheep scrapie- and BSE-affected control tissues. Biotinylated secondary antibodies and the ABC complex were used at 1 : 200.
A range of primary antibodies recognizing specific amino acid sequences of the prion protein were selected according to observations made previously (Jeffrey et al., 2001b). Most antibodies recognized segments of the N-terminal domain of the flexible tail of PrP, with two antibodies recognizing regions of the globular domain and C terminus of the protein (Riek et al., 1997
). The antibodies used and the sequences to which they were raised (where known) are shown in Table 2
. Primary antibodies were diluted at 1 : 400 (P4), 1 : 1000 (FH11, BG4, R145), 1 : 6000 (505) or 1 : 8000 (521, R486).
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RESULTS |
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LRS
Follicular dendritic cells (FDCs).
Within the light zone of secondary follicles, PrPd accumulation associated with FDC processes was observed with all N- and C-terminal antibodies in both scrapie-affected (Fig. 1) and BSE-affected (Fig. 2
) sheep. The intensity of FDC labelling was variable across different follicles of the same or different lymph nodes, but was maintained across the different antibodies. Within this series, FDC labelling of lymph nodes of BSE-infected sheep was often less intense than in scrapie-affected animals.
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Other LRS immunolabelling.
In addition to the above FDC and TBM patterns of PrPd accumulation in follicles, other immunolabelling patterns were found in the spleens. Several antibodies, particularly P4, gave PrP immunolabelling in the marginal zone of the white pulp and in individual cells within the peri-arteriolar lymphoid sheath. These immunolabelling deposits took the form of single, intense, intracytoplasmic granules, similar to that seen in some follicular TBMs, and were detected in all scrapie and BSE sheep, irrespective of genotype or source of infection (data not shown).
CNS
Glial cells.
Intense granular deposits of immunolabelling were found in close association with microglial or astrocytic nuclei and were interpreted as intracytoplasmic accumulations of PrPd in glial cells. They were apparent at several neuroanatomical sites in all scrapie-affected sheep, irrespective of breed or PrP genotype, though their magnitude varied depending on the infecting source. Intraglial PrPd was not observed in any of these scrapie-affected sheep with FH11 or BG4, but it was detected with all of the other antibodies tested (Fig. 4, Table 2
). Irrespective of their breed or route of infection, sheep experimentally infected with BSE showed intraglial PrPd accumulation with C-terminal antibodies and with antibody 505 (Fig. 5
), but not with any of the remaining N-terminal antibodies (i.e. FH11, BG4, P4 and 521) (Fig. 5
, Table 2
).
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DISCUSSION |
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The present results differ from our previous observations in one important respect. In our previous studies, no TBM labelling was detected with antibodies recognizing downstream sequences of the flexible tail near the N terminus of PrPd (BG4, FH11), irrespective of LRS tissue or TSE source. In addition, BSE-infected LRS tissues did not show any TBM labelling with antibodies to upstream sequences (those nearest the globular domain) of the flexible tail of the PrP molecule (P4, 521, 505). The present results are in complete agreement with those previous observations as far as the dark zone of the secondary follicles is concerned, so that fundamental differences between BSE and scrapie are maintained on the basis of the different PrP immunoreactivity of dark zone TBMs. However, morphologically distinct forms of TBM labelling were detectable in this paper in the light zone of the follicles with all N-terminal antibodies regardless of the infectious agent or source, albeit with reduced intensity when compared with C-terminal antibody labelling. We attribute this difference in PrPd labelling patterns to subtle changes in pretreatments and equipment performance between our previous and current IHC protocols and therefore suggest that the precise morphological appearance of the TBM labelling may vary according to tissue processing methods and IHC techniques, due to preservation or unmasking of particular epitopes.
The biology of normal secondary follicles suggests that TBMs phagocytose apoptotic B cells and fragments of the FDC processes with associated trapped antigenantibody complexes. Our current IHC results suggest that in scrapie- and BSE-infected secondary follicles there is a progressive fragmentation and digestion of PrPd within macrophage endosomes and lysosomes. Thus, full-length PrPd (recognized by all antibodies) released by infected FDCs in the light zone of the follicles would be internalized by TBMs. While still in the light zone, TBMs would accumulate PrPd either without any enzymatic cleavage and digestion occurring or with it involving only the most downstream sequences of the molecule, so that the remaining protein would still be detected by all antibodies used in our study. We also infer that these initial events are very similar in scrapie and experimental sheep BSE. TBMs within the dark zone show further progressive PrPd fragmentation and digestion of internalized PrPd. This progressive fragmentation would initially involve the amino acid sequences recognized by FH11 and BG4 antibodies, a common step in both scrapie and sheep BSE infections. While PrPd digestion of scrapie-infected macrophages would stop at this point, it would continue in BSE-infected sheep TBMs, so that the sequences recognized by P4, 521 and 505 antibodies would be removed.
Enzymatic digestion of intracellular PrPd would also occur in the brain, as revealed by the failure of N-terminal antibodies to label intraneuronal and intraglial PrPd accumulations. However, according to our IHC results, differences in intracellular PrPd truncation between scrapie and sheep BSE are also present. Thus, BSE-associated intraneuronal PrPd would be of shorter molecular length than scrapie-associated PrPd, as revealed by the differential reactivity to P4 antibody. Furthermore, intraglial PrPd of BSE-infected sheep would also lack the epitopes recognized by the 521 antibody, suggesting it to be not only shorter than its scrapie counterpart, but also than BSE intraneuronal PrPd. Although specific results are not shown in this paper, extracellular PrPd accumulations in the brain of sheep appear to be full-length, irrespective of the infecting agent, as they were recognized by all antibodies used.
Taking together the LRS and CNS observations derived from this study, we would like to conclude the following (Fig. 7): (i) Extracellular PrPd is full-length protein, both in the LRS (FDC-derived) and in the CNS and both in scrapie- and BSE-infected sheep. (ii) Intracellular PrPd in light zone TBMs is either full-length or is truncated at some point in the initial sequence of the flexible tail (downstream of the epitope(s) within the amino acid sequence 5460). No differences in this respect were found between BSE- and scrapie-affected sheep. (iii) All other intracellular PrPd in sheep scrapie examined so far (TBMs in the dark zone, neurones and glial cells) is truncated at some point approximately within the 6089 amino acid sequence. (iv) Conversely, intracellular PrPd in BSE-affected sheep seems to be truncated at different levels depending on the specific cell type; it would be within the 89104 amino acid sequence in neurones, within the 94105 sequence in glial cells and within the 100111 sequence in dark zone TBMs. It will always be shorter than its scrapie counterpart, a notion that is in agreement with Western blotting studies showing that the molecular mass of brain-derived PrPres from ovine BSE is lower than that from ovine scrapie (Stack et al., 2002
).
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The differences in intracellular truncation of PrPd between scrapie and ovine BSE most probably reflect differences in the conformation of the respective full-length extracellular PrPd. This argument would explain why PrPd produced by a single cell type (e.g. ovine FDCs) is truncated differently when phagocytosed by another single cell type (i.e. ovine TBMs). Moreover, according to our IHC results, the conformational differences between scrapie-derived and BSE-associated PrPd would lie in the upstream segment of the N terminus of the PrPd molecule, i.e. the sequence reacting with P4, 521 and 505 antibodies. However, following the same argument, the different immunolabelling affinities of intracellular PrPd related to the cell type involved (light and dark zone TBMs in scrapie; light zone TBMs, dark zone TBMs, glial cells and neurones in BSE) are more difficult to reconcile with differences in conformation. These cell-related differences in intracytoplasmic truncation are more likely due to variation in the extent of enzymatic cleavage and digestion of PrPd between different cell types, at least within those in the same tissues, either LRS or CNS.
In conclusion, the results of the present study agree with our previous observations suggesting that different conformers of PrPd are produced following infection of sheep with either BSE or different scrapie sources. The precise amino acid at which truncation of these conformationally different PrPd proteins occurs cannot be accurately determined from the present study. In sheep BSE in particular, PrPd molecules of different sizes were inferred from variations in peptide labelling patterns in different cell types or in cells in different evolutionary stages. These variants may be due to differences in enzymatic digestion of PrPd within the endosomallysosomal compartments of different cell types, but could also be due to the production of specific PrPd conformers in different infected cell types.
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ACKNOWLEDGEMENTS |
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Received 12 September 2002;
accepted 27 November 2002.