Analysis of Prion Strains by PrPSc Profiling in Sporadic Creutzfeldt–Jakob Disease

Background Prion diseases are a group of invariably fatal neurodegenerative disorders affecting humans and a wide range of mammals. An essential part of the infectious agent, termed the prion, is composed of an abnormal isoform (PrPSc) of a host-encoded normal cellular protein (PrPC). The conversion of PrPC to PrPSc is thought to play a crucial role in the development of prion diseases and leads to PrPSc deposition, mainly in the central nervous system. Sporadic Creutzfeldt–Jakob disease (sCJD), the most common form of human prion disease, presents with a marked clinical heterogeneity. This diversity is accompanied by a molecular signature which can be defined by histological, biochemical, and genetic means. The molecular classification of sCJD is an important tool to aid in the understanding of underlying disease mechanisms and the development of therapy protocols. Comparability of classifications is hampered by disparity of applied methods and inter-observer variability. Methods and Findings To overcome these difficulties, we developed a new quantification protocol for PrPSc by using internal standards on each Western blot, which allows for generation and direct comparison of individual PrPSc profiles. By studying PrPSc profiles and PrPSc type expression within nine defined central nervous system areas of 50 patients with sCJD, we were able to show distinct PrPSc distribution patterns in diverse subtypes of sCJD. Furthermore, we were able to demonstrate the co-existence of more than one PrPSc type in individuals with sCJD in about 20% of all patients and in more than 50% of patients heterozygous for a polymorphism on codon 129 of the gene encoding the prion protein (PRNP). Conclusion PrPSc profiling represents a valuable tool for the molecular classification of human prion diseases and has important implications for their diagnosis by brain biopsy. Our results show that the co-existence of more than one PrPSc type might be influenced by genetic and brain region–specific determinants. These findings provide valuable insights into the generation of distinct PrPSc types.


A B S T R A C T
Background Prion diseases are a group of invariably fatal neurodegenerative disorders affecting humans and a wide range of mammals. An essential part of the infectious agent, termed the prion, is composed of an abnormal isoform (PrP Sc ) of a host-encoded normal cellular protein (PrP C ). The conversion of PrP C to PrP Sc is thought to play a crucial role in the development of prion diseases and leads to PrP Sc deposition, mainly in the central nervous system. Sporadic Creutzfeldt-Jakob disease (sCJD), the most common form of human prion disease, presents with a marked clinical heterogeneity. This diversity is accompanied by a molecular signature which can be defined by histological, biochemical, and genetic means. The molecular classification of sCJD is an important tool to aid in the understanding of underlying disease mechanisms and the development of therapy protocols. Comparability of classifications is hampered by disparity of applied methods and inter-observer variability.

Methods and Findings
To overcome these difficulties, we developed a new quantification protocol for PrP Sc by using internal standards on each Western blot, which allows for generation and direct comparison of individual PrP Sc profiles. By studying PrP Sc profiles and PrP Sc type expression within nine defined central nervous system areas of 50 patients with sCJD, we were able to show distinct PrP Sc distribution patterns in diverse subtypes of sCJD. Furthermore, we were able to demonstrate the co-existence of more than one PrP Sc type in individuals with sCJD in about 20% of all patients and in more than 50% of patients heterozygous for a polymorphism on codon 129 of the gene encoding the prion protein (PRNP).

Introduction
Transmissible spongiform encephalopathies or prion diseases are neurodegenerative disorders characterized by posttranslational conversion and cerebral accumulation of a pathological isoform (PrP Sc ) of a host-encoded membraneassociated glycoprotein (cellular prion protein, PrP C ) [1]. These diseases include scrapie in sheep, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids, and sporadic, genetic, or acquired human prion diseases [2]. The most common human prion disease, the sporadic form of Creutzfeldt-Jakob disease (sCJD), is clinically characterized by rapidly progressive dementia, neurologic dysfunction including myoclonic involuntary movements, and, finally, a terminal state of severe cognitive impairment leading to death within months from the onset of clinical symptoms [3]. Various clinical phenotypes can be observed, e.g., patients with ataxia opposed to dementia as the initial symptom or patients with anopsia as a prominent clinical feature. This marked clinical heterogeneity observed in sCJD is reflected by the presence of diverse sCJD types [4] and is not yet fully understood.
It is known that various isolates, or strains, of prions may be propagated within genetically identical hosts, leading to distinct clinical and pathological features [5,6]. The apparent diversity of human prion diseases may, at least in part, be attributed to the presence of distinct prion strains in affected individuals. In agreement with this, distinct clinical phenotypes of human prion diseases present with diverse deposition patterns of PrP Sc in addition to disparate biochemical properties of PrP Sc [7,8]. Since nucleic acids do not copurify with prion infectivity and prion strains do not seem to be encoded by differences in PrP primary structure, the hypothesis that PrP Sc itself may encode strain-specific phenotypic properties within its tertiary or even quaternary structure has to be considered [9,10]. This assumption is supported by the presence of strain-specific N-terminal cleavage of PrP Sc upon Western blot analysis following limited digestion with proteinase K [9].
Another important factor in phenotype variability is the host genotype of the gene encoding the prion protein (PRNP). It is well established that the polymorphism at codon 129, encoding either methionine or valine in PRNP, influences the disease phenotype [8].
Thus, the classification of human prion diseases is based on the clinical presentation of the affected individual, PRNP status in concert with neuropathological findings, and the biochemical analysis of PrP Sc [11][12][13]. Previous studies have focused on the assessment of the fragment sizes of unglycosylated PrP Sc and the ratios of the three glycoforms of PrP Sc as seen on Western blot following protease treatment [12,13].
Although there is some disparity in the exact number of mobilities of the unglycosylated PrP Sc fragment, researchers agree that this fragment principally migrates between 19 and 21 kDa [12,13]. Since the resolution of one-dimensional gel electrophoresis is limited, it is likely that additional species of N-terminal fragments will be identified if more accurate techniques, such as two-dimensional immunoblot or mass spectrometry, are utilized. In fact, recent studies using twodimensional gel electrophoresis of human sCJD brain homogenates strongly suggest that this is the case [14,15]. In order to simplify comparability of our study to published work, we decided to distinguish two principal mobilities of unglycosylated PrP Sc i.e., 21 kDa (high) and 19 kDa (low), thereby accepting that both the high and the low PrP Sc fragments show a certain degree of variation.
In agreement with the fact that the predominant PrP Sc type may be indicative of the sCJD type, previous studies have demonstrated that the co-existence of more than one PrP Sc type is a rare event [8]. In the light of these data, the finding that the co-existence of more than one PrP Sc type within one individual occurs in 30% of all patients if multiple regions of the brain are investigated [16] came as a surprise. Because of the limited sample size in the latter study, the question of the true incidence of co-existence of more that one PrP Sc type within one individual has remained unanswered.
It has been shown that the spatial analysis of histopathological changes often referred to as the lesion profile may provide a tool to discriminate various clinically diverse types of prion diseases [8,17]. A major drawback of this method, however, is the considerable inter-observer variability. Thus it has been virtually impossible to compare published lesion profiles [8,13].
Here, we assess the distribution of PrP Sc types in nine central nervous system regions in a cohort of 50 patients with sCJD, using PrP Sc profiling. Furthermore, we address the question of co-existence of more than one PrP Sc type in sCJDaffected individuals.

Selection of Patients
Cases in this study were derived from an unselected series of patients with clinically, genetically, and neuropathologically proven sCJD (Tables 1 and 2). All tissue specimens originated from patients referred to the Swiss National Reference Centre for Prion Diseases, and the specimens were processed according to established guidelines regarding safety and ethics. Frozen brain tissue was stored at À80 8C and samples were taken from the following brain regions: frontal, parietal, occipital, and temporal cortex, and the
To quantify PrP Sc , serial dilutions of a standard sCJD brain homogenate (33, 16.5, and 8.25 lg of protein) were used on each Western blot to generate a standard curve. Quantification was performed only if the square of the correlation coefficient of the standard curve was above 0.93 (r 2 . 0.93).

PRNP Analysis
Genomic DNA was extracted from the buffy-coat fraction of peripheral blood or from brain tissue. The complete PRNP open reading frame was analyzed using established methods [20].

Neuropathology
Tissue from selected patients (n ¼ 32) was fixed in 10% formalin. Tissue blocks from frontal, parietal, occipital, and temporal cortex, and the putamen, thalamus, midbrain, medulla oblongata, and cerebellum, localized adjacent to the specimens used for biochemical analysis, were selected, decontaminated for 1 h with 98% formic acid and embedded in paraffin. Sections (3 lm) were subjected to conventional staining and to immunostaining for glial fibrillary acidic protein (Dako), and for PrP (3F4), upon hydrolytic autoclaving according to published protocols [20].
Spongiosis was evaluated on a 0-4 scale (not detectable, mild, moderate, severe, and status spongiosus). Gliosis and PrP Sc content were scored on a 0-3 scale (not detectable, mild, moderate, severe). PrP Sc accumulation patterns were described as synaptic, perivacuolar, patchy, or plaque-like. Averaging of the three scores resulted in the value that was employed in order to obtain the lesion profile for individual patients [8]. Histological analysis was performed by two independent investigators blinded to clinical data, codon-129 status, and PrP biochemistry.

Reliable Quantification of PrP Sc Using Internal Standards
Routinely, quantification of PrP Sc is performed by comparing the signal intensities on Western blot analysis following limited proteinase K digestion. While this method is highly specific and objective, it does not allow for the comparison of samples run on separate gels owing to variations in the blotting process, antibody binding efficiencies, or differences in visualization reaction. In order to circumvent these problems, we developed a system whereby these variables are compensated for by quantifying the signal intensity of a serially diluted PrP Sc standard on every Western blot. The resulting standard curve is then used to quantify PrP Sc content in defined brain regions. The reliability and feasibility of this method was assessed by analyzing nine different brain areas on a cohort of 50 patients with sCJD (see Table 1; Figure 1). Standard curves were accepted only if the square of the correlation coefficient was above 0.93. Our PrP Sc standard is available to the scientific community and should facilitate comparison of PrP Sc quantities present in defined samples.

Molecular Analysis of sCJD Using PrP Sc Profiling
Nine defined brain areas of 50 subjects with sCJD were analyzed. They showed a mean age of 69.1 y (6 8.5) and a gender ratio of 28 males to 22 females. Patients were grouped according to both their genotype at codon 129 of the PRNP gene and to their predominant PrP Sc type (Table 1) Analysis of the relative amounts of PrP Sc in various brain regions revealed distinct distribution patterns, which seem to be dependent on the type of PrP Sc and on the host's codon-129 polymorphism. Cases with a slower-migrating unglycosylated band of PrP Sc (;21 kDa) and homozygosity for methionine on codon 129 (MM type 1) harbor significant amounts of PrP Sc in cortical regions (frontal, parietal, temporal, and occipital) and the thalamus, smaller and variable amounts in the putamen and cerebellum, and very low or undetectable amounts in the midbrain and medulla oblongata (Figure 1). PrP Sc quantities for this cohort are listed in Table 2. In contrast, cases with a faster-migrating unglycosylated band of PrP Sc (;19 kDa) and homozygosity for valine on codon 129 (VV type 2) had only scant amounts of PrP Sc in the cerebral cortex, but showed marked PrP Sc deposition within the putamen, thalamus, the midbrain, the cerebellum and, to a lesser extent, in the medulla oblongata, although this was clearly detectable (Figure1). PrP Sc quantities for this cohort are listed in Table 2.
Whereas the deposition patterns described above were relatively uniform in the respective groups, the picture is not so obvious for patients heterozygous for methionine or valine at codon 129. In this group, patients with a slower-migrating unglycosylated band of PrP Sc (;21 kDa, MV1) exhibited deposition patterns reminiscent of MM1 individuals. However, PrP Sc deposition was more abundant in the temporal than in the occipital lobe, and deposits within the putamen showed greater variability (Figure 1).
Patients with a faster-migrating unglycosylated band of PrP Sc (;19 kDa, MV2) exhibited abundant PrP Sc deposition in the putamen, thalamus, midbrain, and cerebellum, clearly detectable deposits in the medulla, and, in addition, variable deposits within the cerebral hemispheres ( Figure 1). PrP Sc quantities for these two cohorts and for patients either homozygous for methionine (MM2, n ¼ 2) or valine (VV1, n ¼ 1) are listed in Table 2.
Because this method possesses some similarities with lesion profiling, usually used to discriminate between sCJD types or between prion strains in rodents, we have termed this method ''PrP Sc profiling'' [8,17].

High Proportion of PrP Sc Type Co-Existence in Patients Expressing MV at Codon 129
Studies on relatively small cohorts of patients with sCJD evidenced the co-occurrence of different PrP Sc types within one individual [16]. We analyzed nine distinct brain regions of 50 patients with sCJD, resulting in a total of 450 analyzed specimens. In patients where routine SDS-PAGE analysis suggested possible co-occurrence of distinct PrP Sc types, high-resolution SDS-urea-PAGE was performed (n ¼ 13). A total of nine sCJD individuals showed non-ambiguous cooccurrence of distinct PrP Sc types as evidenced by the presence of a faster-and a slower-migrating unglycosylated band of PrP Sc , either in different brain areas or within one brain region (Figure 2). In the remainder of the cases (n ¼ 41), only a single PrP Sc type could be detected in all nine different brain areas. In cases where more than one PrP Sc type was observed, the topological distribution of distinct strain types revealed no obvious pattern (Table 3). However, the cerebral hemispheres appeared to be less affected than the thalamus, putamen, cerebellum, midbrain, and medulla oblongata. Whereas co-occurrence of both PrP Sc types was found in only two cases within the frontal lobe, the midbrain was affected in three cases, the medulla in four cases, and the putamen, thalamus, and cerebellum each in five cases.
The most striking feature of patients harboring two PrP Sc types was their association with the methionine/valine polymorphism at codon 129. Six out of 11 methionine/valine heterozygotes displayed both PrP Sc types, whereas only three of 32 individuals homozygous for methionine produced more than one PrP Sc type. In the groups of patients expressing valine/valine on codon 129, we were not able to detect more than one PrP Sc type.

Neuropathological Analysis
The analysis of the neuropathological lesion pattern was performed according to published protocols [8,13] with the purpose to: (i) evaluate whether immunohistochemical PrP staining and histological lesions (gliosis, spongiosis) correlate with the absolute amount of biochemically assessed PrP Sc , and (ii) compare whether lesion profiles generated by histological assessment parallel biochemically defined PrP Sc profiles. In order to facilitate these analyses, patients with sCJD were subdivided into following groups: MM1, MV1, MV2, VV2 [12]. Patients presenting with multiple PrP Sc types were allocated according to the dominating PrP Sc type.
The MM1 group (n ¼ 20) showed marked involvement of the frontal and temporal cerebral cortex and slightly fewer lesions in the cerebellum, whereas the putamen, thalamus, midbrain, and medulla oblongata showed relatively modest pathological alterations (see Figure 1).
In the MV1 cohort (n ¼ 4), a similar lesion profile could be observed ( Figure 1). The observation that this group showed a greater variability might in part be attributable to the smaller sample size. MV2-predominant patients (n ¼ 4) exhibited a consistently high degree of neuropathological lesions with relative sparing of the occipital cortex and the cerebellum.
In the VV2 group (n ¼ 4), pathological alterations were most pronounced in the cerebellum and in the cerebral cortex, as well as in the thalamus and midbrain. The medulla oblongata did not show a prominent involvement (Table S1).
The correlation between histological lesions as assessed by lesion profiling and the presence of PrP Sc evaluated by PrP Sc profiling was not striking. Although brain regions with abundant PrP Sc tend to show severe histopathological lesions, the reverse does not always seem to be the case. Brain regions with massive histopathological lesions (i.e., cortical regions in VV2 patients) show only scarce depositions of PrP Sc .
The comparison of lesion profiles to PrP Sc profiles demonstrates that the patterns obtained within the abovementioned groups are only roughly concurrent. Whereas PrP Sc profiles show marked differences between investigated regions, lesion profiles tend to show a more homogeneous picture with fewer marked regional differences.
In cases in which multiple PrP Sc types could be observed, we investigated whether the predominant deposition pattern of PrP Sc was associated with a particular biochemical type of PrP Sc . Areas with a predominance of PrP Sc type 1 (;21-kDa unglycosylated band of PrP Sc ) displayed diffuse, synaptic PrP immunoreactivity, whereas areas with a PrP Sc type 2 (;19-kDa unglycosylated band of PrP Sc ), showed mainly perivacuolar or plaque-like deposits of PrP (Figure 3).

Discussion
The precise classification of sCJD by molecular, histopathological, and clinical parameters is important for understanding the underlying pathogenesis, unraveling possible etiological causes, and devising efficient therapeutic protocols. Several classification systems for human prion diseases have been proposed in the past [8,13]. Although the exact number of molecular subtypes is still a matter of debate, there is a consensus that these may be differentiated on the basis of clinical data, histopathological analysis, codon-129 status of PRNP, and biochemical analysis of PrP Sc . The approach described here represents a new method for the discrimination of molecular subtypes of human prion diseases. By quantifying PrP Sc in nine brain regions of patients with sCJD, we were able to determine the PrP Sc profile of 50 patients with sCJD. Because individual measurements are calibrated to internal standards, this method allows, for the first time, for absolute quantification of PrP Sc in prion-diseased individuals, and therefore enables the direct comparison of individual PrP Sc profiles, excluding inter-observer and methodological variations. Detailed analysis of different molecular subtypes of sCJD confirmed the feasibility of this method and showed subtype-specific differences in the regional distribution of PrP Sc . Besides advancing the molecular analysis of sCJD, these data demonstrate that if confirmation of sCJD by brain biopsy is indispensable, it should only be carried out taking into account the sCJD subtype. In patients belonging to the MM1 groups [8], high amounts of PrP Sc are present in all cortical areas and would favor a cortical biopsy site. In patients belonging to the VV2 group [8], minimal amounts of PrP Sc in the cerebral cortex would designate the cerebellum or the thalamus as sites for successful confirmation of the disease by biopsy.
An earlier study that attempted to assess the distribution of PrP Sc within the central nervous system was performed only on a small patient cohort and did not employ internal PrP Sc standards-thus impeding the direct comparison of analyses [16]. In other studies, lesion profiles were based on histological scores [8,13,17]. Although this histological, score-based method has certain advantages and can be carried out on fixed tissue, we believe that PrP Sc profiling will be a valuable tool for prion research. Since both our work and other studies show that there is no strict correlation between histopathological alterations and the presence of PrP Sc , one could assume that PrP Sc profiling is superior since it measures PrP Sc , which is believed to constitute an essential component of prion infectivity [4,16].
Even though experiments in rodents performed some years ago demonstrated the phenomenon of multiple PrP Sc types within one animal [21], incongruity regarding the frequency of co-occurrence of different PrP Sc types in sCJD patients still endures. Initial studies suggested that this is a relatively rare event, occurring in less than 5% of patients [8]. This low incidence may, at least partially, be attributed to the fact that these analyses are routinely performed on a limited range of distinct brain regions per patient [8,13]. The fact that regionspecific presence of distinct PrP Sc types may occur in sCJD was highlighted by a publication investigating ten defined regions within the central nervous system in 14 patients with sCJD [16]. The authors found more than one PrP Sc type in five individuals and hypothesized that the co-occurrence of more than one PrP Sc type could be the rule, rather than the exception, if the entire central nervous system was investigated. In our analysis of nine distinct central-nervoussystem regions in 50 patients with sCJD using a standardized protocol [22], we detected more than one PrP Sc type in nine individuals. The observation that most patients harboring two PrP Sc types are codon-129 methionine/valine heterozygotes stresses the significance of this polymorphism in the replicative cycle of PrP Sc .
Analysis of the spatial distribution of PrP Sc types revealed that the putamen, thalamus, medulla oblongata, and cerebellum are prone to accommodate multiple PrP Sc types simultaneously, whereas this was rarely observed in the cerebral cortex. The fact that central nervous system regions which simultaneously harbor two PrP Sc types are complex structures containing diverse subsets of neuronal cells indicates that susceptibility of target cells may, at least partially, determine the type of PrP Sc that is produced. This finding is compatible with the target-cell hypothesis of prionstrain diversity [23,24].
In conclusion, the present study has introduced a new method for the characterization of human prion diseases. The approach described here will aid in understanding the molecular diversity of this disease entity. Furthermore, these data imply that the subsets of cells present in specific brain areas in concert with the polymorphism on codon 129 of PRNP may have a fundamental role in determining which PrP Sc type is preferentially replicated. Table S1. Histological Assessment of the VV2 Subgroup of Patients Cases 1 to 4: spongiosis is scored on a scale from 0-4; astrogliosis and PrP immunoreactivity are scored on a scale from 0-3.