Ultrastructural Immuno-localization of Synthetic Prion Protein Peptide Antibodies in 87V Murine Scrapie

NEURODEGENERATION, Vol. 5, pp 101–109 (1996)

Ultrastructural Immuno-localization of Synthetic Prion Protein Peptide Antibodies in 87V Murine Scrapie M. Jeffrey,1 C.M. Goodsir,1 N. Fowler,1 J. Hope,2 M.E. Bruce2 and P.A. McBride2 1

Lasswade Veterinary Laboratory, Bush Estate, Penicuik, Midlothian EH26 0SA; 2Institute for Animal Health, BBSCRC and MRC Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, Midlothian EH9 3JF

Disease specific forms of a host encoded cell surface sialoglycoprotein called prion protein (PrP) accumulate during the incubation period of the transmissible spongiform encephalopathies. A 33–35 kDa disease specific form of PrP is partially resistant to protease digestion whereas the normal form of PrP can be completely digested. Proteinase K digestion of the murine disease specific form of PrP produces diverse forms of low molecular weight PrP, some of which are N-terminally truncated at amino acid residue 49 or 57 within the octapeptide repeat segment. Amyloid plaques are a pathological feature of many of the transmissible spongiform encephalopathies and are composed of PrP. Using synthetic peptide antibodies to the N-terminus of PrP (which is not present in truncated disease specific PrP) and antibodies to the protease resistant fraction of PrP we have immunostained plaques and pre-amyloid deposits in the brains of mice, experimentally infected with the 87V strain of scrapie, for examination by light and electron microscopy. Classical fibrillar amyloid deposits in plaques as well as pre-amyloid deposits were both immunostained by antibodies to the N-terminus of PrP and to the protease resistant core of the PrP molecule. This suggests that both N-terminal and core amino acid residues are present in disease specific PrP released from scrapie infected cells in vivo. The results also suggest that N-terminal truncation of PrP may not be essential for formation of amyloid fibrils.  1996 Academic Press Limited

Key words: ultrastructure, immunogold, prion protein, scrapie, amyloid

which avoid proteolysis, migrates with a Mr of between 33–35 kDa (Hope et al., 1986). A fraction of the abnormal murine form of PrP is truncated in vivo at amino acid residue 49 or 57 within the octapeptide repeat region and the truncated proteins have Mr between 23 and 25 kDa or 26 and 29 kDa respectively (Hope et al., 1988). The size of the molecular weight variants of disease specific PrP and the site of PrPSc truncation differ slightly depending on the host species infected and sequence of the PrP protein (Oesch et al., 1985). Previous studies in kuru and GSS using synthetic antibodies to amino acid residues 23–37 of the human PrP molecule (and which are not present in the truncated PrPSc protein) have shown PrP immunoreactivity only in plaque periphery whereas immunoreaction using antibodies to residues 90–104 was detected throughout the plaque (Hashimoto et al., 1992, Kitamoto et al., 1991). A similar result was obtained for

SCRAPIE is a naturally occurring neurological disease of sheep. It is archetypical of a group of progressive transmissible spongiform encephalopathies (TSE) which in humans include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS) and kuru. TSE are characterized by the accumulation of an abnormal form of a cell surface sialoglycoprotein called prion protein (PrP), this being essential for infection (DeArmond, 1993). The abnormal disease specific form of PrP is partially protease resistant (PrPSc) whereas normal cell surface PrP (PrPC) can be completely degraded by proteases. PrP extracted from both infected and normal brains, using techniques

Correspondence to: M. Jeffrey Received 7 December 1995; revised and accepted for publication 22 January 1996 1055-8330/96/010101 1 9 $18.00/0

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a different GSS family when using antibodies to different N-terminal and core peptides of the protease resistant PrP molecule (Giaccone et al., 1992). Together these results suggest that exogenous proteases react with pre-amyloid forms of PrP in the periphery of plaques converting them into amyloid fibrils composed of the protease resistant core of PrP. We have previously studied the sub-cellular localization of PrP in 87V and ME7 murine scrapie and have shown, by immunogold electron microscopy, that disease specific accumulations of PrP follow the release of PrP from neurons (or parts of their processes) which then diffuses through the neuropil prior to aggregation into fibrils ( Jeffrey et al., 1994a, b, c). In 87V scrapie many of the plaques identified at light microscopy have few or no observable amyloid fibrils when investigated by electron microscopy (Jeffrey et al., 1994b). By using immunogold electron microscopy with synthetic PrP antibodies we determined the composition of amyloid and pre-amyloid PrP deposits in 87V murine scrapie with respect to N-terminal and core amino acid residues.

Materials and Methods Experimental design Appropriate brain regions from VM/Dk-sinc S7 mice terminally ill following infection with the 87V strain of scrapie and demonstrating plaque, diffuse granular or perineuronal types of disease specific PrP accumulation, were immunostained and examined at light and electron microscopy. The antibodies used were 1A8, a high titre polyclonal antiserum raised to murine scrapie associated fibrils (Farquhar et al., 1993), which we have used in previous immunogold studies (Jeffrey et al., 1994 a–c), and a panel of peptide specific antisera. The 1A8 antibody recognizes large areas of the PrP molecule, particularly amino acid residues 89–114, 114–131, 140–182, 187–234 (Langeveld et al., 1993). Two peptide specific antibodies recognize the N-terminal sequence of amino-acids which is removed by the action of proteases on disease specific PrP. P2 recognizes hamster PrP residues 15–40 (Barry et al.,1988); R24 is a polyclonal rabbit antiserum recognizing residues 22–37. Three other polyclonal rabbit antisera which recognize the same amino acids of the protease resistant core of disease specific PrP were also used. These sera, R27, R30 and R34, are polyclonal rabbit antisera raised to amino acids 89–103. This region is structurally important because it contains the last three amino-acids of the octa-repeat region and forms the new N-terminus after partial degradation of PrP by proteinase K. For details of production of R24, R27, R30, R34 see Caughey et al. (1991). As none of the above antibodies can distinguish between scrapie specific PrP (as determined by partial protease resistance) and normal cell surface PrP, it is inappropriate to use the designation PrPSc for the immunolocalized PrP found in scrapie brains studied here. Accordingly, we use the term

PrPD to describe immunolocalized PrP which is of unknown protease sensitivity but is present in an abnormal morphological form or location and is thus disease specific.

Light microscopical immunohistochemistry Brains of four VM mice, terminally ill with 87V scrapie, and two controls were fixed by immersion in 10% buffered formalin for 24 h. Five micron-thick wax sections of cerebral cortex, hippocampus, thalamus or striatum were immunostained by the peroxidase anti-peroxidase or avidin-biotin methods using the 1A8 antibody or each of the peptide specific antibodies. In addition, four scrapie infected and two control VM mice were perfused with 3% paraformaldehyde and 1% glutaraldehyde. One mm cubes of brain tissue were taken from four sites, the frontal cortex, hippocampus, subthalamus and thalamus. Tissue blocks were postfixed in 1% osmium tetroxide, dehydrated and embedded in araldite. Thick sections were cut and stained by toluidine blue to find classical plaques. In addition, 1 µm sections were cut and then etched (deplasticized) with saturated sodium ethoxide for up to 45 min. Endogenous peroxidase was blocked and sections de-osmicated with 3% hydrogen peroxide in methanol for 10 min and then pretreated with 98% formic acid for 30 min. The peroxidase anti-peroxidase immunohistochemical staining method using 1A8 anti-PrP serum, incubated overnight at 4°C at a dilution of 1:400, was then applied to the etched and pre-treated sections. Using these methods as standard, tissue blocks containing appropriate patterns of disease specific PrP accumulation were identified and tested with the panel of peptide specific antibodies. Immunostaining for PrP was not achieved using P2 or R24 antisera using the methods described above. Nor was satisfactory staining achieved using tissues perfused with periodic acid/lysine/paraformaldehyde mixtures or formalin and embedded in araldite. Good immunolabelling was, however, achieved for both P2 and R24 on routine formalin fixed tissues embedded in paraffin wax. In addition, preembedding techniques produced good immunolabelling with both P2 and R24 (vide infra). The primary antibodies were used as above at the following dilutions: R24 at 1:50, R27 at 1:100, R30 at 1:200, R34 at 1:200, P2 at 1:100

Ultrastructural immunohistochemistry Serial 1 µm and 80 nm sections were taken from blocks previously identified as containing, on immunostained sections, plaque or peri-neuronal staining patterns. Eighty nm sections placed on 400 mesh nickel grids treated with G-Pen (Kiyota International, Elk Grove Village, IL) were etched in sodium periodate for 60 min. Endogenous peroxidase was blocked and sections de-osmicated with 3% hydrogen peroxide in methanol for 10 min. Antigen expression was enhanced with formic acid treatment (98%) for 10 min. Residual aldehyde groups were quenched with 0.2 M glycine in PBS, pH 7.4 for 3 min. Primary antibody was then applied (at a dilution of 1:400, 1A8; 1:200, R30 and R34; 1:100, R27 and P2 or 1:50, R24) in incubation buffer at room temperature for 1 h. After rinsing, sections were incubated with Extravidin 10 nm colloidal gold diluted 1:10 in incubation

Ultrastructural immuno-localization in 87V murine scrapie buffer for 1 h. Sections were also stained with 1 nm gold and silver enhanced by the IGSS method. Grids were post-fixed with 2.5% glutaraldehyde in PBS and counterstained with uranyl acetate and lead citrate. The epitopes of antibodies P2 and R24 were not preserved in araldite embedded tissues. However, immunostaining with P2 and R24 was found in tissues that had been embedded in paraffin wax following fixation in 10% formalin, or mixed aldehydes or periodic acid/lysine/paraformaldehyde. To determine the subcellular localization of P2 and R24 antibodies two separate methods were employed. Five µm thick sections of paraffin wax embedded tissues were immunostained as above. Appropriate areas of sections were then cut out on the glass slide and infiltrated with araldite and placed on a stub. Sections were then cut from the section at 1 µm, mesa’s selected and then cut at 70 nm, counterstained in uranyl acetate and lead citrate and mounted on a 400 mesh nickel grid. In addition, brains from affected mice and controls were fixed in 1% glutaraldehyde and 3% paraformaldehyde mixture, cut at 50 µm and immunostained by pre-embedding protocols as follows. Brain tissue slices obtained from control mice and mice terminally ill with 87V scrapie were fixed in 3% paraformaldehyde/1% glutaraldehyde in Karlsson and Schultz buffer. Fifty µm thick vibratome sections were cut through the whole brain. These tissue slices were processed for routine pre-embedding methods (Totterdell et al., 1992). In addition sections were pre-incubated with 98% formic acid for 10 min and endogenous peroxidase was blocked with a solution containing 10% methanol and 3% hydrogen peroxide in phosphate buffer for 5 min. The primary antibody (R24, P2 or 1A8) was incubated overnight at 4°C and the 1 nm immunogold gold particles were used at a dilution of 1:50 for 3 h at room temperature. The sections were post-fixed with 2.5% glutaraldehyde. The silver amplification step was observed with a dissecting microscope and the reaction was stopped when plaques and diffuse areas of immunostaining became visible. Sections were gold toned with 0.05% gold chloride in distilled water. Selected areas of immunostaining were then cut out on the glass slide and infiltrated with araldite and placed on a stub. Serial sections were cut through the entire block, counterstained and examined by electron microscopy.

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Figure 1. Amyloid plaque in the pyramidal neuron layer of the hippocampus showing marked immuno-staining. Peroxidase antiperoxidase (PAP) staining P2 antibody 31120.

Results The distribution of disease specific PrP in the 87V model is well described (Bruce et al., 1989; Jeffrey et al., 1994a, b, c). In this rodent model of scrapie numerous plaques are found (mainly in the cerebral cortex and hippocampus), as well as diffuse granular accumulations and a distinctive form of perineuronal PrP accumulation. At light microscopy all five peptide antisera, including both N-terminal antibodies, stained many plaques in the cerebral cortex and hippocampus (Fig. 1) with fewer numbers being found in the thalamus. On ultrastructural examination, all antisera, including the N-terminal antisera, immunostained the amyloid fibrils of classical plaques (Figs 2–4). Primitive plaques

Figure 2. Classical amyloid plaques showing intense immunolabelling of bundles of amyloid fibrils. Post-embedding immunogold-silver method 1A8 polyclonal antibody; scale bar 5 3500 nm.

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Figure 3. Periphery of classical amyloid plaque immuno-stained with the R30 peptide antibody to core PrP amino acids. There is intense staining of bundles of amyloid fibrils (large arrows). In addition, at the extreme periphery of the plaque, there is immunolabelling, within the extracellular space, not associated with fibrillar material (arrowheads). Post embedding immunogold-silver method with R30(recognizing residues 89–103); scale bar 5 2000 nm.

Figure 4. Part of a classical amyloid plaque, immunostained with P2 antibody to N-terminal PrP residues. The antibody strongly recognizes bundles of amyloid fibrils. Pre-embedding 1 nm immunogold-silver with P2 (recognizing residues 15–40); scale bar 5 1500 nm.

with sparse fibrils and extracellular pre-amyloid forms of PrP located between cell processes were also stained by all peptide antisera, including those directed against N-terminal amino-acids (not shown). At light microscopy, a small number of plaques, particularly those adjacent to the internal capsule, showed in some runs an immunostaining of the periphery while the core remained unstained (Fig. 5). This pattern of immunostaining was found for R27, R30 and R34 antibodies, as well as for R24 and P2 antibodies but was never observed with 1A8. However, this immunostaining pattern was so rare that we were unable to examine it by electron microscopy. Immunolabelling of the lateral hypothalamus with the 1A8 antiserum showed a perineuronal pattern of disease specific PrP accumulation. When viewed in immunolabelled sections prepared for electron microscopy, the PrP immunostaining was associated with preamyloid forms of PrP located in the extracellular space and around neurites and glial cell processes surrounding individual neurons. Although the sub-

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Figure 5. Plaques adjacent to the internal capsule showing a peripheral arrangement of immunolabelling using N-terminal PrP peptide antiserum. The plaque cores are R24 antibody, PAP staining, 31000.

Figure 6. Lateral hypothalamus showing the presence of N-terminal PrP residues within distinctive (pre-amyloid) perineuronal disease specific PrP accumulations (arrows) R24 antibody PAP staining, 31000.

Discussion cellular detail of sections lifted from wax embedded material was, as anticipated, quite poor, we were nevertheless able to see that these PrP accumulations were not present as amyloid fibrils. All five peptide specific antisera gave the same result as the 1A8 antiserum. N-terminal antisera and core antisera recognized equally well perineuronal patterns of PrP accumulation in sections stained for light microscopic examination (Fig. 6). In the thalamus and in the CA2 region of the hippocampus, widespread areas of intense, granular immunostaining were seen at light microscopy (Fig. 7A). In these areas there was marked loss of neurones with an accumulation of amorphous electron dense material and structures in an expanded extracellular space. Immunostaining with the 1A8 antibody showed that these filaments were composed of PrP (Fig. 7B). These individual and loosely grouped amyloid filaments contain N-terminal residues of PrP as shown by immunostaining with P2 and R24 antisera (Fig. 8A, B).

Amyloid plaques are one of the hallmarks of the TSE and contain fibrils composed of PrP. In addition, accumulations of PrP are found in a variety of other morphological forms in each of the TSE. Alzheimer’s disease (AD) is also characterized by the formation of amyloid plaques composed of a host encoded protein, amyloid β peptide (Aβ), generated by the proteolytic processing of β amyloid precursor protein (βAPP) (Kang et al., 1987). Three major isoforms of βAPP are formed by alternative exon splicing two of which are found throughout the body with a further isoform, βAPP695, being expressed mainly in the CNS. Two βAPP processing pathways are recognized: secretory cleavage at or near the cell surface and re-internalization and lysosomal targeting ( Golde et al., 1992; Haass et al., 1992). The amyloid fibrils of AD are mainly composed of a truncated 42-amino-acid peptide (Aβ) derived from βAPP which can assemble spontaneously. Thus, in AD the role for proteolysis in the aetiology of amyloid plaques is well established but a role

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(A)

(B)

Figure 7. (A) Thalamus with marked, localized, granular PrP immunolabelling at light microscopy. Post embedding PAP with 1A8 antibody, 31400. (B) Fibrillar structures (arrowheads) and amorphous deposits within the expanded extracellular space are immunoreactive for PrP. Pre-embedding 1 nm immunogold-silver method with 1A8 antibody; scale bar 5 1000 nm.

Ultrastructural immuno-localization in 87V murine scrapie

(A)

(B)

Figure 8. (A) Ultrastructural appearance of the thalamus where there was marked localized granular PrP immuno-labelling at light microscopy as in Fig. 7A. There is immunolabelling of structures within the extracellular space but cell bodies and processes including myelinated axons are unstained. (B) Individual fibrils, small bundles of fibrils (arrows) and amorphous electron dense material (arrowheads) show immunostaining for N-terminal PrP residues. Pre-embedding 1 nm immunogold-silver method with P2 antiserum; scale bar 5 1000 nm.

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108 of PrP truncation in plaque formation in the spongiform encephalopathies is not so clear. The present observations show that peptides with N-terminal amino acids containing sequences 15–40, as well as the proteinase resistant core amino acids containing sequences 89–103, of disease specific PrP are present in the extracellular space around scrapie infected cells and are also in mature amyloid fibrils of 87V murine scrapie. These findings suggest that proteolytic processing of PrP is not essential for the formation of amyloid fibrils. Previous biochemical studies of the molecular properties of murine PrP have shown that both full length and truncated PrP can be isolated from scrapie infected brains in vitro (Hope et al., 1986). Our present studies show that this may also be true for PrPD deposits visualized in vivo since both pre-amyloid and fibrillar deposits of PrPD contain (at least a proportion of) PrP molecules possessing N-terminal as well as core amino-acid sequences. We suggest that the disease specific PrP when released from scrapie infected cells contains predominantly N-terminal amino acid residues and that PrP probably aggregates initially as a full length protein. Our observations suggest that, at least in this disease model, truncation of the first 49 or 57 amino-acids of the N-terminal sequence of murine PrP is not essential for the formation of amyloid fibrils in vivo. This does not however preclude the requirement for a prior transformation of normal PrP into a modified scrapie specific conformational state as indicated by the prion hypothesis (Prusiner, 1992; DeArmond & Prusiner, 1993). Our findings showing the presence of N-terminal amino acid sequences within plaque amyloid fibrils of mice are in contrast to those of three previous studies of amyloid deposits in human TSE. The distribution of PrP N-terminal amino acids was studied in amyloid plaques of patients with GSS and CJD where distinctive ring patterns of peripheral immunostaining were found. In the amyloid plaques of the Indiana kindred of patients with GSS (which have a mutation at codon 198 of the PrP gene (Hsiao et al., 1992)) the same P2 N-terminal antibody, as in the present study, and a C-terminal peptide antibody (raised against residues 220–232 of the hamster derived amino acid sequence of PrP) were used to immunostain classical and multicentric plaques. Using the N-terminal antibody P2 only the periphery of the plaques was stained; the plaque core was unstained. Ultrastructurally the N terminal antibody P2 recognized amorphous non-fibrillar material at the periphery of the plaque (Giaccone

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et al., 1992) whereas antibodies raised to residues 90–102 recognized amyloid filaments in the plaque core. Patterns of immunostaining found when antibodies to the N-terminus, or the proteinase resistant core, of PrP were used to immunolabel plaques in the brains of Japanese patients with GSS (Kitamoto et al., 1991) and in kuru (Hashimoto et al., 1992) were similar to those described by Giaccone et al. (1992) for the GSS Indiana kindred. The plaque core stained with antibodies to PrPres (the proteinase resistant core of the PrPSc protein) while the periphery stained with N terminal PrP antibodies. However, Kitamoto et al. (1991) also noted that some small congophilic kurutype plaque cores stained using N terminal PrP antibodies. In this present study of murine scrapie only a very small number of plaques showed (inconsistently) a ‘ring-type’of staining of PrP with both N-terminal antibodies and the peptide antibodies to the protease resistant core of PrP showing this pattern. Why should the amyloid filaments in the cores of most PrP plaques in scrapie infected mice react with N-terminal PrP antibodies in contrast to findings previously reported for humans with TSE (Kitamoto et al., 1991; Hashimoto et al., 1992; Giaccone et al., 1992)? Differences in the cellular processing of PrP produced by patients with PrP gene mutations (as in GSS where the PrP peptides expressed in the Indiana kindred are those encoded by the mutant allele (Tagliavini et al., 1994)), might account for the differing immunohistochemical staining patterns in GSS when compared with murine scrapie. However, neither patients with kuru nor the mice used in our present studies have any known abnormality of the PrP gene and it should therefore be anticipated that the formation of amyloid in both would share a similar pattern of aggregation of PrP. However, the period of clinical disease in both GSS and kuru is more protracted than that which occurs in scrapie infected mice and PrP might therefore accumulate in the brains of people with GSS and kuru over a considerably longer period. We therefore suggest that PrP is initially released into the extracellular space as a protein containing both N-terminal and core amino acid sequences and that truncation of PrP is not a pre-requisite for its incorporation into fibrils or other aggregates. However, once aggregated into fibrils the amyloid may be subject to the action of exogenous proteases which remove N-terminal residues of PrP leaving behind the protease resistant core. Previous biochemical studies have suggested that only small amounts (about 5%) of PrP extracted from murine scrapie brains is present as truncated pro-

Ultrastructural immuno-localization in 87V murine scrapie

tein (Hope et al., 1986). It will be interesting to examine other disease models which have longer or shorter periods of incubation or clinical disease as to whether there is evidence for increased truncation of PrP in plaques of a greater chronological age than that found in the 87V plaques investigated in this study. At present, we have no clear explanation for the rare instances where an absence of staining was found at the core of amyloid plaques when these were immunostained with antibodies to PrP 89–103, but the inconsistency with which this pattern of staining occurred suggests that technical reasons should not be ruled out.

Acknowledgements We are grateful to Dr C. Ingham and to Sue Hood for help in developing the pre-embedding immunostaining methods employed in this study. We are gratefull to Dr S.B. Pruisiner for the gift of the P2 antibody and to Byron Caughey for the gifts of the R24, R27, R30 and R34 antibodies.

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109 native processing into amyloid-bearing fragments. Nature 357:500–503 Hashimoto K, Mannen T, Nukina N (1992) Immunohistochemical study of kuru plaques using antibodies against synthetic prion protein peptides. Acta Neuropathol 83:613–617 Hope J, Multhaup G, Reekie LJD, Kimberlin RH, Beyreuther K (1988) Molecular pathology of scrapie associated fibril protein (PrP) in mouse brain affected by the ME7 strain of scrapie. Eur J Biochem 172:271–277 Hope J, Morton LJD, Farquhar CF, Multhaup G, Beyreuther K, Kimberlin RH (1986) The major protein of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP). EMBO 5:2591–2597 Hsiao K, Dlouhy SR, Farlow MR, Ghetti B, Cass C, Da Costa M, Coneally PM, Hodes ME, Prusiner S B (1992) Mutant prion proteins in Gerstmann-Straussler-Scheinker disease with neurofibrillary tangles. Nature Genetics 1:68–71 Jeffrey M, Goodsir CM, Bruce ME, McBride PA, Scott JR, Halliday WG (1994a) Correlative light and electron microscopy studies of PrP localisation in 87V scrapie. Brain Res 656:329–343 Jeffrey M, Goodsir CM, Bruce ME, McBride PA, Farquhar C (1994b) Morphogenesis of amyloid plaques in 87V murine scrapie. Neuropath and Appl Neurobiol 20:535–542 Jeffrey M, Goodsir CM, Bruce ME, McBride PA, Fowler N, Scott JR (1994c) Murine scrapie-infected neurons in vivo release excess PrP into the extracellular space. Neurosci Lett 174:39–42 Kang J., Lemaire H-G, Unterbeck A, Salbaum JM, Masters CL, Grzeschik K-H, Multhaup G, Beyreuther K, Muller-Hill B (1987) The precursor of Alzheimer’s disease amyloid A4 protein re-sembles a cell surface receptor. Nature Gen 325:733–736 Kitamoto T, Muramoto T, Hilbich C, Beyreuther K, Tateishi J (1991) N-terminal sequence of prion protein is also integrated into kuru plaques in patients with Gerstmann- Straussler-syndrome. Brain Res 545:319–321 Langeveld JPM, Farquhar CF, Pocchiari M, Birkett C, Bostock C, Meloen RH (1993) Antigenic sites of bovine prion protein. In Bradley R, Marchant B (eds) Transmissible Spongiform Encephalopathies. Proceedings of a Consultation on BSE. Commission of the European Communities, Brussels, 14–15 September 1993 Oesch B, Westaway D, Walchi M, McKinley MP, Kent SBH, Aebersold R, Barry RA, Teplow DB, Tempst DB, Hood LE, Prusiner S B, Weissmann C (1985) A cellular gene encodes scrapie PrP 27–30 protein. Cell 40:735–746 Prusiner SB (1992) Prion Biology. In Prusiner SB, Collinge J, Powell J, Anderton B (eds). Prion Diseases of Humans and Animals. Ellis Horwood, New York, pp 533–567 Tagliavini F, Prelli F, Porro M, Rossi G, Giaccone G, Farlow MR, Dlouhy SR, Ghetti B, Bugiani O, Frangione B (1994) Amyloid fibrils in Gerstmann-Straussler-Scheinker disease (Indiana and Swedish kindreds) express only PrP peptides encoded by the mutant allele. Cell 79:695–703 Totterdell S, Ingham CA, Bolam JP (1992) Immunocytochemistry I: pre-embedding staining in Experimental Neuroanatomy: A practical approach. Oxford University Press; Oxford pp 103–127