Influence of host and prion strain on patological prion protein chemical and physical properties: a comparative study in humans and other species.

Le encefalopatie spongiformi trasmissibili (EST), o malattie da prioni, sono patologie neurodegenerative che colpiscono l'uomo e gli altri mammiferi. Possono essere sporadiche, genetiche o acquisite attraverso fonti contaminate. Le EST più comuni sono la malattia di Creutzfeldt-Jakob (CJD) nell’uomo, lo scrapie in pecore e capre, l'encefalopatia spongiforme bovina (BSE) nei bovini e la malattia del deperimento cronico (CWD) nei cervi e negli alci. Tutte le EST sono caratterizzate dall'accumulo di PrPSc, una forma misfolded della proteina cellulare PrPC. La PrPSc è insolubile nei detergenti, parzialmente resistente alla proteolisi e altamente arricchita in struttura secondaria a foglietto β. Per ogni EST, si può distinguere un certo numero di ceppi prionici, che danno luogo a diversi fenotipi dopo l'inoculazione in ospiti singenici. Secondo l'ipotesi della protein only, in base alla quale i prioni sono composti esclusivamente da PrPSc, i ceppi prionici sono determinati da diverse conformazioni della PrPSc. Evidenze sperimentali crescenti indicano che i ceppi prionici possono essere identificati mediante le proprietà biochimiche della PrPSc. In questo studio, abbiamo analizzato alcune di esse (dimensioni del frammento principale dopo proteolisi, presenza e tipo di frammenti troncati aggiuntivi, grado di resistenza alla proteolisi e grado di solubilizzazione indotta dal calore) in 11 ceppi di EST naturali e nelle trasmissioni ai bank voles (CJD sporadica), alle pecore (BSE) e all’uomo (BSE). E’ risultato che una specifica combinazione di queste proprietà distingue la PrPSc in ciascun ceppo prionico nell'ospite naturale. Inoltre, confrontando i ceppi nei loro ospiti naturali e dopo trasmissione ad altre specie, abbiamo dimostrato che tutte queste proprietà di PrPSc sono significativamente influenzate dall'ospite. Infine, abbiamo riportato che tra le proprietà analizzate la termostabilità è quella che correla meglio con la “virulenza” del ceppo. Quindi, questi dati aggiungono informazioni nel quadro affascinante delle conformazioni della PrPSc e del loro rapporto con i ceppi prionici.Transmissible spongiform encephalopathies (TSEs), or prion diseases, are neurodegenerative disorders that affect humans and other mammals. They can be sporadic, genetic or acquired through contaminated sources. The most common TSEs are Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goat, bovine spongiform encephalopathy (BSE) in cattle and chronic wasting disease (CWD) in deer and elk. All TSEs are characterised by the accumulation of PrPSc, a misfolded form of the cellular protein PrPC. PrPSc is insoluble in detergents, partially resistant to proteolysis and highly enriched in β-sheet secondary structure. For each TSE, a number of prion strains can be distinguished, which give rise to different phenotypes after inoculation in syngeneic hosts. According to the protein-only hypothesis, which postulates that prions are composed exclusively of PrPSc, prion strains are enciphered in PrPSc conformation. Increasing evidence indicates that prion strains can be identified by means of PrPSc biochemical properties. We studied a selected pool of PrPSc biochemical properties (core fragment size after proteolysis, presence and type of additional truncated fragments, degree of resistance to proteolysis and degree of heat-induced solubilisation) in 11 naturally occurring TSE strains. In addition, we investigated sporadic CJD after transmission to bank voles and BSE after transmission to sheep and humans (e.g. variant CJD or human BSE). We found that a specific combination of biochemical PrPSc properties distinguishes each prion strain in the natural host. By comparing prion strains in natural hosts and after transmission to other species, we demonstrated that all PrPSc properties we analysed are also significantly influenced by the host. Finally, we show that among the properties analysed PrPSc thermo-stability is the one that correlates best with strain “virulence” after transmission in a compatible host genotype. Thus, the present data add insights into the intriguing puzzle of PrPSc conformations and their relationship to prion strains

Analysis of conformational stability of abnormal prion protein aggregates across the spectrum of Creutzfeldt-Jakob disease prions

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

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

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

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

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

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

Abstract 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

Additional file 1: Figure S1. of Atypical Creutzfeldt-Jakob disease with PrP-amyloid plaques in white matter: molecular characterization and transmission to bank voles show the M1 strain signature

Western blot analysis of np-CJDMM1 and p-CJDMM1 (case #1) subcortical white matter. FC: frontal cortex; PC: parietal cortex. (a) Electrophoretic mobility of PK-digested PrPSc (i.e. PrP27–30) after separation in a 7 cm long gel. Blot was probed with the primary antibody 3F4. (b) CTF13 analysis after PrP deglycosylation with PNGase F. Blot was probed with the primary antibody SAF60. Relative molecular masses are expressed in kDa. Percentages (mean ± standard deviation) of CTF13 are referred to the total PrPSc amount: np-CJDMM1 = 12.8 ± 5.0, p-CJDMM1 = 14.1 ± 2.9. (TIFF 824 kb

Additional file 4: Table S2. of Atypical Creutzfeldt-Jakob disease with PrP-amyloid plaques in white matter: molecular characterization and transmission to bank voles show the M1 strain signature

Relative amounts of PrPSc fragments in samples from p-CJDMM1 and np-CJDMM1. Values represent the percentage (mean ± standard deviation) of fragments referred to the total PrPSc amount. Differences were not statistically significant (Student’s t test). (DOCX 13 kb

Additional file 5: Table S3. of Atypical Creutzfeldt-Jakob disease with PrP-amyloid plaques in white matter: molecular characterization and transmission to bank voles show the M1 strain signature

PrPSc glycoform ratio in p-CJDMM1 and np-CJDMM1. Values represent the percentage (mean ± standard deviation) of glycoforms referred to the total PrPSc amount. D: diglycosylated, M: monoglycosylated, U: unglycosylated PrPSc. (DOCX 13 kb