Unraveling the key to the resistance of canids to prion diseases.

One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different in vitro and in vivo models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the Leporidae and Equidae families. Here we undertake in vitro and in vivo approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis in silico, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel transgenic mouse model carrying this substitution and these mice showed complete resistance to disease despite intracerebral challenge with three different mouse prion strains (RML, 22L and 301C) known to cause disease in wild-type mice. These findings suggest that dog D163 amino acid is primarily, if not totally, responsible for the prion resistance of canids.

The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy.

The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27-30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27-30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27-30 molecule as ~17.7 Å. Together, the data indicate a four-rung ß-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.

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.

The effect of ß2-α2 loop mutation on amyloidogenic properties of the prion protein.

Recent studies revealed that elk-like S170N/N174T mutation in mouse prion protein (moPrP), which results in an increased rigidity of ß2-α2 loop, leads to a prion disease in transgenic mice. Here we characterized the effect of this mutation on biophysical properties of moPrP. Despite similar thermodynamic stabilities of wild type and mutant proteins, the latter was found to have markedly higher propensity to form amyloid fibrils. Importantly, this effect was observed even under fully denaturing conditions, indicating that the increased conversion propensity of the mutant protein is not due to loop rigidity but rather results from greater amyloidogenic potential of the amino acid sequence within the loop region of S170N/N174T moPrP.

Plasmacytoid dendritic cells sequester high prion titres at early stages of prion infection.

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.

Cryo-immunogold electron microscopy for prions: toward identification of a conversion site.

Prion diseases are caused by accumulation of an abnormally folded isoform (PrP(Sc)) of the cellular prion protein (PrP(C)). The subcellular distribution of PrP(Sc) and the site of its formation in brain are still unclear. We performed quantitative cryo-immunogold electron microscopy on hippocampal sections from mice infected with the Rocky Mountain Laboratory strain of prions. Two antibodies were used: R2, which recognizes both PrP(C) and PrP(Sc); and F4-31, which only detects PrP(C) in undenatured sections. At a late subclinical stage of prion infection, both PrP(C) and PrP(Sc) were detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24-fold) was found on the early/recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrP(Sc) may be oligomeric, protease-sensitive PrP(Sc).

Native, amyloid fibrils and beta-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly.

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and beta-oligomers) of recombinant human PrP (90-231 and 121-231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, beta-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that beta-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.

Cellular prion protein electron microscopy: attempts/limits and clues to a synaptic trait. Implications in neurodegeneration process.

Prion diseases are caused by an infectious agent constituted by a rogue protein called prion (PrP Sc) of neuronal origin (PrP c) and are exemplified by Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. Considerable efforts have been made to understand the cerebral damage caused by these diseases but a clear comprehensive view cannot be achieved without defining the neurophysiological function of PrP c. This lack of information is in part attributable to our ignorance of the precise localization of PrP c in the brain neuronal cell. One relevant option to explore this aspect is to undertake PrP immunohistochemistry at the electron-microscopy level, knowing that this challenge raises major technical constraints. In describing the attempts and restrictions of the various approaches used, we review here the efforts that have been invested in this particular field of prionology. The common result emerging from these contributions is that the synapse could be the site at which PrP c exerts its critical activity. This location suggests, in the perspective of synaptic regulation, that PrP c can be assigned multiple biological functions and supports the novel concept that prion-like changes are involved in long-term memory formation. The synaptic trait of PrP c and PrP Sc suggests that synapse loss is the key event in neuronal death. Interestingly, synaptic alterations are also considered to be predominant in the pathophysiological mechanism in Alzheimer, Parkinson and Huntington diseases. All these brain disorders, characterized by the formation of a specific amyloid protein of synaptic origin, can be classified under the heading of amyloidogenic synaptopathies.

Reversible monomer-oligomer transition in human prion protein.

The structure and the dissociation reaction of oligomers Pr(Poligo) from reduced human prion huPrP(C)(23-231) have been studied by (1)H-NMR and tryptophan fluorescence spectroscopy at varying pressure, along with circular dichroism and atomic force microscopy. The 1H-NMR and fluorescence spectral feature of the oligomer is consistent with the notion that the N-terminal residues including all seven Trp residues, are free and mobile, while residues 105 approximately 210, comprising the AGAAAAGA motif and S1-Loop-HelixA-Loop-S2-Loop-HelixC, are engaged in intra- and/ or inter-molecular interactions. By increasing pressure to 200 MPa, the oligomers tend to dissociate into monomers which may be identified with PrP(C*), a rare metastable form of PrP(C) stabilized at high pressure (Kachel et al., BMC Struct Biol 6:16). The results strongly suggest that the oligomeric form PrP(oligo) is in dynamic equilibrium with the monomeric forms via PrP(C*), namely huPrP(C)[left arrow over right arrow]huPrP(C*)[left arrow over right arrow]huPrP(oligo).

Instability of familial spongiform encephalopathy-related prion mutants.

We examined the influence of D177N (D178N in humans) mutation on the conformational stability of the S2 region of moPrP(C) with varying pHs by using the SDSL-ESR technique. The ESR spectrum of D177N at pH 7.5 was narrower than that of Y161R1, referred to as WT( *). The ESR spectrum of D177N did not change when pH in the solution decreased to pH 4.0. Our results suggested that the disappearance of a salt bridge (D177-R163) induced the increase in the instability of S2 region. Moreover, the line shape of the ESR spectrum obtained from H176S neighboring the salt bridge linked to the S2 region was similar to D177N. These results indicate that the protonation of H176 is strongly associated with the stability of S2 region. These findings are important for understanding the mechanism by which the disruption of the salt bridge in the S2 region forms the pathogenic PrP(Sc) structure in hereditary prion disease.

Influence of phosphorus dendrimers on the aggregation of the prion peptide PrP 185-208.

Inhibition of fibril assembly is a potential therapeutic strategy in prion diseases. The effect of cationic phosphorous dendrimers on the aggregation process of the prion peptide PrP 185-208 was studied using a spectrofluorometric assay with thioflavin T (ThT) and Fourier transformed infrared spectroscopy in order to monitor the kinetics of the process and the changes in the peptide secondary structure. The results show that phosphorous dendrimers are able to clearly interfere with PrP 185-208 aggregation process by both slowing down the formation of aggregates (by causing a decrease of the nucleation rate) and by lowering the final amount of amyloid fibrils, a common hallmark of conformational diseases. The dendrimers effect on the aggregation process would imply their interaction with peptide monomers and oligomers during the nucleation phase.

The configuration of the Cu2+ binding region in full-length human prion protein.

The cellular prion protein (PrP(C)) is a Cu(2+) binding protein connected to the outer cell membrane. The molecular features of the Cu(2+) binding sites have been investigated and characterized by spectroscopic experiments on PrP(C)-derived peptides and the recombinant human full-length PrP(C )(hPrP-[23-231]). The hPrP-[23-231] was loaded with (63)Cu under slightly acidic (pH 6.0) or neutral conditions. The PrP(C)/Cu(2+)-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu(2+) structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu(2+) coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show marked influence on the configuration of copper binding to PrP(C).

Structural properties of prion protein protofibrils and fibrils: an experimental assessment of atomic models.

Decades after the prion protein was implicated in transmissible spongiform encephalopathies, the structure of its toxic isoform and its mechanism of toxicity remain unknown. By gathering available experimental data, albeit low resolution, a few pieces of the prion puzzle can be put in place. Currently, there are two fundamentally different models of a prion protofibril. One has its building blocks derived from a molecular dynamics simulation of the prion protein under amyloidogenic conditions, termed the spiral model. The other model was constructed by threading a portion of the prion sequence through a beta-helical structure from the Protein Data Bank. Here we compare and contrast these models with respect to all of the available experimental information, including electron micrographs, symmetries, secondary structure, oligomerization interfaces, enzymatic digestion, epitope exposure, and disaggregation profiles. Much of this information was not available when the two models were introduced. Overall, we find that the spiral model is consistent with all of the experimental results. In contrast, it is difficult to reconcile several of the experimental observables with the beta-helix model. While the experimental constraints are of low resolution, in bringing together the previously disconnected experiments, we have developed a clearer picture of prion aggregates. Both the improved characterization of prion aggregates and the existing atomic models can be used to devise further experiments to better elucidate the misfolding pathway and the structure of prion protofibrils.

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.

Retrovirus infection strongly enhances scrapie infectivity release in cell culture.

Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrPSc (conformationally altered isoform of cellular prion protein (PrPC); Sc for scrapie), a partially protease-resistant isoform of the PrPC. PrPSc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrPSc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrPC, PrPSc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent.

Putative aggregation initiation sites in prion protein.

Misfolded prion protein, PrPSc, is believed to be the pathogenic agens in transmissible spongiform encephalopathies. Little is known about the autocatalytic misfolding process. Looking at the intrinsic properties of short sequence stretches, such as conformational flexibility and the tendency to populate extended conformers, we have examined the aggregation behaviour of various peptides within the region 106-157 of the sequence of human prion protein. We observed fast aggregation for the peptide containing residues I138-I-H-F141. This sequence, which is presented at the surface of cellular prion protein, PrPC, in an almost beta-sheet-like conformation, is therefore an ideal anchor-point for initial intermolecular contacts leading to oligomerization. We further report that the aggregation propensity of the neurotoxic peptide 106-126 appears to be centred in its termini and not in the central, alanine-rich sequence (A113-G-AAAA-G-A120).

Prion protein is a component of the multimolecular signaling complex involved in T cell activation.

In this study we analyzed the interaction of prion protein PrP(C) with components of glycosphingolipid-enriched microdomains in lymphoblastoid T cells. PrP(C) was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrP(C) is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrP(C) and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP-70 was immunoprecipitated by anti-PrP after T cell activation. These results demonstrate that PrP(C) interacts with ZAP-70, suggesting that PrP(C) is a component of the multimolecular signaling complex within microdomains involved in T cell activation.

Echigo-1: a panencephalopathic strain of Creutzfeldt-Jakob disease. II. Ultrastructural studies in hamsters.

In this and a companion paper we present immunohistochemical and ultrastructural data on hamsters infected with the Echigo-1 strain of Creutzfeldt-Jakob disease. Ultrastructurally, two types of vacuoles were readily discriminated in the brain: the grey matter vacuoles of spongiform change and intramyelin vacuoles. The vacuoles were always membrane-bound; the membranes were single or double. The axons were entirely missing from the plane of the sections or, if visible, were shrunken and attached to the innermost layer of the myelin. It was noteworthy that some vacuoles indented cell bodies or processes and thus were reminiscent of the intraneuronal vacuoles typical for natural scrapie, BSE and CWD in ungulates and cervids but not of the vacuoles encountered in rodent models of scrapie and CJD. We also noticed vacuoles distending myelinated fibres in which the axons had become dystrophic. Some axons underwent Wallerian degeneration while others met the criteria for dystrophic neuritis. Both alterations existed in the same areas. Typical dystrophic neurites contained abnormal subcellular organelles, mainly electron-dense lysosomal inclusions. Other neurites contained numerous multi-vesicular bodies and autophagic vacuoles. Nuclear paracrystalline rod-like inclusions were occasionally visible in neurons while other inclusions comprised spiroplasma-like inclusions in synaptic boutons. The robust cellular reactions consisted of reactive astrocytes and macrophages filled with cellular debris. It is of note that complex autophagic vacuoles were observed in the cytoplasm of neurons.

The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein.

The mode of internalization of glycosylphosphatidylinositol-anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid 'raft' domains to enter non-raft membrane, from which it enters coated pits. The N-terminal domain (residues 23-107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH(2)-KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed.

Structural changes of the prion protein in lipid membranes leading to aggregation and fibrillization.

Prion diseases are associated with a major refolding event of the normal cellular prion protein, PrP(C), where the predominantly alpha-helical and random coil structure of PrP(C) is converted into a beta-sheet-rich aggregated form, PrP(Sc). Under normal physiological conditions PrP(C) is attached to the outer leaflet of the plasma membrane via a GPI anchor, and it is plausible that an interaction between PrP and lipid membranes could be involved in the conversion of PrP(C) into PrP(Sc). Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of beta-PrP to model lipid membranes and compares the structural changes in alpha- and beta-PrP induced upon membrane binding. beta-PrP binds to negatively charged POPG membranes and to raft membranes composed of DPPC, cholesterol, and sphingomyelin. Binding of beta-PrP to raft membranes results in substantial unfolding of beta-PrP. This membrane-associated largely unfolded state of PrP is slowly converted into fibrils. In contrast, beta-PrP and alpha-PrP gain structure with POPG membranes, which instead leads to amorphous aggregates. Furthermore, binding of beta-PrP to POPG has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents, whereas raft vesicles are not destabilized upon binding of beta-PrP.