Atypical Creutzfeldt-Jakob disease with PrP-amyloid plaques in white matter: molecular characterization and transmission to bank voles show the M1 strain signature.

Amyloid plaques formed by abnormal prion protein (PrPSc) aggregates occur with low frequency in Creutzfeldt-Jakob disease, but represent a pathological hallmark of three relatively rare disease histotypes, namely variant CJD, sporadic CJDMV2K (methionine/valine at PRNP codon 129, PrPSc type 2 and kuru-type amyloid plaques) and iatrogenic CJDMMiK (MM at codon 129, PrPSc of intermediate type and kuru plaques). According to recent studies, however, PrP-amyloid plaques involving the subcortical and deep nuclei white matter may also rarely occur in CJDMM1 (MM at codon 129 and PrPSc type 1), the most common CJD histotype.To further characterize the phenotype of atypical CJDMM1 with white matter plaques (p-CJDMM1) and unravel the basis of amyloid plaque formation in such cases, we compared clinical and histopathological features and PrPSc physico-chemical properties between 5 p-CJDMM1 and 8 typical CJDMM1 brains lacking plaques. Furthermore, transmission properties after bioassay in two genetic lines of bank voles were also explored in the two groups.All 5 p-CJDMM1 cases had a disease duration longer than one year. Three cases were classified as sporadic CJDMM1, one as sporadic CJDMM1 + 2C and one as genetic CJDE200K-MM1. Molecular mass, protease sensitivity and thermo-solubilization of PrPSc aggregates did not differ between p-CJDMM1 and classical CJDMM1 cases. Likewise, transmission properties such as incubation time, lesion profile and PrPSc properties in bank voles also matched in the two groups.The present data further define the clinical-pathologic phenotype of p-CJDMM1, definitely establish it as a distinctive CJD histotype and demonstrate that PrP-plaque formation in this histotype is not a strain-specific feature. Since cases lacking amyloid plaques may also manifest a prolonged (i.e. > than one year) disease course, unidentified, host-specific factors likely play a significant role, in addition to disease duration, in generating white matter PrP-amyloid plaques in p-CJDMM1.

Analysis of Conformational Stability of Abnormal Prion Protein Aggregates across the Spectrum of Creutzfeldt-Jakob Disease Prions.

UNLABELLED: The wide phenotypic variability of prion diseases is thought to depend on the interaction of a host genotype with prion strains that have self-perpetuating biological properties enciphered in distinct conformations of the misfolded prion protein PrP(Sc) This concept is largely based on indirect approaches studying the effect of proteases or denaturing agents on the physicochemical properties of PrP(Sc) aggregates. Furthermore, most data come from studies on rodent-adapted prion strains, making current understanding of the molecular basis of strains and phenotypic variability in naturally occurring diseases, especially in humans, more limited. To fill this gap, we studied the effects of guanidine hydrochloride (GdnHCl) and heating on PrP(Sc) aggregates extracted from 60 sporadic Creutzfeldt-Jakob disease (CJD) and 6 variant CJD brains. While denaturation curves obtained after exposure of PrP(Sc) to increasing GdnHCl concentrations showed similar profiles among the 7 CJD types analyzed, PrP(Sc) exposure to increasing temperature revealed significantly different and type-specific responses. In particular, MM1 and VV2, the most prevalent and fast-replicating CJD types, showed stable and highly resistant PrP(Sc) aggregates, whereas VV1, a rare and slowly propagating type, revealed unstable aggregates that easily dissolved at low temperature. Taken together, our results indicate that the molecular interactions mediating the aggregation state of PrP(Sc), possibly enciphering strain diversity, are differently targeted by GdnHCl, temperature, and proteases. Furthermore, the detected positive correlation between the thermostability of PrP(Sc) aggregates and disease transmission efficiency makes inconsistent the proposed hypothesis that a decrease in conformational stability of prions results in an increase in their replication efficiency. IMPORTANCE: Prion strains are defined as infectious isolates propagating distinctive phenotypic traits after transmission to syngeneic hosts. Although the molecular basis of prion strains is not fully understood, it is largely accepted that variations in prion protein conformation drive the molecular changes leading to the different phenotypes. In this study, we exposed abnormal prion protein aggregates encompassing the spectrum of human prion strains to both guanidine hydrochloride and thermal unfolding. Remarkably, while exposure to increasing temperature revealed significant strain-specific differences in the denaturation profile of the protein, treatment with guanidine hydrochloride did not. The findings suggest that thermal and chemical denaturation perturb the structure of prion protein aggregates differently. Moreover, since the most thermostable prion protein types were those associated with the most prevalent phenotypes and most rapidly and efficiently transmitting strains, the results suggest a direct correlation between strain replication efficiency and the thermostability of prion protein aggregates.

Transmission properties of atypical Creutzfeldt-Jakob disease: a clue to disease etiology?

UNLABELLED: The genotype at polymorphic codon 129 of the PRNP gene has a profound influence on both phenotypic expression and prion strain susceptibility in humans. For example, while the most common sporadic Creutzfeldt-Jakob disease (CJD) subtype, sporadic CJD-MM1 (M1 strain), induces a single phenotype after experimental transmission regardless of the codon 129 genotype of the recipient animal, the phenotype elicited by sporadic CJD-VV2 (V2 strain), the second most common subtype, varies according to the host codon 129 genotype. In particular, the propagation of the V2 strain in codon 129 methionine homozygotes has been linked only to acquired forms of CJD such as plaque-type dura mater graft-associated CJD (dCJD), a subgroup of iatrogenic CJD with distinctive phenotypic features, but has never been observed in sporadic CJD cases. In the present report, we describe atypical CJD cases carrying codon 129 methionine homozygosity, in a neurosurgeon and in a patient with a medical history of neurosurgery without dural grafting, showing the distinctive phenotypic features and transmission properties of plaque-type dCJD. These findings raise the possibility that the two cases, previously thought to represent sporadic CJD, might actually represent acquired CJD caused by infection with the V2 strain. Thus, careful analyses of phenotypic features and transmission properties in atypical cases may be useful to distinguish acquired from sporadic cases of CJD. IMPORTANCE: Susceptibility to and phenotypic expression of Creutzfeldt-Jakob disease (CJD) depend on both the prion strain and genotype at polymorphic codon 129 of the PRNP gene. For example, propagation of the second most common sporadic CJD strain (V2 strain) into codon 129 methionine homozygotes has been linked to plaque-type dura mater graft-associated CJD (dCJD), a subgroup of iatrogenic CJD with distinctive phenotypic features, but has never been observed in sporadic CJD. In the present report, we describe atypical CJD cases in a neurosurgeon and in a patient with a medical history of neurosurgery without dural grafting, showing the distinctive phenotypic features and transmission properties of plaque-type dCJD. These findings raise the possibility that the two cases, previously considered to represent sporadic CJD, might actually represent acquired CJD caused by infection with the V2 strain.

Analyses of protease resistance and aggregation state of abnormal prion protein across the spectrum of human prions.

Prion diseases are characterized by tissue accumulation of a misfolded, ß-sheet-enriched isoform (scrapie prion protein (PrP(Sc))) of the cellular prion protein (PrP(C)). At variance with PrP(C), PrP(Sc) shows a partial resistance to protease digestion and forms highly aggregated and detergent-insoluble polymers, two properties that have been consistently used to distinguish the two proteins. In recent years, however, the idea that PrP(Sc) itself comprises heterogeneous species has grown. Most importantly, a putative proteinase K (PK)-sensitive form of PrP(Sc) (sPrP(Sc)) is being increasingly investigated for its possible role in prion infectivity, neurotoxicity, and strain variability. The study of sPrP(Sc), however, remains technically challenging because of the need of separating it from PrP(C) without using proteases. In this study, we have systematically analyzed both PK resistance and the aggregation state of purified PrP(Sc) across the whole spectrum of the currently characterized human prion strains. The results show that PrP(Sc) isolates manifest significant strain-specific differences in their PK digestion profile that are only partially explained by differences in the size of aggregates, suggesting that other factors, likely acting on PrP(Sc) aggregate stability, determine its resistance to proteolysis. Fully protease-sensitive low molecular weight aggregates were detected in all isolates but in a limited proportion of the overall PrP(Sc) (i.e. <10%), arguing against a significant role of slowly sedimenting PK-sensitive PrP(Sc) in the biogenesis of prion strains. Finally, we highlight the limitations of current operational definitions of sPrP(Sc) and of the quantitative analytical measurements that are not based on the isolation of a fully PK-sensitive PrP(Sc) form.

Atypical neuropathological sCJD-MM phenotype with abundant white matter Kuru-type plaques sparing the cerebellar cortex.

We describe an atypical neuropatholgical phenotype of sporadic Creutzfeldt-Jakob disease (sCJD) in a 64-year-old man presenting with a 5-month history of rapidly progressive dementia, comprising behavioral disturbances, memory complaints, disorientation and language alterations. MRI showed diffuse atrophy and hyperintensities in parietal, occipital, temporal and frontal cortices and left caudate nucleus on T2-weighted and fluid-attenuated inversion recovery images. No typical EEG alterations were observed. Repeated 14-3-3 assay was positive after a first negative test. Neuropathology showed classical CJD changes with small cortical foci of large confluent vacuoles and relatively well-preserved cerebellar cortex. The most striking feature was the presence of abundant Kuru-type plaques in both cerebral cortex and subcortical white matter. Sparse Kuru-type plaques were also seen in cerebellum, although only in white matter. Immunohistochemistry showed, in addition to unicentric plaques, diffuse synaptic and patchy perivacuolar, as well as plaque-like and periaxonal pathological prion protein deposits (PrP(res) ). Western blot studies demonstrated the co-occurrence of PrP(res) types 1 and 2 in frontal cortex and a relatively weak type 2 signal in cerebellum. PRNP genotyping revealed methionine homozygosity at codon 129 and excluded mutations. This case shows a previously undescribed combination of histopathological features which preclude its classification according to the current phenotypic and molecular sCJD classification. The observation demonstrates that Kuru-type amyloid plaques mainly involving the cerebral white matter may also occur in sCJD cases with short clinical course and the co-existence of PrP(res) types 1 and 2. This case further highlights the complexity of the correlations between histopathological phenotype and PrP(res) isotype in prion diseases.

Prion protein misfolding, strains, and neurotoxicity: an update from studies on Mammalian prions.

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders affecting humans and other mammalian species. The central event in TSE pathogenesis is the conformational conversion of the cellular prion protein, PrP(C), into the aggregate, ß -sheet rich, amyloidogenic form, PrP(Sc). Increasing evidence indicates that distinct PrP(Sc) conformers, forming distinct ordered aggregates, can encipher the phenotypic TSE variants related to prion strains. Prion strains are TSE isolates that, after inoculation into syngenic hosts, cause disease with distinct characteristics, such as incubation period, pattern of PrP(Sc) distribution, and regional severity of histopathological changes in the brain. In analogy with other amyloid forming proteins, PrP(Sc) toxicity is thought to derive from the existence of various intermediate structures prior to the amyloid fiber formation and/or their specific interaction with membranes. The latter appears particularly relevant for the pathogenesis of TSEs associated with GPI-anchored PrP(Sc), which involves major cellular membrane distortions in neurons. In this review, we update the current knowledge on the molecular mechanisms underlying three fundamental aspects of the basic biology of prions such as the putative mechanism of prion protein conversion to the pathogenic form PrP(Sc) and its propagation, the molecular basis of prion strains, and the mechanism of induced neurotoxicity by PrP(Sc) aggregates.

Consensus classification of human prion disease histotypes allows reliable identification of molecular subtypes: an inter-rater study among surveillance centres in Europe and USA.

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, PrP(Sc) (i.e. type 1 migrating at 21 kDa and type 2 at 19 kDa). We previously demonstrated that PrP(Sc) 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 PrP(Sc) 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 PrP(Sc) typing, histopathological analysis permits reliable disease classification with high interlaboratory accuracy.

Molecular pathology, classification, and diagnosis of sporadic human prion disease variants.

Human prion diseases are a unique group of transmissible neurodegenerative diseases that occur as sporadic, familial or acquired disorders and show a wide range of phenotypic variation. The latter has been attributed to the existence of distinct strains of the agent or prion, and the genetic background of the host, namely the primary sequence of the gene encoding the prion protein, which is the site of mutations and polymorphisms. The characterization of distinct isoforms of the abnormal prion protein in the brain of affected patients, which has been shown to correlate with the disease phenotype, has recently led to the concept of molecular strain typing, in which the different prion protein isoforms or "types", possibly enciphering the strain variability in their conformation, may serve as surrogate markers for individual prion strains. In sporadic Creutzfeldt-Jakob disease, the most common human prion disease, there are at least six distinct clinico-pathological disease phenotypes that largely correlate at a molecular level with two prion protein types with distinctive physicochemical properties and the genotype at the methionine/valine polymorphic codon 129 in the prion protein gene. Recent results of transmission studies indicate that five prion strains with distinctive biological properties can be isolated from these six disease variants. It has also been shown that about a third of sporadic cases show a mixed phenotype and the co-occurrence of prion protein types. The origin of prion strains and their co-occurrence as well as the mechanisms underlying the strain-specific neuronal targeting remain largely unexplained and their understanding constitute, together with the development of successful therapies and more sensitive and specific clinical biomarkers, the major challenges that this disease poses for the future.

Genetic Creutzfeldt-Jakob disease and fatal familial insomnia: insights into phenotypic variability and disease pathogenesis.

Human prion diseases are a group of rare neurodegenerative disorders characterized by the conversion of the constitutively expressed prion protein, PrP(C), into an abnormally aggregated isoform, called PrP(Sc). While most people who develop a prion disease have no identifiable cause and a few acquire the disease through an identified source of infection, about 10-15% of patients are affected by a genetic form and carry either a point mutation or an insertion of octapeptide repeats in the prion protein gene. Prion diseases show the highest extent of phenotypic heterogeneity among neurodegenerative disorders and comprise three major disease entities with variable though overlapping phenotypic features: Creutzfeldt-Jakob disease (CJD), fatal insomnia and the Gerstmann-Sträussler-Scheinker syndrome. Both CJD and fatal insomnia are fully transmissible diseases, a feature that led to the isolation and characterization of different strains of the agent or prion showing distinctive clinical and neuropathological features after transmission to syngenic animals. Here, we review the current knowledge of the effects of the pathogenic mutations linked to genetic CJD and fatal familial insomnia on the prion protein metabolism and physicochemical properties, the disease phenotype and the strain characteristics. The data derived from studies in vitro and from those using cell and animal models are compared with those obtained from the analyses of the naturally occurring disease. The extent of phenotypic variation in genetic prion disease is analyzed in comparison to that of the sporadic disease, which has recently been the topic of a systematic and detailed characterization.