PrP Sc Conformation Research Articles

Species barrier as molecular basis for adaptation of synthetic prions with N-terminally truncated PrP.

Mammalian prions are neurotropic pathogens formed from PrPSc assemblies, a misfolded variant of the host-encoded prion protein PrPC. Multiple PrPSc conformations or strains self-propagate in host populations or mouse models of prion diseases, exhibiting distinct biological and biochemical phenotypes. Constrained interactions between PrPSc and PrPC conformations confer species specificity and regulate cross-species transmission. The pathogenicity of fibrillar assemblies derived from bacterially expressed recombinant PrP (rPrP) has been instrumental in demonstrating the protein-only nature of prions. Yet, their ability to encode different strains and transmit between species remains poorly studied, hampering their use in exploring structure-to-strain relationships. Fibrillar assemblies from rPrP with hamster, mouse, human, and bovine primary structures were generated and tested for transmission and adaptation in tg7 transgenic mice expressing hamster PrPC. All assemblies, except the bovine ones, were fully pathogenic on the primary passage, causing clinical disease, PrPSc brain deposition, and spongiform degeneration. They exhibited divergent adaptation processes and strain properties upon subsequent passage. Assemblies of hamster origin propagated without apparent need for adaptation, those of mouse origin adapted abruptly, and those of human origin required serial passages for optimal fitness. Molecular analyses revealed the presence of endogenously truncated PrPSc species in the resulting synthetic strains that lack the 90-140 amino acid region considered crucial for infectivity. In conclusion, rPrP assemblies provide a facile means of generating novel prion strains with adaptative/evolutive properties mimicking genuine prions. The PrP amino acid backbone is sufficient to encode different strains with specific adaptative properties, offering insights into prion transmission and strain diversity.

Abnormal synaptic architecture in iPSC-derived neurons from a multi-generational family with genetic Creutzfeldt-Jakob disease

Genetic prion diseases are caused by mutations in PRNP, which encodes the prion protein (PrPC). Why these mutations are pathogenic, and how they alter the properties of PrPC are poorly understood. We have consented and accessed 22 individuals of a multi-generational Israeli family harboring the highly penetrant E200K PRNP mutation and generated a library of induced pluripotent stem cells (iPSCs) representing nine carriers and four non-carriers. iPSC-derived neurons from E200K carriers display abnormal synaptic architecture characterized by misalignment of postsynaptic NMDA receptors with the cytoplasmic scaffolding protein PSD95. Differentiated neurons from mutation carriers do not produce PrPSc, the aggregated and infectious conformer of PrP, suggesting that loss of a physiological function of PrPC may contribute to the disease phenotype. Our study shows that iPSC-derived neurons can provide important mechanistic insights into the pathogenesis of genetic prion diseases and can offer a powerful platform for testing candidate therapeutics.

Adaptation of the protein misfolding cyclic amplification (PMCA) technique for the screening of anti-prion compounds.

Prion diseases result from the misfolding of the physiological prion protein (PrPC) to a pathogenic conformation (PrPSc). Compelling evidence indicates that prevention and/or reduction of PrPSc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrPSc conformations (or strains) associated with disease represents a major problem when identifying anti-prion compounds. Efforts to identify strain-specific anti-prion molecules are limited by the lack of biologically relevant high-throughput screening platforms to interrogate compound libraries. Here, we describe adaptations to the protein misfolding cyclic amplification (PMCA) technology (able to faithfully replicate PrPSc strains) that increase its throughput to facilitate the screening of anti-prion molecules. The optimized PMCA platform includes a reduction in sample and reagents, as well as incubation/sonication cycles required to efficiently replicate and detect rodent-adapted and cervid PrPSc strains. The visualization of PMCA products was performed via dot blots, a method that contributed to reduced processing times. These technical changes allowed us to evaluate small molecules with previously reported anti-prion activity. This proof-of-principle screening was evaluated for six rodent-adapted prion strains. Our data show that these compounds targeted either none, all or some PrPSc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrPSc strains. Further analyses of a small compound library against deer prions demonstrate the potential of this new PMCA format to identify strain-specific anti-prion molecules. The data presented here demonstrate the use of the PMCA technique in the selection of prion strain-specific anti-prion compounds.

Conformational diversity in purified prions produced in vitro.

Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the serial protein misfolding cyclic amplification (sPMCA) technique do not display this same diversity in vitro. This discrepancy has left a gap in our understanding of how conformational diversity arises at the molecular level in both types of prions. Here, we use continuous shaking instead of sPMCA to generate conformationally diverse purified prions in vitro. Using this approach, we show for the first time that wild type prions initially seeded by different native strains can propagate as metastable PrPSc conformers with distinguishable strain properties in purified reactions containing a single active cofactor. Propagation of these metastable PrPSc conformers requires appropriate shaking conditions, and changes in these conditions cause all the different PrPSc conformers to converge irreversibly into the same single conformer as that produced in sPMCA reactions. We also use continuous shaking to show that two mutant PrP molecules with different pathogenic point mutations (D177N and E199K) adopt distinguishable PrPSc conformations in reactions containing pure protein substrate without cofactors. Unlike wild-type prions, the conformations of mutant prions appear to be dictated by substrate sequence rather than seed conformation. Overall, our studies using purified substrates in shaking reactions show that wild-type and mutant prions use fundamentally different mechanisms to generate conformational diversity at the molecular level.

General Method of Quantifying the Extent of Methionine Oxidation in the Prion Protein

The normal cellular prion protein (PrPC) and its infectious conformer, PrPSc, possess a disproportionately greater amount of methionines than would be expected for a typical mammalian protein. The thioether of methionine can be readily oxidized to the corresponding sulfoxide, which means that oxidation of methionine can be used to map the surface of the conformation of PrPC or PrPSc, as covalent changes are retained after denaturation. We identified a set of peptides (TNMK, MLGSAMSR, LLGSAMSR, PMIHFGNDWEDR, ENMNR, ENMYR, IMER, MMER, MIER, VVEQMCVTQYQK, and VVEQMCITQYQR) that contains every methionine in sheep, cervid, mouse, and bank vole PrP. Each is the product of a tryptic digestion and is suitable for a multiple reaction monitoring (MRM) based analysis. The peptides chromatograph well. The oxidized and unoxidized peptides containing one methionine readily separate. The unoxidized, two singly oxidized, and doubly oxidized forms of the MLGSAMSR and MMER peptides are also readily distinguishable. This approach can be used to determine the surface exposure of each methionine by measuring its oxidation after reaction with added hydrogen peroxide.

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.

Understanding Intra-Species and Inter-Species Prion Conversion and Zoonotic Potential Using Protein Misfolding Cyclic Amplification.

Prion diseases are fatal neurodegenerative disorders that affect humans and animals, and can also be transmitted from animals to humans. A fundamental event in prion disease pathogenesis is the conversion of normal host prion protein (PrPC) to a disease-associated misfolded form (PrPSc). Whether or not an animal prion disease can infect humans cannot be determined a priori. There is a consensus that classical bovine spongiform encephalopathy (C-type BSE) in cattle transmits to humans, and that classical sheep scrapie is of little or no risk to human health. However, the zoonotic potential of more recently identified animal prion diseases, such as atypical scrapie, H-type and L-type BSE and chronic wasting disease (CWD) in cervids, remains an open question. Important components of the zoonotic barrier are (i) physiological differences between humans and the animal in question, (ii) amino acid sequence differences of the animal and human PrPC, and (iii) the animal prion strain, enciphered in the conformation of PrPSc. Historically, the direct inoculation of experimental animals has provided essential information on the transmissibility and compatibility of prion strains. More recently, cell-free molecular conversion assays have been used to examine the molecular compatibility on prion replication and zoonotic potential. One such assay is Protein Misfolding Cyclic Amplification (PMCA), in which a small amount of infected tissue homogenate, containing PrPSc, is added as a seed to an excess of normal tissue homogenate containing PrPC, and prion conversion is accelerated by cycles of incubation and ultrasonication. PMCA has been used to measure the molecular feasibility of prion transmission in a range of scenarios using genotypically homologous and heterologous combinations of PrPSc seed and PrPC substrate. Furthermore, this method can be used to speculate on the molecular profile of PrPSc that might arise from a zoonotic transmission. We discuss the experimental approaches that have been used to model both the intra- and inter-species molecular compatibility of prions, and the factors affecting PrPc to PrPSc conversion and zoonotic potential. We conclude that cell-free prion protein conversion assays, especially PMCA, are useful, rapid and low-cost approaches for elucidating the mechanisms of prion propagation and assessing the risk of animal prions to humans.

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

Environmental and host factors that contribute to prion strain evolution.

Prions are novel pathogens that are composed entirely of PrPSc, the self-templating conformation of the host prion protein, PrPC. Prion strains are operationally defined as a heritable phenotype of disease that are encoded by strain-specific conformations of PrPSc. The factors that influence the relative distribution of strains in a population are only beginning to be understood. For prions with an infectious etiology, environmental factors, such as strain-specific binding to surfaces and resistance to weathering, can influence which strains are available for transmission to a naïve host. Strain-specific differences in efficiency of infection by natural routes of infection can also select for prion strains. The host amino acid sequence of PrPC has the greatest effect on dictating the repertoire of prion strains. The relative abundance of PrPC, post-translational modifications of PrPC and cellular co-factors involved in prion conversion can also provide conditions that favor the prevalence of a subset of prion strains. Additionally, prion strains can interfere with each other, influencing the emergence of a dominant strain. Overall, both environmental and host factors may influence the repertoire and distribution of strains within a population.

Neuropathology of Animal Prion Diseases.

Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are a fatal group of infectious, inherited and spontaneous neurodegenerative diseases affecting human and animals. They are caused by the conversion of cellular prion protein (PrPC) into a misfolded pathological isoform (PrPSc or prion- proteinaceous infectious particle) that self-propagates by conformational conversion of PrPC. Yet by an unknown mechanism, PrPC can fold into different PrPSc conformers that may result in different prion strains that display specific disease phenotype (incubation time, clinical signs and lesion profile). Although the pathways for neurodegeneration as well as the involvement of brain inflammation in these diseases are not well understood, the spongiform changes, neuronal loss, gliosis and accumulation of PrPSc are the characteristic neuropathological lesions. Scrapie affecting small ruminants was the first identified TSE and has been considered the archetype of prion diseases, though atypical and new animal prion diseases continue to emerge highlighting the importance to investigate the lesion profile in naturally affected animals. In this report, we review the neuropathology and the neuroinflammation of animal prion diseases in natural hosts from scrapie, going through the zoonotic bovine spongiform encephalopathy (BSE), the chronic wasting disease (CWD) to the newly identified camel prion disease (CPD).

The Size and Stability of Infectious Prion Aggregates Fluctuate Dynamically during Cellular Uptake and Disaggregation.

Prion diseases arise when PrPSc, an aggregated, infectious, and insoluble conformer of the normally soluble mammalian prion protein, PrPC, catalyzes the conversion of PrPC into more PrPSc, which then accumulates in the brain leading to disease. PrPSc is the primary, if not sole, component of the infectious prion. Despite the stability and protease insensitivity of PrPSc aggregates, they can be degraded after cellular uptake. However, how cells disassemble and degrade PrPSc is poorly understood. In this work, we analyzed how the protease sensitivity and size distribution of PrPSc aggregates from two different mouse-adapted prion strains, 22L, that can persistently infect cells and 87V, that cannot, changed during cellular uptake. We show that within the first 4 h following uptake large PrPSc aggregates from both prion strains become less resistant to digestion by proteinase K (PK) through a mechanism that is dependent upon the acidic environment of endocytic vesicles. We further show that during disassembly, PrPSc aggregates from both strains become more resistant to PK digestion through the apparent removal of protease-sensitive PrPSc, with PrPSc from the 87V strain disassembled more readily than PrPSc from the 22L strain. Taken together, our data demonstrate that the sizes and stabilities of PrPSc from different prion strains change during cellular uptake and degradation, thereby potentially impacting the ability of prions to infect cells.

Short and sweet: How glycans impact prion conversion, cofactor interactions, and cross-species transmission.

Prion diseases are transmissible and incurable neurodegenerative disorders of humans and animals. Similar to other neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, prion diseases are characterized by protein aggregation in the central nervous system (CNS). The key molecular event in prion disease is the conformational conversion of the cellular prion protein, PrPC, into a misfolded and aggregated conformer, PrPSc, which templates further PrPC misfolding [1]. Although this same protein misfolding event occurs in all prion diseases, affected individuals expressing identical PrPC sequences can exhibit strikingly heterogeneous clinical and pathological phenotypes. These phenotypic differences have been linked to distinct PrPSc conformations, known as strains, yet the source of prion strain diversity is incompletely understood. One potential contributor to strain diversity lies in the posttranslational modifications (PTMs) on PrPC, which add a layer of structural complexity to an otherwise highly conserved protein. PrPC is posttranslationally modified by the covalent linkage of (i) 0 to 2 N-linked glycans at positions 181 and 197 (human PrP) and (ii) a glycosylphosphatidylinositol (GPI) moiety, which anchors PrPC in the outer leaflet of the plasma membrane [2,3]. The N-linked glycans on PrPC are branched [bi- (51%), tri- (32%), or tetra-antennary (17%)] and terminally sialylated, predominantly via alpha 2,6 linkages [4–6]. While necessary for regulating protein interactions and function, PTMs can profoundly modulate the pathogenesis of neurodegenerative diseases. For example, aberrant hyperphosphorylation of tau protein leads to tau detachment from microtubules and fibrillization into neurofibrillary tangles, a pathologic hallmark of Alzheimer’s disease. Prion diseases are no different, and the presence of PrP PTMs can markedly alter both the disease phenotype and transmission barrier. Here we explore how PrP PTMs impact prion conversion, cross-species transmission, the neuroinflammatory response, and PrP interaction with cofactors.

Intrinsic disorder and phase transitions: Pieces in the puzzling role of the prion protein in health and disease.

After four decades of prion protein research, the pressing questions in the literature remain similar to the common existential dilemmas. Who am I? Some structural characteristics of the cellular prion protein (PrPC) and scrapie PrP (PrPSc) remain unknown: there are no high-resolution atomic structures for either full-length endogenous human PrPC or isolated infectious PrPSc particles. Why am I here? It is not known why PrPC and PrPSc are found in specific cellular compartments such as the nucleus; while the physiological functions of PrPC are still being uncovered, the misfolding site remains obscure. Where am I going? The subcellular distribution of PrPC and PrPSc is wide (reported in 10 different locations in the cell). This complexity is further exacerbated by the eight different PrP fragments yielded from conserved proteolytic cleavages and by reversible post-translational modifications, such as glycosylation, phosphorylation, and ubiquitination. Moreover, about 55 pathological mutations and 16 polymorphisms on the PrP gene (PRNP) have been described. Prion diseases also share unique, challenging features: strain phenomenon (associated with the heterogeneity of PrPSc conformations) and the possible transmissibility between species, factors which contribute to PrP undruggability. However, two recent concepts in biochemistry-intrinsically disordered proteins and phase transitions-may shed light on the molecular basis of PrP's role in physiology and disease.

Improving the Predictive Value of Prion Inactivation Validation Methods to Minimize the Risks of Iatrogenic Transmission With Medical Instruments.

Prions are pathogenic infectious agents responsible for fatal, incurable neurodegenerative diseases in animals and humans. Prions are composed exclusively of an aggregated and misfolded form (PrPSc) of the cellular prion protein (PrPC). During the propagation of the disease, PrPSc recruits and misfolds PrPC into further PrPSc. In human, iatrogenic prion transmission has occurred with incompletely sterilized medical material because of the unusual resistance of prions to inactivation. Most commercial prion disinfectants validated against the historical, well-characterized laboratory strain of 263K hamster prions were recently shown to be ineffective against variant Creutzfeldt-Jakob disease human prions. These observations and previous reports support the view that any inactivation method must be validated against the prions for which they are intended to be used. Strain-specific variations in PrPSc physico-chemical properties and conformation are likely to explain the strain-specific efficacy of inactivation methods. Animal bioassays have long been used as gold standards to validate prion inactivation methods, by measuring reduction of prion infectivity. Cell-free assays such as the real-time quaking-induced conversion (RT-QuIC) assay and the protein misfolding cyclic amplification (PMCA) assay have emerged as attractive alternatives. They exploit the seeding capacities of PrPSc to exponentially amplify minute amounts of prions in biospecimens. European and certain national medicine agencies recently implemented their guidelines for prion inactivation of non-disposable medical material; they encourage or request the use of human prions and cell-free assays to improve the predictive value of the validation methods. In this review, we discuss the methodological and technical issues regarding the choice of (i) the cell-free assay, (ii) the human prion strain type, (iii) the prion-containing biological material. We also introduce a new optimized substrate for high-throughput PMCA amplification of human prions bound on steel wires, as translational model for prion-contaminated instruments.

Modeling PrPSc Generation Through Deformed Templating.

Deformed templating is the process by which self-replicating protein conformations with a given cross-β folding pattern can seed formation of an alternative self-replicating state with different cross-β folding pattern. In particular, uninfectious but propagative PrP amyloid can transform into a bona fide infectious conformer, PrPSc through deformed templating. The process can take many rounds of replication (if taking place in vitro) or even several passages of the evolving PrP conformers through successive brains if in vivo, through experimental transmission. In all cases, deformed templating involves a forced conversion in which there is a mismatch between the template and the substrate and/or the templating environment, typically a recombinant PrP amyloid, adept at converting recombinant PrP under denaturing conditions (e.g., presence of chaotropic agents), encountering a glycosylated, GPI-anchored PrPC substrate under physiological conversion conditions. Deformed templating is characterized by emergence of intermediate conformers that exhibit biochemical characteristics that are intermediate between those of the initial PrP amyloid and the final PrPSc conformers. Here, we took advantage of the recent elucidation of the structure of a PrP amyloid by cryo-EM and the availability of a physically plausible atomistic model of PrPSc that we have recently proposed. Using modeling and Molecular Dynamics (MD) approaches, we built a complete molecular modelization of deformed templating, including an atomistic model of a glycosylated intermediate conformer and a modified model of PrPSc. Among other unanticipated outcomes, our results show that fully glycosylated PrP can be stacked in-register, and how 4-rung β-solenoid (4RβS) PrP architectures can share key structural motifs with parallel-in register intermolecular sheet (PIRIBS) PrP amyloids. Our results shed light on the mechanisms of prion replication.

Quantifying the Role of Lysine in Prion Replication by Nano-LC Mass Spectrometry and Bioassay.

Prions propagate by a template driven process, inducing the normal cellular isoform (PrPC) to adopt the prion (PrPSc) conformation. In PrPC, the positions of lysines are highly conserved and strongly influence prion propagation. In this study, covalent modification was used to quantitate the role of lysines in the PrPSc template that drives prion replication. The ε-amino group of lysines in the PrPSc (hamster-adapted scrapie Sc237) template was acetylated by either acetic anhydride (Ac2O) or the N-hydroxysuccinimide ester of acetic acid (Ac-NHS). The extent of lysine acetylation in PrPSc was quantitated by mass spectrometry or Western blot-based analysis. Identical samples were bioassayed to quantitate the loss of infectivity associated with lysine acetylation. The reduction of infectivity at the highest reagent concentration was approximately 90% (∼10-fold). Ten of the eleven prion lysines were acetylated to a greater extent (25−400-fold) than the observed loss of infectivity. Only one lysine, at position 220 (K220), had a reactivity that is consistent with the loss of infectivity. Although lysines are highly conserved and play a crucial role in converting PrPC into the PrPSc conformation, once that conformation is adopted, the lysines present in the PrPSc template play only a limited role in prion replication. In principle, this approach could be used to clarify the role of other amino acids in the replication of prions and other prion-like protein misfolding diseases.

Identification of a homology-independent linchpin domain controlling mouse and bank vole prion protein conversion

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrPSc, onto the cell-expressed conformer, PrPC. Here we study the conversion of PrPC to PrPSc using a recombinant mouse PrPSc conformer (mouse protein-only recPrPSc) as a unique tool that can convert bank vole but not mouse PrPC substrates in vitro. Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrPC substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrPSc. Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrPC substrates to be converted by Mo protein-only recPrPSc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion.

Cofactor and glycosylation preferences for in vitroprion conversion are predominantly determined by strain conformation.

Prion diseases are caused by the misfolding of a host-encoded glycoprotein, PrPC, into a pathogenic conformer, PrPSc. Infectious prions can exist as different strains, composed of unique conformations of PrPSc that generate strain-specific biological traits, including distinctive patterns of PrPSc accumulation throughout the brain. Prion strains from different animal species display different cofactor and PrPC glycoform preferences to propagate efficiently in vitro, but it is unknown whether these molecular preferences are specified by the amino acid sequence of PrPC substrate or by the conformation of PrPSc seed. To distinguish between these two possibilities, we used bank vole PrPC to propagate both hamster or mouse prions (which have distinct cofactor and glycosylation preferences) with a single, common substrate. We performed reconstituted sPMCA reactions using either (1) phospholipid or RNA cofactor molecules, or (2) di- or un-glycosylated bank vole PrPC substrate. We found that prion strains from either species are capable of propagating efficiently using bank vole PrPC substrates when reactions contained the same PrPC glycoform or cofactor molecule preferred by the PrPSc seed in its host species. Thus, we conclude that it is the conformation of the input PrPSc seed, not the amino acid sequence of the PrPC substrate, that primarily determines species-specific cofactor and glycosylation preferences. These results support the hypothesis that strain-specific patterns of prion neurotropism are generated by selection of differentially distributed cofactors molecules and/or PrPC glycoforms during prion replication.

Crossing Species Barriers Relies on Structurally Distinct Prion Assemblies and Their Complementation.

Prion replication results from the autocatalytic templated assisted conversion of the host-encoded prion protein PrPC into misfolded, polydisperse PrPSc conformers. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Within and between prion strains, the biological activity (replicative efficacy and specific infectivity) of PrPSc assemblies is size dependent and thus reflects an intrinsic structural heterogeneity. The contribution of such PrPSc heterogeneity across species prion adaptation, which is believed to be based on fit adjustment between PrPSc template(s) and host PrPC, has not been explored. To define the structural-to-fitness PrPSc landscape, we measured the relative capacity of size-fractionated PrPSc assemblies from different prion strains to cross mounting species barriers in transgenic mice expressing foreign PrPC. In the absence of a transmission barrier, the relative efficacy of the isolated PrPSc assemblies to induce the disease is like the efficacy observed in the homotypic context. However, in the presence of a transmission barrier, size fractionation overtly delays and even abrogates prion pathogenesis in both the brain and spleen tissues, independently of the infectivity load of the isolated assemblies. Altering by serial dilution PrPSc assembly content of non-fractionated inocula aberrantly reduces their specific infectivity, solely in the presence of a transmission barrier. This suggests that synergy between structurally distinct PrPSc assemblies in the inoculum is requested for crossing the species barrier. Our data support a mechanism whereby overcoming prion species barrier requires complementation between structurally distinct PrPSc assemblies. This work provides key insight into the "quasispecies" concept applied to prions, which would not necessarily rely on prion substrains as constituent but on structural PrPSc heterogeneity within prion population.

Novel Strain of the Chronic Wasting Disease Agent Isolated From Experimentally Inoculated Elk With LL132 Prion Protein

Chronic wasting disease (CWD) is a fatal, progressive disease that affects cervid species, including Rocky mountain elk (Cervus elaphus nelsoni). There are 2 allelic variants in the elk prion protein gene: L132 (leucine) and M132 (methionine). Following experimental oral challenge with the CWD agent incubation periods are longest in LL132 elk, intermediate in ML132 elk, and shortest in MM132 elk. In order to ascertain whether such CWD-infected elk carry distinct prion strains, groups of Tg12 mice that express M132 elk prion protein were inoculated intracranially with brain homogenate from individual CWD-infected elk of various genotypes (LL132, LM132, or MM132). Brain samples were examined for microscopic changes and assessment of the biochemical properties of disease-associated prion protein (PrPSc). On first passage, mice challenged with LL132 elk inoculum had prolonged incubation periods and greater PrPSc fibril stability compared to mice challenged with MM132 or LM132 inoculum. On second passage, relative incubation periods, western blot profiles, and neuropathology were maintained. These results suggest that the CWD prion isolated from LL132 elk is a novel CWD strain and that M132 PrPC is able to propagate some biophysical properties of the L132 PrPSc conformation.