Laura Pirisinu

Chronic Wasting Disease in Bank Voles: Characterisation of the Shortest Incubation Time Model for Prion Diseases

In order to assess the susceptibility of bank voles to chronic wasting disease (CWD), we inoculated voles carrying isoleucine or methionine at codon 109 (Bv109I and Bv109M, respectively) with CWD isolates from elk, mule deer and white-tailed deer. Efficient transmission rate (100%) was observed with mean survival times ranging from 156 to 281 days post inoculation. Subsequent passages in Bv109I allowed us to isolate from all CWD sources the same vole-adapted CWD strain (Bv109ICWD), typified by unprecedented short incubation times of 25–28 days and survival times of ∼35 days. Neuropathological and molecular characterisation of Bv109ICWD showed that the classical features of mammalian prion diseases were all recapitulated in less than one month after intracerebral inoculation. Bv109ICWD was characterised by a mild and discrete distribution of spongiosis and relatively low levels of protease-resistant PrPSc (PrPres) in the same brain regions. Despite the low PrPres levels and the short time lapse available for its accumulation, end-point titration revealed that brains from terminally-ill voles contained up to 108,4 i.c. ID50 infectious units per gram. Bv109ICWD was efficiently replicated by protein misfolding cyclic amplification (PMCA) and the infectivity faithfully generated in vitro, as demonstrated by the preservation of the peculiar Bv109ICWD strain features on re-isolation in Bv109I. Overall, we provide evidence that the same CWD strain was isolated in Bv109I from the three-cervid species. Bv109ICWD showed unique characteristics of “virulence”, low PrPres accumulation and high infectivity, thus providing exceptional opportunities to improve basic knowledge of the relationship between PrPSc, neurodegeneration and infectivity.

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

Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.

Gerstmann-Sträussler-Scheinker disease subtypes efficiently transmit in bank voles as genuine prion diseases.

Gerstmann-Sträussler-Scheinker disease (GSS) is an inherited neurodegenerative disorder associated with mutations in the prion protein gene and accumulation of misfolded PrP with protease-resistant fragments (PrPres) of 6–8 kDa. With the exception of a few GSS cases characterized by co-accumulation of PrPres of 21 kDa, efforts to transmit GSS to rodents have been unsuccessful. As a result, GSS subtypes exclusively associated with 6–8 kDa PrPres have often been considered as non-transmissible proteinopathies rather than true prion diseases. We show that GSS with P102L, A117V and F198S mutations transmit efficiently and produce distinct pathological phenotypes in bank voles (M. glareolus), irrespective of the presence of 21 kDa PrPres in the inoculum, demonstrating that GSS is a genuine prion disease characterized by both transmissibility and strain variation.

Small Ruminant Nor98 Prions Share Biochemical Features with Human Gerstmann-Sträussler-Scheinker Disease and Variably Protease-Sensitive Prionopathy

Prion diseases are classically characterized by the accumulation of pathological prion protein (PrPSc) with the protease resistant C-terminal fragment (PrPres) of 27–30 kDa. However, in both humans and animals, prion diseases with atypical biochemical features, characterized by PK-resistant PrP internal fragments (PrPres) cleaved at both the N and C termini, have been described. In this study we performed a detailed comparison of the biochemical features of PrPSc from atypical prion diseases including human Gerstmann-Sträussler-Scheinker disease (GSS) and variably protease-sensitive prionopathy (VPSPr) and in small ruminant Nor98 or atypical scrapie. The kinetics of PrPres production and its cleavage sites after PK digestion were analyzed, along with the PrPSc conformational stability, using a new method able to characterize both protease-resistant and protease-sensitive PrPSc components. All these PrPSc types shared common and distinctive biochemical features compared to PrPSc from classical prion diseases such as sporadic Creutzfeldt-Jakob disease and scrapie. Notwithstanding, distinct biochemical signatures based on PrPres cleavage sites and PrPSc conformational stability were identified in GSS A117V, GSS F198S, GSS P102L and VPSPr, which allowed their specific identification. Importantly, the biochemical properties of PrPSc from Nor98 and GSS P102L largely overlapped, but were distinct from the other human prions investigated. Finally, our study paves the way towards more refined comparative approaches to the characterization of prions at the animal–human interface.

Molecular discrimination of sheep bovine spongiform encephalopathy from scrapie.

Sheep CH1641-like transmissible spongiform encephalopathy isolates have shown molecular similarities to bovine spongiform encephalopathy (BSE) isolates. We report that the prion protein PrPSc from sheep BSE is extremely resistant to denaturation. This feature, combined with the N-terminal PrPSc cleavage, allowed differentiation of classical scrapie, including CH1641-like, from natural goat BSE and experimental sheep BSE.

PrPC Governs Susceptibility to Prion Strains in Bank Vole, While Other Host Factors Modulate Strain Features.

ABSTRACT 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 PrPC (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 PrPSc types propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrPC modulate prion strain features. IMPORTANCE The 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 PrPC sequence may affect the selection of the substrain replicating in the animal model.

Biochemical characterization of prion strains in bank voles.

Prions exist as different strains exhibiting distinct disease phenotypes. Currently, the identification of prion strains is still based on biological strain typing in rodents. However, it has been shown that prion strains may be associated with distinct PrPSc biochemical types. Taking advantage of the availability of several prion strains adapted to a novel rodent model, the bank vole, we investigated if any prion strain was actually associated with distinctive PrPSc biochemical characteristics and if it was possible to univocally identify strains through PrPSc biochemical phenotypes. We selected six different vole-adapted strains (three human-derived and three animal-derived) and analyzed PrPSc from individual voles by epitope mapping of protease resistant core of PrPSc (PrPres) and by conformational stability and solubility assay. Overall, we discriminated five out of six prion strains, while two different scrapie strains showed identical PrPSc types. Our results suggest that the biochemical strain typing approach here proposed was highly discriminative, although by itself it did not allow us to identify all prion strains analyzed.

Oral pravastatin prolongs survival time of scrapie-infected mice.

Statins are potent inhibitors of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase in the cholesterol-biosynthesis pathway. They are either lipophilic (e.g. simvastatin) or hydrophilic [e.g. pravastatin (PRV)] compounds, considered mainly for long-term treatment of hypercholesterolaemic individuals. Beneficial effects of statins are not related exclusively to their lipid-lowering action; they also possess cholesterol-independent, pleiotropic effects (e.g. anti-inflammatory and antioxidant). Recent studies revealed that simvastatin treatment increased survival significantly in scrapie-infected mice. Although PRV treatment results in measurable drug levels in the mouse brain, the anti-prion effect of this compound has not been investigated. Therefore, we aimed to test the potential therapeutic action of PRV in a murine scrapie model. Our study showed that high-dose and long-term oral PRV treatment prolonged survival times of strain 139A scrapie-infected mice significantly (194 versus 177 days) in the absence of any obvious toxicity, suggesting that protective effects of statins may be independent of absolute solvent or water solubility of the drug.

Effect of PrP genotype and route of inoculation on the ability of discriminatory Western blot to distinguish scrapie from sheep bovine spongiform encephalopathy.

Procedures for discriminating scrapie from bovine spongiform encephalopathy (BSE) in sheep are relevant to ascertain whether BSE has entered the sheep population. This study was aimed at investigating whether the suitability of an official EU discriminative method is affected by the sheep PrP genotype and route of infection.

Cofactors influence the biological properties of infectious recombinant prions

Prion diseases are caused by a misfolding of the cellular prion protein (PrP) to a pathogenic isoform named PrPSc. Prions exist as strains, which are characterized by specific pathological and biochemical properties likely encoded in the three-dimensional structure of PrPSc. However, whether cofactors determine these different PrPSc conformations and how this relates to their specific biological properties is largely unknown. To understand how different cofactors modulate prion strain generation and selection, Protein Misfolding Cyclic Amplification was used to create a diversity of infectious recombinant prion strains by propagation in the presence of brain homogenate. Brain homogenate is known to contain these mentioned cofactors, whose identity is only partially known, and which facilitate conversion of PrPC to PrPSc. We thus obtained a mix of distinguishable infectious prion strains. Subsequently, we replaced brain homogenate, by different polyanionic cofactors that were able to drive the evolution of mixed prion populations toward specific strains. Thus, our results show that a variety of infectious recombinant prions can be generated in vitro and that their specific type of conformation, i.e., the strain, is dependent on the cofactors available during the propagation process. These observations have significant implications for understanding the pathogenesis of prion diseases and their ability to replicate in different tissues and hosts. Importantly, these considerations might apply to other neurodegenerative diseases for which different conformations of misfolded proteins have been described.