Anti-Prion Activity of a Panel of Aromatic Chemical Compounds: In Vitro and In Silico Approaches

The prion protein (PrP) is implicated in the Transmissible Spongiform Encephalopathies (TSEs), which comprise a group of fatal neurodegenerative diseases affecting humans and other mammals. Conversion of cellular PrP (PrP^C) into the scrapie form (PrP^Sc) is the hallmark of TSEs. Once formed, PrP^Sc aggregates and catalyzes PrP^C misfolding into new PrP^Sc molecules. Although many compounds have been shown to inhibit the conversion process, so far there is no effective therapy for TSEs. Besides, most of the previously evaluated compounds failed in vivo due to poor pharmacokinetic profiles. In this work we propose a combined in vitro/in silico approach to screen for active anti-prion compounds presenting acceptable drugability and pharmacokinetic parameters. A diverse panel of aromatic compounds was screened in neuroblastoma cells persistently infected with PrP^Sc (ScN2a) for their ability to inhibit PK-resistant PrP (PrP^Res) accumulation. From ∼200 compounds, 47 were effective in decreasing the accumulation of PrP^Res in ScN2a cells. Pharmacokinetic and physicochemical properties were predicted in silico, allowing us to obtain estimates of relative blood brain barrier permeation and mutagenicity. MTT reduction assays showed that most of the active compounds were non cytotoxic. Compounds that cleared PrP^Res from ScN2a cells, were non-toxic in the MTT assay, and presented a good pharmacokinetic profile were investigated for their ability to inhibit aggregation of an amyloidogenic PrP peptide fragment (PrP^109–149). Molecular docking results provided structural models and binding affinities for the interaction between PrP and the most promising compounds. In summary, using this combined in vitro/in silico approach we have identified new small organic anti-scrapie compounds that decrease the accumulation of PrP^Res in ScN2a cells, inhibit the aggregation of a PrP peptide, and possess pharmacokinetic characteristics that support their drugability. These compounds are attractive candidates for prion disease therapy.     

The octarepeat region of prion protein,but not the TM1 domain,is important for the antioxidant effect of prion protein

The cellular prion protein (PrP^c) plays a crucial role in the pathogenesis of prion diseases, but its physiological function is far from understood. Several candidate functions have been proposed including binding and internalization of metal ions, a superoxide dismutase-like activity, regulation of cellular antioxidant activities, and signal transduction. The transmembrane (TM1) region of PrP^c (residues 110–135) is particularly interesting because of its very high evolutionary conservation. We investigated a possible role of TM1 in the antioxidant defense, by assessing the impact of overexpressing wt-PrP or deletion mutants in N₂A mouse neuroblastoma cells on intracellular reactive oxygen species (ROS) levels. Under conditions of oxidative stress, intracellular ROS levels were significantly lowered in cells overexpressing either wild-type PrP^c (wt-PrP) or a deletion mutant affecting TM1 (Δ8TM1-PrP), but, as expected, not in cultures overexpressing a deletion mutant lacking the octapeptide region (Δocta-PrP). Overexpression of wt-PrP, Δ8TM1-PrP, or Δocta-PrP did not affect basal ROS levels. Interestingly, the mitochondrial membrane potential was significantly lowered in Δocta-PrP-transfected cultures in the absence of oxidative stress. We conclude that the protective effect of PrP^c against oxidative stress involves the octarepeat region but not the TM1 domain nor the high-affinity copper binding site described for human residues His96/His111.     

Reactive oxygen species-mediated beta-cleavage of the prion protein in the cellular response to oxidative stress

The cellular prion protein (PrP(C)) is critical for the development of prion diseases. However, the physiological role of PrP(C) is less clear, although a role in the cellular resistance to oxidative stress has been proposed. PrP(C) is cleaved at the end of the copper-binding octapeptide repeats through the action of reactive oxygen species (ROS), a process termed beta-cleavage. Here we show that ROS-mediated beta-cleavage of cell surface PrP(C) occurs within minutes and was inhibited by the hydroxyl radical quencher dimethyl sulfoxide and by an antibody against the octapeptide repeats. A construct of PrP lacking the octapeptide repeats, PrPDeltaoct, failed to undergo ROS-mediated beta-cleavage, as did two mutant forms of PrP, PG14 and A116V, associated with human prion diseases. As compared with cells expressing wild type PrP, when challenged with H2O2 and Cu2+, cells expressing PrPdeltaoct, PG14, or A116V had reduced viability and glutathione peroxidase activity and increased intracellular free radicals. Thus, lack of ROS-mediated beta-cleavage of PrP correlated with the sensitivity of the cells to oxidative stress. These data indicate that the beta-cleavage of PrP(C) is an early and critical event in the mechanism by which PrP protects cells against oxidative stress.     

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP^C) from a mainly α-helical to a β-sheet rich PrP-scrapie (PrP^Sc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP^Sc, nor the normal function of PrP^C is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP^C mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP^C increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP^C on ferritin iron content is enhanced by stimulating PrP^C endocytosis, and reversed by cross-linking PrP^C on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP^102L decreases ferritin iron content significantly relative to PrP^C expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP^C nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP^C in cellular iron uptake and transport to ferritin, and dysfunction of PrP^C as a significant contributing factor of brain iron imbalance in prion disorders.     

Review of studies that have used knockout mice to assess normal function of prion protein under immunological or pathophysiological stress

Deletion of cellular isoform of prion protein (PrP^C) increases neuronal predisposition to damage by modulating apoptosis and the negative consequences of oxidative stress. In vivo studies have demonstrated that PrP^C‐deficient mice are more prone to seizure, depression, and induction of epilepsy and experience extensive cerebral damage following ischemic challenge or viral infection. In addition, adenovirus‐mediated overexpression of PrP^C reduces brain damage in rat models of cerebral ischemia. In experimental autoimmune encephalomyelitis, PrP^C‐deficient mice reportedly have a more aggressive disease onset and less clinical improvement during the chronic phase than wild‐type mice mice. In mice given oral dextran sulfate, PrP^C has a potential protective role against inflammatory bowel disease. PrP^C‐deficient mice demonstrate significantly greater increases in blood glucose concentrations after intraperitoneal injection of glucose than wild‐type mice. Further in vivo challenges to PrP gene‐deficient models and conditional knockout models with siRNA and in vivo administration of PrP‐ligating agents may assist in refining knowledge of the lymphoid function of PrP^C and predicting the effects of anti‐PrP treatment on the immune system. Together, these findings indicate that PrP^C may have multiple neuroprotective and anti‐inflammatory roles, which explains why this protein is so widely expressed.     

Prion Seeding Activities of Mouse Scrapie Strains with Divergent PrPSc Protease Sensitivities and Amyloid Plaque Content Using RT-QuIC and eQuIC

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrP^Sc) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrP^Sc propagation involves conversion from its normal isoform, PrP^C, by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrP^Sen) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrP^Sc (PrP^Res). Scrapie brain dilutions up to 10⁻⁸ and 10⁻¹³ were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrP^Res levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrP^C. Although the brains of 263K-affected mice had little immunoblot-detectable PrP^Res, RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrP^Res strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrP^Res levels. We also found that eQuIC, which incorporates a PrP^Sc immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrP^Sc.     

Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion

Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrP^C into the infectious misfolded isoform PrP^Sc. Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrP^Sc destabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121–231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders.     

Prion Protein Protects against Renal Ischemia/Reperfusion Injury

The cellular prion protein (PrP^C), a protein most noted for its link to prion diseases, has been found to play a protective role in ischemic brain injury. To investigate the role of PrP^C in the kidney, an organ highly prone to ischemia/reperfusion (IR) injury, we examined wild-type (WT) and PrP^C knockout (KO) mice that were subjected to 30-min of renal ischemia followed by 1, 2, or 3 days of reperfusion. Renal dysfunction and structural damage was more severe in KO than in WT mice. While PrP was undetectable in KO kidneys, Western blotting revealed an increase in PrP in IR-injured WT kidneys compared to sham-treated kidneys. Compared to WT, KO kidneys exhibited increases in oxidative stress markers heme oxygenase-1, nitrotyrosine, and N^ε-(carboxymethyl)lysine, and decreases in mitochondrial complexes I and III. Notably, phosphorylated extracellular signal-regulated kinase (pERK) staining was predominantly observed in tubular cells from KO mice following 2 days of reperfusion, a time at which significant differences in renal dysfunction, histological changes, oxidative stress, and mitochondrial complexes between WT and KO mice were observed. Our study provides the first evidence that PrP^C may play a protective role in renal IR injury, likely through its effects on mitochondria and ERK signaling pathways.     

Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

In prion diseases, the posttranslational modification of host-encoded prion protein PrP^c yields a high β-sheet content modified protein PrP^sc, which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP^c to PrP^sc. Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs—ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity.     

Prion protein—Semisynthetic prion protein (PrP) variants with posttranslational modifications

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrP^C) into scrapie prion protein (PrP^Sc) that further propagates PrP^C misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrP^Sc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior invitro and invivo as it provides access to defined site‐selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed.     

Prion infection: Seeded fibrillization or more?

The prion infection is a conversion of host encoded prion protein (PrP) from its cellular isoform PrP^C into the pathological and infectious isoform PrP^Sc; the conversion process was investigated by in vitro studies using recombinant and cellular PrP and natural PrP^Sc. We present a brief summary of the results determined with our in vitro conversion system and the derived mechanistic models. We describe well characterized intermediates and precursor states during the conversion process, kinetic studies of spontaneous and seeded fibrillogenesis and the impact of the membrane environment.Key words: prion protein conversion, seeding, fibril, dimer, precursor state, kinetics, membrane     

Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP^C) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.     

Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein

Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrP^Sc of the host-encoded prion protein (PrP^c). A profound conformational change of PrP^c underlies formation of PrP^Sc and prion propagation involves conversion of PrP^c substrate by direct interaction with PrP^Sc template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI) effects of conversion-incompetent, internally-deleted PrP (ΔPrP) on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1∼H2) which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1∼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrP^C into PrP^Sc. To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale of prion conversion and PrP^Sc recycling and can exert DNI there. This shows that the intracellular trafficking of PrPs is more complex than previously anticipated.     

Octapeptide repeat region of prion protein (PrP) is required at an early stage for production of abnormal prion protein in PrP-deficient neuronal cell line

An abnormal isoform of prion protein (PrP^Sc), which is composed of the same amino acids as cellular PrP (PrP^C) and has proteinase K (PK)-resistance, hypothetically converts PrP^C into PrP^Sc. To investigate the region important for PrP^Sc production, we examined the levels of PrP^Sc in PrP gene-deficient cells (HpL3-4) expressing PrP^C deleted of various regions including the octapeptide repeat region (OR) or hydrophobic region (HR). After Chandler or Obihiro prion infection, PrP^Sc was produced in HpL3-4 cells expressing wild-type PrP^C or PrP^C deleted of HR at an early stage and further reduced to below the detectable level, whereas cells expressing PrP^C deleted of OR showed no PrP^Sc production. The results suggest that OR of PrP^C is required for the early step of efficient PrP^Sc production.     

Selective Processing and Metabolism of Disease-Causing Mutant Prion Proteins

Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP^C). In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP^Sc form) that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP^C to PrP^Sc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP^Sc in familial prion disease.     

Cellular uptake of the prion protein fragment PrP106-126 in vitro

The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrP^c). This pathological isoform (PrP^Sc) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrP^Sc in affected tissue. To investigate the possible uptake mechanisms that may be required for PrP^Sc-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrP^c expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrP^Sc in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.     

The octarepeat region of prion protein, but not the TM1 domain, is important for the antioxidant effect of prion protein

The cellular prion protein (PrP(c)) plays a crucial role in the pathogenesis of prion diseases, but its physiological function is far from understood. Several candidate functions have been proposed including binding and internalization of metal ions, a superoxide dismutase-like activity, regulation of cellular antioxidant activities, and signal transduction. The transmembrane (TM1) region of PrP(c) (residues 110-135) is particularly interesting because of its very high evolutionary conservation. We investigated a possible role of TM1 in the antioxidant defense, by assessing the impact of overexpressing wt-PrP or deletion mutants in N(2)A mouse neuroblastoma cells on intracellular reactive oxygen species (ROS) levels. Under conditions of oxidative stress, intracellular ROS levels were significantly lowered in cells overexpressing either wild-type PrP(c) (wt-PrP) or a deletion mutant affecting TM1 (Delta8TM1-PrP), but, as expected, not in cultures overexpressing a deletion mutant lacking the octapeptide region (Deltaocta-PrP). Overexpression of wt-PrP, Delta8TM1-PrP, or Deltaocta-PrP did not affect basal ROS levels. Interestingly, the mitochondrial membrane potential was significantly lowered in Deltaocta-PrP-transfected cultures in the absence of oxidative stress. We conclude that the protective effect of PrP(c) against oxidative stress involves the octarepeat region but not the TM1 domain nor the high-affinity copper binding site described for human residues His96/His111.     

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.