The role of rafts in the fibrillization and aggregation of prions

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrP(C)) to the disease-specific form (PrP(Sc)). The transition from PrP(C) to PrP(Sc) involves a major conformational change, resulting in amorphous aggregates and/or fibrillar amyloid deposits. Here several lines of evidence implicating membranes in the conversion of PrP are reviewed with a particular emphasis on the role of lipid rafts in the conformational transition of prion proteins. New correlations between in vitro biophysical studies and findings from cell biology work on the role of rafts in prion conversion are highlighted and a mechanism for the role of rafts in prion conversion is proposed.     

The prion protein and lipid rafts

Prions are the causative agent of the transmissible spongiform encephalopathies, such as Creutzfeldt-Jakob disease in humans. In these prion diseases the normal cellular form of the prion protein (PrP(C)) undergoes a post-translational conformational conversion to the infectious form (PrP(Sc)). PrP(C) associates with cholesterol- and glycosphingolipid-rich lipid rafts through association of its glycosyl-phosphatidylinositol (GPI) anchor with saturated raft lipids and through interaction of its N-terminal region with an as yet unidentified raft associated molecule. PrP(C) resides in detergent-resistant domains that have different lipid and protein compositions to the domains occupied by another GPI-anchored protein, Thy-1. In some cells PrP(C) may endocytose through caveolae, but in neuronal cells, upon copper binding to the N-terminal octapeptide repeats, the protein translocates out of rafts into detergent-soluble regions of the plasma membrane prior to endocytosis through clathrin-coated pits. The current data suggest that the polybasic region at its N-terminus is required to engage PrP(C) with a transmembrane adaptor protein which in turn links with the clathrin endocytic machinery. PrP(C) associates in rafts with a variety of signalling molecules, including caveolin-1 and Fyn and Src tyrosine kinases. The clustering of PrP(C) triggers a range of signal transduction processes, including the recruitment of the neural cell adhesion molecule to rafts which in turn promotes neurite outgrowth. Lipid rafts appear to be involved in the conformational conversion of PrP(C) to PrP(Sc), possibly by providing a favourable environment for this process to occur and enabling disease progression.     

Aggregation and fibrillization of prions in lipid membranes

A key molecular event in prion diseases is the conversion of PrP (prion protein) from its normal cellular form (PrP(c)) into the disease-specific form (PrP(Sc)). The transition from PrP(c) to PrP(Sc) involves a major conformational change, resulting in amorphous aggregates and/or fibrillar amyloid deposits. Here, we review several lines of evidence implicating membranes in the conversion of PrP, and summarize recent results from our own work on the role of lipid membranes in conformational transitions of prion proteins. By establishing new correlations between in vivo biological findings with in vitro biophysical results, we propose a role for lipid rafts in prion conversion, which takes into account the structural heterogeneity of PrP in different lipid environments.     

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.     

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.     

Evolving views in prion glycosylation: functional and pathological implications

Prion diseases form a group of neurodegenerative disorders with the unique feature of being transmissible. These diseases involve a pathogenic protein, called PrP(Sc) for the scrapie isoform of the cellular prion protein (PrP(C)) which is an abnormally-folded counterpart of PrP(C). Many questions remain unresolved concerning the function of PrP(C) and the mechanisms underlying prion replication, transmission and neurodegeneration. PrP(C) is a glycosyl-phosphatidylinositol-anchored glycoprotein expressed at the cell surface of neurons and other cell types. PrP(C) may be present as distinct isoforms depending on proteolytic processing (full length and truncated), topology(GPI-anchored, transmembrane or soluble) and glycosylation (non- mono- and di-glycosylated). The present review focuses on the implications of PrP(C) glycosylation as to the function of the normal protein, the cellular pathways of conversion into PrP(Sc), the diversity of prion strains and the related selective neuronal targeting.     

Altered prion protein glycosylation in the aging mouse brain

The normal cellular prion protein (PrP(C)) is a glycoprotein with two highly conserved potential N-linked glycosylation sites. All prion diseases, whether inherited, infectious or sporadic, are believed to share the same pathogenic mechanism that is based on the conversion of the normal cellular prion protein (PrP(C)) to the pathogenic scrapie prion protein (PrP(Sc)). However, the clinical and histopathological presentations of prion diseases are heterogeneous, depending not only on the strains of PrP(Sc) but also on the mechanism of diseases, such as age-related sporadic vs. infectious prion diseases. Accumulated evidence suggests that N-linked glycans on PrP(C) are important in disease phenotype. A better understanding of the nature of the N-linked glycans on PrP(C) during the normal aging process may provide new insights into the roles that N-linked glycans play in the pathogenesis of prion diseases. By using a panel of 19 lectins in an antibody-lectin enzyme-linked immunosorbent assay (ELISA), we found that the lectin binding profiles of PrP(C) alter significantly during aging. There is an increasing prevalence of complex oligosaccharides on the aging PrP(C), which are features of PrP(Sc). Taken together, this study suggests a link between the glycosylation patterns on PrP(C) during aging and PrP(Sc).     

Monoacylated cellular prion protein modifies cell membranes, inhibits cell signaling, and reduces prion formation

Prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into a disease related, protease-resistant isoform (PrP(Sc)). In these studies, a cell painting technique was used to introduce PrP(C) to prion-infected neuronal cell lines (ScGT1, ScN2a, or SMB cells). The addition of PrP(C) resulted in increased PrP(Sc) formation that was preceded by an increase in the cholesterol content of cell membranes and increased activation of cytoplasmic phospholipase A(2) (cPLA(2)). In contrast, although PrP(C) lacking one of the two acyl chains from its glycosylphosphatidylinositol (GPI) anchor (PrP(C)-G-lyso-PI) bound readily to cells, it did not alter the amount of cholesterol in cell membranes, was not found within detergent-resistant membranes (lipid rafts), and did not activate cPLA(2). It remained within cells for longer than PrP(C) with a conventional GPI anchor and was not converted to PrP(Sc). Moreover, the addition of high amounts of PrP(C)-G-lyso-PI displaced cPLA(2) from PrP(Sc)-containing lipid rafts, reduced the activation of cPLA(2), and reduced PrP(Sc) formation in all three cell lines. In addition, ScGT1 cells treated with PrP(C)-G-lyso-PI did not transmit infection following intracerebral injection to mice. We propose that that the chemical composition of the GPI anchor attached to PrP(C) modified the local membrane microenvironments that control cell signaling, the fate of PrP(C), and hence PrP(Sc) formation. In addition, our observations raise the possibility that pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases.     

Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.     

Novel heparan mimetics potently inhibit the scrapie prion protein and its endocytosis

During prion diseases the normal prion protein PrP(C) is refolded into an abnormal conformer PrP(Sc). We have studied the PrP(Sc) inhibiting activity of a library of synthetic heparan mimetic (HM) biopolymers. HMs are chemically derived dextrans obtained by successive substitutions with carboxymethyl, benzylamide, and sulfate groups on glucose residues. Some HMs eliminated PrP(Sc) from prion-infected cells after a 5 day course at 100 ng/ml and were 15 x potent than pentosan sulfate in this system. The anti-PrP(Sc) activity of HMs correlated with the degree of sulfation but was increased by benzylamidation. HMs did not reduce the synthesis of PrP(C) nor its attachment to lipid rafts, but instead blocked its conversion into PrP(Sc). The anti-PrP(Sc) HMs also prevented the uptake of prion rods by cultured cells. HMs may thus block the interaction of PrP(Sc) with a putative cellular receptor, possibly heparan sulfate. HMs provide an attractive chemical approach for the synthesis of TSE therapeutic and prophylactic reagents.     

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.     

Delineating common molecular mechanisms in Alzheimer's and prion diseases

The structure of the infectious agent responsible for prion diseases has not been fully characterized, but evidence points to a beta-rich conformer of the host-encoded prion protein. Amyloid-beta peptide (Abeta), a proteolytic fragment generated from the amyloid precursor protein, has been implicated as the toxic molecule involved in the pathogenesis of Alzheimer's disease. The mechanism of Abeta toxicity might be mediated through the coordination of redox-active transition-metal ions such as copper leading to the generation of reactive oxygen species, coupled with the propensity to interact with lipid bilayers. Key sequence and chemical similarities between prion protein (PrP) and Abeta indicate that similar therapeutic strategies might be applicable for the treatment of Alzheimer's and prion diseases.     

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.     

Dominant-negative inhibition of prion formation diminished by deletion mutagenesis of the prion protein

Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrP(C) act as dominant-negative, inhibitors of PrP(Sc) formation (K. Kaneko et al., Proc. Natl. Acad. Sci. USA 94:10069-10074, 1997). Trafficking of substituted PrP(C) to caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrP(C) are responsible for dominant-negative inhibition of PrP(Sc) formation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrP(C) to PrP(Sc). We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrP(C) molecule and that N-terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrP(C) to an auxiliary molecule that participates in PrP(Sc) formation remains to be established.     

Prion propagation in cells expressing PrP glycosylation mutants

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.     

In vivo conversion of cellular prion protein to pathogenic isoforms, as monitored by conformation-specific antibodies

The central event in prion disease is thought to be conformational conversion of the cellular isoform of prion protein (PrP(C)) to the insoluble isoform PrP(Sc). We generated polyclonal and monoclonal antibodies by immunizing PrP(C)-null mice with native PrP(C). All seven monoclonal antibodies (mAbs) immunoprecipitated PrP(C), but they immunoprecipitated PrP(Sc) weakly or not at all, thereby indicating preferential reactivities to PrP(C) in solution. Immunoprecipitation using these mAbs revealed a marked loss of PrP(C) in brains at the terminal stage of illness. Histoblot analyses using these polyclonal antibodies in combination of pretreatment of blots dissociated PrP(C) and PrP(Sc) in situ and consistently demonstrated the decrease of PrP(C) at regions where PrP(Sc) accumulated. Interestingly, same mAbs showed immunohistochemical reactivities to abnormal isoforms. One group of mAbs showed reactivity to materials that accumulated in astrocytes, while the other group did so to amorphous plaques in neuropil. Epitope mapping indicated that single mAbs have reactivities to multiple epitopes, thus implying dual specificities. This suggests the importance of octarepeats as a part of PrP(C)-specific conformation. Our observations support the notion that loss of function of PrP(C) may partly underlie the pathogenesis of prion diseases. The conversion of PrP(C) to PrP(Sc) may involve multiple steps at different sites.     

The prion protein unstructured N-terminal region is a broad-spectrum molecular sensor with diverse and contrasting potential functions

The physiological function of the prion protein (PrP(C) ) and its conversion into its infectious form (PrP(Sc) ) are central issues to understanding the pathogenesis of prion diseases. The N-terminal moiety of PrP(C) (NH(2) -PrP(C) ) is an unstructured region with the characteristic of interacting with a broad range of partners. These interactions endow PrP(C) with multifunctional and sometimes contrasting capabilities, including neuroprotection and neurotoxicity. Recently, binding of β-sheet rich conformers to NH(2) -PrP(C) demonstrated a probable neurotoxic function for PrP(C) in Alzheimer's disease. NH(2) -PrP(C) also enhances the propagation of prions in vivo and is the target of the most potent antiprion compounds. Another level of complexity is provided by endoproteolysis and release of most of NH(2) -PrP(C) into the extracellular space. Further studies will be necessary to understand how NH(2) -PrP(C) regulates the physiological function of PrP(C) and how it is involved in the corruption of its normal function in diseases.     

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.     

PrP(C) association with lipid rafts in the early secretory pathway stabilizes its cellular conformation

The pathological conversion of cellular prion protein (PrP(C)) into the scrapie prion protein (PrP(Sc)) isoform appears to have a central role in the pathogenesis of transmissible spongiform encephalopathies. However, the identity of the intracellular compartment where this conversion occurs is unknown. Several lines of evidence indicate that detergent-resistant membrane domains (DRMs or rafts) could be involved in this process. We have characterized the association of PrP(C) to rafts during its biosynthesis. We found that PrP(C) associates with rafts already as an immature precursor in the endoplasmic reticulum. Interestingly, compared with the mature protein, the immature diglycosylated form has a different susceptibility to cholesterol depletion vs. sphingolipid depletion, suggesting that the two forms associate with different lipid domains. We also found that cholesterol depletion, which affects raft-association of the immature protein, slows down protein maturation and leads to protein misfolding. On the contrary, sphingolipid depletion does not have any effect on the kinetics of protein maturation or on the conformation of the protein. These data indicate that the early association of PrP(C) with cholesterol-enriched rafts facilitates its correct folding and reinforce the hypothesis that cholesterol and sphingolipids have different roles in PrP metabolism.     

Trapping prion protein in the endoplasmic reticulum impairs PrPC maturation and prevents PrPSc accumulation

The conversion of the normal cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is a key feature of prion diseases. The pathogenic mechanisms and the subcellular sites of the conversion are complex and not completely understood. In particular, little is known on the role of the early compartment of the secretory pathway in the processing of PrP(C) and in the pathogenesis of prion diseases. In order to interfere with the intracellular traffic of endogenous PrP(C) we have generated two anti-prion single chain antibody fragments (scFv) directed against different epitopes, each fragment tagged either with a secretory leader or with the ER retention signal KDEL. The stable expression of these constructs in PC12 cells allowed us to study their specific effects on the synthesis, maturation, and processing of endogenous PrP(C) and on PrP(Sc) formation. We found that ER-targeted anti-prion scFvs retain PrP(C) in the ER and inhibit its translocation to the cell surface. Retention in the ER strongly affects the maturation and glycosylation state of PrP(C), with the appearance of a new aberrant endo-H sensitive glycosylated species. Interestingly, ER-trapped PrP(C) acquires detergent insolubility and proteinase K resistance. Furthermore, we show that ER-targeted anti-prion antibodies prevent PrP(Sc) accumulation in nerve growth factor-differentiated PC12 cells, providing a new tool to study the molecular pathology of prion diseases.