Beyond PrPres Type 1/Type 2 Dichotomy in Creutzfeldt-Jakob Disease

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrPres) identified on Western blotting (type 1 or type 2). These biochemically distinct PrPres types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrPres in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain areas from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrPres and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrPSc) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrPres were identified. Despite this, the other two biochemical assays found that PrPSc from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrPSc subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrPres pattern. The identification of four different PrPSc biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrPres isoform provides an alternative biochemical definition of PrPSc diversity and new insight in the perception of Human TSE agents variability.


Introduction
Transmissible spongiform encephalopathies (TSE) are neurodegenerative disorders affecting a large spectrum of mammalian species that share similar characteristics, including a long incubation period (which in man may be measured in decades) and a progressive clinical course resulting in death [1].
The most common form of human TSE is an idiopathic disorder named sporadic Creutzfeldt-Jakob disease (sCJD). sCJD is not a uniform disorder in terms of its clinical and neuropathological phenotype. It remains unclear whether this variability is related to variations in the causative TSE agent strains, or to the influence of the methionine/valine polymorphism at codon 129 of the PRNP [2,3].
A key event in the pathogenesis of TSE is the conversion of the normal cellular prion protein (PrP C , which is encoded by the PRNP gene) into an abnormal disease-associated isoform (PrP Sc ) in tissues of infected individuals. Conversion of PrP C into PrP Sc is a post-translational process involving structural modifications of the protein and resulting in a higher b-sheet content [4]. PrP C is completely degraded after controlled digestion with proteinase K (PK) in the presence of detergents. PrP Sc is N-terminally truncated under such conditions, resulting in a PK resistant core, termed PrP res [5]. PrP res , also named PrP 27-30, is a disease marker for TSE and the presence of PrP Sc seems to correlate with infectivity [5,6]. According to the prion hypothesis, PrP Sc is the infectious agent in TSE [7] and, in the last decades, several lines of evidence have indicated that particular biochemical properties of PrP Sc , such as solubility in N-lauroylsarcosine, PK resistance and electromobility in western blotting (WB) can be used to distinguish between different prion agents or strains [8,9].
In sCJD, two major PrP res types have been described by WB: in type 1 PrP res , the unglycosylated fragment is 21 kDa, while in type 2, the apparent molecular weight of this unglycosylated fragment is 19 kDa [3]. Protein N-terminal sequencing revealed that type 2 isoform derives from preferential cleavage of the protein during PK digestion at amino acid 97, while in type 1 preferential cleavage occurs at amino acid 82 [10]. sCJD cases can be subclassified according the PrP res isoform and the PRNP codon 129 methionine (M)/valine (V) polymorphism, resulting in 6 major subypes: MM1, MM2, MV1, MV2, VV1 and VV2. Interestingly, these subtypes appear to carry distinct pathological and clinical features, [2,3], and it has been proposed that type 1 and type 2 isoforms in sCJD might correspond to different TSE agent strains. However, the description of PrP res isoforms which appear to be distinct from type 1 and type 2, and the increasing number of reports describing the coexistence of type 1 and type 2 PrP res in different areas or the same area in the brain from a single sCJD patient, calls into questions the subclassification system described above in sCJD [11][12][13][14]. Here, in a large group of cases including 41 sCJD and 12 iCJD patients, we confirmed that type 1 and type 2 PrP res can be observed as a mixture in a substantial number of patients. However, using two novel assays described here, PrP Sc from these patients with mixed PrP res types are homogeneous irrespective of the brain area considered. Moreover, based on these novel PrP Sc biochemical properties, four distinct subgroups were observed in our cohort of sCJD patients. Similar findings were observed in iCJD cases from two countries and differing sources of infection.

Cases Studied
A total of 41 French cases of sCJD, each of which had frozen tissue (2-4 g) available from preferentially 5 brain regions: (occipital, temporal and frontal cortex, cerebellum and the caudate nucleus), were included in this study. All six currently defined classes of s-CJD patients (MM1-MM2-MV1-MV2-VV1-VV2) were represented in our panel (Table 1). Moreover, 12 cases of iatrogenic CJD (iCJD), linked to contamination by growth hormone (GH) or dura mater grafts, from patients originating either from United Kingdom (UK) or France, were also investigated ( Table 1). None of the patients had a familial history of prion disease and, in each case, the entire PRNP coding sequence was analyzed, either by denaturating gradient gel electrophoresis and/or direct sequencing. All patients died from CJD during the period 1997-2004. Additionally, five cases of Alzheimer's disease were included as non-CJD controls.
In all cases, informed consent for research was obtained and the material used had appropriate ethical approval for use in this project.

Tissue Homogenate Preparation
For each sample, a 20% brain homogenate (weight/volume) in 5% glucose was prepared using a high-speed homogenizer (TeSeE Precess 48 system). The homogenates were then filtered through a 20 Gauge needle before storage at 280uC.

Western Blot PrP sc Banding Pattern
Various factors have been reported to influence the results of PrP res analysis by WB, including tissue pH and the effect of Cu 2+ ions [15][16][17]. In order to limit these factors, each homogenate was diluted a 100-fold in a single non-CJD control brain homogenate prior to further investigation.
A WB kit (TeSeE WB kit Bio-Rad) was used following the manufacturer's recommendations.

PrP Sc Resistance ELISA
PrP Sc detection was carried out using sandwich ELISA test (TeSeE CJD, Bio-Rad) used following the manufacturer's recommendations. The assay protocol includes a preliminary purification of the PrP Sc (TeSeE purification kit) consisting in (i) digestion of PrP C with PK, (ii) precipitation of PrP Sc by buffer B and centrifugation, (iii) denaturation of PrP Sc in buffer C at 100uC, before immuno-enzymatic detection. In this ELISA, the capture antibody 3B5 recognizes the octarepeat region of PrP [19], while the detection antibody 12F10 binds to the core part of the protein [18].
PK resistance of the PrP Sc portion recognized in the ELISA test was determined by measurement of the ELISA specific signal recovered from a series of homogenate aliquots digested with different concentrations of PK in buffer A9 reagent (TeSeE Sheep/ Goat purification kit). Each sample was first diluted in normal brain homogenate (between 100-and 10,000-fold) until obtaining a signal between 1.5 and 2 absorbance units after digestion with 50 mg/ml of PK. Triplicate of equilibrated samples were then submitted to a PK digestion with concentrations ranging from 50 to 500 mg/ml, before PrP Sc precipitation and ELISA detection. Results were expressed as the percentage of residual signal when compared to the 50 mg/ml PK digestion (lowest PK concentration). In each assay, two standardized controls (scrapie and BSE

Author Summary
Prion diseases are transmissible neurodegenerative disorders characterized by accumulation of an abnormal isoform (PrP Sc ) of a host-encoded protein (PrP C ) in affected tissues. According to the prion hypothesis, PrP Sc alone constitutes the infectious agent. Sporadic Creutzfeldt-Jakob disease (sCJD) is the commonest human prion disease. Although considered as a spontaneous disorder, the clinicopathological phenotype of sCJD is variable and substantially influenced by the methionine/valine polymorphism at codon 129 of the prion protein gene (PRNP). Based on these clinicopathological and genetic criteria, a subclassification of sCJD has been proposed. Here, we used two new biochemical assays that identified four distinct biochemical PrP Sc subgroups in a cohort of 41 sCJD cases. These subgroups correlate with the current sCJD subclassification and could therefore represent distinct prion strains. Iatrogenic CJD (iCJD) occurs following presumed accidental human-to-human sCJD transmission. Our biochemical investigations on 12 iCJD cases from different countries found the same four subgroups as in sCJD. However, in contrast to the sCJD cases, no particular correlation between the PRNP codon 129 polymorphism and biochemical PrP Sc phenotype could be established in iCJD cases. This study provides an alternative biochemical definition of PrP Sc diversity in human prion diseases and new insights into the perception of agent variability.  Express the PK concentration for which, when increasing PK concentration, the ELISA PrP Sc signal reach an arbitrary cut-off value set at 20% of the signal observed with a PK concentration of 50 mg/ml. c Express the ratio of ELISA signal obtained (A/A9) after PrP sc PK digestion differential PK digestions in a non-perturbing detergent mixture (A), and a denaturing (SDS) detergent mixture (A9). doi:10.1371/journal.ppat.1000029.t001 from sheep) were used as an internal standard. About 20% of samples were randomly selected and submitted to two independent tests separated in time as to assess inter-assay variation.

''Strain Typing'' ELISA
The ELISA test used in this study was adapted from the Bio-Rad TeSeE test, validated at CEA for EU strain typing studies in ruminants and designed to distinguish BSE in sheep from scrapie. The principle was to measure conformational variations in PrP Sc by applying two differential PK digestions under the modification of detergent conditions (SDS sensitivity). For each sample, PK digestion was performed under two conditions: (i) two aliquots of 250 ml of 20% homogenate were mixed either with 250 ml of A reagent (TeSeE purification kit) containing 20 mg of PK, (ii) with 250 ml of A9 reagent (N-lauroylsarcosine sodium salt 5% (W/V), sodium dodecyl sulfate 5% (W/V) containing 55 mg of PK, All the tubes were then mixed by inversion 10 times and incubated at 37uC (in a water bath) for exactly 15 min. Subsequently, 250 ml of reagent B (Bio-Rad purification kit)/PMSF (final concentration 4 mM) were added, mixed and the tubes were centrifuged for 5 minutes at 20,000 g at 20uC. Supernatants were discarded and tubes dried by inversion onto an absorbent paper for 5 min. Each pellet was denatured for 5 min at 100uC with 25 ml of C reagent (Bio-Rad purification kit). The samples were diluted in 250 mL of R6 buffer containing 4 mM of serine protease inhibitor AEBSF (4-2-aminoethyl benzenesulfonyl fluoride hydrochloride), and, if desired, further serially diluted in R6 buffer. ELISA plates were then incubated for two hours at room temperature and, after three washes, antibody detection (TeSeE CJD, Bio-Rad) was added for two hours at 4uC. The ratio of the absorbance obtained in the two conditions (A/A9) was calculated using appropriate dilutions providing absorbance measurements ranging from 0.5 to 2.5 absorbance units in A conditions. For each plate, the same three control samples (one MM1, one VV2 and one MM GH) were included. To avoid inter-assay variations, final results were expressed as a normalized ratio established by dividing the ratio obtained for the analyzed sample by the one obtained for a VV2 sample selected as standard.

Intra-Individual Variability of WB Banding Pattern
For each sCJD case, PrP res profile was determined from five brain areas using both Sha31 and 8G8 antibodies. A single PrP res type (type 1 or type 2) was observed in investigated brain areas of most of the MM1 (n = 11), and all the areas from MV1 (n = 8), VV1 (n = 1) and MM2 (n = 3) sCJD cases of our panel. However, in several cases initially classified as MM1 (n = 2), and in a majority of VV2 (n = 5) or MV2 (n = 6) cases, some brain areas harboured mixed electrophoretic pattern characterized by two distinct bands at 19 and 21 kDa, indicating the coexistence of PrP res type 1 and type 2 (Table 1 and Figure 1A). Moreover, in individual patients some brain areas were found to be type 1, while another area could be found to be type 2 (Table 1 and Figure 1A). Since both Sha31 and 8G8 gave similar results this phenomenon cannot be attributed to some antibody peculiarity in PrP recognition ( Figure 1B and 1C).
Antibody 12B2 is specific for the amino acid sequence 89-93 that is located N-terminally of the type 2 cleavage site (amino acid 97). In principle, this antibody is unable to recognize type 2 PrP res . Systematic western blotting with 12B2 consistently demonstrated the presence of the 21 kDa band, characteristic for type 1 PrP res , in nearly all type 2 classified samples, regardless of the PRNP codon 129 polymorphism ( Figure 1D). In a limited number of type 2 samples, 12B2 failed to detect a type 1 band ( Figure 1E and 1F, lane 2,3).
Using Sha31 or 8G8, mixed type 1/type 2 PrP res profiles were observed in several iCJD cases (Figure 2A), regardless of their national origin or mode of infection. In most (but not all) samples initially classified as type 2, the 12B2 antibody revealed the presence of a 21 kDa band, characteristic of type 1 PrP res ( Figure 2B).
Together these findings point to the existence of variable amounts of type 1 PrP res molecules in all or nearly all type 2 classified patients ( Table 1).

All Brain Areas from a Single Patient Have Similar PrP Sc Features
In sCJD and iCJD patients who harboured a single WB PrP Sc type in the different brain areas, as assessed by Sha31, a single ELISA PK resistance profile (Table 1 and Figure 3A) and a comparable ratio in strain typing assay (Table 1 and Figure 3B) were observed in all brain areas. Surprisingly, in each patient harbouring both type 1 and 2 PrP res , either in the same or in different brain areas, a single ELISA PK digestion profile (Table 1 and Figure 3C and 3D) and a comparable signal ratio in strain typing assay (Table 1 and Figure 3B) was also observed, irrespective of region assayed.
MM1 and VV2 samples but also MM2 and VV1 samples, which harboured similar apparent PrP Sc content (as assessed by ELISA) were artificially mixed in different proportions. Using WB, a mixed type 1+2 profile could, or could not, be observed depending on the mixture proportions ( Figure 3E and 3F). Both PrP Sc resistance ELISA assay ( Figure 3G and 3H) and strain typing ELISA (not shown) were able to discriminate the different mixtures from the original isolates and from each other. These results clearly demonstrate that the uniformity of PrP Sc biochemical properties, as demonstrated by both PrP Sc resistance ELISA and strain typing ELISA, in patients harbouring different PrP res isoforms cannot be attributed to a lack of discriminative power of these techniques.
Together, these data strongly indicate that, despite possible variations in PrP res type on WB analysis, patients with either sCJD or iCJD appear to harbour a single PrP Sc isoform in their brain.

Four Distinct PrP Sc Biochemical Signatures Are Observed in iCJD and sCJD Patients
According to the results from PrP Sc PK resistance assay and strain typing ELISAs, sCJD patients could be split into four groups ( Table 1, and Figure 3A and 3B). The first group was characterized by a strong PK resistance ( Figure 3A) and a low ratio in strain typing assay ( Figure 3B). Group 1 could be readily differentiated from Group 2 which showed a higher sensitivity regarding PK digestion, as well as an increased signal ratio in strain typing assay, when compared to Group 1. Two other PrP Sc groups were also observed. Group 3 harboured an intermediate PK lability in the PrP Sc resistance ELISA and ratio in the strain typing ELISA, when compared to Group 1 and 2. Group 4 had a very high PK-sensitivity and ratio in the strain typing ELISA. No overlapping in PK resistance profile or ratio value in strain typing assay were observed between the four determined groups (Table 1).  . PrP Sc signal intensity was measured after PK digestion into two different detergent solutions. Normalized A/A9 ratio was calculated for each sample (see Methods section). MM1 and MV1 had a low ratio indicating an absence of alteration of PrP sc PK sensitivity linked to the modification detergent digestion conditions. This ratio was higher in MV2, VV2, MV1+2, and VV1+2, while, in the unique VV1 case, an intermediate ratio was observed. In MM2 patients, the huge ratio indicated a strong increase in PK sensitivity by modification of detergent conditions. (C, D) PK resistance Group1 was composed of sCJD MM and MV patients, harbouring predominantly type 1 PrP res while Group 2 consisted in VV and MV patients harbouring predominantly type 2 or type 1+2 PrP res . Groups 3 and 4 were respectively composed with VV1 and MM2 patients from our sCJD panel.
Striking differences were observed in the PrP Sc properties between the different iCJD cases and all four groups relying on PrP Sc signatures observed in sCJD cases were identified (Table 1, and Figures 3B and 4).
As it might have been expected from sCJD cases observations, Group 1 PrP Sc properties was identified in MM1 UK dura mater graft patients (n = 2) ( Figure 4A) while Group 2 PrP Sc features were observed in UK VV2 (n = 2) ( Figure 4D) and MV2 (n = 1) ( Figure 4B) GH patients. Surprisingly, a typical Group 2 PrP Sc signature was also observed in one out of the three MV1 French GH patients (type 1 in all brain areas). Meanwhile, all investigated MM1 and two out of the three MV1 French GH cases ( Figure 4A) harboured identical PrP Sc properties than Group 3 sCJD ( Figure 4E). Finally, a Group 4 sCJD PrP Sc signature ( Figure 4F) was observed, using both PrP Sc resistance ELISA ( Figure 4E) and strain typing ELISA (Figure 3B), in a French dura mater VV1 case (n = 1), which harboured a type 1 PrP res WB profile in every investigated area.
Taken together, these observations support the concept that, in iCJD patients, variability in the PrP Sc biochemical properties is not related to the route of infection or the PRNP codon 129 genotype. It also indirectly suggests that the range of different PrP Sc properties observed in iCJD might be related to those in the source of infection (likely to have been a sCJD case).

Coexistence of Different PrP res Types in the Same Subject
In this study, detection, by WB, of the coexistence of two PrP res types in about 30% (13/41) of cases is consistent with already published data [12,14]. This observation could suggest the existence in brain from a single patient of different abnormal PrP species. Although two main PK cleavage sites are associated with PrP res type 1 and type 2 (respectively amino acid 82 and 97), N-terminal sequencing revealed in all investigated cases the presence of a whole spectrum of overlapping cleavage sites. Moreover in a part of investigated cases this technique demonstrated the presence (i) of variable but consistent level of type 1 PrP res in patients classified type 2 using WB and (ii) in some patient classified type 1, of low amount of type 2 PrP res [10]. These observations could suggest that, rather than a pure type 1 or type 2 PrP res , PK digestion of a PrP Sc specific conformer generate variable mixture of PrP res fragments (with presence of dominant or sub dominant type 1 or type 2 PrP res ), which WB usually failed to reveal accurately because its intrinsic technical limits [14]. Antibodies either harbouring higher affinity to PrP (like Sha31) [18] or probing specifically type 1 PrP res (like 12B2) [20], now allow a better perception of such mixture. However, investigations carried out using artificial mixture of type 1 and type 2 brain homogenate, even using high affinity anti-PrP antibodies, clearly indicate the current limits of WB discriminative power [14]. Together, these data suggest that WB analysis of PrP res on its own could be misleading for adequate discrimination between PrP Sc variants in CJD.
Both PrP Sc PK resistance ELISA and strain typing ELISA are based on the characterization the N terminal part of the PrP Sc PK digestion either by increasing PK amount or modifying detergent conditions. While WB profile could be compared to a snapshot picture of PrP res fragments generated by PK digestion process, these assays reflect the dynamics of the PK cleavage rather than its final result (different forms of PrP res ). Consequently they could provide different but also more accurate perception of the PrP Sc conformers.
Our findings from the PrP Sc capture immunoassays clearly indicate that in a single patient, irrespective of brain area, sCJD associated PrP Sc displays uniform biochemical properties, regardless of the regional variation of type 1 and type 2 isoforms determined by WB. Such findings support the idea of the presence of a specific TSE agent in each brain and the accumulation of a single associated PrP Sc conformer.

sCJD Classification
Because the limited size of our cohort of cases, an in depth comparison between the PrP Sc signature (as established in this study) and the Parchi classification system is not possible.
However, despite this limitation, two major groups were identified in our panel according to the PrP Sc properties. The first major group was constituted with patients harbouring a highly PK resistant PrP Sc (MM1 and MV1 patients). The second group included patients harboring a PK labile PrP Sc (VV2 and MV2 patients). Using both lesion profile and clinical parameters [2], two major forms of sCJD are commonly recognized. The first sCJD form, named ''classical'', is characterized by a ''rapid evolution'' (usually around 4 months), and affects most of the MM1 and MV1 patients. The second sCJD form, named ''atypical'', affects VV2 and MV2 with a longer symptomatic evolution (usually longer than 6 months) and a late dementia. Despite inter-individual variations, sCJD Groups 1 and 2, as we defined them on biochemical criteria were consistent with this classification.
Both VV1 and MM2 sCJD cases are extremely rare; they respectively represent 1% and 4% of the identified sCJD cases. According to the literature, these patients have clinical features and lesion profiles that are very different from other sCJD patients [2]. However, in our study as in previously published studies, WB did not identify any distinct biochemical difference from other type 1 and type 2 cases. In contrast, both the strain typing ELISA and PrP Sc resistance assays clearly differentiated these cases from Group 1 and Group 2 cases. This finding, which is consistent with clinico/pathological observations carried out in patients, could indicate that there are indeed differences in PrP Sc that distinguish these VV1 and MM2 cases from other sCJD groups. assay in three areas from a (C) VV (triangles) or MM (circles) patient and in (D) a MV (triangles) patient harbouring distinct PrP Sc WB type in their different brain areas. Artificial mixtures of MM2/VV1 or MM1/VV2 samples were prepared. All homogenates were first equilibrated by dilution into negative brain homogenate to obtain an equal PrP Sc   Although prion strains can only be identified definitively by bioassay, molecular in vitro tools to characterize PrP Sc are more and more widely used for the rapid identification of particular agents, such as BSE in cattle, sheep, rodent and humans (vCJD) [20,21]. This has come to be termed ''molecular strain typing'' and although widely employed, the exact relationship between PrP Sc biochemistry and the biological properties of the agents responsible remain to be determined. In sCJD, the presence of four distinct PrP Sc biochemical forms apparently correlated to clinico-pathological phenotypes as defined by Parchi et al. [2] could be an indication of the involvement of different TSE agents.
iCJD cases are a consequence of accidental human to human TSE transmission, most likely representing transmission of sCJD. The identification in iCJD cases of the four PrP Sc signatures identified in sCJD is consistent with the existence of distinct prions associated with these biochemical forms.
Three examples of human-to-human transmission of variant CJD through blood transfusion have now been identified. While all blood donors were MM at codon 129 PRNP, the recipients had either a MM (n = 2) or a MV genotype (n = 1). Despite this genotype difference there appears to have been conservation of the disease phenotype and PrP res type in all ''secondary'' vCJD cases [22][23][24][25]. These observations could suggest that in case of interhuman transmission, difference in donor/recipient genotype could result in un-altered abnormal PrP signature.
Our identification of MM GH iCJD cases harbouring similar PrP Sc signature as a VV1 sCJD case or of a VV dura mater iCJD case similar to MM2 sCJD might indicate preservation of a specific PrP Sc biochemical signature after human to human transmission between individuals of different codon 129 genotypes.
Treatment with extracts of GH contaminated by CJD has lead to a high number of iCJD cases in France and the UK. The codon 129 genotypes of the affected individuals in the two countries differ, with the French cohort predominantly MM and MV and the British cohort MV and VV [26]. In the absence of any clear explanation for this finding, it was suggested that it might be due to contamination of different batches of GH with different prion strains from individuals of differing PRNP codon 129 genotypes. Our identification of different biochemical forms of PrP Sc in GH French patients and in UK patients is consistent with this hypothesis. The variability observed within the French GH cases could signify involvement of different prion strains, consistent with multiple contaminated GH batches in the French epidemic.

Conclusion
The identification in this study of different PrP Sc species in CJD patients with the same PRNP polymorphism at codon 129 and WB PrP res profile offers a new perspective on our understanding of the relationship between PrP biochemistry, prion disease phenotype and agent strain. We highlight two novel approaches to analysing PrP Sc in sCJD and iCJD and offer evidence that these analyses provide potentially-strain associated information, which appears to be lacking from the conventional WB assay.