Protease-Sensitive Scrapie Prion Protein in Aggregates of Heterogeneous Sizes

Chronic wasting disease (CWD) is a naturally occurring, fatal neurodegenerative disease of cervids. The potential for swine to serve as hosts for the agent of CWD is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following experimental oral or intracranial inoculation. Crossbred piglets were assigned to three groups, intracranially inoculated (n ϭ 20), orally inoculated (n ϭ 19), and noninoculated (n ϭ 9). At approximately the age at which commercial pigs reach market weight, half of the pigs in each group were culled ("market weight" groups). The remaining pigs ("aged" groups) were allowed to incubate for up to 73 months postinoculation (mpi). Tissues collected at necropsy were examined for disease-associated prion protein (PrP Sc ) by Western blotting (WB), antigen capture enzyme immunoassay (EIA), immunohistochemistry (IHC), and in vitro real-time quaking-induced conversion (RT-QuIC). Brain samples from selected pigs were also bioassayed in mice expressing porcine prion protein. Four intracranially inoculated aged pigs and one orally inoculated aged pig were positive by EIA, IHC, and/or WB. By RT-QuIC, PrP Sc was detected in lymphoid and/or brain tissue from one or more pigs in each inoculated group. The bioassay was positive in four out of five pigs assayed. This study demonstrates that pigs can support low-level amplification of CWD prions, although the species barrier to CWD infection is relatively high. However, detection of infectivity in orally inoculated pigs with a mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity. IMPORTANCEWe challenged domestic swine with the chronic wasting disease agent by inoculation directly into the brain (intracranially) or by oral gavage (orally). Disease-associated prion protein (PrP Sc ) was detected in brain and lymphoid tissues from intracranially and orally inoculated pigs as early as 8 months of age (6 months postinoculation). Only one pig developed clinical neurologic signs suggestive of prion disease. The amount of PrP Sc in the brains and lymphoid tissues of positive pigs was small, especially in orally inoculated pigs. Regardless, positive results obtained with orally inoculated pigs suggest that it may be possible for swine to serve as a reservoir for prion disease under natural conditions.

Section: Discussionsupporting

confidence: 67%

Experimental Transmission of the Chronic Wasting Disease Agent to Swine after Oral or Intracranial Inoculation

Moore

Greenlee

Kondru

et al. 2017

“…The S1 fractions prepared by centrifugation of 20% brain homogenates at 8,000 ϫ g for 5 min at 4°C were incubated with an equal volume of 2% sarcosyl for 30 min on ice. Samples were loaded atop a 10 to 60% step sucrose gradient and centrifuged at 200,000 ϫ g for 1 h at 4°C, as described by Tzaban et al (39). Eleven fractions were collected from the top of the tube and processed for Western blot analysis of PrP.…”

Section: Novel Cjd Prion Strainmentioning

confidence: 99%

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

Galeno

Bari

Nonno

et al. 2017

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.

“…For example, it is largely believed that the heterogeneity in the fragment profile of proteinase K (PK)-digested PrP Sc , which distinguishes at least some of the known prion strains, is the direct consequence of PrP Sc aggregates having distinct conformations (17)(18)(19)(20)(21). Similarly, sedimentation profiles and protease sensitivity have been used as indirect markers of PrP Sc structure and have shown a correlation with strain-specific transmission properties (22)(23)(24)(25)(26)(27). More recently, studies with rodent-adapted, cloned prion strains demonstrated that also the conformational stability of PrP Sc , measured indirectly either by inducing a progressive denaturation of the protein with the chaotropic salt guanidine hydrochloride (GdnHCl) or by exposing the protein to increasing temperatures in the presence of sodium dodecyl sulfate (SDS), may vary among different strains (28)(29)(30).…”

mentioning

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 ...