Properties Of PrPSc Research Articles

Novel histotypes of sporadic Creutzfeldt–Jakob disease linked to 129MV genotype

The MV1 and MV2 subtypes of sporadic Creutzfeldt–Jakob disease (sCJD) are linked to the heterozygous methionine (M)/valine (V) polymorphism at codon 129 of the prion protein (PrP) gene. MV2 is phenotypically heterogeneous, whereas MV1, due to its low prevalence, is one of the least well characterized subtypes. In this study, we investigated the biochemical properties of PrPSc and phenotypic expression of cases diagnosed as sCJD MV1 and MV2. We describe four MV2 histotypes: 2C, with cortical (C) coarse pathology; 2K, with kuru (K) plaque deposits; 2C-K, with co-existing C and K histotypic features; and the novel histotype 2C-PL that mimics 2C in the cerebral cortex and cerebellum, but exhibits plaque-like (PL) PrP deposits in subcortical regions (e.g., basal nuclei, thalamus and midbrain). Histotype prevalence is highest for 2C-K (55%), intermediate for 2C (31%), and lowest for 2C-PL and 2K (7%). Nearly every MV2 case expressed both PrPSc types, with T2 being the predominant type (“MV2-1”). MV1 cases typically show a rapid disease course (≤ 4 months), and feature the 1C histotype, phenotypically identical to sCJDMM1. Co-existing PrPSc types, with T1 significantly exceeding T2 (“MV1-2”), are detected in patients diagnosed as MV1 with longer disease courses. We observed four histotypes among MV1-2 cases, including two novel histotypes: 1V, reminiscent of sCJDVV1; 1C-2C, resembling sCJDMM1-2 with predominant MM1 histotypic component; and novel histotypes 1C-2PL and 1C-2K, overall mimicking 1C in the cerebral cortex, but harboring T2 and plaque-like PrP deposits in subcortical regions (1C-2PL), and T2 and kuru plaques in the cerebellum (1C-2K). Lesion profiles of 1C, 1V, and 1C-2C are similar, but differ from 1C-2PL and 1C-2K, as the latter two groups show prominent hippocampal and nigral degeneration. We believe that the novel “C-PL” histotypes are distinct entities rather than intermediate forms between “C” and “C-K” groups, and that 1C-2PL and 1C-2K histotypes may be characterized by different T1 variants of the same size.

Prion strains: shining new light on old concepts.

Prion diseases are a group of inevitably fatal neurodegenerative disorders affecting numerous mammalian species, including humans. The existence of heritable phenotypes of disease in the natural host suggested that prions exist as distinct strains. Transmission of sheep scrapie to rodent models accelerated prion research, resulting in the isolation and characterization of numerous strains with distinct characteristics. These strains are grouped into categories based on the incubation period of disease in different strains of mice and also by how stable the strain properties were upon serial passage. These classical studies defined the host and agent parameters that affected strain properties, and, prior to the advent of the prion hypothesis, strain properties were hypothesized to be the result of mutations in a nucleic acid genome of a conventional pathogen. The development of the prion hypothesis challenged the paradigm of infectious agents, and, initially, the existence of strains was difficult to reconcile with a protein-only agent. In the decades since, much evidence has revealed how a protein-only infectious agent can perform complex biological functions. The prevailing hypothesis is that strain-specific conformations of PrPSc encode prion strain diversity. This hypothesis can provide a mechanism to explain the observed strain-specific differences in incubation period of disease, biochemical properties of PrPSc, tissue tropism, and subcellular patterns of pathology. This hypothesis also explains how prion strains mutate, evolve, and adapt to new species. These concepts are applicable to prion-like diseases such as Parkinson's and Alzheimer's disease, where evidence of strain diversity is beginning to emerge.

Prion protein with a mutant N-terminal octarepeat region undergoes cobalamin-dependent assembly into high–molecular weight complexes

The cellular prion protein (PrPC) has a C-terminal globular domain and a disordered N-terminal region encompassing five octarepeats (ORs). Encounters between Cu(II) ions and four OR sites produce interchangeable binding geometries; however, the significance of Cu(II) binding to ORs in different combinations is unclear. To understand the impact of specific binding geometries, OR variants were designed that interact with multiple or single Cu(II) ions in specific locked coordinations. Unexpectedly, we found that one mutant produced detergent-insoluble, protease-resistant species in cells in the absence of exposure to the infectious prion protein isoform, scrapie-associated prion protein (PrPSc). Formation of these assemblies, visible as puncta, was reversible and dependent upon medium formulation. Cobalamin (Cbl), a dietary cofactor containing a corrin ring that coordinates a Co3+ ion, was identified as a key medium component, and its effect was validated by reconstitution experiments. Although we failed to find evidence that Cbl interacts with Cu-binding OR regions, we instead noted interactions of Cbl with the PrPC C-terminal domain. We found that some interactions occurred at a binding site of planar tetrapyrrole compounds on the isolated globular domain, but others did not, and N-terminal sequences additionally had a marked effect on their presence and position. Our studies define a conditional effect of Cbl wherein a mutant OR region can act in cis to destabilize a globular domain with a wild type sequence. The unexpected intersection between the properties of PrPSc's disordered region, Cbl, and conformational remodeling events may have implications for understanding sporadic prion disease that does not involve exposure to PrPSc.

Phenotypic diversity of genetic Creutzfeldt\u2013Jakob disease: a histo-molecular-based classification

The current classification of sporadic Creutzfeldt–Jakob disease (sCJD) includes six major clinicopathological subtypes defined by the physicochemical properties of the protease-resistant core of the pathologic prion protein (PrPSc), defining two major PrPSc types (i.e., 1 and 2), and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein gene (PRNP). How these sCJD subtypes relate to the well-documented phenotypic heterogeneity of genetic CJD (gCJD) is not fully understood. We analyzed molecular and phenotypic features in 208 individuals affected by gCJD, carrying 17 different mutations, and compared them with those of a large series of sCJD cases. We identified six major groups of gCJD based on the combination PrPSc type and codon 129 genotype on PRNP mutated allele, each showing distinctive histopathological characteristics, irrespectively of the PRNP associated mutation. Five gCJD groups, named M1, M2C, M2T, V1, and V2, largely reproduced those previously described in sCJD subtypes. The sixth group shared phenotypic traits with the V2 group and was only detected in patients carrying the E200K-129M haplotype in association with a PrPSc type of intermediate size (“i”) between type 1 and type 2. Additional mutation-specific effects involved the pattern of PrP deposition (e.g., a “thickened” synaptic pattern in E200K carriers, cerebellar “stripe-like linear granular deposits” in those with insertion mutations, and intraneuronal globular dots in E200K-V2 or -M”i”). A few isolated cases linked to rare PRNP haplotypes (e.g., T183A-129M), showed atypical phenotypic features, which prevented their classification into the six major groups. The phenotypic variability of gCJD is mostly consistent with that previously found in sCJD. As in sCJD, the codon 129 genotype and physicochemical properties of PrPSc significantly correlated with the phenotypic variability of gCJD. The most common mutations linked to CJD appear to have a variable and overall less significant effect on the disease phenotype, but they significantly influence disease susceptibility often in a strain-specific manner. The criteria currently used for sCJD subtypes can be expanded and adapted to gCJD to provide an updated classification of the disease with a molecular basis.

Pathologic and biochemical characterization of PrPSc from elk with PRNP polymorphisms at codon 132 after experimental infection with the chronic wasting disease agent

BackgroundThe Rocky Mountain elk (Cervus elaphus nelsoni) prion protein gene (PRNP) is polymorphic at codon 132, with leucine (L132) and methionine (M132) allelic variants present in the population. In elk experimentally inoculated with the chronic wasting disease (CWD) agent, different incubation periods are associated with PRNP genotype: LL132 elk survive the longest, LM132 elk are intermediate, and MM132 elk the shortest. The purpose of this study was to investigate potential mechanisms underlying variations in incubation period in elk of different prion protein genotypes. Elk calves of three PRNP genotypes (n = 2 MM132, n = 2 LM132, n = 4 LL132) were orally inoculated with brain homogenate from elk clinically affected with CWD.ResultsElk with longer incubation periods accumulated relatively less PrPSc in the brain than elk with shorter incubation periods. PrPSc accumulation in LM132 and MM132 elk was primarily neuropil-associated while glial-associated immunoreactivity was prominent in LL132 elk. The fibril stability of PrPSc from MM132 and LM132 elk were similar to each other and less stable than that from LL132 elk. Real-time quaking induced conversion assays (RT-QuIC) revealed differences in the ability of PrPSc seed from elk of different genotypes to convert recombinant 132 M or 132 L substrate.ConclusionsThis study provides further evidence of the importance of PRNP genotype in the pathogenesis of CWD of elk. The longer incubation periods observed in LL132 elk are associated with PrPSc that is more stable and relatively less abundant at the time of clinical disease. The biochemical properties of PrPSc from MM132 and LM132 elk are similar to each other and different to PrPSc from LL132 elk. The shorter incubation periods in MM132 compared to LM132 elk may be the result of genotype-dependent differences in the efficiency of propagation of PrPSc moieties present in the inoculum. A better understanding of the mechanisms by which the polymorphisms at codon 132 in elk PRNP influence disease pathogenesis will help to improve control of CWD in captive and free-ranging elk populations.

Atypical Creutzfeldt-Jakob disease with PrP-amyloid plaques in white matter: molecular characterization and transmission to bank voles show the M1 strain signature

Amyloid plaques formed by abnormal prion protein (PrPSc) aggregates occur with low frequency in Creutzfeldt-Jakob disease, but represent a pathological hallmark of three relatively rare disease histotypes, namely variant CJD, sporadic CJDMV2K (methionine/valine at PRNP codon 129, PrPSc type 2 and kuru-type amyloid plaques) and iatrogenic CJDMMiK (MM at codon 129, PrPSc of intermediate type and kuru plaques). According to recent studies, however, PrP-amyloid plaques involving the subcortical and deep nuclei white matter may also rarely occur in CJDMM1 (MM at codon 129 and PrPSc type 1), the most common CJD histotype.To further characterize the phenotype of atypical CJDMM1 with white matter plaques (p-CJDMM1) and unravel the basis of amyloid plaque formation in such cases, we compared clinical and histopathological features and PrPSc physico-chemical properties between 5 p-CJDMM1 and 8 typical CJDMM1 brains lacking plaques. Furthermore, transmission properties after bioassay in two genetic lines of bank voles were also explored in the two groups.All 5 p-CJDMM1 cases had a disease duration longer than one year. Three cases were classified as sporadic CJDMM1, one as sporadic CJDMM1 + 2C and one as genetic CJDE200K-MM1. Molecular mass, protease sensitivity and thermo-solubilization of PrPSc aggregates did not differ between p-CJDMM1 and classical CJDMM1 cases. Likewise, transmission properties such as incubation time, lesion profile and PrPSc properties in bank voles also matched in the two groups.The present data further define the clinical-pathologic phenotype of p-CJDMM1, definitely establish it as a distinctive CJD histotype and demonstrate that PrP-plaque formation in this histotype is not a strain-specific feature. Since cases lacking amyloid plaques may also manifest a prolonged (i.e. > than one year) disease course, unidentified, host-specific factors likely play a significant role, in addition to disease duration, in generating white matter PrP-amyloid plaques in p-CJDMM1.

Secondary-structure prediction revisited: Theoretical β-sheet propensity and coil propensity represent structures of amyloids and aid in elucidating phenomena involved in interspecies transmission of prions.

Prions are unique infectious agents, consisting solely of abnormally-folded prion protein (PrPSc). However, they possess virus-like features, including strain diversity, the ability to adapt to new hosts and to be altered evolutionarily. Because prions lack genetic material (DNA and RNA), these biological phenomena have been attributed to the structural properties of PrPSc. Therefore, many structural models of the structure of PrPSc have been proposed based on the limited structural information available, regardless of the incompatibility with high-resolution structural analysis. Recently hypothesized models consist solely of β-sheets and intervening loops/kinks; i.e. parallel in-register β-sheet and β-solenoid models. Owing to the relative simplicity of these structural models of PrPSc, we hypothesized that numerical conversion of the primary structures with a relevant algorithm would enable quantitative comparison between PrPs of distinct primary structures. We therefore used the theoretical values of β-sheet (Pβ) and random-coil (Pc) propensity calculated by secondary structure prediction with a neural network, to analyze interspecies transmission of prions. By reviewing experiments in the literature, we ascertained the biological relevance of Pβ and Pc and found that these classical parameters surprisingly carry substantial information of amyloid structures. We also demonstrated how these parameters could aid in interpreting and explaining phenomena in interspecies transmissions. Our approach can lead to the development of a versatile tool for investigating not only prions but also other amyloids.

Biochemical Characterization of Prions.

Prion disease or transmissible spongiform encephalopathies are characterized by the presence of the abnormal form of the prion protein (PrPSc). The pathological and transmissible properties of PrPSc are enciphered in its secondary and tertiary structures. Since it's well established that different strains of prions are linked to different conformations of PrPSc, biochemical characterization of prions seems a preliminary but reliable approach to detect, analyze, and compare prion strains. Experimental biochemical procedures might be helpful in distinguishing PrPSc physicochemical properties and include resistance to proteinase K (PK) digestion, insolubility in nonionic detergents, PK-resistance under denaturing conditions and sedimentation properties in sucrose gradients. This biochemical approach has been extensively applied in human prion disorders and subsequently expanded for PrPSc characterization in animals. In particular, in sporadic Creutzfedlt-Jakob disease (sCJD) PrPSc is characterized by two main glycotypes conventionally named Type 1 and Type 2, based on the apparent gel migration at 21 and 19kDa of the PrPSc PK-resistant fragment. An additional PrPSc type was identified in sCJD characterized by an unglycosylated dominant glycoform pattern and in 2010 a variably protease-sensitive prionopathy (VPSPr) was reported showing a PrPSc with an electrophoretic ladder like pattern. Additionally, the presence of PrPSc truncated fragments completes the electrophoretic characterization of different prion strains. By two-dimensional (2D) electrophoretic analysis additional PrPSc pattern was identified, since this procedure provides information about the isoelectric point and the different peptides length related to PK cleavage, as well as to glycosylation extent or GPI anchor presence. We here provide and extensive review on PrPSc biochemical analysis in human and animal prion disorders. Further, we show that PrPSc glycotypes observed in CJD share similarities with PrPSc in bovine spongiform encephalopathy forms (BSE).

Prion Strain Diversity.

Prion diseases affect a wide range of mammal species and are caused by a misfolded self-propagating isoform (PrPSc) of the normal prion protein (PrPC). Distinct strains of prions exist and are operationally defined by differences in a heritable phenotype under controlled experimental transmission conditions. Prion strains can differ in incubation period, clinical signs of disease, tissue tropism, and host range. The mechanism by which a protein-only pathogen can encode strain diversity is only beginning to be understood. The prevailing hypothesis is that prion strain diversity is encoded by strain-specific conformations of PrPSc; however, strain-specific cellular cofactors have been identified in vitro that may also contribute to prion strain diversity. Although much progress has been made on understanding the etiological agent of prion disease, the relationship between the strain-specific properties of PrPSc and the resulting phenotype of disease in animals is poorly understood.

Inhibition of human prion neuropeptide PrP106-126 aggregation by hexacoordinated ruthenium complexes

Prion disease is a neurodegenerative disorder that can occur among humans and other animals. The aberrant isoform of prion protein PrPSc has been identified as the infectious agent. The neuropeptide PrP106-126 has been widely used as a suitable model to study the biological and physiochemical properties of PrPSc. PrP106-126 shares several physicochemical and biological properties with PrPSc, including cellular toxicity, fibrillogenesis, and membrane-binding affinity. Ruthenium complexes are commonly employed in anti-cancer studies due to their low cellular toxicity. In this study, six hexacoordinated ruthenium complexes with different molecular configurations were used to investigate their effects on PrP106-126 aggregation inhibition. Results revealed that the interaction between the complexes and the peptide included metal coordination and hydrophobic interaction mainly. Those complexes with aromatic structure displayed better inhibitory effects, although they only had a common binding affinity to PrP106-126. This study provided better understanding on the interaction of metal complexes with PrP106-126 and paved the way for potential Ru-based metallodrugs against prion diseases.

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.

Assessment of strain-specific PrP(Sc) elongation rates revealed a transformation of PrP(Sc) properties during protein misfolding cyclic amplification.

Prion replication is believed to consist of two components, a growth or elongation of infectious isoform of the prion protein (PrPSc) particles and their fragmentation, a process that provides new replication centers. The current study introduced an experimental approach that employs Protein Misfolding Cyclic Amplification with beads (PMCAb) and relies on a series of kinetic experiments for assessing elongation rates of PrPSc particles. Four prion strains including two strains with short incubation times to disease (263K and Hyper) and two strains with very long incubation times (SSLOW and LOTSS) were tested. The elongation rate of brain-derived PrPSc was found to be strain-specific. Strains with short incubation times had higher rates than strains with long incubation times. Surprisingly, the strain-specific elongation rates increased substantially for all four strains after they were subjected to six rounds of serial PMCAb. In parallel to an increase in elongation rates, the percentages of diglycosylated PrP glycoforms increased in PMCAb-derived PrPSc comparing to those of brain-derived PrPSc. These results suggest that PMCAb selects the same molecular features regardless of strain initial characteristics and that convergent evolution of PrPSc properties occurred during in vitro amplification. These results are consistent with the hypothesis that each prion strain is comprised of a variety of conformers or ‘quasi-species’ and that change in the prion replication environment gives selective advantage to those conformers that replicate most effectively under specific environment.

Inactivation of Template-Directed Misfolding of Infectious Prion Protein by Ozone

Misfolded prions (PrP(Sc)) are well known for their resistance to conventional decontamination processes. The potential risk of contamination of the water environment, as a result of disposal of specified risk materials (SRM), has raised public concerns. Ozone is commonly utilized in the water industry for inactivation of microbial contaminants and was tested in this study for its ability to inactivate prions (263K hamster scrapie = PrP(Sc)). Treatment variables included initial ozone dose (7.6 to 25.7 mg/liter), contact time (5 s and 5 min), temperature (4°C and 20°C), and pH (pH 4.4, 6.0, and 8.0). Exposure of dilute suspensions of the infected 263K hamster brain homogenates (IBH) (0.01%) to ozone resulted in the in vitro destruction of the templating properties of PrP(Sc), as measured by the protein misfolding cyclic amplification (PMCA) assay. The highest levels of prion inactivation (≥4 log(10)) were observed with ozone doses of 13.0 mg/liter, at pH 4.4 and 20°C, resulting in a CT (the product of residual ozone concentration and contact time) value as low as 0.59 mg · liter(-1) min. A comparison of ozone CT requirements among various pathogens suggests that prions are more susceptible to ozone degradation than some model bacteria and protozoa and that ozone treatment may be an effective solution for inactivating prions in water and wastewater.

Sc237 hamster PrPSc and Sc237-derived mouse PrPSc generated by interspecies in vitro amplification exhibit distinct pathological and biochemical properties in tga20 transgenic mice

Prions are the infectious agents responsible for transmissible spongiform encephalopathy, and are primarily composed of the pathogenic form (PrP(Sc)) of the host-encoded prion protein (PrP(C)). Recent studies have revealed that protein misfolding cyclic amplification (PMCA), a highly sensitive method for PrP(Sc) detection, can overcome the species barrier in several xenogeneic combinations of PrP(Sc) seed and PrP(C) substrate. Although these findings provide valuable insight into the origin and diversity of prions, the differences between PrP(Sc) generated by interspecies PMCA and by in vivo cross-species transmission have not been described. This study investigated the histopathological and biochemical properties of PrP(Sc) in the brains of tga20 transgenic mice inoculated with Sc237 hamster scrapie prion and PrP(Sc) from mice inoculated with Sc237-derived mouse PrP(Sc), which had been generated by interspecies PMCA using Sc237 as seed and normal mouse brain homogenate as substrate. Tga20 mice overexpressing mouse PrP(C) were susceptible to Sc237 after primary transmission. PrP(Sc) in the brains of mice inoculated with Sc237-derived mouse PrP(Sc) and in the brains of mice inoculated with Sc237 differed in their lesion profiles and accumulation patterns, Western blot profiles, and denaturant resistance. In addition, these PrP(Sc) exhibited distinctive virulence profiles upon secondary passage. These results suggest that different in vivo and in vitro environments result in propagation of PrP(Sc) with different biological properties.

A molecular switch controls interspecies prion disease transmission in mice

Transmissible spongiform encephalopathies are lethal neurodegenerative disorders that present with aggregated forms of the cellular prion protein (PrPC), which are known as PrPSc. Prions from different species vary considerably in their transmissibility to xenogeneic hosts. The variable transmission barriers depend on sequence differences between incoming PrPSc and host PrPC and additionally, on strain-dependent conformational properties of PrPSc. The beta2-alpha2 loop region within PrPC varies substantially between species, with its structure being influenced by the residue types in the 2 amino acid sequence positions 170 (most commonly S or N) and 174 (N or T). In this study, we inoculated prions from 5 different species into transgenic mice expressing either disordered-loop or rigid-loop PrPC variants. Similar beta2-alpha2 loop structures correlated with efficient transmission, whereas dissimilar loops correlated with strong transmission barriers. We then classified literature data on cross-species transmission according to the 170S/N polymorphism. Transmission barriers were generally low between species with the same amino acid residue in position 170 and high between those with different residues. These findings point to a triggering role of the local beta2-alpha2 loop structure for prion transmissibility between different species.

Live Cell Fluorescence Resonance Energy Transfer Predicts an Altered Molecular Association of Heterologous PrPSc with PrPC

Prion diseases result from the accumulation of a misfolded isoform (PrP(Sc)) of the normal host prion protein (PrP(C)). PrP(Sc) propagates by templating its conformation onto resident PrP(C) to generate new PrP(Sc). Although the nature of the PrP(Sc)-PrP(C) complex is unresolved, certain segments or specific residues are thought to feature critically in its formation. The polymorphic residue 129 is one such site under considerable study. We combined transmission studies with a novel live cell yeast-based fluorescence resonance energy transfer (FRET) system that models the molecular association of PrP in a PrP(Sc)-like state, as a way to explore the role of residue 129 in this process. We show that a reduction in efficiency of prion transmission between donor PrP(Sc) and recipient PrP(C) that are mismatched at residue 129 correlates with a reduction in FRET between PrP-129M and PrP-129V in our yeast model. We further show that this effect depends on the different secondary structure propensities of Met and Val, rather than the specific amino acids. Finally, introduction of the disease-associated P101L mutation (mouse- equivalent) abolished FRET with wild-type mouse PrP, whereas mutant PrP-P101L displayed high FRET with homologous PrP-P101L, as long as residue 129 matched. These studies provide the first evidence for a physical alteration in the molecular association of PrP molecules differing in one or more residues, and they further predict that the different secondary structure propensities of Met and Val define the impaired association observed between PrP(Sc) and PrP(C) mismatched at residue 129.

In Vivo Generation of Neurotoxic Prion Protein: Role for Hsp70 in Accumulation of Misfolded Isoforms

Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrPC) converts into a misfolded isoform (PrPSc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrPC; in older flies, PrP misfolds, acquires biochemical and structural properties of PrPSc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrPSc-specific conformational epitopes. In contrast to PrPSc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrPSc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrPSc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrPSc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.

Mouse-Adapted Ovine Scrapie Prion Strains Are Characterized by Different Conformers of PrPSc

The agent responsible for prion disease may exist in different forms, commonly referred to as strains, with each carrying the specific information that determines its own distinct biological properties, such as incubation period and lesion profile. Biological strain typing of ovine scrapie isolates by serial passage in conventional mice has shown some diversity in ovine prion strains. However, this biological diversity remains poorly supported by biochemical prion strain typing. The protein-only hypothesis predicts that variation between different prion strains in the same host is manifest in different conformations adopted by PrPSc. Here we have investigated the molecular properties of PrPSc associated with two principal Prnp(a) mouse-adapted ovine scrapie strains, namely, RML and ME7, in order to establish biochemical prion strain typing strategies that may subsequently be used to discriminate field cases of mouse-passaged ovine scrapie isolates. We used a conformation-dependent immunoassay and a conformational stability assay, together with Western blot analysis, to demonstrate that RML and ME7 PrPSc proteins show distinct biochemical and physicochemical properties. Although RML and ME7 PrPSc proteins showed similar resistance to proteolytic digestion, they differed in their glycoform profiles and levels of proteinase K (PK)-sensitive and PK-resistant isoforms. In addition, the PK-resistant core (PrP27-30) of ME7 was conformationally more stable following exposure to guanidine hydrochloride or Sarkosyl than was RML PrP27-30. Our data show that mouse-adapted ovine scrapie strains can be discriminated by their distinct conformers of PrPSc, which provides a basis to investigate their diversity at the molecular level.

Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP(C), into a misfolded, protease-resistant form, PrP(Sc). Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106-120 from the so-called amyloidogenic core of PrP (residues 106-126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65 degrees C. Under partially denaturing conditions (65 degrees C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65 degrees C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65 degrees C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65 degrees C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP(Sc) can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106-120 of PrP and does not require other cellular factors or a PrP(Sc) template.

Effects of Different Experimental Conditions on the PrPSc Core Generated by Protease Digestion: IMPLICATIONS FOR STRAIN TYPING AND MOLECULAR CLASSIFICATION OF CJD

The discovery of molecular subtypes of the pathological prion protein PrPSc has provided the basis for a novel classification of human transmissible spongiform encephalopathies (TSEs) and a potentially powerful method for strain typing. However, there is still a significant disparity regarding the understanding and nomenclature of PrPSc types. In addition, it is still unknown whether a specific PrPSc type is associated with each TSE phenotypic variant. In sporadic Creutzfeldt-Jakob disease (sCJD), five disease phenotypes are known, but only two major types of PrPSc, types 1 and 2, have been consistently reproduced. We further analyzed PrPSc properties in sCJD and variant CJD using a high resolution gel electrophoresis system and varying experimental conditions. We found that pH varies among CJD brain homogenates in standard buffers, thereby influencing the characteristics of protease-treated PrPSc. We also show that PrPSc type 1 and type 2 are heterogeneous species which can be further distinguished into five molecular subtypes that fit the current histopathological classification of sCJD variants. Our results shed light on previous disparities in PrPSc typing, provide a refined classification of human PrPSc types, and support the notion that the pathological TSE phenotype is related to PrPSc structure.