Red-Backed Vole Brain Promotes Highly Efficient In Vitro Amplification of Abnormal Prion Protein from Macaque and Human Brains Infected with Variant Creutzfeldt-Jakob Disease Agent

Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrP^TSE) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrP^TSE in tissues and blood. Macaque vCJD PrP^TSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrP^TSE. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrP^TSE. Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrP^TSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrP^TSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrP^TSE was more permissive than human PrP^TSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrP^TSE from brains of humans and macaques with vCJD. PrP^TSE signals were reproducibly detected by Western blot in dilutions through 10^-12 of vCJD-infected 10% brain homogenates. This is the first report showing PrP^TSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrP^TSE in vCJD-infected human and macaque blood.     

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

The development of variant Creutzfeldt-Jakob disease (vCJD) in three recipients of non-leukoreduced red blood cells from asymptomatic donors who subsequently developed the disease has confirmed existing concerns about the possible spread of transmissible spongiform encephalopathies (TSEs) via blood products. In addition, the presence of disease-associated misfolded prion protein (PrP^TSE), generally associated with infectivity, has been demonstrated in the blood of vCJD patients. However, its origin and distribution in this biological fluid are still unknown. Various studies have identified cellular prion protein (PrP^C) among the protein cargo in human blood-circulating extracellular vesicles released from endothelial cells and platelets, and exosomes isolated from the conditioned media of TSE-infected cells have caused the disease when injected into experimental mice. In this study, we demonstrate the detection of PrP^TSE in extracellular vesicles isolated from plasma samples collected during the preclinical and clinical phases of the disease from mice infected with mouse-adapted vCJD and confirm the presence of the exosomal marker Hsp70 in these preparations.     

Plasminogen-Based Capture Combined with Amplification Technology for the Detection of PrPTSE in the Pre-Clinical Phase of Infection

Background

Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrP^TSE) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrP^TSE concentrations in the femtomolar range.

Methodology/Principal Findings

We have developed a three-step assay that firstly captures PrP^TSE from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrP^TSE detection by western blot. We achieved a PrP^TSE capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrP^TSE in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrP^TSE in human plasma spiked with a 10⁻⁸ dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP^TSE in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.

Conclusion/Significance

We have developed a sensitive and specific amplification assay allowing the detection of PrP^TSE in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrP^TSE in blood of patients displaying positivity in large scale screening tests.     

Dissociation of Prion Protein Amyloid Seeding from Transmission of a Spongiform Encephalopathy

Misfolding and aggregation of proteins are common pathogenic mechanisms of a group of diseases called proteinopathies. The formation and spread of proteinaceous lesions within and between individuals were first described in prion diseases and proposed as the basis of their infectious nature. Recently, a similar “prion-like” mechanism of transmission has been proposed in other neurodegenerative diseases such as Alzheimer''s disease. We investigated if misfolding and aggregation of corrupted prion protein (PrP^TSE) are always associated with horizontal transmission of disease. Knock-in transgenic mice (101LL) expressing mutant PrP (PrP-101L) that are susceptible to disease but do not develop any spontaneous neurological phenotype were inoculated with (i) brain extracts containing PrP^TSE from healthy 101LL mice with PrP plaques in the corpus callosum or (ii) brain extracts from mice overexpressing PrP-101L with neurological disease, severe spongiform encephalopathy, and formation of proteinase K-resistant PrP^TSE. In all instances, 101LL mice developed PrP plaques in the area of inoculation and vicinity in the absence of clinical disease or spongiform degeneration of the brain. Importantly, 101LL mice did not transmit disease on serial passage, ruling out the presence of subclinical infection. Thus, in both experimental models the formation of PrP^TSE is not infectious. These results have implications for the interpretation of tests based on the detection of protein aggregates and suggest that de novo formation of PrP^TSE in the host does not always result in a transmissible prion disease. In addition, these results question the validity of assuming that all diseases due to protein misfolding can be transmitted between individuals.     

Experimental Verification of a Traceback Phenomenon in Prion Infection

The clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD) correlate with the allelotypes (M or V) of the polymorphic codon 129 of the human prion protein (PrP) gene and the electrophoretic mobility patterns of abnormal prion protein (PrP^Sc). Transmission of sCJD prions to mice expressing human PrP with a heterologous genotype (referred to as cross-sequence transmission) results in prolonged incubation periods. We previously reported that cross-sequence transmission can generate a new prion strain with unique transmissibility, designated a traceback phenomenon. To verify experimentally the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into mice expressing human PrP with the 129M/M genotype. These 129M/M mice showed altered neuropathology and a novel PrP^Sc type after a long incubation period. We then passaged the brain homogenate from the 129M/M mouse inoculated with sCJD-VV2 prions into other 129M/M or 129V/V mice. Despite cross-sequence transmission, 129V/V mice were highly susceptible to these prions compared to the 129M/M mice. The neuropathology and PrP^Sc type of the 129V/V mice inoculated with the 129M/M mouse-passaged sCJD-VV2 prions were identical to those of the 129V/V mice inoculated with sCJD-VV2 prions. Moreover, we generated for the first time a type 2 PrP^Sc-specific antibody in addition to type 1 PrP^Sc-specific antibody and discovered that drastic changes in the PrP^Sc subpopulation underlie the traceback phenomenon. Here, we report the first direct evidence of the traceback in prion infection.Creutzfeldt-Jakob disease (CJD) is a lethal transmissible neurodegenerative disease caused by an abnormal isoform of prion protein (PrP^Sc), which is converted from the normal cellular isoform (PrP^C) (1, 23). The genotype (M/M, M/V, or V/V, where M and V are allelotypes) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of PrP^Sc in the brain are major determinants of the clinicopathological phenotypes of sporadic CJD (sCJD) (15-18). Type 1 and type 2 PrP^Sc are distinguishable according to the size of the proteinase K-resistant core of PrP^Sc (PrP^res) (21 and 19 kDa, respectively), reflecting differences in the proteinase K cleavage site (at residues 82 and 97, respectively) (15, 18). According to this molecular typing system, sCJD can be classified into six subgroups (MM1, MM2, MV1, MV2, VV1, or VV2).The homology of the PrP genes between inoculated animals and the inoculum determines the susceptibility to prion infection. Transmission of sCJD prions to mice expressing human PrP with a nonhomologous genotype (referred to as cross-sequence transmission) results in a relatively long incubation period (10, 12). Meanwhile, the cross-sequence transmission can generate a new prion strain. Transmission of sCJD-VV2 prions to mice expressing human PrP with the 129M/M genotype generates unusual PrP^res intermediate in size between type 1 and type 2 (10). We have designated this unusual PrP^res with an upward size shift (Sh+) from the inoculated type 2 template MM[VV2]2^Sh+ PrP^res, where the notation is of the following form: host genotype [type of inoculated prion] type of generated PrP^res.Similar to the MM[VV2]2^Sh+ PrP^res, the intermediate-sized PrP^res has been observed in the plaque-type of dura mater graft-associated CJD (p-dCJD) (10, 13). Furthermore, a transmission study using p-dCJD prions revealed that PrP-humanized mice with the 129V/V genotype were highly susceptible to p-dCJD prions despite cross-sequence transmission (10). In addition, these 129V/V mice inoculated with p-dCJD prions produced type 2 PrP^res (10). These findings suggest that p-dCJD could be caused by cross-sequence transmission of sCJD-VV2 prions to individuals with the 129M/M genotype. We have designated this phenomenon “traceback.” The traceback phenomenon was discovered for the first time by a transmission study using variant CJD (vCJD) prions (2). Mice expressing bovine PrP were highly susceptible to vCJD prions because vCJD was caused by cross-sequence transmission of bovine spongiform encephalopathy prions to human. These findings suggest that a traceback study can be a powerful tool to identify the origin of prions (2, 10, 11). However, the traceback phenomenon has not been verified experimentally despite the abundant circumstantial evidence described above.To verify the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into PrP-humanized mice with the 129M/M genotype as an experimental model of p-dCJD. Thereafter, we inoculated these MM[VV2]2^Sh+ prions into PrP-humanized mice with the 129M/M or 129V/V genotype and compared the incubation period, neuropathology, and the type of PrP^res in the brain. Here, we report the first direct evidence of the traceback in prion infection.     

Inherited Prion Disease A117V Is Not Simply a Proteinopathy but Produces Prions Transmissible to Transgenic Mice Expressing Homologous Prion Protein

Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene (PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP^Sc), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP (^CtmPrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP^Sc was demonstrated in the brains of recipient transgenic mice. This PrP^Sc rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of ^CtmPrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.     

Structural Insights into Alternate Aggregated Prion Protein Forms

The conversion of the cellular form of the prion protein (PrP^C) to an abnormal, alternatively folded isoform (PrP^Sc) is the central event in prion diseases or transmissible spongiform encephalopathies. Recent studies have demonstrated de novo generation of murine prions from recombinant prion protein (recPrP) after inoculation into transgenic and wild-type mice. These so-called synthetic prions lead to novel prion diseases with unique neuropathological and biochemical features. Moreover, the use of recPrP in an amyloid seeding assay can specifically detect and amplify various strains of prions. We employed this assay in our experiments and analyzed in detail the morphology of aggregate structures produced under defined chemical constraints. Our results suggest that changes in the concentration of guanidine hydrochloride can lead to different kinetic traces in a typical thioflavin T(ThT) assay. Morphological and structural analysis of these aggregates by atomic force microscopy indicates a variation in the structure of the PrP molecular assemblies.In particular, ThT positive PrP aggregates produced from rec mouse PrP residues 89 to 230 lead to mostly oligomeric structures at low concentrations of guanidine hydrochloride, while more amyloidal structures were observed at higher concentrations of the denaturant. These findings highlight the presence of numerous and complex pathways in deciphering prion constraints for infectivity and toxicity.     

Optimization of the photodynamic inactivation of prions by a phthalocyanine photosensitizer: The crucial involvement of singlet oxygen

Prion disorders are fatal neurodegenerative diseases caused by the autocatalytic conversion of a natively occurring prion protein (PrP^C) into its misfolded infectious form (PrP^TSE). The proven resistance of PrP^TSE to common disinfection procedures increases the risk of prion transmission in medical settings. Herein, we present the effective photodynamic inactivation (PDI) of prions by disulfonated hydroxyaluminum phthalocyanine (AlPcOH(SO₃)₂) utilizing two custom‐built red light sources. The treatment eliminates PrP^TSE signal in infectious mouse brain homogenate with efficiency that depends on light intensity but has a low effect on the overall protein content. Importantly, singlet oxygen (O₂(¹Δ_g)) is the only species significantly photogenerated by AlPcOH(SO₃)₂, and it is responsible for the PDI of prions. More intensive light conditions show not only higher O₂(¹Δ_g) production but also decreases in AlPcOH(SO₃)₂ photostability. Our findings suggest that PDI by AlPcOH(SO₃)₂‐generated O₂(¹Δ_g) represents a promising approach for prion inactivation that may be useful in future decontamination strategies for delicate medical tools.     

Can plants serve as a vector for prions causing chronic wasting disease?

Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrP^TSE) for 24 h in three replicate studies with PrP^TSE in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrP^C) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrP^TSE) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrP^TSE from the roots to the stem to be detectable by the methods employed. Undigested PrP^TSE did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits.     

Multiorgan Detection and Characterization of Protease-Resistant Prion Protein in a Case of Variant CJD Examined in the United States

Background

Variant Creutzfeldt–Jakob disease (vCJD) is a prion disease thought to be acquired by the consumption of prion-contaminated beef products. To date, over 200 cases have been identified around the world, but mainly in the United Kingdom. Three cases have been identified in the United States; however, these subjects were likely exposed to prion infection elsewhere. Here we report on the first of these subjects.

Methodology/Principal Findings

Neuropathological and genetic examinations were carried out using standard procedures. We assessed the presence and characteristics of protease-resistant prion protein (PrP^res) in brain and 23 other organs and tissues using immunoblots performed directly on total homogenate or following sodium phosphotungstate precipitation to increase PrP^res detectability. The brain showed a lack of typical spongiform degeneration and had large plaques, likely stemming from the extensive neuronal loss caused by the long duration (32 months) of the disease. The PrP^res found in the brain had the typical characteristics of the PrP^res present in vCJD. In addition to the brain and other organs known to be prion positive in vCJD, such as the lymphoreticular system, pituitary and adrenal glands, and gastrointestinal tract, PrP^res was also detected for the first time in the dura mater, liver, pancreas, kidney, ovary, uterus, and skin.

Conclusions/Significance

Our results indicate that the number of organs affected in vCJD is greater than previously realized and further underscore the risk of iatrogenic transmission in vCJD.     

Mapping the Prion Protein Using Recombinant Antibodies

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrP^C) into a pathogenic isoform (PrP^Sc). The occurrence of PrP^C on the cell surface and PrP^Sc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp^0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrP^C on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrP^C and PrP^Sc, while epitopes associated with most of the antibodies in the panel were present on PrP^C but absent from PrP^Sc.     

Infrared Microspectroscopy Detects Protein Misfolding Cyclic Amplification (PMCA)-induced Conformational Alterations in Hamster Scrapie Progeny Seeds

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP^C) into Proteinase K-resistant, infectious PrP particles (PrP^TSE), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP^C substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP^C substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.     

Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrP^Sc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). Stable variations in PrP^Sc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrP^Sc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrP^Sc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrP^Sc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrP^Sc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrP^Sc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrP^Sc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.     

Enzymatic Formulation Capable of Degrading Scrapie Prion under Mild Digestion Conditions

The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrP^Sc signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrP^Sc at 65°C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50°C over 2 h revealed the progressive attenuation of PrP^Sc intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrP^Sc infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1) (p-value = 0.008 at 95% confidence interval). This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions.     

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

During prion infection, the normal, protease-sensitive conformation of prion protein (PrP^C) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP^Sc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP^Sc in prion-infected brain homogenates as an initiating seed to convert PrP^C and trigger the self-propagation of PrP^Sc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP^Sc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP^Sc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP^Sc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.     

Co-existence of Distinct Prion Types Enables Conformational Evolution of Human PrPSc by Competitive Selection

The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion proteins (PrP^Sc). The molecular mechanism responsible for the adaptation, mutation, and evolution of prions observed in cloned cells and upon crossing the species barrier remains unsolved. Using biophysical techniques and conformation-dependent immunoassays in tandem, we isolated two distinct populations of PrP^Sc particles with different conformational stabilities and aggregate sizes, which frequently co-exist in the most common human prion disease, sporadic Creutzfeldt-Jakob disease. The protein misfolding cyclic amplification replicates each of the PrP^Sc particle types independently and leads to the competitive selection of those with lower initial conformational stability. In serial propagation with a nonglycosylated mutant PrP^C substrate, the dominant PrP^Sc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to its lowest stability. Cumulatively, the data show that sporadic Creutzfeldt-Jakob disease PrP^Sc is not a single conformational entity but a dynamic collection of two distinct populations of particles. This implies the co-existence of different prions, whose adaptation and evolution are governed by the selection of progressively less stable, faster replicating PrP^Sc conformers.     

Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation

In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrP^Sc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrP^Sc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrP^Sc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrP^C by TACE α-secretase, which physiologically precludes PrP^Sc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrP^Sc. In mice challenged with prions, inhibition of ROCK also lowered brain PrP^Sc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrP^Sc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases.     

Immunopurification of Pathological Prion Protein Aggregates

Background

Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP^C) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrP^Sc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP^C molecules. A great deal of effort has been devoted to developing protocols for purifying PrP^Sc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP^Sc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases.

Principal Findings

We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP^Sc and mutant PrP aggregates at electrophoretic homogeneity. PrP^Sc purified from prion-infected mice was able to seed misfolding of PrP^C in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons.

Significance

The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.     

Direct Detection of Disease Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N Terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.Key Words: BSE, scrapie and vCJD Diagnosis, DELFIA®, novel N terminal antibodies