1 Department of Neurological Science, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Sendai 980-8575, Japan
2 CREST, JST (Japan Science and Technology), Kawaguchi, Japan
3 Sakai City Institute of Public Health, Sakai, Japan
4 Prion Research Project, Ministry of Health, Labour and Welfare, Japan
5 School of Humanities for Environmental Policy and Technology, Himeji Institute of Technology, Himeji, Japan
6 CJD Surveillance Unit, Western General Hospital, Edinburgh, UK
7 Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, Sapporo, Japan
8 Department of Neurology and Neurobiology of Ageing, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan
9 Kohnodai Hospital, National Center of Neurology and Psychiatry, Ichikawa, Japan
10 Department of Laboratory Animal Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Correspondence
Tetsuyuki Kitamoto (at address 1)
kitamoto{at}mail.cc.tohoku.ac.jp
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ABSTRACT |
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Present address: The First Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Japan.
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INTRODUCTION |
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In order to examine dCJD subtypes further, we carried out a larger-scale clinicopathological analysis and typing of protease-resistant PrP (PrPSc) from cases of dCJD. Along with differences among subtypes with respect to several clinicopathological aspects, we discovered that np-dCJD was associated with a protease-resistant C-terminal PrP fragment of 1112 kDa, while dCJD with plaque-type PrP deposits (plaque-type dCJD, p-dCJD) was not. The type (or subtype)-specific association of the PrP fragment was also observed in subjects with other prion diseases, thus arguing for a relationship with the pathogenesis. The present study provides clinical, pathological and biochemical evidence that supports the need for the subtyping of dCJD.
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METHODS |
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Histological analysis.
Formalin-fixed paraffin-embedded brain sections were subjected to histological analysis, including haematoxylin/eosin staining and PrP immunohistochemistry using the 3F4 monoclonal antibody (mAb), as previously described (Kascsak et al., 1987; Kitamoto et al., 1992
).
Prion protein analysis.
Cerebral tissues from patients and unaffected subjects (Tables 1 and 3) were homogenized in buffer (50 mM Tris/HCl, pH 8·0, 10 mM NaCl, 10 mM MgCl2) containing Complete Protease Inhibitor cocktail (Roche) and DNase I and incubated at 37 °C for 1 h. After adding Sarkosyl to a final concentration of 10 %, the samples were centrifuged at 17 000 g for 30 min to obtain the supernatants (brain lysates). An aliquot of each brain lysate was mixed at a 4 : 1 ratio with 5x Laemmli's sample buffer, incubated at 100 °C for 10 min and used for Western blots (brain lysate samples). Proteins in another aliquot of brain lysates were precipitated with methanol/chloroform and resuspended in 20 mM Tris/HCl, pH 8·0, containing 1 % Sarkosyl. The resuspended samples were then treated with proteinase K (PK, 25 µg ml-1) at 37 °C for 1 h. The protease-digested samples were mixed at a 2 : 1 ratio with 3x Laemmli's sample buffer, incubated at 100 °C and used for Western blots (PrPSc samples). Anti-PrP antibodies used for the analysis included mAbs 3F4, 6H4 (Prionics), #71 and #2065. Antibodies #71 and #2065 were raised against the recombinant human PrP peptides of residues 23230 and 122230, respectively. The core of the epitope for #71 was mapped to residues 215220 (Muramoto et al., 2000
), while #2065 required residues 171216 for its binding (T. Muramoto, T. Tanaka, N. Kitamoto & T. Kitamoto, unpublished data). Signals were developed using the enhanced chemiluminescence system (Amersham Pharmacia).
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To examine the solubility of PrP in brain lysate from sporadic CJD (sCJD), brain lysate from an sCJD case was subjected to PK digestion or left untreated at 37 °C for 1 h. After inactivating PK by adding Pefabloc (Roche), the samples were centrifuged at 453 000 g for 1 h at 4 °C. Proteins in this supernatant were precipitated with methanol/chloroform, and the pellet was sonicated in 20 mM Tris/HCl, pH 8·0, containing 10 % Sarkosyl and then recentrifuged to obtain the second pellet. Proteins from the first supernatant and in the second pellet were denatured in 1x Laemmli's sample buffer, deglycosylated and analysed by Western blot.
Informed consent.
Clinical information, peripheral blood and brain tissue from patients and unaffected subjects were obtained with informed consent for research use.
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RESULTS |
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Cerebral specimens from all 13 cases had the pathological characteristics of CJD as described below. Six cases without plaques were categorized as np-dCJD and the other seven with plaques as p-dCJD (Table 1).
Clinical findings
Clinical profiles are summarized in Tables 1 and 2. There was no significant difference between the two subtypes in the latent period (the period from surgical operation to the onset of CJD) or in the duration of CJD (Table 2
). Many np-dCJD and p-dCJD cases presented initially with ataxia as reported (Table 1
) (Hoshi et al., 2000
; Lang et al., 1998
). Mental deterioration such as disorientation or memory disturbance most often followed ataxia. To search for differences among subtypes, we then focused on findings that were well documented and typical of CJD and found that the subtypes differed significantly on three points (Table 2
). There was an absence or late occurrence of myoclonus and PSDs on electroencephalography (EEG) in p-dCJD. PSDs never appeared on the EEGs from five subjects with p-dCJD and three p-dCJD cases did not have myoclonus. The third difference was slow progression of neurological dysfunction in p-dCJD, which was represented by a relatively late transition into the state of akinetic mutism.
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Neuropathological findings
Brain weight was more preserved in p-dCJD than in np-dCJD: 877±102 g (mean±SD) in np-dCJD (n=6) and 1155±147 g in p-dCJD (n=6) (P<0·01).
Typical CJD pathology was observed in cerebral tissues from all 13 cases. Included were spongiform changes, neuronal loss and synaptic-type diffuse PrP deposits in the grey matter and astrocytic gliosis in the grey and white matter. All seven p-dCJD cases contained plaque-type PrP deposits in the cerebral grey matter (Fig. 1A, B). In addition, unique PrP deposits fringing neuronal cell bodies and processes were found in the cerebral grey matter of five (#1, 2, 3, 4, 7) p-dCJD cases (Fig. 1B
). Such perineuronal deposits were not observed in any case of np-dCJD.
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Typing of PrPSc fragments
To search for a biochemical marker that would reveal differences among dCJD subtypes, we typed PrPSc from three np-dCJD and five p-dCJD cases as well as 15 sCJD cases (Tables 1 and 3) (Collinge et al., 1996
; Parchi et al., 1996
). Western blot analysis of PrPSc samples with mAb 3F4 revealed three major PrPSc species of 2130 kDa from all np-dCJD and p-dCJD cases (Fig. 2
A). These PrPSc species had the same molecular masses as those from sCJD cases with the codon 129 Met/Met (sCJD MM1) and were judged to be of Parchi's PrPSc type 1; they converged into a band at 21 kDa after deglycosylation, as previously reported (Fig. 2A
) (Parchi et al., 1996
). In constrast, PrPSc from an sCJD case with 129 Val/Val (sCJD VV2) was of 1928 kDa; it converged into a band at 19 kDa after deglycosylation and was judged to be of Parchi's PrPSc type 2 (Fig. 2A
) (Parchi et al., 1996
). This case had plaque-type PrP deposits in the cerebrum (data not shown) (Miyazono et al., 1992
; Parchi et al., 1996
, 1999
; de Silva et al., 1994
). Three sCJD cases with 129 Val/Met were split into one case of the PrPSc type 1 (sCJD VM1) and two of the PrPSc type 2 (sCJD VM2) (Fig. 2A
). These two subgroups were also pathologically distinct, as previously reported: sCJD VM1 was without PrP plaques, while sCJD VM2 had PrP plaques in the cerebrum (data not shown) (Miyazono et al., 1992
; Parchi et al., 1996
, 1999
; de Silva et al., 1994
).
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Deglycosylation did not alter the size of fPrP1112 but did intensify the signal (Fig. 3). It was apparent that the fPrP signal on the blot was derived from the non-glycosylated species and that the glycosylated species of fPrP1112 also existed and contributed to the fPrP signal but only after deglycosylation. These notions were supported by data on the location of fPrP1112 in the entire amino acid sequence of PrP (Fig. 4
). Since fPrP1112 was detected by mAb #71 but not by mAbs 3F4 or 6H4, it seemed likely that it was the C-terminal fragment that has been processed resulting in the loss of the epitopes for mAbs 3F4 (residues 109112) (Kascsak et al., 1987
) and 6H4 (residues 144152). In accordance with this, fPrP1112 was recognized by #2065, another mAb against the C-terminus of PrP. Judging from the location of the epitope for these mAbs, fPrP must encompass residues 171220. Thus, fPrP1112 probably retains the two intrinsic N-glycosylation sites (residues 181 and 197).
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To determine whether other types of prion disease are associated with fPrP1112, we analysed PrPSc samples from various types of prion disease (Table 3). The PrPSc type determined by mAb 3F4 was type 1 in CJD with the E200K mutation (CJD E200K) and type 2 in the thalamic form of sCJD (sCJD-T) (Martin, 1975
) and variant CJD (vCJD), in agreement with previous reports (Fig. 3
; data not shown) (Parchi et al., 1997
, 1999
, 2000
). In addition, we discovered that PrPSc from CJD with the M232R mutation (CJD M232R) was of type 1 (Figs 2A and 3
). fPrP1112 was detected by mAb #71 in CJD E200K, CJD M232R and sCJD-T but not in vCJD (Fig. 3
; data not shown). As was the case in dCJD and sCJD, the presence (or absence) of fPrP1112 in the PrPSc sample was consistent among cases affected by the same type of disease (Table 3
).
To examine whether fPrP1112 was a product of protease digestion in vitro, we analysed brain lysates from prion disease patients [six cases of sCJD MM1, three of p-dCJD, two of sCJD-T and sCJD VM2, and one each of np-dCJD, sCJD VV2, CJD E200K, CJD M232R (M/M) and vCJD] and unaffected subjects. Intriguingly, the PrP fragment of 1112 kDa was detected by mAb #71 in brain lysates from the patients (n=11) associated with fPrP1112, but not from the patients (n=7) or control subjects (n=8) unassociated with fPrP1112 (Fig. 5A; Table 3
). The PrP fragment in the brain lysates (fPrP-in-lysate) was indistinguishable from fPrP1112 with respect to the immunoreactivity to anti-PrP antibodies (Fig. 5A
), molecular mass (Fig. 5B
) and solubility: both fPrP1112 and fPrP-in-lysate were totally insoluble in 10 % Sarkosyl.
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DISCUSSION |
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Because np-dCJD and p-dCJD cases showed no differences in the ORF of PrP genes, the most plausible explanation for the origin of subtypes may be differences in the properties of prions in the contaminating dura grafts. Additional information, e.g. the type of the contaminating PrPSc or the PrP genotype of the donor, will be required to answer this question.
In searching for biochemical differences among dCJD subtypes, we found no difference in PrPSc typing by mAb 3F4: both subtypes had the PrPSc type 1 (Parchi et al., 1996). This is consistent with reports of Collinge's PrPSc type 2 in dCJD cases (Collinge et al., 1996
; Parchi et al., 1997
). However, our studies with anti-PrP C-terminal antibodies revealed a protease-resistant C-terminal fragment, designated fPrP1112, that can differentiate between the subtypes: its presence is associated with np-dCJD but not with p-dCJD. fPrP1112 is distinct from the 3F4-recognizable PrP fragments of 7 or 11 kDa that are major components of amyloid from particular types of GerstmannSträusslerScheinker disease (GSS) (Tagliavini et al., 1991
, 2001
) and is also distinct from the 8 kDa PrP fragment with ragged N and C termini that was detected in proteinase K-treated brain lysate fractions from GSS with the P102L mutation (GSS P102L) (Parchi et al., 1998
). fPrP1112 resembles, and may be the same as, the 13 or 12 kDa C-terminal PrP fragments that were detected in proteinase K-treated brain lysate fractions from a group of GSS P102L cases with a distinctive phenotype (Parchi et al., 1998
) and CJD E200K cases (Capellari et al., 2000
).
It is important to determine whether fPrP1112 is a product of protease digestion in vitro or is generated by CJD-associated processes in vivo. Our data argue for the latter possibility. First, the fPrP-in-lysate that was indistinguishable from fPrP1112 was detected in all cases associated with fPrP1112. Secondly, neither fPrP1112 nor fPrP-in-lysate was detected in unaffected controls. Thirdly, both fPrP1112 and fPrP-in-lysate were detected in a disease type-specific manner. There may be at least three possibilities for the origin of fPrP1112: it may be derived from processing of full-length PrPSc or the abnormal isoform of shorter PrP species, like the C1 fragment (Chen et al., 1995), or it might be the abnormal isoform of an 1112 kDa physiological counterpart that is detectable only when it is converted and accumulates in the brain. It is a possibility that some fPrP1112 molecules were produced by proteolytic cleavage of longer PrPSc species during post-mortem time or during sample preparation. However, the observed disease-type specificity of fPrP1112 makes such non-specific processes unlikely as the main or exclusive origin of fPrP1112.
The disease-type-specific association of fPrP1112 suggests a causal relationship with pathological processes that are intrinsic to the disease type, e.g. the structure of PrPSc. In the present study, the coincidence of the formation of PrP plaques and the absence of fPrP1112 was observed in several types (p-dCJD, sCJD VM2, sCJD VV2 and vCJD) of diseases with the PrPSc type 2 (Table 2). This raises the possibility that the two phenomena are linked. However, this notion is challenged by the fact that the fPrP1112-like fragment has been associated with a subtype of GSS P102L (Parchi et al., 1998
). It is possible that the origin of PrP plaques and their relationship with pathogenesis could be heterogeneous between various prion diseases.
The relationship between fPrP1112 and infectivity/prions remains to be determined. There are no experimental data to show that mutant PrPs with such a large N-terminal truncation as fPrP1112 can propagate prions by themselves (Fischer et al., 1996; Shmerling et al., 1998
; Supattapone et al., 2001
). However, preliminary results from our transmission studies that are currently under way suggest a link between transmissibility and fPrP1112. Brain homogenates from four CJD cases (sCJD MM1, sCJD VM1, np-dCJD and CJD M232R) with type 1 PrPSc and fPrP1112 were inoculated into transgenic mice expressing mouse/human chimeric PrP (Kitamoto et al., 2002
; T. Kitamoto, S. Mohri & I. Miyoshi, unpublished data). The diseases were transmitted with the incubation period ranging from 140 to 180 days. In contrast, transmissions from three p-dCJD cases with type 1 PrPSc but without fPrP1112 were unsuccessful for over 600 days after inoculation. These data imply a relationship of fPrP with the infectious properties of prions.
The relationships of fPrP with the phenotype of human prion diseases are distinct from those of other sources of phenotypic variations such as PrPSc types or PrP genotypes and support the view that the phenotypic heterogeneity of the diseases is related, at least in part, to the formation of different types of disease-specific PrP species and/or fragments thereof. The present data may contribute to the understanding of the processing of disease-specific PrP species, a novel aspect in the pathogenesis of human prion diseases.
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ACKNOWLEDGEMENTS |
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Received 17 March 2003;
accepted 16 June 2003.