Five phenotypically distinct subtypes have been identified in sporadic Creutzfeldt–Jakob disease (sCJD), based on the methionine/valine polymorphic genotype of codon 129 of the prion protein (PrP) gene and the presence of either one of the two protease K-resistant scrapie prion protein (PrPSc) types identified as 1 and 2. The infrequent co-existence of both PrPSc types in the same case has been known for a long time. Recently, it has been reported, using type-specific antibodies, that the PrPSc type 1 is present in all cases of sCJD carrying PrPSc type 2. The consistent co-occurrence of both PrPSc types complicates the diagnosis and the current classification of sCJD, and has implications for the pathogenesis of naturally occurring prion diseases. In the present study, we investigated the prevalence of PrPSc types 1 and 2 co-occurrence, along with its effects on the disease phenotype and PrPSc strain characteristics, comparatively analysing 34 cases of sCJD, all methionine homozygous at codon 129 of the PrP gene (sCJDMM). To minimize overestimating the prevalence of the sCJDMM cases carrying PrPSc types 1 and 2 (sCJDMM1-2), we used proteinase K concentrations designed to hydrolyse all fragments resulting from an incomplete digestion, while preserving the protease-resistant PrPSc core. Furthermore, we used several antibodies to maximize the detection of both PrPSc types. Our data show that sCJDMM cases associated exclusively with either PrPSc type 1 (sCJDMM1) or PrPSc type 2 (sCJDMM2) do exist; we estimate that they account for approximately 56% and 5% of all the sCJDMM cases, respectively; while in 39% of the cases, both PrPSc types 1 and 2 are present together (sCJDMM1-2) either mixed in the same anatomical region or separate in different regions. Clinically, sCJDMM1-2 had an average disease duration intermediate between the other two sCJDMM subtypes. The histopathology was also intermediate, except for the cerebellum where it resembled that of sCJDMM1. These features, along with the PrP immunostaining pattern, offer a diagnostic clue. We also observed a correlation between the disease duration and the prevalence of PrPSc type 2 and sCJDMM2 phenotypes. The use of different antibodies and of the conformational stability immunoassay indicated that the co-existence of types 1 and 2 in the same anatomical region may confer special conformational characteristics to PrPSc types 1 and 2. All of these findings indicate that sCJDMM1-2 should be considered as a separate entity at this time.
prion protein; prion disease; co-existence; conformation; sporadic Creutzfeldt–Jakob disease
The origin, range, and structure of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD), are largely unknown. To investigate the molecular mechanism responsible for the broad phenotypic variability of sCJD, we analyzed the conformational characteristics of protease-sensitive and protease-resistant fractions of the pathogenic prion protein (PrPSc) using novel conformational methods derived from a conformation-dependent immunoassay (CDI). In 46 brains of patients homozygous for polymorphisms in the PRNP gene and exhibiting either Type 1 or Type 2 western blot pattern of the PrPSc, we identified an extensive array of PrPSc structures that differ in protease sensitivity, display of critical domains, and conformational stability. Surprisingly, in sCJD cases homozygous for methionine or valine at codon 129 of the PRNP gene, the concentration and stability of protease-sensitive conformers of PrPSc correlated with progression rate of the disease. These data indicate that sCJD brains exhibit a wide spectrum of PrPSc structural states, and accordingly argue for a broad spectrum of prion strains coding for different phenotypes. The link between disease duration, levels, and stability of protease-sensitive conformers of PrPSc suggests that these conformers play an important role in the pathogenesis of sCJD.
Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common human prion disease worldwide. This neurodegenerative disease, which is transmissible and invariably fatal, is characterized by the accumulation of an abnormally folded isoform (PrPSc) of a host-encoded protein (PrPC), predominantly in the brain. Most researchers believe that PrPSc is the infectious agent and five or six subtypes of sCJD have been identified. Whether or not these subtypes represent distinct strains of sCJD prions is debated in the context of the extraordinary variability of sCJD phenotypes, frequent co-occurrence of different PrPSc fragments in the same brain, and the fact that up to 90% of protease-sensitive PrPSc eludes the conventional analysis because it is destroyed by protease treatment. Using novel conformational methods, we identified within each clinical and pathological category an array of PrPSc structures that differ in protease-sensitivity, display of critical domains, and conformational stability. Each of these features offers evidence of a distinct conformation. The link between the rate at which the disease progresses, on the one hand, and the concentration and stability of protease-sensitive conformers of PrPSc on the other, suggests that these conformers play an important role in how the disease originates and progresses.
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.
Prion diseases are transmissible neurodegenerative disorders characterized by accumulation of an abnormal isoform (PrPSc) of a host-encoded protein (PrPC) in affected tissues. According to the prion hypothesis, PrPSc 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 PrPSc 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 PrPSc phenotype could be established in iCJD cases. This study provides an alternative biochemical definition of PrPSc diversity in human prion diseases and new insights into the perception of agent variability.
Sporadic Creutzfeldt-Jakob disease is classified according to the genotype at polymorphic codon 129 (M or V) of the prion protein (PrP) gene and the type (1 or 2) of abnormal isoform of PrP (PrPSc) in the brain. The most complicated entity in the current classification system is MV2, since it shows wide phenotypic variations, i.e., MV2 cortical form (MV2C), MV2 with kuru plaques (MV2K), or a mixed form (MV2K + C). To resolve their complicated pathogenesis, we performed a comprehensive analysis of the three MV2 subgroups based on histopathological, molecular, and transmission properties.
In histopathological and molecular analyses, MV2C showed close similarity to MM2 cortical form (MM2C) and could be easily discriminated from the other MV2 subgroups. By contrast, MV2K and MV2K + C showed the same molecular type and the same transmission type, and the sole difference between MV2K and MV2K + C was the presence of cortical pathology characteristic of MV2C/MM2C. The remarkable molecular feature of MV2K or MV2K + C was a mixture of type 2 PrPSc and intermediate type PrPSc, which shows intermediate electrophoretic mobility between types 1 and 2 PrPSc. Modeling experiments using PrP-humanized mice indicated that MV2K contains a mixture of intermediate type PrPSc with the 129M genotype (Mi PrPSc) and type 2 PrPSc with the 129V genotype (V2 PrPSc) that originated from V2 PrPSc, whereas MV2C + K may also contain type 2 PrPSc with the 129M genotype and cortical pathology (M2C PrPSc) that lacks infectivity to the PrP-humanized mice in addition to Mi and V2 PrPSc.
Taken together, the present study suggests that the phenotypic heterogeneity of MV2 stems from their different PrPSc origin(s).
Creutzfeldt-Jakob disease; Prion protein; Classification; Humanized knock-in mouse
The current classification of human sporadic prion diseases recognizes six major phenotypic subtypes with distinctive clinicopathological features, which largely correlate at the molecular level with the genotype at the polymorphic codon 129 (methionine, M, or valine, V) in the prion protein gene and with the size of the protease-resistant core of the abnormal prion protein, PrPSc (i.e. type 1 migrating at 21 kDa and type 2 at 19 kDa). We previously demonstrated that PrPSc typing by Western blotting is a reliable means of strain typing and disease classification. Limitations of this approach, however, particularly in the interlaboratory setting, are the association of PrPSc types 1 or 2 with more than one clinicopathological phenotype, which precludes definitive case classification if not supported by further analysis, and the difficulty of fully recognizing cases with mixed phenotypic features. In this study, we tested the inter-rater reliability of disease classification based only on histopathological criteria. Slides from 21 cases covering the whole phenotypic spectrum of human sporadic prion diseases, and also including two cases of variant Creutzfeldt–Jakob disease (CJD), were distributed blindly to 13 assessors for classification according to given instructions. The results showed good-to-excellent agreement between assessors in the classification of cases. In particular, there was full agreement (100 %) for the two most common sporadic CJD subtypes and variant CJD, and very high concordance in general for all pure phenotypes and the most common subtype with mixed phenotypic features. The present data fully support the basis for the current classification of sporadic human prion diseases and indicate that, besides molecular PrPSc typing, histopathological analysis permits reliable disease classification with high interlaboratory accuracy.
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).
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.
Internal standards allow meaningful comparison of PrPSc profiles from different origins. Applying the new method to 50 postmortem samples from patients with sporadic CJD reveals substantial differences in molecular pathology.
The mammalian prions replicate by converting cellular prion protein (PrPC) into pathogenic conformational isoform (PrPSc). 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 PrPSc on conversion of PrPC in vitro using PrPSc 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) PrPSc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrPSc. The tight correlation between conversion potency of small oligomers of human sPrPSc observed in vitro and duration of the disease suggests that sPrPSc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.
Mammalian prion diseases were originally characterized by accumulation of protease-resistant prion protein (PrPSc), often forming large amyloid deposits and fibrils. However, the apparent absence of protease-resistant PrPSc or amyloid fibrils in growing number of prion diseases raised several fundamental questions; specifically, whether presumably protease-sensitive forms of PrPSc exist as distinct conformers; and whether they comprise the initial steps in prion replication or are related to the alternative misfolding pathway generating noninfectious aggregates. We investigated the conformational characteristics of protease sensitive conformers of PrPSc and their role in the pathogenesis of sporadic Creutzfeldt-Jakob disease (sCJD). Using two different in vitro prion protein (PrPC) conversion techniques in tandem with biophysical methods, we identified small oligomers of protease sensitive PrPSc present in sCJD brains as the most potent initiators of PrPC conversion. Their concentration and conformational stability determine the distinctly different replication potency of PrPSc in individual isolates of sCJD and each of these characteristics correlates tightly with duration of the disease. These features argue for a broad range of distinct prion strains causing the sCJD and imply that small oligomers of protease sensitive conformers of pathogenic prion protein are encoding incubation time and progression rate of the disease.
Variably protease sensitive prionopathy (VPSPr) is a recently described, sporadic human prion disease that is pathologically and biochemically distinct from the currently recognised sporadic Creutzfeldt-Jakob disease (sCJD) subtypes. The defining biochemical features of the abnormal form of the prion protein (PrPSc) in VPSPr are increased sensitivity to proteolysis and the presence of an N- and C-terminally cleaved ~8 kDa protease resistant PrPSc (PrPres) fragment. The biochemical and neuropathological profile of VPSPr has been proposed to resemble either Gerstmann–Sträussler–Scheinker syndrome (GSS) or familial CJD with the PRNP-V180I mutation. However, in some cases of VPSPr two protease resistant bands have been observed in Western blots that co-migrate with those of type 2 PrPres, suggesting that a proportion of the PrPSc present in VPSPr has properties similar to those of sCJD.
Here, we have used conformation dependent immunoassay to confirm the presence of PrPSc in VPSPr that is more protease sensitive compared with sCJD. However, CDI also shows that a proportion of PrPSc in VPSPr resists PK digestion of its C-terminus, distinguishing it from GSS associated with ~8 kDa PrPres, and showing similarity to sCJD. Intensive investigation of a single VPSPr case with frozen tissue from multiple brain regions shows a broad, region-specific spectrum of protease sensitivity and differential stability of PrPSc in the absence of PK treatment. Finally, using protein misfolding cyclic amplification and real-time quaking induced conversion, we show that VPSPr PrPSc has the potential to seed conversion in vitro and that seeding activity is dispersed through a broad range of aggregate sizes. We further propose that seeding activity is associated with the ~19 and ~23 kDa PrPres rather than the ~8 kDa fragment.
Therefore, PrPSc in VPSPr is heterogeneous in terms of protease sensitivity and stability to denaturation with the chaotrope GdnHCl and includes a proportion with similar properties to that found in sCJD.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-014-0152-4) contains supplementary material, which is available to authorized users.
Variably protease resistant prionopathy; VPSPr; Prion; PrP; PrPSc; Gerstmann–Sträussler–Scheinker syndrome (GSS); PMCA; RT-QuIC
Prion diseases are classically characterized by the accumulation of pathological prion protein (PrPSc) with the protease resistant C-terminal fragment (PrPres) of 27–30 kDa. However, in both humans and animals, prion diseases with atypical biochemical features, characterized by PK-resistant PrP internal fragments (PrPres) cleaved at both the N and C termini, have been described. In this study we performed a detailed comparison of the biochemical features of PrPSc from atypical prion diseases including human Gerstmann-Sträussler-Scheinker disease (GSS) and variably protease-sensitive prionopathy (VPSPr) and in small ruminant Nor98 or atypical scrapie. The kinetics of PrPres production and its cleavage sites after PK digestion were analyzed, along with the PrPSc conformational stability, using a new method able to characterize both protease-resistant and protease-sensitive PrPSc components. All these PrPSc types shared common and distinctive biochemical features compared to PrPSc from classical prion diseases such as sporadic Creutzfeldt-Jakob disease and scrapie. Notwithstanding, distinct biochemical signatures based on PrPres cleavage sites and PrPSc conformational stability were identified in GSS A117V, GSS F198S, GSS P102L and VPSPr, which allowed their specific identification. Importantly, the biochemical properties of PrPSc from Nor98 and GSS P102L largely overlapped, but were distinct from the other human prions investigated. Finally, our study paves the way towards more refined comparative approaches to the characterization of prions at the animal–human interface.
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 (PrPSc) 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.
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 PrPSc 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 PrPSc 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.
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.
Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrPC) converts into a misfolded isoform (PrPSc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrPC; in older flies, PrP misfolds, acquires biochemical and structural properties of PrPSc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrPSc-specific conformational epitopes. In contrast to PrPSc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrPSc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrPSc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrPSc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.
Creutzfeldt-Jakob disease is a type of dementia caused by the deposition of the prion protein in the brain. This disorder belongs to a unique class of degenerative diseases that includes mad-cow disease in bovine and scrapie in sheep. An abnormal form of the prion protein is not only responsible for the disease in several mammals, but is also an infectious agent that can transmit the disease within or across species. To shed light on how the prion protein changes from its normal to the disease-causing form, we expressed the prion protein from hamster in transgenic flies. We observed that the prion protein progressively converts to the pathological form and induces neuronal loss in the brain. Thus, the prion protein experiences its typical transition from normal to disease-causing form in flies. This behavior gave us the opportunity to investigate whether other proteins can regulate such transition. We found that the stress-related protein Hsp70 prevents the accumulation of abnormal prion protein and prevents neuronal loss. We also determined that Hsp70 directly interacts with the prion protein in specific membrane domains. Overall, our studies provide new insight into the mechanisms that regulate the accumulation of abnormal prion protein. This discovery could have therapeutic applications in treating these devastating disorders.
Prions, the infectious agents responsible for transmissible spongiform encephalopathies, consist mainly of the misfolded prion protein (PrPSc). The unique mechanism of transmission and the appearance of a variant form of Creutzfeldt–Jakob disease, which has been linked to consumption of prion-contaminated cattle meat, have raised concerns about public health. Evidence suggests that variant Creutzfeldt–Jakob disease prions circulate in body fluids from people in whom the disease is silently incubating.
To investigate whether PrPSc can be detected in the urine of patients with variant Creutzfeldt–Jakob disease, we used the protein misfolding cyclic amplification (PMCA) technique to amplify minute quantities of PrPSc, enabling highly sensitive detection of the protein. We analyzed urine samples from several patients with various transmissible spongiform encephalopathies (variant and sporadic Creutzfeldt–Jakob disease and genetic forms of prion disease), patients with other degenerative or nondegenerative neurologic disorders, and healthy persons.
PrPSc was detectable only in the urine of patients with variant Creutzfeldt–Jakob disease and had the typical electrophoretic profile associated with this disease. PrPSc was detected in 13 of 14 urine samples obtained from patients with variant Creutzfeldt–Jakob disease and in none of the 224 urine samples obtained from patients with other neurologic diseases and from healthy controls, resulting in an estimated sensitivity of 92.9% (95% confidence interval [CI], 66.1 to 99.8) and a specificity of 100.0% (95% CI, 98.4 to 100.0). The PrPSc concentration in urine calculated by means of quantitative PMCA was estimated at 1×10−16 g per milliliter, or 3×10−21 mol per milliliter, which extrapolates to approximately 40 to 100 oligomeric particles of PrPSc per milliliter of urine.
Urine samples obtained from patients with variant Creutzfeldt–Jakob disease contained minute quantities of PrPSc. (Funded by the National Institutes of Health and others.)
Interspecies transmission of the transmissible spongiform encephalopathies (TSEs), or prion diseases, can result in the adaptation and selection of TSE strains with an expanded host range and increased virulence such as in the case of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease. To investigate TSE strain adaptation, we serially passaged a biological clone of transmissible mink encephalopathy (TME) into Syrian golden hamsters and examined the selection of distinct strain phenotypes and conformations of the disease-specific isoform of the prion protein (PrPSc). The long-incubation-period drowsy (DY) TME strain was the predominate strain, based on the presence of its strain-specific PrPSc following interspecies passage. Additional serial passages in hamsters resulted in the selection of the hyper (HY) TME PrPSc strain-dependent conformation and its short incubation period phenotype unless the passages were performed with a low-dose inoculum (e.g., 10−5 dilution), in which case the DY TME clinical phenotype continued to predominate. For both TME strains, the PrPSc strain pattern preceded stabilization of the TME strain phenotype. These findings demonstrate that interspecies transmission of a single cloned TSE strain resulted in adaptation of at least two strain-associated PrPSc conformations that underwent selection until one type of PrPSc conformation and strain phenotype became predominant. To examine TME strain selection in the absence of host adaptation, hamsters were coinfected with hamster-adapted HY and DY TME. DY TME was able to interfere with the selection of the short-incubation HY TME phenotype. Coinfection could result in the DY TME phenotype and PrPSc conformation on first passage, but on subsequent passages, the disease pattern converted to HY TME. These findings indicate that during TSE strain adaptation, there is selection of a strain-specific PrPSc conformation that can determine the TSE strain phenotype.
Human prion diseases are fatal neurodegenerative disorders associated with an accumulation of PrPSc in the central nervous system (CNS). Of the human prion diseases, sporadic Creutzfeldt-Jakob disease (sCJD), which has no known origin, is the most common form while variant CJD (vCJD) is an acquired human prion disease reported to differ from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. Peripheral tissue involvement in prion disease, as judged by PrPSc accumulation in the tonsil, spleen, and lymph node has been reported in vCJD as well as several animal models of prion diseases. However, this distribution of PrPSc has not been consistently reported for sCJD. We reexamined CNS and non-CNS tissue distribution and levels of PrPSc in both sCJD and vCJD. Using a sensitive immunoassay, termed SOFIA, we also assessed PrPSc levels in human body fluids from sCJD as well as in vCJD-infected humanized transgenic mice (Tg666). Unexpectedly, the levels of PrPSc in non-CNS human tissues (spleens, lymph nodes, tonsils) from both sCJD and vCJD did not differ significantly and, as expected, were several logs lower than in the brain. Using protein misfolding cyclic amplification (PMCA) followed by SOFIA, PrPSc was detected in cerebrospinal fluid (CSF), but not in urine or blood, in sCJD patients. In addition, using PMCA and SOFIA, we demonstrated that blood from vCJD-infected Tg666 mice showing clinical disease contained prion disease-associated seeding activity although the data was not statistically significant likely due to the limited number of samples examined. These studies provide a comparison of PrPSc in sCJD vs. vCJD as well as analysis of body fluids. Further, these studies also provide circumstantial evidence that in human prion diseases, as in the animal prion diseases, a direct comparison and intraspecies correlation cannot be made between the levels of PrPSc and infectivity.
A national system for surveillance of prion diseases (PrDs) was established in Japan in April 1999. Here, we analyzed the relationships among prion protein gene (PRNP) mutations and the clinical features, cerebrospinal fluid (CSF) markers, and pathological characteristics of the major genotypes of genetic PrDs (gPrDs). We retrospectively analyzed age at onset and disease duration; the concentrations and incidences of 14-3-3 protein, tau protein, and abnormal prion protein (PrPSc) in the CSF of 309 gPrD patients with P102L, P105L, E200K, V180I, or M232R mutations; and brain pathology in 32 autopsied patients. Three clinical phenotypes were seen: rapidly progressive Creutzfeldt-Jakob disease (CJD), which included 100% of E200K cases, 70% of M232R, and 21% of P102L; slowly progressive CJD, which included 100% of V180I and 30% of M232R; and Gerstmann-Sträussler-Scheinker disease, which included 100% of P105L and 79% of P102L. PrPSc was detected in the CSF of more than 80% of patients with E200K, M232R, or P102L mutations but in only 39% of patients with V180I. V180I was accompanied by weak PrP immunoreactivity in the brain. Patients negative for PrPSc in the CSF were older at disease onset than positive patients. Patients with mutations associated with high 14-3-3 protein levels in the CSF typically had synaptic deposition of PrP in the brain and a rapid course of disease. The presence of small PrP protein fragments in brain homogenates was not correlated with other clinicopathological features. Positivity for PrPSc in the CSF may reflect the pathological process before or at disease onset, or abnormality in the secretion or metabolism of PrPSc. The amount of 14-3-3 protein in the CSF likely indicates the severity of the pathological process and accompanying neuronal damage. These characteristic features of the CSF in cases of gPrD will likely facilitate accurate diagnosis and clinicopathological study of the various disease subtypes.
Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrPSc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). Stable variations in PrPSc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrPSc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrPSc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrPSc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrPSc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrPSc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrPSc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.
Prions are infectious agents causing irremediably fatal neurodegenerative diseases in human and in farmed or wild animals. They are thought to be formed from abnormally folded assemblies (PrPSc) of the host-encoded prion protein (PrPC). Different PrPSc conformational variants associated with distinct biological phenotypes, or ‘strains,’ can propagate in the same host. To gain some structural information on the physical relationship between packing order (i.e. quaternary structure) and the strain-specific biological information, we previously subjected PrPSc assemblies from prion strains classified as fast or slow (according to their survival time in susceptible laboratory animals) to sedimentation velocity ultracentrifugation experiments. For the fast strains specifically, the most infectious assemblies sedimented slowly and partitioned from the bulk of PrPSc macromolecular complexes. By changing the solubilization and sedimentation conditions, we established here that a small PrPSc aggregation size and not a low density accounts for these hydrodynamic properties. We further showed that these small assemblies resist proteolytic digestion and outcompete by several orders of magnitude the larger-size assemblies in cell-free prion conversion assays. Thus PrPSc quaternary structure appears to be a determining factor of prion replication dynamics. For certain strains, a discrete subset of PrPSc assemblies appears to be the best template for prion replication.
Prion infectivity and its molecular marker, the pathological prion protein PrPSc, accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrPSc was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles). Here, we address the question of whether prions target the skin and show widespread PrPSc deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrPSc was detected before the onset of symptoms, but the bulk of skin-associated PrPSc accumulated in the clinical phase. PrPSc was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrPSc in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrPSc by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission.
Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases affecting the central nervous system. According to the prion hypothesis, TSEs are caused by proteinaceous infectious particles (“prions”) that consist essentially of PrPSc, an aberrant form of the prion protein with a pathologically altered folding and/or aggregation structure. Scrapie of sheep, chronic wasting disease (CWD) of deer, bovine spongiform encephalopathy (BSE) of cattle, and variant Creutzfeldt-Jakob disease (vCJD) of humans are prominent examples of acquired prion diseases. To further pinpoint the peripheral tissues that could serve as reservoirs of prions in the mammalian body and from which these pathogens could be potentially disseminated into the environment and transmitted to other individuals, we examined the skin of hamsters perorally challenged with scrapie and of naturally infected scrapie sheep for the presence of PrPSc. We show that PrPSc can accumulate in the skin at late stages of incubation, and that the protein is located primarily in small nerve fibres within this organ. The question of whether the skin may also provide a reservoir for prions in CWD, BSE, or vCJD, and the role of the skin in relation to the natural transmission of scrapie in the field needs further investigation.
Insertion of 144-base pair (bp) containing six extra octapeptide repeats between residues 51 and 91 of prion protein (PrP) gene is associated with inherited prion diseases. Most cases linked to this insertion examined by Western blotting showed detectable proteinase K-resistant PrPSc (rPrPSc) resembling PrPSc type 1 and type 2 in sporadic Creutzfeldt-Jakob disease (sCJD), or PrP7-8 in Gerstmann-Sträussler-Scheinker disease. However, cases lacking detectable rPrPSc also have been reported. Which PrP conformer is associated with neuropathological changes in the cases without detectable rPrPSc remains to be determined. Here we report that while all six but one subjects with the 144-bp insertion mutations examined display the pathognomonic PrP patches in the cerebellum, one of them exhibits no detectable typical rPrPSc even in PrPSc-enriched preparations. Instead, a large amount of abnormal PrP is captured from this case by gene 5 protein and sodium phosphotungstate, reagents that have been proved to specifically capture abnormal PrP. All captured abnormal PrP from the cerebellum and other brain regions is virtually sensitive to PK-digestion (termed sPrPSc). The presence of the predominant sPrPSc but absence of rPrPSc in this 144-bp insertion-linked inherited CJD case suggests that mutant sPrPSc is the main component of the PrP deposit patches and sPrPSc is sufficient to cause neurotoxicity and prion disease.
Prions; protease-sensitive prions; prion disease; insertion mutation; neuropathology; Western blotting; phenotype
Six clinico-pathological phenotypes of sporadic Creutzfeldt–Jakob disease have been characterized which correlate at the molecular level with the type (1 or 2) of the abnormal prion protein, PrPTSE, present in the brain and with the genotype of polymorphic (methionine or valine) codon 129 of the prion protein gene. However, to what extent these phenotypes with their corresponding molecular combinations (i.e. MM1, MM2, VV1 etc.) encipher distinct prion strains upon transmission remains uncertain. We studied the PrPTSE type and the prion protein gene in archival brain tissues from the National Institutes of Health series of transmitted Creutzfeldt–Jakob disease and kuru cases, and characterized the molecular and pathological phenotype in the affected non-human primates, including squirrel, spider, capuchin and African green monkeys. We found that the transmission properties of prions from the common sporadic Creutzfeldt–Jakob disease MM1 phenotype are homogeneous and significantly differ from those of sporadic Creutzfeldt–Jakob disease VV2 or MV2 prions. Animals injected with iatrogenic Creutzfeldt–Jakob disease MM1 and genetic Creutzfeldt–Jakob disease MM1 linked to the E200K mutation showed the same phenotypic features as those infected with sporadic Creutzfeldt–Jakob disease MM1 prions, whereas kuru most closely resembled the sporadic Creutzfeldt–Jakob disease VV2 or MV2 prion signature and neuropathology. The findings indicate that two distinct prion strains are linked to the three most common Creutzfeldt–Jakob disease clinico-pathological and molecular subtypes and kuru, and suggest that kuru may have originated from cannibalistic transmission of a sporadic Creutzfeldt–Jakob disease of the VV2 or MV2 subtype.
prion diseases; neuropathology; neurodegenerative disorders; phenotype; strain typing
Dura mater graft-associated Creutzfeldt-Jakob disease (dCJD) can be divided into two subgroups that exhibit distinct clinical and neuropathological features, with the majority represented by a non-plaque-type of dCJD (np-dCJD) and the minority by a plaque-type of dCJD (p-dCJD). The two distinct phenotypes of dCJD had been considered to be unrelated to the genotype (methionine, M or valine, V) at polymorphic codon 129 of the PRNP gene or type (type 1 or type 2) of abnormal isoform of prion protein (PrPSc) in the brain, while these are major determinants of clinicopathological phenotypes of sporadic CJD (sCJD). The reason for the existence of two distinct subgroups in dCJD had remained elusive. Recent progress in research of the pathogenesis of dCJD has revealed that two distinct subgroups of dCJD are caused by infection with different PrPSc strains from sCJD, i.e., np-dCJD caused by infection with sCJD-MM1/MV1, and p-dCJD caused by infection with sCJD-VV2 or -MV2. These studies have also revealed previously unrecognized problems as follows: (i) the numbers of p-dCJD patients may increase in the future, (ii) the potential risks of secondary infection from dCJD, particularly from p-dCJD, may be considerable, and (iii) the effectiveness of the current PrPSc decontamination procedures against the PrPSc from p-dCJD is uncertain. To prevent secondary infection from p-dCJD, the establishment of effective decontamination procedures is an urgent issue. In this review, we summarize the past and future problems surrounding dCJD.
Creutzfeldt-Jakob disease; Prion protein; Dura mater grafts; Humanized knock-in mouse
The coexistence of different molecular types of classical protease-resistant prion protein in the same individual have been described, however, the simultaneous finding of these with the recently described protease-sensitive variant or variably protease-sensitive prionopathy has, to the best of our knowledge, not yet been reported.
A 74-year-old Caucasian woman showed a sporadic Creutzfeldt–Jakob disease clinical phenotype with reactive depression, followed by cognitive impairment, akinetic-rigid Parkinsonism with pseudobulbar syndrome and gait impairment with motor apraxia, visuospatial disorientation, and evident frontal dysfunction features such as grasping, palmomental reflex and brisk perioral reflexes. She died at age 77.
Neuropathological findings showed: spongiform change in the patient’s cerebral cortex, striatum, thalamus and molecular layer of the cerebellum with proteinase K-sensitive synaptic-like, dot-like or target-like prion protein deposition in the cortex, thalamus and striatum; proteinase K-resistant prion protein in the same regions; and elongated plaque-like proteinase K-resistant prion protein in the molecular layer of the cerebellum. Molecular analysis of prion protein after proteinase K digestion revealed decreased signal intensity in immunoblot, a ladder-like protein pattern, and a 71% reduction of PrPSc signal relative to non-digested material. Her cerebellum showed a 2A prion protein type largely resistant to proteinase K. Genotype of polymorphism at codon 129 was valine homozygous.
Molecular typing of prion protein along with clinical and neuropathological data revealed, to the best of our knowledge, the first case of the coexistence of different protease-sensitive prion proteins in the same patient in a rare case that did not fulfill the current clinical diagnostic criteria for either probable or possible sporadic Creutzfeldt–Jakob disease. This highlights the importance of molecular analyses of several brain regions in order to correctly diagnose rare and atypical prionopathies.
Creutzfeldt–Jakob; Prion; Protease resistance; Resistant PrP; Sensitive PrP; Variably Protease Sensitive Prionopathy
The clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD) correlate with the allelotypes (M or V) of the polymorphic codon 129 of the human prion protein (PrP) gene and the electrophoretic mobility patterns of abnormal prion protein (PrPSc). Transmission of sCJD prions to mice expressing human PrP with a heterologous genotype (referred to as cross-sequence transmission) results in prolonged incubation periods. We previously reported that cross-sequence transmission can generate a new prion strain with unique transmissibility, designated a traceback phenomenon. To verify experimentally the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into mice expressing human PrP with the 129M/M genotype. These 129M/M mice showed altered neuropathology and a novel PrPSc type after a long incubation period. We then passaged the brain homogenate from the 129M/M mouse inoculated with sCJD-VV2 prions into other 129M/M or 129V/V mice. Despite cross-sequence transmission, 129V/V mice were highly susceptible to these prions compared to the 129M/M mice. The neuropathology and PrPSc type of the 129V/V mice inoculated with the 129M/M mouse-passaged sCJD-VV2 prions were identical to those of the 129V/V mice inoculated with sCJD-VV2 prions. Moreover, we generated for the first time a type 2 PrPSc-specific antibody in addition to type 1 PrPSc-specific antibody and discovered that drastic changes in the PrPSc subpopulation underlie the traceback phenomenon. Here, we report the first direct evidence of the traceback in prion infection.
To define the clinicopathologic, genetic, and pathogenic prion protein (PrPSc) characteristics associated with a novel mutation of the prion protein gene (PRNP).
The coding segment of PRNP from the proband and family members was sequenced and the brain of the proband was histologically studied. The Western blot profile of the proteinase K (PK) resistant fraction of PrPSc, an approximation of its conformation, or “PrPSc-type,” was determined.
We detected a novel mutation at codon 105 of PRNP that results in a serine (S) substitution of proline (P) (P105S), in a young woman who developed progressive aphasia, behavioral changes, dementia, and parkinsonism, lasting 10 years to her death. Histopathologic findings included an intense focus of multicentric PrP-plaques within the hippocampus, punctate plaques scattered throughout the cerebellum, and intense spongiform degeneration focally within the putamen, suggesting a variant of Gerstmann-Sträussler-Scheinker syndrome (GSS). However, PrPSc-typing revealed two PK-resistant PrPSc fragments (∼21 and 26 kDa), a pattern not previously detected in GSS.
This mutation is the third sequence variation at codon 105 of PRNP. The unusual phenotype and PrPSc-type distinguishes this genetic prion disease from typical Gerstmann-Sträussler-Scheinker syndrome and other codon 105 substitutions, suggesting that, in addition to the loss of proline at this position, the PrPSc conformation and phenotype is dependent on the specific amino acid substitution.
= familial Creutzfeldt-Jakob disease;
= familial fatal insomnia;
= frontotemporal dementia;
= Gerstmann-Sträussler-Scheinker syndrome;
= proteinase K.
Scrapie in sheep and goats has been known for more than 250 years and belongs nowadays to the so-called prion diseases that also include e.g. bovine spongiform encephalopathy in cattle (BSE) and Creutzfeldt-Jakob disease in humans. According to the prion hypothesis, the pathological isoform (PrPSc) of the cellular prion protein (PrPc) comprises the essential, if not exclusive, component of the transmissible agent. Currently, two types of scrapie disease are known - classical and atypical/Nor98 scrapie. In the present study we examine 24 cases of classical and 25 cases of atypical/Nor98 scrapie with the sensitive PET blot method and validate the results with conventional immunohistochemistry. The sequential detection of PrPSc aggregates in the CNS of classical scrapie sheep implies that after neuroinvasion a spread from spinal cord and obex to the cerebellum, diencephalon and frontal cortex via the rostral brainstem takes place. We categorize the spread of PrPSc into four stages: the CNS entry stage, the brainstem stage, the cruciate sulcus stage and finally the basal ganglia stage. Such a sequential development of PrPSc was not detectable upon analysis of the present atypical/Nor98 scrapie cases. PrPSc distribution in one case of atypical/Nor98 scrapie in a presumably early disease phase suggests that the spread of PrPSc aggregates starts in the di- or telencephalon. In addition to the spontaneous generation of PrPSc, an uptake of the infectious agent into the brain, that bypasses the brainstem and starts its accumulation in the thalamus, needs to be taken into consideration for atypical/Nor98 scrapie.
The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrPSc) of the normal and ubiquitous prion protein PrPC. The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp0/0) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrPSc or for both PrPC and PrPSc. PrPSc-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrPSc-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrPSc and PrPC, did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrPSc elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrPSc-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.