A New Method for the Characterization of Strain-Specific Conformational Stability of Protease-Sensitive and Protease-Resistant PrPSc

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In 2007, we reported a patient with an atypical form of CreutzfeldtJakob disease (CJD) heterozygous for methionine-valine (MV) at codon 129 who showed a novel pathological prion protein (PrP TSE ) conformation with an atypical glycoform (AG) profile and intraneuronal PrP deposition. In the present study, we further characterize the conformational properties of this pathological prion protein (PrP TSE MV AG ), showing that PrP TSE MV AG is composed of multiple conformers with biochemical properties distinct from those of PrP TSE type 1 and type 2 of MV sporadic CJD (sCJD). Experimental transmission of CJD-MV AG to bank voles and genetargeted transgenic mice carrying the human prion protein gene (TgHu mice) showed unique transmission rates, survival times, neuropathological changes, PrP TSE deposition patterns, and PrP TSE glycotypes that are distinct from those of sCJD-MV1 and sCJD-MV2. These biochemical and experimental data suggest the presence of a novel prion strain in CJD-MV AG .IMPORTANCE Sporadic Creutzfeldt-Jakob disease is caused by the misfolding of the cellular prion protein, which assumes two different major conformations (type 1 and type 2) and, together with the methionine/valine polymorphic codon 129 of the prion protein gene, contribute to the occurrence of distinct clinical-pathological phenotypes. Inoculation in laboratory rodents of brain tissues from the six possible combinations of pathological prion protein types with codon 129 genotypes results in the identification of 3 or 4 strains of prions. We report on the identification of a novel strain of Creutzfeldt-Jakob disease isolated from a patient who carried an abnormally glycosylated pathological prion protein. This novel strain has unique biochemical characteristics, does not transmit to humanized transgenic mice, and shows exclusive transmission properties in bank voles. The identification of a novel human prion strain improves our understanding of the pathogenesis of the disease and of possible mechanisms of prion transmission.

Section: Discussionmentioning

confidence: 99%

Prion Strain Characterization of a Novel Subtype of Creutzfeldt-Jakob Disease

Galeno

Bari

Nonno

et al. 2017

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“…The conformational stability and solubility assay (CSSA) was done as previously described (26). Briefly, aliquots of brain homogenates (6% [wt/vol] in 100 mM Tris-HCl, pH 7.4) were added with an equal volume of 100 mM Tris-HCl (pH 7.4), 4% Sarkosyl and incubated for 1 h at 37°C with gentle shaking.…”

Section: Methodsmentioning

confidence: 99%

PrP ^C Governs Susceptibility to Prion Strains in Bank Vole, While Other Host Factors Modulate Strain Features

Espinosa

Nonno

Bari

et al. 2016

Bank vole is a rodent species that shows differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origins were assayed both in bank vole expressing methionine at codon 109 (Bv109M) and in transgenic mice expressing physiological levels of bank vole PrP C (the BvPrP-Tg407 mouse line). This work is the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic-mouse model and in its natural counterpart. The results showed very similar transmission properties in both the natural species and the transgenic-mouse model, demonstrating the key role of the PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrP Sc types propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrP C modulate prion strain features. IMPORTANCEThe differential susceptibility of bank voles to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is controlled by the vole PrP sequence alone rather than by other species-specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that host factors other than the PrP C sequence may affect the selection of the substrain replicating in the animal model. Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in small ruminants. The prion agent consists mainly, if not entirely, of PrP Sc , which is an abnormal isoform of the host-encoded prion cellular protein, PrP C (1-4). Prion propagation is considered to occur in a two-step process in which aggregates of PrP Sc bind new PrP C monomers and then induce their conformational change into PrP Sc . The conversion into PrP Sc results in changes in physical properties, such as decreased solubility in nondenaturing detergents, an increase in ␤-pleated-sheet content, and increased resistance to proteolytic degradation.The occurrence of different TSE strains has been described in several species. Upon experimental transmission, TSE strains show distinctive incubation periods and neuropathological features that are maintained after subpassage in the same species. These strains correspond to different PrP Sc conformations, according to the prion hypothesis (5). Prion strain transmission among different species seems to be driven by the differences in the PrP amino acid sequences and by the prion strain being transmitted (6). Transgenic-mouse models null for the murine Prnp gene and expressing PrP C from another species (such as bovine, ovine, porcine, or human), have been used in the prion research field as useful tools to characterize prion strains and to learn about the susceptibility of the species t...

“…In particular, a systematic analysis of the conformational stability of PrP Sc aggregates across the spectrum of human prions is still lacking. Previous studies focused on the comparison between sCJD subtypes MM1 and MM2 (31)(32)(33) or between variant CJD (vCJD) and the most common sCJD type MM1 (34). Furthermore, only the unfolding induced by GdnHCl was addressed, whereas the thermostability of PrP Sc has never been explored in CJD.…”

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confidence: 99%

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

Cescatti

Saverioni

Capellari

et al. 2016

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. IMPORTANCEPrion 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. P rion diseases are invariably fatal neurodegenerative disorders of humans ...