Allelic origin of protease-sensitive and protease-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease with the P102L mutation

Gerstmann-Str?ussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP(Sc), consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrP(Sc) isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrP(Sc) has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrP(Sc) allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrP(Sc) molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrP(Sc) quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.     

Aberrant metal binding by prion protein in human prion disease

Human prion diseases are characterized by the conversion of the normal prion protein (PrP(C)) into a pathogenic isomer (PrP(Sc)). Distinct PrP(Sc) conformers are associated with different subtypes of prion diseases. PrP(C) binds copper and has antioxidation activity. Changes in metal-ion occupancy can lead to significant decline of the antioxidation activity and changes in conformation of the protein. We studied the trace element status of brains from patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found a decrease of up to 50% of copper and an increase in manganese of approximately 10-fold in the brain tissues from sCJD subjects. We have also studied the metal occupancy of PrP in sCJD patients. We observed striking elevation of manganese and, to a lesser extent, of zinc accompanied by significant reduction of copper bound to purified PrP in all sCJD variants, determined by the PrP genotype and PrP(Sc) type, combined. Both zinc and manganese were undetectable in PrP(C) preparations from controls. Copper and manganese changes were pronounced in sCJD subjects homozygous for methionine at codon 129 and carrying PrP(Sc) type-1. Anti-oxidation activity of purified PrP was dramatically reduced by up to 85% in the sCJD variants, and correlated with increased in oxidative stress markers in sCJD brains. These results suggest that altered metal-ion occupancy of PrP plays a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sCJD variants, they may contribute to the diversity of PrP(Sc) and disease phenotype in sCJD. Finally, this study also presented two potential approaches in the diagnosis of CJD; the significant increase in brain manganese makes it potentially detectable by MRI, and the binding of manganese by PrP in sCJD might represent a novel diagnostic marker.     

Beyond PrP9res) 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 (PrP(res)) identified on Western blotting (type 1 or type 2). These biochemically distinct PrP(res) types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrP(res) 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 PrP(res) and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrP(Sc)) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrP(res) were identified. Despite this, the other two biochemical assays found that PrP(Sc) from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrP(Sc) 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 PrP(res) pattern. The identification of four different PrP(Sc) biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrP(res) isoform provides an alternative biochemical definition of PrP(Sc) diversity and new insight in the perception of Human TSE agents variability.     

pH-dependent prion protein conformation in classical Creutzfeldt-Jakob disease.

In transmissible spongiform encephalopathies, the cellular prion protein (PrP(C)) undergoes a conformational change from a prevailing alpha-helical structure to a beta-sheet-rich, protease-resistant isoform, termed PrP(Sc). PrP(C) has two characteristics: a high affinity for Cu(2+) and a strong pH-dependent conformation. Lines of evidence indicate that PrP(Sc) conformation is dependent on copper and that acidic conditions facilitate the conversion of PrP(C) --> PrP(Sc). In each species, PrP(Sc) exists in multiple conformations, which are associated with different prion strains. In sporadic Creutzfeldt-Jakob disease (sCJD), different biochemical types of PrP(Sc) have been identified according to the size of the protease-resistant fragments, patterns of glycosylation, and the metal-ion occupancy. Based on the site of cleavage produced by proteinase K, we investigated the conformational stability of PrP(Sc) under acidic, neutral, and basic conditions in 42 sCJD subjects. Our study shows that only one type of sCJD PrP(Sc), associated with the classical form, shows a pH-dependent conformation, whereas two other biochemical PrP(Sc) types, detected in distinct sCJD phenotypes, are unaffected by pH variations. This novel approach demonstrates the presence of three types of PrP(Sc) in sCJD.     

Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC

The mammalian prions replicate by converting cellular prion protein (PrP^C) into pathogenic conformational isoform (PrP^Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP^Sc on conversion of PrP^C in vitro using PrP^Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP^Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP^Sc. The tight correlation between conversion potency of small oligomers of human sPrP^Sc observed in vitro and duration of the disease suggests that sPrP^Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.     

Biochemical and strain properties of CJD prions: complexity versus simplicity

Prions, the agents responsible for transmissible spongiform encephalopathies, are infectious proteins consisting primarily of scrapie prion protein (PrP(Sc)), a misfolded, β-sheet enriched and aggregated form of the host-encoded cellular prion protein (PrP(C)). Their propagation is based on an autocatalytic PrP conversion process. Despite the lack of a nucleic acid genome, different prion strains have been isolated from animal diseases. Increasing evidence supports the view that strain-specific properties may be enciphered within conformational variations of PrP(Sc). In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most frequent form of prion diseases and has demonstrated a wide phenotypic and molecular spectrum. In contrast, variant Creutzfeldt-Jakob disease (vCJD), which results from oral exposure to the agent of bovine spongiform encephalopathy, is a highly stereotyped disease, that, until now, has only occurred in patients who are methionine homozygous at codon 129 of the PrP gene. Recent research has provided consistent evidence of strain diversity in sCJD and also, unexpectedly enough, in vCJD. Here, we discuss the puzzling biochemical/pathological diversity of human prion disorders and the relationship of that diversity to the biological properties of the agent as demonstrated by strain typing in experimental models.     

Novel differences between two human prion strains revealed by two-dimensional gel electrophoresis

The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP(Sc)), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrP(Sc) type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrP(Sc) in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrP(Sc) form is overrepresented and carries glycans that are different from those present in the PrP(Sc) of sFI. Because the altered glycans are detectable only in the PrP(Sc) and not in the normal or cellular PrP (PrP(C)), they are likely to result from preferential conversion to PrP(Sc) of rare PrP(C) glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.     

Prion protein with an E200K mutation displays properties similar to those of the cellular isoform PrP(C)

Creutzfeldt-Jakob disease (CJD) in Libyan Jews, linked to the E200K mutation in PRNP (E200KCJD), is the most prevalent of the inherited prion diseases. As other prion diseases, E200KCJD is characterized by the brain accumulation of PrP(Sc), a pathologic conformational isoform of a normal glycoprotein denominated PrP(C). To investigate whether the E200K mutation is enough to de novo confer PrP(Sc) properties to mutant PrP, as suggested by experiments in Chinese hamster ovary cells, we examined the biochemical behavior of E200KPrP in brains and fibroblasts from sporadic as well as homozygous and heterozygous E200KCJD patients, asymptomatic transgenic mice carrying the E200K mutation, as well as in normal and scrapie-infected mouse neuroblastoma cells expressing E200KPrP. E200KPrP was examined for protease sensitivity, solubility in detergents, releasibility by phosphoinositol phospholypase-C and localization in cholesterol enriched membrane microdomains (rafts). In all tissues except in brains of CJD patients and ScN2a cells, E200KPrP displayed properties similar to those of PrP(C). Our results indicate that the E200K mutation does not automatically convey the properties of PrP(Sc) to new PrP molecules. A conversion process occurs mainly in the prion disease affected brain, suggesting the presence of a tissue-specific or age-dependent factor, in accord with the late onset nature of inherited CJD.     

Identification of distinct N-terminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes

In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules.     

Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes

The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.     

Protease-resistant prions selectively decrease Shadoo protein

The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc) causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho), a protein that resembles the flexibly disordered N-terminal domain of PrP(C), were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrP(Sc) in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc). Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc). Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc) during prion disease.     

Identification of I137M and other mutations that modulate incubation periods for two human prion strains

We report here the transmission of human prions to 18 new transgenic (Tg) mouse lines expressing 8 unique chimeric human/mouse prion proteins (PrP). Extracts from brains of two patients, who died of sporadic Creutzfeldt-Jakob disease (sCJD), contained either sCJD(MM1) or sCJD(VV2) prion strains and were used for inocula. Mice expressing chimeric PrP showed a direct correlation between expression level and incubation period for sCJD(MM1) prions irrespective of whether the transgene encoded methionine (M) or valine (V) at polymorphic residue 129. Tg mice expressing chimeric transgenes encoding V129 were unexpectedly resistant to infection with sCJD(VV2) prions, and when transmission did occur, it was accompanied by a change in strain type. The transmission of sCJD(MM1) prions was modulated by single amino acid reversions of each human PrP residue in the chimeric sequence. Reverting human residue 137 in the chimeric transgene from I to M prolonged the incubation time for sCJD(MM1) prions by more than 100 days; structural analyses suggest a profound change in the orientation of amino acid side chains with the I→M mutation. These findings argue that changing the surface charge in this region of PrP greatly altered the interaction between PrP isoforms during prion replication. Our studies contend that strain-specified replication of prions is modulated by PrP sequence-specific interactions between the prion precursor PrP(C) and the infectious product PrP(Sc).     

Allelic Origin of Protease-Sensitive and Protease-Resistant Prion Protein Isoforms in Gerstmann-Str?ussler-Scheinker Disease with the P102L Mutation

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP^Sc, consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrP^Sc isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrP^Sc has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrP^Sc allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrP^Sc molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrP^Sc quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.     

PRNP contains both intronic and upstream regulatory regions that may influence susceptibility to Creutzfeldt-Jakob Disease

The Prion protein (PrP) plays a central role in Creutzfeldt-Jakob Disease (CJD) and other transmissible spongiform encephalopathies (TSEs). Mutations in the protein coding region of the human PrP gene (PRNP), which have been proposed to alter the stability of the PrP protein, have been linked to a number of forms of TSE. However, the majority of CJD cases are not associated with mutations in the PRNP coding region and alternative mechanisms must therefore underlie susceptibility to these forms of CJD. Transgenic mice, that over- or under-express PrP genes, have shown a correlation between the level of PrP gene expression and the incubation time of disease. Polymorphisms that lead to alterations in human PRNP gene expression, could therefore be candidates for influencing susceptibility of an individual to CJD. In order to investigate this hypothesis, we have defined an upstream and intronic regulatory region of the PRNP gene. Sequencing of these regions in controls, sporadic CJD (sCJD) and variant CJD (vCJD) patients has identified three polymorphisms, all of which are more common in sCJD patients than controls. Our data suggests that polymorphisms in the regulatory region of the PRNP gene may be a risk factor for CJD.     

GFP-tagged PrP supports compromised prion replication in transgenic mice

The ability of green fluorescent protein (GFP)-prion protein (PrP) fusions to support prion propagation has not been demonstrated. Here, we show that while transgenic mice expressing PrP tagged at its amino terminus with enhanced GFP, referred to as EGFPrP-N, supported prion replication, disease onset was prolonged, the brains of diseased mice did not exhibit typical disease neuropathology and disease-associated EGFPrP-N lacked the full spectrum of biochemical properties normally associated with PrP(Sc). Co-expression of wild-type PrP and EGFPrP-N substantially reduced prion incubation times and resulted in accumulation of protease-resistant EGFPrP(Sc)-N in the brains of transgenic mice as well as chronically infected cultured cells, suggesting that wild-type PrP rescued a compromised amino terminal function in EGFPrP-N. While our results show that EGFPrP(C)-N adopts a conformation necessary for the production of infectious prions, the synergistic interaction of wild-type and EGFPrP-N underscores the importance of the amino terminus in modulating prion pathogenesis.     

The strain-encoded relationship between PrP replication, stability and processing in neurons is predictive of the incubation period of disease

Prion strains are characterized by differences in the outcome of disease, most notably incubation period and neuropathological features. While it is established that the disease specific isoform of the prion protein, PrP(Sc), is an essential component of the infectious agent, the strain-specific relationship between PrP(Sc) properties and the biological features of the resulting disease is not clear. To investigate this relationship, we examined the amplification efficiency and conformational stability of PrP(Sc) from eight hamster-adapted prion strains and compared it to the resulting incubation period of disease and processing of PrP(Sc) in neurons and glia. We found that short incubation period strains were characterized by more efficient PrP(Sc) amplification and higher PrP(Sc) conformational stabilities compared to long incubation period strains. In the CNS, the short incubation period strains were characterized by the accumulation of N-terminally truncated PrP(Sc) in the soma of neurons, astrocytes and microglia in contrast to long incubation period strains where PrP(Sc) did not accumulate to detectable levels in the soma of neurons but was detected in glia similar to short incubation period strains. These results are inconsistent with the hypothesis that a decrease in conformational stability results in a corresponding increase in replication efficiency and suggest that glia mediated neurodegeneration results in longer survival times compared to direct replication of PrP(Sc) in neurons.     

Cross-sequence transmission of sporadic Creutzfeldt-Jakob disease creates a new prion strain

The genotype (methionine or valine) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of abnormal isoform of PrP (PrP(Sc)) are major determinants of the clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD). Here we found that the transmission of sCJD prions from a patient with valine homozygosity (129V/V) and type 2 PrP(Sc) (sCJD-VV2 prions) to mice expressing human PrP with methionine homozygosity (129M/M) generated unusual PrP(Sc) intermediate in size between type 1 and type 2. The intermediate type PrP(Sc) was seen in all examined dura mater graft-associated CJD cases with 129M/M and plaque-type PrP deposits (p-dCJD). p-dCJD prions and sCJD-VV2 prions exhibited similar transmissibility and neuropathology, and the identical type of PrP(Sc) when inoculated into PrP-humanized mice with 129M/M or 129V/V. These findings suggest that p-dCJD could be caused by cross-sequence transmission of sCJD-VV2 prions.     

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrP^Sc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion.

Case presentation

We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrP^Sc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrP^Sc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine.

Conclusions

A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

     

Chronic wasting disease of elk and deer and Creutzfeldt-Jakob disease: comparative analysis of the scrapie prion protein

Chronic wasting disease (CWD), a transmissible prion disease that affects elk and deer, poses new challenges to animal and human health. Although the transmission of CWD to humans has not been proven, it remains a possibility. If this were to occur, it is important to know whether the "acquired" human prion disease would show a phenotype including the scrapie prion protein (PrP(Sc)) features that differ from those associated with human sporadic prion disease. In this study, we have compared the pathological profiles and PrP(Sc) characteristics in brains of CWD-affected elk and deer with those in subjects with sporadic Creutzfeldt-Jakob disease (CJD), as well as CJD-affected subjects who might have been exposed to CWD, using histopathology, immunohistochemistry, immunoblotting, conformation stability assay, and N-terminal protein sequencing. Spongiform changes and intense PrP(Sc) staining were present in several brain regions of CWD-affected animals. Immunoblotting revealed three proteinase K (PK)-resistant bands in CWD, representing different glycoforms of PrP(Sc). The unglycosylated PK-resistant PrP(Sc) of CWD migrated at 21 kDa with an electrophoretic mobility similar to that of type 1 human PrP(Sc) present in sporadic CJD affecting subjects homozygous for methionine at codon 129 (sCJDMM1). N-terminal sequencing showed that the PK cleavage site of PrP(Sc) in CWD occurred at residues 82 and 78, similar to that of PrP(Sc) in sCJDMM1. Conformation stability assay also showed no significant difference between elk CWD PrP(Sc) and the PrP(Sc) species associated with sCJDMM1. However, there was a major difference in glycoform ratio of PrP(Sc) between CWD and sCJDMM1 affecting both subjects potentially exposed to CWD and non-exposed subjects. Moreover, PrP(Sc) of CWD exhibited a distinct constellation of glycoforms distinguishable from that of sCJDMM1 in two-dimensional immunoblots. These findings underline the importance of detailed PrP(Sc) characterization in trying to detect novel forms of acquired prion disease.     

Preclinical deposition of pathological prion protein in muscle of experimentally infected primates

Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and animals. A central step in disease progression is the accumulation of a misfolded form (PrP(Sc)) of the host encoded prion protein (PrP(C)) in neuronal and non-neuronal tissues. The involvement of peripheral tissues in preclinical states increases the risk of accidental transmission. On the other hand, detection of PrP(Sc) in non-neuronal easy-accessible compartments such as muscle may offer a novel diagnostic tool. Primate models have proven invaluable to investigate prion diseases. We have studied the deposition of PrP(Sc) in muscle and central nervous system of rhesus monkeys challenged with sporadic Creutzfeldt-Jakob disease (sCJD), variant CJD (vCJD) and bovine spongiform encephalopathy (BSE) in preclinical and clinical stage using biochemical and morphological methods. Here, we show the preclinical presence of PrP(Sc) in muscle and central nervous system of rhesus monkeys experimentally infected with vCJD.