Scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies.

Denaturation studies with guanidine HCl (GdnHCl) were performed to test the relationship between scrapie infectivity and properties of scrapie-associated prion protein (PrP(Sc)). Large GdnHCl-induced reductions in infectivity were associated with the irreversible elimination of both the proteinase K resistance and apparent self-propagating converting activity of PrP(Sc). In intermediate GdnHCl concentrations that stimulate converting activity and partially disaggregate PrP(Sc), both scrapie infectivity and converting activity were associated with residual partially protease-resistant multimers of PrP(Sc).     

pH-dependent prion protein conformation in classical Creutzfeldt-Jakob disease.

In transmissible spongiform encephalopathies, the cellular prion protein (PrP(C)) undergoes a conformational change from a prevailing alpha-helical structure to a beta-sheet-rich, protease-resistant isoform, termed PrP(Sc). PrP(C) has two characteristics: a high affinity for Cu(2+) and a strong pH-dependent conformation. Lines of evidence indicate that PrP(Sc) conformation is dependent on copper and that acidic conditions facilitate the conversion of PrP(C) --> PrP(Sc). In each species, PrP(Sc) exists in multiple conformations, which are associated with different prion strains. In sporadic Creutzfeldt-Jakob disease (sCJD), different biochemical types of PrP(Sc) have been identified according to the size of the protease-resistant fragments, patterns of glycosylation, and the metal-ion occupancy. Based on the site of cleavage produced by proteinase K, we investigated the conformational stability of PrP(Sc) under acidic, neutral, and basic conditions in 42 sCJD subjects. Our study shows that only one type of sCJD PrP(Sc), associated with the classical form, shows a pH-dependent conformation, whereas two other biochemical PrP(Sc) types, detected in distinct sCJD phenotypes, are unaffected by pH variations. This novel approach demonstrates the presence of three types of PrP(Sc) in sCJD.     

An aggregation-specific enzyme-linked immunosorbent assay: detection of conformational differences between recombinant PrP protein dimers and PrP(Sc) aggregates

The conversion of the normal cellular prion protein, PrP(C), into the protease-resistant, scrapie PrP(Sc) aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrP(Sc) aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrP(Sc) aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease.     

Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes

The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.     

Monoclonal antibody against a peptide of human prion protein discriminates between Creutzfeldt-Jacob's disease-affected and normal brain tissue

Current methods for diagnosing transmissible spongiform encephalopathies rely on the degradation of the cellular prion protein (PrP(C)) and the subsequent detection of the protease-resistant remnant of the pathological prion isoform PrP(Sc) by antibodies that react with all forms of PrP. We report on a monoclonal antibody, V5B2, raised against a peptide from the C-terminal part of PrP, which recognizes an epitope specific to PrP(Sc). In cryostat sections from Creutzfeldt-Jacob's disease (CJD) patients' brains, V5B2 selectively labels various deposits of PrP(Sc) without any pretreatment for removal of PrP(C). V5B2 does not bind to non-CJD brain samples or to recombinant PrP, either in its native or denatured form. Specificity for PrP is confirmed by a sandwich enzyme-linked immunosorbent assay utilizing V5B2, which discriminates between CJD and normal samples without proteinase K treatment, and by immunoprecipitation from CJD brain homogenate. The PrP(Sc)-specific epitope is disrupted by denaturation. We conclude that the C-terminal part of PrP in disease-associated PrP(Sc) aggregates forms a structural epitope whose conformation is distinct from that of PrP(C).     

Aggregation of cellular prion protein is initiated by proximity-induced dimerization

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP(C)) into an altered disease-associated conformation generally designated PrP(Sc), abnormal deposition of PrP(Sc) aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP(C) aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP(C) molecules may trigger its aggregation in neuronal cells. A chimeric PrP(C) composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP(C) were created, and transfected in N2a cells. Similar to PrP(C), Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP(C), both dimerization and oligomerization of PrP(C) resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP(C) molecules may constitute a minimal and sufficient molecular event leading to PrP(C) aggregation and extracellular deposition.     

Acidic pH and detergents enhance in vitro conversion of human brain PrPC to a PrPSc-like form

In the presence of a low concentration of denaturants or detergents, acidic pH triggers a conformational transition of alpha-helices into beta-sheets in recombinant prion protein (PrP), likely mimicking some aspects of the transformation of host-encoded normal cellular PrP (PrP(C)) into its pathogenic isoform (PrP(Sc)). Here we observed the effects of acidic pH and guanidine hydrochloride (GdnHCl) on the physicochemical and structural properties of PrP(C) derived from normal human brain and determined the ability of the acid/GdnHCl-treated PrP to form a proteinase K (PK)-resistant species in the absence and presence of PrP(Sc) template. After treatment with 1.5 m GdnHCl at pH 3.5, PrP(C) from normal brain homogenates was converted into a detergent-insoluble form similar to PrP(Sc). Unlike PrP(Sc), however, the treated brain PrP(C) was protease-sensitive and retained epitope accessibility to monoclonal antibodies 3F4 and 6H4. Brain PrP(C) treated with acidic pH/GdnHCl acquired partial PK resistance upon further treatment with low concentrations of sodium dodecyl sulfate (SDS). Formation of this PrP(Sc)-like isoform was greatly enhanced by incubation with trace quantities of PrP(Sc) from Creutzfeldt-Jakob disease brain. Acid/GdnHCl-treated brain PrP may constitute a "recruitable intermediate" in PrP(Sc) formation. Further structural rearrangement seems essential for this species to acquire PK resistance, which can be promoted by the presence of a PrP(Sc) template.     

Influence of the N-terminal domain on the aggregation properties of the prion protein

Prion diseases appear to be caused by the aggregation of the cellular prion protein (PrP(C)) into an infectious form denoted PrP(Sc). The in vitro aggregation of the prion protein has been extensively investigated, yet many of these studies utilize truncated polypeptides. Because the C-terminal portion of PrP(Sc) is protease-resistant and retains infectivity, it is assumed that studies on this fragment are most relevant. The full-length protein can be distinguished from the truncated protein because it contains a largely structured, alpha-helical, C-terminal region in addition to an N terminus that is unstructured in the absence of metal ion binding. Herein, the in vitro aggregation of a truncated portion of the prion protein (PrP 90-231) and a full-length version (PrP 23-231) were compared. In each case, concentration-dependent aggregation was analyzed to discern whether it proceeds by a nucleation-dependent pathway. Both protein constructs appear to aggregate via a nucleated polymerization with a small nucleus size, yet the later steps differ. The full-length protein forms larger aggregates than the truncated protein, indicating that the N terminus may mediate higher-order aggregation processes. In addition, the N terminus has an influence on the assembly state of PrP before aggregation begins, causing the full-length protein to adopt several oligomeric forms in a neutral pH buffer. Our results emphasize the importance of studying the full-length protein in addition to the truncated forms for in vitro aggregation studies in order to make valid hypotheses about the mechanisms of prion aggregation and the distribution of aggregates in vivo.     

Hydration and packing effects on prion folding and beta-sheet conversion. High pressure spectroscopy and pressure perturbation calorimetry studies

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we compare the stability against pressure and the thermomechanical properties of the alpha-helical and beta-sheet conformations of recombinant murine prion protein, designated as alpha-rPrP and beta-rPrP, respectively. High temperature induces aggregates and a large gain in intermolecular antiparallel beta-sheet (beta-rPrP), a conformation that shares structural similarity with PrP(Sc). alpha-rPrP is highly stable, and only pressures above 5 kilobars (1 kilobar = 100 MegaPascals) cause reversible denaturation, a process that leads to a random and turnrich conformation with concomitant loss of alpha-helix, as measured by Fourier transform infrared spectroscopy. In contrast, aggregates of beta-rPrP are very sensitive to pressure, undergoing transition into a dissociated species that differs from the denatured form derived from alpha-rPrP. The higher susceptibility to pressure of beta-rPrP can be explained by its less hydrated structure. Pressure perturbation calorimetry supports the view that the accessible surface area of alpha-rPrP is much higher than that of beta-rPrP, which explains the lower degree of hydration of beta-rPrP. Our findings shed new light on the mechanism of prion conversion and show how water plays a prominent role. Our results allow us to propose a volume and free energy diagram of the different species involved in the conversion and aggregation. The existence of different folded conformations as well as different denatured states of PrP may explain the elusive character of its conversion into a pathogenic form.     

Single particle analysis of manganese-induced prion protein aggregates

Prion diseases are characterized by the conversion of the cellular prion protein (PrP(C)) to a disease-specific aggregated isoform (PrP(Sc)). We have shown that Mn(2+) ions amplify aggregation, whereas Cu(2+) has an inhibitory effect. To characterize Mn(2+)-induced aggregates, we used cross-correlation analysis as well as scanning for intensely fluorescent targets in an SDS-dependent aggregation assay with fluorescently labeled PrP. We found that the effect of Mn(2+) was mainly due to the association of preformed PrP oligomers to larger aggregates, rapidly reversible by EDTA, and independent of the histidine-dependent copper-binding sites of PrP, suggesting that Mn(2+) induces reversible intermolecular binding. In contrast, the inhibitory effect of Cu(2+) required binding to histidine-containing binding sites, indicating that binding of copper affects the structure of PrP(C) which in turn modifies the susceptibility to manganese and the ability to aggregate. These findings suggest that copper and manganese may also affect prion propagation in vivo.     

Affinity-tagged miniprion derivatives spontaneously adopt protease-resistant conformations

An abridged PrP molecule of 106 amino acids designated PrP106 can form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His(6)) affinity tags to selective sites within PrP106 resulted unexpectedly in new PrP proteins that spontaneously adopted protease-resistant conformations when expressed in neuroblastoma cells and Tg mice. Acquisition of protease resistance depended on the length, charge, and placement of the affinity tag. Introduction of the disease-linked mutation E200K into the sequence of PrP106(140/6His) increased the recovery of protease-resistant PrP fivefold, whereas introduction of the mutations C213A and Delta214-220 did not affect the recovery of protease-resistant PrP. Treatment of cultured cells expressing affinity-tagged PrP106 mutants with polypropyleneimine dendrimer rendered these proteins sensitive to protease digestion in a manner similar to wild-type PrP(Sc). We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrP(Sc). These proteins might be useful tools in the identification of new disease-causing mutations as well as for screening compounds for therapeutic efficacy.     

Protease-resistant and detergent-insoluble prion protein is not necessarily associated with prion infectivity.

PrPSc, an abnormal isoform of PrPC, is the only known component of the prion, an agent causing fatal neurodegenerative disorders such as bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD). It has been postulated that prion diseases propagate by the conversion of detergent-soluble and protease-sensitive PrPC molecules into protease-resistant and insoluble PrPSc molecules by a mechanism in which PrPSc serves as a template. We show here that the chemical chaperone dimethyl sulfoxide (Me2SO) can partially inhibit the aggregation of either PrPSc or that of its protease-resistant core PrP27-30. Following Me2SO removal by methanol precipitation, solubilized PrP27-30 molecules aggregated into small and amorphous structures that did not resemble the rod configuration observed when scrapie brain membranes were extracted with Sarkosyl and digested with proteinase K. Interestingly, aggregates derived from Me2SO-solubilized PrP27-30 presented less than 1% of the prion infectivity obtained when the same amount of PrP27-30 in rods was inoculated into hamsters. These results suggest that the conversion of PrPC into protease-resistant and detergent-insoluble PrP molecules is not the only crucial step in prion replication. Whether an additional requirement is the aggregation of newly formed proteinase K-resistant PrP molecules into uniquely structured aggregates remains to be established.     

Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.     

In vitro self-propagation of recombinant PrPSc-like conformation generated in the yeast cytoplasm

Self-propagation is characteristic property for a prion conformation. Previous studies revealed that prion protein expressed in the cytoplasm gained a PrP(Sc)-like conformation. However, it remains unclear whether the PrP(Sc)-like conformation has the self-propagating property. We found that PrP partially purified from yeast cytoplasm formed amyloid fiber like structures, and we found that the PrP(Sc)-like conformation is able to convert normal PrP(C) in the brain homogenate to a proteinase K-resistant conformation. These results suggest that yeast cytoplasm expressed recombinant PrP(Sc)-like conformation has the characteristic self-propagating property of a prion, which may have implications in the pathogenesis of sporadic and inherited prion diseases.     

Detection of Bovine Spongiform Encephalopathy-Specific PrPSc by Treatment with Heat and Guanidine Thiocyanate

The conversion of a ubiquitous cellular protein (PrP(C)), an isoform of the prion protein (PrP), to the pathology-associated isoform PrP(Sc) is one of the hallmarks of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE). Accumulation of PrP(Sc) has been used to diagnose BSE. Here we describe a quantitative enzyme-linked immunosorbent assay (ELISA) that involves antibodies against epitopes within the protease-resistant core of the PrP molecule to measure the amount of PrP in brain tissues from animals with BSE and normal controls. In native tissue preparations, little difference was found between the two groups. However, following treatment of the tissue with heat and guanidine thiocyanate (Gh treatment), the ELISA discriminated BSE-specific PrP(Sc) from PrP(C) in bovine brain homogenates. PrP(Sc) was identified by Western blot, centrifugation, and protease digestion experiments. It was thought that folding or complexing of PrP(Sc) is most probably reversed by the Gh treatment, making hidden antigenic sites accessible. The digestion experiments also showed that protease-resistant PrP in BSE is more difficult to detect than that in hamster scrapie. While the concentration of PrP(C) in cattle is similar to that in hamsters, PrP(Sc) sparse in comparison. The detection of PrP(Sc) by a simple physicochemical treatment without the need for protease digestion, as described in this study, could be applied to develop a diagnostic assay to screen large numbers of samples.     

Lactoferrin induces cell surface retention of prion protein and inhibits prion accumulation

Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrP(C)) into its pathogenic, amyloidogenic isoform (PrP(Sc)) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin (TF) family, which accumulates in the amyloid deposits in the brain in neurodegenerative disorders, such as Alzheimer's disease and Pick's disease. In the present study, we have examined the effects of LF on PrP(Sc) formation by using cell culture models. Bovine LF inhibited PrP(Sc) accumulation in scrapie-infected cells in a time- and dose-dependent manner, whereas TF was not inhibitory. Bioassays of LF-treated cells demonstrated prolonged incubation periods compared with non-treated cells indicating a reduction of prion infectivity. LF mediated the cell surface retention of PrP(C) by diminishing its internalization and was capable of interacting with PrP(C) in addition to PrP(Sc). Furthermore, LF partially inhibited the formation of protease-resistant PrP as determined by the protein misfolding cyclic amplification assay. Our results suggest that LF has multifunctional antiprion activities.     

Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease

In prion disease, the abnormal conformer of the cellular prion protein, PrP(Sc), deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP(Sc) to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrP(Sc) C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrP(Sc) or PrP27-30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrP(Sc) in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrP(Sc) and indicate that the characterization of truncated PrP(Sc) forms may further improve molecular typing in CJD.     

Molecular distinction between pathogenic and infectious properties of the prion protein

Tg(PG14) mice express a prion protein (PrP) with a nine-octapeptide insertion associated with a human familial prion disease. These animals spontaneously develop a fatal neurodegenerative disorder characterized by ataxia, neuronal apoptosis, and accumulation in the brain of an aggregated and weakly protease-resistant form of mutant PrP (designated PG14(spon)). Brain homogenates from Tg(PG14) mice fail to transmit disease after intracerebral inoculation into recipient mice, indicating that PG14(spon), although pathogenic, is distinct from PrP(Sc), the infectious form of PrP. In contrast, inoculation of Tg(PG14) mice with exogenous prions of the RML strain induces accumulation of PG14(RML), a PrP(Sc) form of the mutant protein that is infectious and highly protease resistant. Like PrP(Sc), both PG14(spon) and PG14(RML) display conformationally masked epitopes in the central and octapeptide repeat regions. However, these two forms differ profoundly in their oligomeric states, with PG14(RML) aggregates being much larger and more resistant to dissociation. Our analysis provides new molecular insight into an emerging puzzle in prion biology, the discrepancy between the infectious and neurotoxic properties of PrP.     

DNA converts cellular prion protein into the beta-sheet conformation and inhibits prion peptide aggregation

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.     

Multiple biochemical similarities between infectious and non-infectious aggregates of a prion protein carrying an octapeptide insertion

A nine-octapeptide insertion in the prion protein (PrP) gene is associated with an inherited form of human prion disease. Transgenic (Tg) mice that express the mouse homolog of this mutation (designated PG14) spontaneously accumulate in their brains an insoluble and weakly protease-resistant form of the mutant protein. This form (designated PG14(Spon)) is highly neurotoxic, but is not infectious in animal bioassays. In contrast, when Tg(PG14) mice are inoculated with the Rocky Mountain Laboratory (RML) strain of prions, they accumulate a different form of PG14 PrP (designated PG14(RML)) that is highly protease resistant and infectious in animal transmission experiments. We have been interested in characterizing the molecular properties of PG14(Spon) and PG14(RML), with a view to identifying features that determine two, apparently distinct properties of PrP aggregates: their infectivity and their pathogenicity. In this paper, we have subjected PG14(Spon) and PG14(RML) to a panel of assays commonly used to distinguish infectious PrP (PrP(Sc)) from cellular PrP (PrP(C)), including immobilized metal affinity chromatography, precipitation with sodium phosphotungstate, and immunoprecipitation with PrP(C)- and PrP(Sc)-specific antibodies. Surprisingly, we found that aggregates of PG14(Spon) and PG14(RML) behave identically to each other, and to authentic PrP(Sc), in each of these biochemical assays. PG14(Spon) however, in contrast to PG14(RML) and PrP(Sc), was unable to seed the misfolding of PrP(C) in an in vitro protein misfolding cyclic amplification reaction. Collectively, these results suggest that infectious and non-infectious aggregates of PrP share common structural features accounting for their toxicity, and that self-propagation of PrP involves more subtle molecular differences.