Mammalian prions: Tracking the infectious entities

Protein misfolding is central to the pathogenesis of several neurodegenerative disorders. Among these disorders, prion diseases are unique because they are transmissible. The conversion of the host-encoded GPI-anchored PrP protein into a structurally altered form is crucially associated with the infectious and neurotoxic properties of the resulting abnormal PrP. Many lines of evidence indicate that distinct aggregated forms with different size and protease resistance are produced during prion multiplication. The recent isolation of various subsets of abnormal PrP, along with the improved biochemical tools and infectivity detection assays have shed light on the diversity of abnormal PrP protein and may give insights into the features of the more infectious subsets of abnormal PrP.Key words: prions, aggregates, infectivity, EST, PrP protein     

Dual conformation of H2H3 domain of prion protein in mammalian cells

The concept of prion is applied to protein modules that share the ability to switch between at least two conformational states and transmit one of these through intermolecular interaction and change of conformation. Although much progress has been achieved through the understanding of prions from organisms such as Saccharomyces cerevisiae, Podospora anserina, or Aplysia californica, the criteria that qualify a protein module as a prion are still unclear. In addition, the functionality of known prion domains fails to provide clues to understand the first identified prion, the mammalian infectious prion protein, PrP. To address these issues, we generated mammalian cellular models of expression of the C-terminal two helices of PrP, H2 and H3, which have been hypothesized, among other models, to hold the replication and conversion properties of the infectious PrP. We found that the H2H3 domain is an independent folding unit that undergoes glycosylations and glycosylphosphatidylinositol anchoring similar to full-length PrP. Surprisingly, in some conditions the normally folded H2H3 was able to systematically go through a conversion process and generate insoluble proteinase K-resistant aggregates. This structural switch involves the assembly of amyloid structures that bind thioflavin S and oligomers that are reactive to A11 antibody, which specifically detects protein oligomers from neurological disorders. Overall, we show that H2H3 is a conformational switch in a cellular context and is thus suggested to be a candidate for the conversion domain of PrP.     

Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange

A fragment of the prion protein, PrP(89–143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89–143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102–109 and 117–136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.     

Infectious prions and proteinopathies

Transmissible spongiform encephalopathies (TSEs) are caused by an infectious agent that is thought to consist of only misfolded and aggregated prion protein (PrP). Unlike conventional micro-organisms, the agent spreads and propagates by binding to and converting normal host PrP into the abnormal conformer, increasing the infectious titre. Synthetic prions, composed of refolded fibrillar forms of recombinant PrP (rec-PrP) have been generated to address whether PrP aggregates alone are indeed infectious prions. In several reports, the development of TSE disease has been described following inoculation and passage of rec-PrP fibrils in transgenic mice and hamsters. However in studies described here we show that inoculation of rec-PrP fibrils does not always cause clinical TSE disease or increased infectious titre, but can seed the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). These data are reminiscent of the “prion-like” spread of misfolded protein in other models of neurodegenerative disease following inoculation of transgenic mice with pre-formed amyloid seeds. Protein misfolding, even when the protein is PrP, does not inevitably lead to the development of an infectious TSE disease. It is possible that most in vivo and in vitro produced misfolded PrP is not infectious and that only a specific subpopulation is associated with infectivity and neurotoxicity.     

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.     

Generation of a new form of human PrP(Sc) in vitro by interspecies transmission from cervid prions

Prion diseases are infectious neurodegenerative disorders that affect humans and animals and that result from the conversion of normal prion protein (PrP(C)) into the misfolded prion protein (PrP(Sc)). Chronic wasting disease (CWD) is a prion disorder of increasing prevalence within the United States that affects a large population of wild and captive deer and elk. Determining the risk of transmission of CWD to humans is of utmost importance, considering that people can be infected by animal prions, resulting in new fatal diseases. To study the possibility that human PrP(C) can be converted into the misfolded form by CWD PrP(Sc), we performed experiments using the protein misfolding cyclic amplification technique, which mimics in vitro the process of prion replication. Our results show that cervid PrP(Sc) can induce the conversion of human PrP(C) but only after the CWD prion strain has been stabilized by successive passages in vitro or in vivo. Interestingly, the newly generated human PrP(Sc) exhibits a distinct biochemical pattern that differs from that of any of the currently known forms of human PrP(Sc). Our results also have profound implications for understanding the mechanisms of the prion species barrier and indicate that the transmission barrier is a dynamic process that depends on the strain and moreover the degree of adaptation of the strain. If our findings are corroborated by infectivity assays, they will imply that CWD prions have the potential to infect humans and that this ability progressively increases with CWD spreading.     

Modeling and analysis of prion dynamics in the presence of a chaperone

Prions are infectious agents and are polymers called PrP(Sc)-Prion protein scrapies, of a normal protein, a monomer called PrP(c)-Prion protein cellular. These PrP(Sc)s cause TSEs-transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE) in cattle, scrapies in sheep, Kuru and Creutzfeld-Jacob diseases in humans. Cellular molecular chaperones, which are ubiquitous, stress-induced proteins, and newly found chemical and pharmacological chaperones have been found to be effective in preventing misfolding of different disease-causing proteins, essentially reducing the severity of several neurodegenerative disorders and many other protein-misfolding diseases. In this work, we propose a model for the replication of prions by nucleated polymerization in the presence of a chaperone. According to this model, the biological processes of coagulation, splitting and the inhibitory effects of the chaperone can be described by a coupled system consisting of ordinary differential equations and a partial differential equation. The model is converted into a system of ordinary differential equations and the equilibrium points are computed and their stability is studied. We give a numerical simulation of the model and we find that a disease free state can be achieved in the presence of a chaperone. The duration of the disease free state is found to increase with the amount of chaperone and this amount of chaperone can be computed from the model.     

Isolation from cattle of a prion strain distinct from that causing bovine spongiform encephalopathy

To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical profile of abnormal protease-resistant prion protein (PrP(res)) isolated from brains of affected animals or humans. However, alternate PrP(res) signatures in cattle have recently been discovered through large-scale screening. To test whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrP(res) of higher apparent molecular mass--called H-type--into transgenic mice expressing bovine or ovine PrP. All mice developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals.     

Molecular analysis of the abnormal prion protein during coinfection of mice by bovine spongiform encephalopathy and a scrapie agent

Molecular features of the proteinase K-resistant prion protein (PrP res) may discriminate among prion strains, and a specific signature could be found during infection by the infectious agent causing bovine spongiform encephalopathy (BSE). To investigate the molecular basis of BSE adaptation and selection, we established a model of coinfection of mice by both BSE and a sheep scrapie strain (C506M3). We now show that the PrP res features in these mice, characterized by glycoform ratios and electrophoretic mobilities, may be undistinguishable from those found in mice infected with scrapie only, including when mice were inoculated by both strains at the same time and by the same intracerebral inoculation route. Western blot analysis using different antibodies against sequences near the putative N-terminal end of PrP res also demonstrated differences in the main proteinase K cleavage sites between mice showing either the BSE or scrapie PrP res profile. These results, which may be linked to higher levels of PrP res associated with infection by scrapie, were similar following a challenge by a higher dose of the BSE agent during coinfection by both strains intracerebrally. Whereas PrP res extraction methods used allowed us to distinguish type 1 and type 2 PrP res, differing, like BSE and scrapie, by their electrophoretic mobilities, in the same brain region of some patients with Creutzfeldt-Jakob disease, analysis of in vitro mixtures of BSE and scrapie brain homogenates did not allow us to distinguish BSE and scrapie PrP res. These results suggest that the BSE agent, the origin of which remains unknown so far but which may have arisen from a sheep scrapie agent, may be hidden by a scrapie strain during attempts to identify it by molecular studies and following transmission of the disease in mice.     

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.     

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.     

PET-blot analysis contributes to BSE strain recognition in C57Bl/6 mice.

Identification of the strain of agent responsible for bovine spongiform encephalopathy (BSE) can be made histologically through the analysis of both distribution and intensity of brain vacuolar lesions after BSE transmission to mouse. Another useful way to distinguish the BSE agent from other prion strains is the study of the distribution of the abnormal prion protein (PrP(res)). For that purpose, paraffin-embedded tissue blot (PET-blot) method was applied on brains from C57Bl/6 mice infected with cattle BSE, experimental sheep BSE, or feline spongiform encephalopathy (FSE) from a cheetah. PrP(res) distribution was comparable, whichever of the three BSE agent sources was considered and was distinct from the PrP(res) distribution in C57Bl/6 mice inoculated with a French scrapie isolate or with a mouse-adapted scrapie strain (C506M3). These data confirm a common origin of infectious agent responsible for the British and French cattle BSE. They also indicate that PET-blot method appears as a precise complementary tool in prion strain studies because it offers easy and quick assessment of the PrP(res) mapping. Advantages and limits of the PET-blot method are discussed and compared with other established and validated methods of strain typing.     

The highways and byways of prion protein trafficking

Prions are defined as infectious agents that comprise only proteins and are responsible for transmissible spongiform encephalopathies (TSEs)--fatal neurodegenerative diseases that affect humans and other mammals and include Creutzfeldt-Jacob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. Prions have been proposed to arise from the conformational conversion of the cellular prion protein PrP(C) to a misfolded form termed PrP(Sc) that precipitates into aggregates and fibrils. The conversion process might be triggered by interaction of the infectious form with the cellular form or it might result from a mutation in the gene encoding PrP(C). Exactly how and where in the cell the interaction and the conversion of PrP(C) to PrP(Sc) occur, however, remain controversial. Recent studies have shed light on the intracellular trafficking of PrP(C), the role of protein mis-sorting and the cellular factors that are thought to be required for the conformational conversion of prion proteins.     

Polythiophenes inhibit prion propagation by stabilizing prion protein (PrP) aggregates

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrP(C) (PrP(Sc)) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits.     

Non-infectious aggregates of the prion protein react with several PrPSc-directed antibodies

The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP^C) into an infectious, aggregated isoform (PrP^Sc) that has a high content of β-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP^Sc but not PrP^C, as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19–33, 89–112, and 136–158) have been previously reported to react specifically with infectious PrP^Sc but not PrP^C. In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.     

Direct evidence of generation and accumulation of β-sheet-rich prion protein in scrapie-infected neuroblastoma cells with human IgG1 antibody specific for β-form prion protein

We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.     

Glycosylphosphatidylinositol anchor-dependent stimulation pathway required for generation of baculovirus-derived recombinant scrapie prion protein

The pathogenic isoform (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrP(Sc) seed catalyzes the structural conversion of endogenous PrP(C) into nascent PrP(Sc) in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrP(res)) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrP(Sc) by PMCA. However, the biological properties of PrP(Sc) obtained by in vivo transmission of recPrP(res) are unique or different from those of PrP(Sc) used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrP(Sc). In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrP(C) substrate for amplification of the mouse scrapie prion strain Chandler, and PrP(Sc) that accumulated in mice inoculated with Bac-PrP(res) had biochemical and pathological properties very similar to those of the PrP(Sc) seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrP(res), the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrP(Sc). Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.     

CJD PrPsc removal by nanofiltration process: application to a therapeutic immunoglobulin solution (Lymphoglobuline).

The characteristic of transmissible spongiform encephalopathies (TSE) is an accumulation of partially protease resistant (PrP(res)) abnormal prion protein (PrP(sc)). This pathological prion protein is very resistant to conventional inactivation methods. The risk of transmission of TSE, such as Creutzfeldt-Jakob disease (CJD), by biopharmaceutical products prepared from human cells must be taken into account. The nanofiltration process has been proved to be effective in removing viruses and scrapie agent. The major advantages of this technique are flexibility and efficacy in removing infectious particles without altering biopharmaceutical characteristics and properties. This study focused on the removal of human PrP(sc) by means of a nanofiltration method after spiking a Lymphoglobuline solution with a CJD brain homogenate. Lymphoglobuline equine anti-human thymocyte immunoglobulin is a selective immunosuppressive agent acting mainly on human T lymphocytes. The therapeutic indications are: immunosuppression for transplantation: prevention and treatment of graft rejection; treatment of aplastic anemia. In our study, CJD homogenate was spiked at three different dilutions (low, moderate and high) in the Lymphoglobuline product. The nanofiltration process was performed on each sample. Using the western blot technique, the PrP(res) signal detected in nanofiltrates was compared to that obtained with a reference scale (dilution series of CJD brain homogenate in Lymphoglobuline detected by western blot and elaborated on 3.3 log). After nanofiltration, the PrP(res) western blot signal was detected with a significant reduction in the less dilute sample, whereas the signal was undetectable in the two other samples. These are the first data in CJD demonstrating a clearance between 1.6 and 3.3 log with a Lymphoglobuline recovery of over 93%. The nanofiltration process confirms its relative efficacy in removing human CJD PrP(sc).     

Experimental approaches to TSE prevention via inhibition of prion formation

Transmissible spongiform encepahalopathies (TSEs) are fatal diseases that damage the central nervous system. TSEs are unique in that they may be inherited, infectious or spontaneous. The central pathogenic agent is thought to be a conformationally distinct form (PrP(Sc;)) of the endogenous prion protein(PrP(c)), which is high in beta-sheet content and is resistant to proteases; infectivity is thought to involve formation of PrP(Sc) via imprinting of abnormal conformation on the normal form of the protein (PrP(c)) by seeds of PrP(Sc). A number of compounds found to inhibit the conversion of PrP(c) to PrP(Sc) have been proposed as therapeutics to halt TSEs.     

Proteasomal dysfunction and endoplasmic reticulum stress enhance trafficking of prion protein aggregates through the secretory pathway and increase accumulation of pathologic prion protein

A conformational change of the cellular prion protein (PrP(c)) underlies formation of PrP(Sc), which is closely associated with pathogenesis and transmission of prion diseases. The precise conformational prerequisites and the cellular environment necessary for this post-translational process remain to be completely elucidated. At steady state, glycosylated PrP(c) is found primarily at the cell surface, whereas a minor fraction of the population is disposed of by the ER-associated degradation-proteasome pathway. However, chronic ER stress conditions and proteasomal dysfunctions lead to accumulation of aggregation-prone PrP molecules in the cytosol and to neurodegeneration. In this study, we challenged different cell lines by inducing ER stress or inhibiting proteasomal activity and analyzed the subsequent repercussion on PrP metabolism, focusing on PrP in the secretory pathway. Both events led to enhanced detection of PrP aggregates and a significant increase of PrP(Sc) in persistently prion-infected cells, which could be reversed by overexpression of proteins of the cellular quality control. Remarkably, upon proteasomal impairment, an increased fraction of misfolded, fully glycosylated PrP molecules traveled through the secretory pathway and reached the plasma membrane. These findings suggest a novel pathway that possibly provides additional substrate and template necessary for prion formation when protein clearance by the proteasome is impaired.