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

In 2007, we reported a patient with an atypical form of Creutzfeldt-Jakob disease (CJD) heterozygous for methionine-valine (MV) at codon 129 who showed a novel pathological prion protein (PrPTSE) 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 (PrPTSE MVAG), showing that PrPTSE MVAG is composed of multiple conformers with biochemical properties distinct from those of PrPTSE type 1 and type 2 of MV sporadic CJD (sCJD). Experimental transmission of CJD-MVAG to bank voles and gene-targeted transgenic mice carrying the human prion protein gene (TgHu mice) showed unique transmission rates, survival times, neuropathological changes, PrPTSE deposition patterns, and PrPTSE 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-MVAGIMPORTANCE 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.

Synthetic scrapie infectivity: interaction between recombinant PrP and scrapie brain-derived RNA.

The key molecular event in human cerebral proteinopathies, which include Alzheimer's, Parkinson's and Huntington's diseases, is the structural conversion of a specific host protein into a ß-sheet-rich conformer. With regards to this common mechanism, it appears difficult to explain the outstanding infectious properties attributed to PrP(Sc), the hallmark of another intriguing family of cerebral proteinopathies known as transmissible spongiform encephalopathies (TSE) or prion diseases. The infectious PrP(Sc) or "prion" is thought to be composed solely of a misfolded form of the otherwise harmless cellular prion protein (PrP(c)). To gain insight into this unique situation, we used the 263K scrapie hamster model to search for a putative PrP(Sc)-associated factor that contributes to the infectivity of PrP(Sc) amyloid. In a rigorously controlled set of experiments that included several bioassays, we showed that originally innocuous recombinant prion protein (recPrP) equivalent to PrP(c) is capable of initiating prion disease in hamsters when it is converted to a prion-like conformation (ß-sheet-rich) in the presence of RNA purified from scrapie-associated fibril (SAF) preparations. Analysis of the recPrP-RNA infectious mixture reveals the presence of 2 populations of small RNAs of approximately 27 and 55 nucleotides. These unprecedented findings are discussed in light of the distinct relationship that may exist between this RNA material and the 2 biological properties, infectivity and strain features, attributed to prion amyloid.

Mutant PrPCJD prevails over wild-type PrPCJD in the brain of V210I and R208H genetic Creutzfeldt-Jakob disease patients.

Creutzfeldt-Jakob disease (CJD) is a neurodegenerative disorder characterized by the deposition of the pathological conformer (PrP(CJD)) of the host encoded cellular prion protein (PrP(C)). In genetic CJD associated with V210I or R208H PrP substitutions, the pathogenic role of mutant residues is still poorly understood. To understand how V210I or R208H PrP mutations facilitate the development of the disease, we determined by mass spectrometry the quantitative ratio of mutant/wild-type PrP(CJD) allotypes in brains from affected subjects. We found that the mutant PrP(CJD) allotypes moderately exceeds of 2- or 3-fold the amount of the wild-type counterpart suggesting that these mutations mainly exert their pathogenic effect on the onset of the pathogenic cascade. Different mechanisms can be hypothesized to explain the pathogenic role of mutant residues: V210I and R208H substitutions can increase the concentration of PrP(C) and the probability to form insoluble aggregates, or they may facilitate the formation of pathological intermediates, or, alternatively, they may increase the affinity for ligands that are involved in the initial phases of PrP(CJD) formation and aggregation. Whatever the mechanism, the enrichment found for the mutated PrP(CJD) species indicates that these altered structures are more prone, with respect to the non-mutated ones, to be captured in the polymerization process either at the onset or during the development of the disease.

Assessment of prion reduction filters in decreasing infectivity of ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" human blood and red blood cells.

BACKGROUND: The safety of red blood cells (RBCs) is of concern because of the occurrence of four transfusion-transmitted variant Creutzfeldt-Jakob disease (vCJD) cases in the United Kingdom. The absence of validated screening tests requires the use of procedures to remove prions from blood to minimize the risk of transmission. These procedures must be validated using infectious prions in a form that is as close as possible to one in blood. STUDY DESIGN AND METHODS: Units of human whole blood (WB) and RBCs were spiked with high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked samples were leukoreduced and then passed through prion-removing filters (Pall Corporation). In another experiment, RBCs from 263K scrapie-infected hamsters were treated as above, and residual infectivity was measured by bioassay. RESULTS: The overall removal of infectivity by the filters from prion-spiked WB and RBCs was approximately two orders of magnitude. No infectivity was detected in filtered hamster RBCs endogenously infected with scrapie. CONCLUSION: The use of prion-removing filters may help to reduce the risk of transfusion-transmitted vCJD. To avoid overestimation of prion removal efficiency in validation studies, it may be more appropriate to use supernates from ultracentrifugation of scrapie-infected hamster brain homogenate rather than the current standard brain homogenates.

Role of proteomics in understanding prion infection.

Transmissible spongiform encephalopathies or prion diseases are fatal neurodegenerative pathologies characterized by the autocatalytic misfolding and polymerization of a cellular glycoprotein (cellular prion protein [PrP(C)]) that accumulates in the CNS and leads to neurodegeneration. The detailed mechanics of PrP(C) conversion to its pathological isoform (PrP(TSE)) are unclear but one or more exogenous factors are likely involved in the process of PrP misfolding. In the last 20 years, proteomic investigations have identified several endogenous proteins that interact with PrP(C), PrP(TSE) or both, which are possibly involved in the prion pathogenetic process. However, current approaches have not yet produced convincing conclusions on the biological value of such PrP interactors. Future advancements in the comprehension of the molecular pathogenesis of prion diseases, in experimental techniques and in data analysis procedures, together with a boost in more productive international collaborations, are therefore needed to improve the understanding on the role of PrP interactors. Finally, the advancement of 'omics' techniques in prion diseases will contribute to the development of novel diagnostic tests and effective drugs.

Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

The pathological form of prion protein (PrP(Sc)), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc) extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP90₋231) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc). In the present study we demonstrate that hPrP90₋231, pre-incubated with 10 mM Ca⁺⁺ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP90₋231 bearing pathogenic mutations (D202N and E200K). We also report that Ca⁺⁺ binding to hPrP90₋231 induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP90₋231 cytotoxicity. Finally, by in silico structural analysis, we propose that Ca⁺⁺ binding to hPrP90₋231 modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.

Comparison of nanofiltration efficacy in reducing infectivity of centrifuged versus ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" albumin solutions.

BACKGROUND: The safety of plasma-derived products is of concern for possible transmission of variant Creutzfeldt-Jakob disease. The absence of validated screening tests requires the use of procedures to remove or inactivate prions during the manufacture of plasma-derived products to minimize the risk of transmission. These procedures need proper validation studies based on spiking human plasma or intermediate fractions of plasma fractionation with prions in a form as close as possible to that present in blood. STUDY DESIGN AND METHODS: Human albumin was spiked with low-speed or high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked albumin was then passed through a cascade of filters from 100 nm down to 20 to 15 nm. Residual infectivity was measured by bioassay. RESULTS: The overall removal of infectivity spiked into albumin through serial nanofiltration steps was 4 to 5 logs using low-speed supernatant and 2 to 3 logs with high-speed supernatant. CONCLUSION: These findings confirm the utility of nanofiltration in removing infectivity from plasma (or other products) spiked with scrapie brain homogenate supernatants. However, efficiency is diminished using supernatants that have been ultracentrifuged to reduce aggregated forms of the infectious agent. Thus, filtration removal data based on experiments using "standard" low-speed centrifugation supernatants might overestimate the amount of prion removal in plasma or urine-derived therapeutic products.

The pathological prion protein forms ionic conductance in lipid bilayer.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative pathologies characterized by the accumulation of amyloid fibrils mainly composed of the pathological isoform of the prion protein (PrP(TSE)). PrP(TSE) pre-amyloid fibrils are supposed to induce neurodegenerative lesions possibly through the alteration of membrane permeability. The effect of PrP(TSE) on cellular membranes has been modeled in vitro by synthetic peptides that are, however, only partially representative of PrP(TSE) isoforms found in vivo. In the present work we show that a synthetic membrane exposed to PrP27-30 extracted from TSE-infected hamster brains changes its permeability because of the formation of molecular pores that alter the conductance of the synthetic lipid bilayer. Synthetic membrane challenged with the recombinant prion peptide PrP90-231 shows a much lower conductance. Elevation of calcium ion concentration not only increases the current amplitude due to the action of both PrP27-30 and PrP90-231 on the membrane, but also amplifies the interaction of PrP90-231 with the lipid bilayer.

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 PrP(Sc), a disease-associated isoform of the host-encoded cellular protein (PrP(C)). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP(Sc). However, PrP(Sc) 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 PrP(Sc) aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP(C) and PrP(Sc) by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrP(Sc) 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 PrP(Sc), we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP(Sc) 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 PrP(Sc) conformational stabilities of protease-resistant and protease-sensitive PrP(Sc) and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.

Efficient transmission and characterization of Creutzfeldt-Jakob disease strains in bank voles.

Transmission of prions between species is limited by the "species barrier," which hampers a full characterization of human prion strains in the mouse model. We report that the efficiency of primary transmission of prions from Creutzfeldt-Jakob disease patients to a wild rodent species, the bank vole (Clethrionomys glareolus), is comparable to that reported in transgenic mice carrying human prion protein, in spite of a low prion protein-sequence homology between man and vole. Voles infected with sporadic and genetic Creutzfeldt-Jakob disease isolates show strain-specific patterns of spongiform degeneration and pathological prion protein-deposition, and accumulate protease-resistant prion protein with biochemical properties similar to the human counterpart. Adaptation of genetic Creutzfeldt-Jakob disease isolates to voles shows little or no evidence of a transmission barrier, in contrast to the striking barriers observed during transmission of mouse, hamster, and sheep prions to voles. Our results imply that in voles there is no clear relationship between the degree of homology of the prion protein of the donor and recipient species and susceptibility, consistent with the view that the prion strain gives a major contribution to the species barrier. The vole is therefore a valuable model to study human prion diversity and, being susceptible to a range of animal prions, represents a unique tool for comparing isolates from different species.