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

The prion protein protease sensitivity, stability and seeding activity in variably protease sensitive prionopathy brain tissue suggests molecular overlaps with sporadic Creutzfeldt-Jakob disease

INTRODUCTION: 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 (PrP(Sc)) in VPSPr are increased sensitivity to proteolysis and the presence of an N- and C-terminally cleaved ~8 kDa protease resistant PrP(Sc) (PrP(res)) 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 PrP(res), suggesting that a proportion of the PrP(Sc) present in VPSPr has properties similar to those of sCJD. RESULTS: Here, we have used conformation dependent immunoassay to confirm the presence of PrP(Sc) in VPSPr that is more protease sensitive compared with sCJD. However, CDI also shows that a proportion of PrP(Sc) in VPSPr resists PK digestion of its C-terminus, distinguishing it from GSS associated with ~8 kDa PrP(res), 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 PrP(Sc) in the absence of PK treatment. Finally, using protein misfolding cyclic amplification and real-time quaking induced conversion, we show that VPSPr PrP(Sc) 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 PrP(res) rather than the ~8 kDa fragment. CONCLUSIONS: Therefore, PrP(Sc) 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

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

La memoria lunga. Eredità culturali d'Abruzzo - 6 documentari brevi

Il format La memoria lunga. Eredità culturali d'Abruzzo è pensato come uno strumento per favorire la conoscenza del patrimonio culturale del territorio montano e rurale d’Abruzzo. La memoria lunga nasce dalla confluenza di ricerca antropologica e cinema, di indagine sul campo, relazione umana e tecnologia audiovisiva, dall'unione di testimonianze, documentazioni di attività, di vita quotidiana, di ambiente e paesaggio. La scelta micro-tematica ha permesso l’approfondimento su scala ridotta, l’apertura di un punto di osservazione ravvicinato sopra un particolare aspetto di un fenomeno, lasciandone la narrazione agli stessi interpreti e protagonisti. Girate in Full HD, le prime sei puntate raccontano aspetti poco noti del vasto patrimonio culturale materiale e immateriale della provincia di Teramo, con un taglio autoriale che cerca di coniugare la narrazione divulgativa e la profondità di osservazione

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, \u3b2 -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