Search

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrP Sc , in the central nervous system of prion-affected humans and animals. PrP Sc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrP Sc . Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrP Sc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrP Sc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrP Sc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.

Prion diseases are fatal neurodegenerative diseases caused by pathogenic misfolding of the prion protein, PrP. They are transmissible between hosts, and sometimes between different species, as with transmission of bovine spongiform encephalopathy to humans. Although PrP is found in a wide range of vertebrates, prion diseases are seen only in certain mammals, suggesting that infectious misfolding was a recent evolutionary development. To explore when PrP acquired the ability to misfold infectiously, we reconstructed the sequences of ancestral versions of PrP from the last common primate, primate-rodent, artiodactyl, placental, bird, and amniote. Recombinant ancestral PrPs were then tested for their ability to form β-sheet aggregates, either spontaneously or when seeded with infectious prion strains from human, cervid, or rodent species. The ability to aggregate developed after the oldest ancestor (last common amniote), and aggregation capabilities diverged along evolutionary pathways consistent with modern-day susceptibilities. Ancestral bird PrP could not be seeded with modern-day prions, just as modern-day birds are resistant to prion disease. Computational modeling of structures suggested that differences in helix 2 could account for the resistance of ancestral bird PrP to seeding. Interestingly, ancestral primate PrP could be converted by all prion seeds, including both human and cervid prions, raising the possibility that species descended from an ancestral primate have retained the susceptibility to conversion by cervid prions. More generally, the results suggest that susceptibility to prion disease emerged prior to ~100 million years ago, with placental mammals possibly being generally susceptible to disease., (© 2022 The Protein Society.)

The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion diseases is neuroinflammation characterized by activation of neuroglia surrounding prion deposition. The cause and effect of this cellular response, however, is unclear. We investigated innate immune defenses against prion infection using primary mixed neuronal and glial cultures. Conditional prion propagation occurred in glial cultures depending on their immune status. Preconditioning of the cells with the toll-like receptor (TLR) ligand, lipopolysaccharide, resulted in a reduction in prion propagation, whereas suppression of the immune responses with the synthetic glucocorticoid, dexamethasone, increased prion propagation. In response to recombinant prion fibrils, glial cells up-regulated TLRs (TLR1 and TLR2) expression and secreted cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6, granulocyte-macrophage colony-stimulating factor, and interferon-β). Preconditioning of neuronal and glial cultures with recombinant prion fibrils inhibited prion replication and altered microglial and astrocytic populations. Our results provide evidence that, in early stages of prion infection, glial cells respond to prion infection through TLR-mediated innate immunity., (© 2016 Wiley Periodicals, Inc.)

The sequence of a host's prion protein (PrP) can affect that host's susceptibility to prion disease and is the primary basis for the species barrier to transmission. Yet within many species, polymorphisms of the prion protein gene (Prnp) exist, each of which can further affect susceptibility or influence incubation period, pathology and phenotype. As strains are defined by these features (incubation period, pathology, phenotype), polymorphisms may also lead to the preferential propagation or generation of certain strains. In our recent study of the mouse Prnp(a) and Prnp(b) polymorphisms (which produced the proteins PrP(a) and PrP(b), respectively), we found differences in aggregation tendency, strain adaptability and conformational variability. Comparing our in vitro data with that of in vivo studies, we found that differing incubation periods between Prnp(a) and Prnp(b) mice can primarily be explained on the basis of faster or more efficient aggregation of PrP(a). In addition, and more importantly, we found that the faithful propagation of strains in Prnp(b) mice can be explained by the ability of PrP(b) to adopt a wider range of conformations. This adaptability allows PrP(b) to successfully propagate the structural features of a seed. In contrast, Prnp(a) mice revert PrP(b) strains into PrP(a) -type strains, and overall they have a narrower distribution of incubation periods. This can be explained by PrP(a) having fewer preferred conformations. We propose that Prnp polymorphisms are one route by which certain prion strains may preferentially propagate. This has significant implications for prion disease, chronic wasting disease (CWD) in particular, as it is spreading through North America. Deer which are susceptible to CWD also carry polymorphisms which influence their susceptibility. If these polymorphisms also preferentially allow strain diversification and propagation, this may accelerate the crossing of species barriers and propagation of the disease up the food chain.

Prion diseases are fatal neurodegenerative disorders associated with the polymerization of the cellular form of prion protein (PrP(C)) into an amyloidogenic β-sheet infectious form (PrP(Sc)). The sequence of host PrP is the major determinant of host prion disease susceptibility. In mice, the presence of allele a (Prnp(a), encoding the polymorphism Leu-108/Thr-189) or b (Prnp(b), Phe-108/Val-189) is associated with short or long incubation times, respectively, following infection with PrP(Sc). The molecular bases linking PrP sequence, infection susceptibility, and convertibility of PrP(C) into PrP(Sc) remain unclear. Here we show that recombinant PrP(a) and PrP(b) aggregate and respond to seeding differently in vitro. Our kinetic studies reveal differences during the nucleation phase of the aggregation process, where PrP(b) exhibits a longer lag phase that cannot be completely eliminated by seeding the reaction with preformed fibrils. Additionally, PrP(b) is more prone to propagate features of the seeds, as demonstrated by conformational stability and electron microscopy studies of the formed fibrils. We propose a model of polymerization to explain how the polymorphisms at positions 108 and 189 produce the phenotypes seen in vivo. This model also provides insight into phenomena such as species barrier and prion strain generation, two phenomena also influenced by the primary structure of PrP.

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype. Author summary: Prion diseases are neurodegenerative diseases that include bovine spongiform encephalopathy (BSE or mad cow disease) in cattle, chronic wasting disease (CWD) in cervids and Creutzfeldt-Jakob disease (CJD) in humans. These diseases are caused by self-propagated misfolding and aggregation of the naturally occurring prion protein. Variations in the structure of prion aggregates are associated with distinct disease phenotypes, but how this prion structural heterogeneity translates into clinical presentation has been difficult to determine, largely because it is technically difficult to isolate and characterize the full range of prion structures from prion-infected brain. Here, we overcame this challenge by using a versatile fractionation technique, one that is strikingly unexplored in neurodegenerative research, and present the most detailed description, to date, of strain-specific prion subpopulations. We found that prion quaternary structure was a major contributor to structural heterogeneity. We also discovered that all prion strains studied underwent a significant structural change resulting in two distinctive subpopulations whose proportions correlated with the strain phenotype. Our work provides new insights into the molecular basis of prion strain variation and is a proof of concept that can be applied to other protein misfolding neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrPSc. The high-resolution structure of the infectious PrPSc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene-maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrPSc attached toSMAlipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes. [ABSTRACT FROM AUTHOR]