Type 1 and type 2 human PrPSc have different aggregation sizes in methionine homozygotes with sporadic, iatrogenic and variant Creutzfeldt–Jakob disease

Atsushi Kobayashi1, Sakae Satoh2, James W. Ironside3, Shirou Mohri4 and Tetsuyuki Kitamoto1

1 Division of CJD Science and Technology, Department of Prion Research, Center for Translational and Advanced Animal Research on Human Diseases, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
2 Planova Division, Asahi Kasei Pharma Corporation, Tokyo, Japan
3 National Creutzfeldt–Jakob Disease Surveillance Unit, Division of Pathology, University of Edinburgh, Western General Hospital, Edinburgh, UK
4 Laboratory of Biomedicine, Center of Biomedical Research, Graduate School of Medical Sciences, Kyusyu University, Fukuoka, Japan

Correspondence
Tetsuyuki Kitamoto
kitamoto{at}mail.tains.tohoku.ac.jp


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In Creutzfeldt–Jakob disease (CJD), the type (type 1 or 2) of abnormal isoform of the prion protein (PrPSc) in the brain and the genotype at codon 129 of the PrP gene are major determinants of clinicopathological phenotype. Little is known about the difference in biochemical properties between the two types of PrPSc, except for the different proteinase K cleavage sites. To investigate the size of aggregates formed by PrPSc types 1 and 2, brain homogenates from various cases of CJD with the same genotype (homozygous for methionine at codon 129) were passed through filters with a mean pore size of 72±4 nm. Type 2 PrPSc was efficiently removed from the filtrates by the filters, in contrast to type 1. Even type 2 PrPSc from a patient without amyloid plaques was removed more efficiently than type 1 from patients with amyloid plaques. These results indicate that type 2 PrPSc has a larger aggregation size than type 1, irrespective of the existence of amyloid plaques.


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Creutzfeldt–Jakob disease (CJD), scrapie and bovine spongiform encephalopathy are lethal transmissible neurodegenerative diseases, caused by an abnormal isoform (PrPSc) of prion protein (PrP), which is converted from the normal cellular isoform, PrPC (Prusiner et al., 1998). PrPSc forms large amyloid aggregates named prion rods or scrapie-associated fibrils (10–20 nm in diameter and 100–200 nm in length), consisting of as many as 1000 PrP molecules, in the brains of scrapie-infected animals and CJD patients (Prusiner et al., 1983; Bockman et al., 1985). It has been proposed that CJD can be classified by the type of PrPSc (named types 1 and 2) and the genotype at codon 129 of the PrP gene, the site of a methionine/valine polymorphism (Parchi et al., 1996, 1997, 1999). On the basis of experimental results in our laboratory, we employ the Parchi et al. (1996) classification of PrPSc subtypes rather than the classification by Collinge et al. (1996). Type 1 and type 2 PrPSc are distinguished by the size of their proteinase K (PK)-resistant core (21 and 19 kDa, respectively), reflecting differences in the PK cleavage site (at residues 82 and 97, respectively) (Parchi et al., 1996, 2000). Moreover, type 2 PrPSc can be subclassified into type 2A and type 2B by the difference in ratio of glycoforms (Parchi et al., 1997); type 2B PrPSc is characteristic of variant CJD (vCJD). Because the amino acid sequences of the two types of PrP are identical, Parchi et al. (1996, 2000) speculated that different PK cleavage sites resulted from the different conformations of PrPSc. On the basis of these findings, we hypothesized that: (i) type 1 and type 2 PrPSc might have distinct aggregation sizes; and (ii) PrPSc from a patient with PrP amyloid plaques in the brain might have a larger aggregation size than PrPSc from a patient with synaptic non-amyloid-type PrP deposits in the brain. To clarify the difference in aggregation size, we filtered brain homogenates from various cases of CJD with the same genotype (homozygous for methionine at codon 129) through virus-removal filters with a mean pore size of 72±4 nm.

Brain tissue was obtained at autopsy from CJD patients with informed consent for research use. The codon 129 genotype and the absence of mutations in the coding region of the PrP gene were determined by sequence analysis (Kitamoto et al., 1993). The diagnoses of CJD and the type of PrPSc were confirmed by neuropathological examination, PrPSc immunohistochemistry and immunoblot analysis as described previously (Kitamoto et al., 1992; Taguchi et al., 2003). All subjects examined in this study were homozygous for methionine at codon 129 of the PrP gene and were classified as follows: sporadic CJD (sCJD) with type 1 PrPSc (MM 1; two cases), or with both type 1 and type 2 PrPSc coexisting (MM 1+2; three cases); the thalamic form of sCJD with type 2A PrPSc (MM 2A; one case); dura mater graft-associated iatrogenic CJD with plaque-type deposits (p-dCJD) with type 1 PrPSc (MM 1 plaque; two cases); and vCJD with type 2B PrPSc (MM 2B; three cases).

Brain tissues were homogenized in lysis buffer [1 % (v/v) Sarkosyl (sodium N-lauroylsarcosine), 50 mM Tris/HCl, pH 8·0] with ten strokes in a glass homogenizer (Asahi Techno Glass) to yield a final concentration of 5 % (w/v). After centrifugation at 1000 g for 10 min at 4 °C, supernatants were subjected to filtration through Planova 75N filters (mean pore size 72±4 nm; Asahi Kasei Pharma) as described previously (Tateishi et al., 2001). Pre-filtration samples (100 µl) were digested with PK (12·5 µg ml–1) for 1 h at 37 °C, mixed with 100 µl of 2x Laemmli's sample buffer and boiled for 15 min at 100 °C. Filtrates (post-filtration samples; 1 ml) were digested with PK (2 µg ml–1) for 1 h at 37 °C, mixed with an equal volume of 4 M NaCl and ultracentrifuged at 453 000 g for 1 h at 4 °C. The pellets were mixed with 50 µl Laemmli's sample buffer and boiled for 15 min at 100 °C. For deglycosylation of PrPSc, samples were treated with peptide N-glycosidase F (New England Biolabs) overnight at 37 °C. Pre-filtration samples [corresponding to 0·25 mg (wet weight) of brain tissue] and filtrates [corresponding to 10 mg (wet weight) of brain tissue] were subjected to 15 % SDS-PAGE and analysed by immunoblotting as described previously (Taguchi et al., 2003).

We reported previously that Planova filters with a membrane pore size of 15 nm or less could remove the infectivity of the scrapie agent (Tateishi et al., 2001). However, Planova 35N filters (mean pore size 35±2 nm) removed most of MM 1 PrPSc at the sensitivity of a Western blot assay (data not shown), whereas 75N filters (mean pore size 72±4 nm) did not. Therefore, we used Planova 75N filters in the present study to distinguish aggregation sizes at the sensitivity of a Western blot assay.

The filtration study using brain homogenates from various cases of CJD (Fig. 1) showed that MM 2 PrPSc was efficiently removed by Planova 75N filters, in contrast to MM 1. Even MM 2A PrPSc from a patient without amyloid plaques was removed more efficiently than MM 1 plaque PrPSc from the p-dCJD cases with amyloid plaques. Only faint bands were observed in the filtrates of MM 2 PrPSc when the blot was overexposed (Fig. 1c). These results appeared to demonstrate a correlation between removal efficiency and the type of PrPSc. However, other possible explanations for these findings included differences in the homogenization efficiency between the various homogenizers used or leakage of PrPSc from the brain homogenates through the membranes as an overload effect. To rule out these possibilities, we used brains from sCJD patients homozygous for methionine at codon 129 with type 1 and type 2 PrPSc coexisting (MM 1+2) and prepared samples from the brain regions where both types were found. The removal efficiency of the two different PrPSc subtypes was compared in these MM 1+2 samples homogenized under identical conditions using the same homogenizers and filtered through the same membranes (Fig. 2). The proportion of MM 2 PrPSc (~19 kDa) was notably decreased in the filtrates (Fig. 2a). Whether or not the proportion of MM 2 PrPSc was higher than MM 1 in the pre-filtration samples, MM 2 was removed more efficiently than MM 1 (Fig. 2b). Therefore, we concluded that MM 2 PrPSc is removed by filtration more efficiently than MM 1 under the same conditions.



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Fig. 1. Removal of each type of PrPSc by Planova 75N filters in various cases of CJD homozygous for methionine at codon 129 with type 1 (MM 1) or type 2 (MM 2) PrPSc. Western blots of pre-filtration samples (a), filtrates (b) and overexposed filtrates (c). All samples were digested with PK. Planova 75N filters removed MM 2 PrPSc more efficiently than MM 1, irrespective of the existence of amyloid plaques. Lanes 1 and 2, sCJD (MM 1); lanes 3 and 4 p-dCJD (MM 1 plaque); lane 5–7, vCJD (MM 2B); lane 8, thalamic form of sCJD (MM 2A).

 


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Fig. 2. Proportion of each type of PrPSc in pre-filtration samples (Pre) and filtrates (Post) of brain homogenates from sCJD patients homozygous for methionine at codon 129 with both type 1 and type 2 PrPSc coexisting (MM 1+2). (a) Western blot after PK digestion and deglycosylation. (b) Relative proportion of each type of PrPSc. Planova 75N filters removed MM 2 PrPSc (~19 kDa) more efficiently than MM 1 (~21 kDa) under the same conditions.

 
In this study, we filtered brain homogenates from various cases of CJD (codon 129 MM; type 1, type 2A, type 2B and type 1+2 PrPSc; with or without amyloid plaques). The two major PrPSc subtypes exhibited different removal efficiencies, but these were not influenced by the existence of amyloid plaques in the brains from which the homogenates were prepared.

These results demonstrated that MM 1 and MM 2 PrPSc have distinct aggregation sizes. PrPSc forms aggregates of heterogeneous sizes ranging from less than 600 kDa (fewer than 20 PrP molecules) to that of prion rods (as many as 1000 PrP molecules) (Tzaban et al., 2002). We speculate that this heterogeneity in aggregation size is the reason why MM 2 PrPSc was not completely separated from MM 1 by the filtration, and why the total content of PrPSc in the filtrates was greatly reduced (the filtrates loaded on SDS-PAGE contained 40 times as much brain material as the pre-filtration samples). Although the distribution and mean of the aggregation sizes were not assessed because of limited variety of the membrane pore size, the present study indicates that MM1 PrPSc aggregates are generally of a small size, while MM2 PrPSc aggregates are all of a larger size and do not contain a fraction of small-sized aggregates.

One of the main biochemical differences between type 1 and type 2 PrPSc is the location of the major PK cleavage site. Type 1 and type 2 PrPSc are distinguished by the size of the PK-resistant core (21 and 19 kDa, respectively), reflecting the differences in the PK cleavage site (residues 82 and 97) (Parchi et al., 1996, 2000). The present study indicates a new biochemical property, i.e. aggregation size, which can discriminate between the two types of PrPSc. Further investigation is needed to confirm whether these findings apply to the other genotypes (heterozygous for methionine/valine and homozygous for valine at codon 129) of CJD.

There might be several explanations for the distinct aggregation sizes, for example conformational differences between PrPSc types 1 and 2, the number of participating molecules and differing interactions with other molecules. Parchi et al. (1996, 2000) also speculated that the different PK cleavage sites resulted from different conformations between the two types of PrPSc. Likewise, differences in monomeric PrPSc conformation may also affect aggregation size.

Our results show that aggregation size is not associated with the existence of amyloid plaques. Puoti et al. (1999) reported that type 2 PrPSc was strictly associated with perivacuolar and plaque-like deposits in brains where both types of PrPSc coexisted. This raised the possibility that the large aggregation size of MM 2 PrPSc might reflect a correlation between type 2 PrPSc and amyloid plaques. However, aggregates from the sCJD case with type 2A PrPSc but without amyloid plaques were removed efficiently by filtration, in contrast to aggregates from the p-dCJD cases with type 1 PrPSc and with amyloid plaques (Satoh et al., 2003). Therefore, we consider that the mechanisms resulting in the formation of PrPSc aggregates and the much larger PrPSc amyloid plaques are different.

To date, several filtration studies using size-exclusion filters have been performed for the purpose of removing PrPSc from blood-derived products and other biopharmaceutical products (Tateishi et al., 1993, 1995, 2001; Van Holten et al., 2002). Previously, we reported that the different strains of transmissible spongiform encephalopathy pathogens (scrapie agent ME7 and Gerstmann–Sträussler–Scheinker syndrome agent Fukuoka-1) showed different removal efficiencies (Tateishi et al., 1995, 2001). In agreement with this previous study, our results showed different removal efficiencies for the different types of PrPSc. Our findings suggest that type 1 PrPSc should be used as the starting material in filtration studies using size-exclusion filters for removal of the CJD agent because of the smaller size of its PrPSc aggregates.

In conclusion, these results indicate that type 2 PrPSc has a larger aggregation size than type 1, irrespective of the existence of amyloid plaques. This is the first demonstration of a direct correlation between the type of PrPSc and the size of PrPSc aggregates in the brain in CJD.


   ACKNOWLEDGEMENTS
 
We thank G. Ishikawa and K. Yoshinari for help with the filtration procedure. This study was supported by a grant from the Organization for Pharmaceutical Safety and Research (S. M. and T. K.). A. K. was supported by the Sugawara fund for the promotion of medicine.


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Received 22 June 2004; accepted 22 September 2004.



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