[Frontiers in Bioscience 16, 151-168, January 1, 2011]
The role of lipid rafts in prion protein biology
Victoria Lewis 1,2, Nigel M. Hooper2
1
TABLE OF CONTENTS
1. ABSTRACT
The conformational conversion of the cellular prion protein, PrPC, to the misfolded isoform PrPSc is the central pathogenic event in the uniquely transmissible neurodegenerative prion diseases. As both PrPC and PrPSc are associated with membranes, the nature of the membrane microenvironment may well play a significant role in both the conformational conversion process as well as the normal functions of PrPC. Within the membrane are various microdomains, areas of distinct lipid and protein composition, the best studied of which are the cholesterol- and sphingolipid-rich lipid rafts. These domains are characterized biochemically by their relative resistance to solubilization in certain detergents at low temperature. In this article we review the evidence for the involvement of lipid rafts in the localization and trafficking of PrPC, in the cellular signaling, neuroprotective and metal binding functions of PrPC, and as sites for the conversion of PrPC to PrPSc and in cell-to-cell prion transmission.
2. INTRODUCTION
The cellular prion protein, PrPC, is a ubiquitously transcribed and expressed glycoprotein, found most abundantly in neuronal tissue (1-3), and localized predominantly at the cell surface (4). The primary physiological function of PrPC has not yet been defined, although interestingly prion proteins from different mammalian species show high sequence identity (5), with many features conserved (6-10) (see Figure 1), implying the evolutionary and biological importance of this protein. Experimental evidence, particularly that pertaining to structural features of the prion protein, its subcellular localization and various binding partners, points to possible roles in cell signaling, neuroprotection and copper transport, amongst others. These aspects of prion protein biology and the significance of the localization of PrPC to cholesterol-rich lipid rafts are reviewed in detail below.
Prion diseases are a group of fatal neurodegenerative diseases that occur naturally in humans and various other mammalian species. Prion diseases are unique, in that they can have either a sporadic, inherited or acquired aetiology, and the transmissible causative agent appears to be a misfolded conformer (named PrPSc ) of the normal cellular prion protein (11, 12). Prion diseases are transmissible both in nature and experimentally to other animals and to cultured cells in vitro, and are often characterized by the accumulation of a partially protease resistant prion protein conformer, known as PrPres. In the context of prion infection, studies have confirmed the presence of PrPC to be an absolute requirement for the development of disease (13-15). However, the role of the specific subcellular localization of each of the prion protein isoforms in prion diseases has not been conclusively established, and the possible implications of this are discussed herein.
3. LIPID RAFTS
The plasma membrane (PM) surrounding mammalian cells is a continuous lipid bilayer composed of hundreds of different sphingolipids, phospholipids and cholesterol. It provides a differentially permeable barrier for regulated passive and active transport of molecules into and out of the cell. Importantly, cellular lipid membranes are structurally dynamic, continually allowing adaptation in response to specific biological processes (16). Lipid rafts, also known as membrane rafts, are specific membrane microdomains composed primarily of liquid ordered cholesterol and sphingolipid, which laterally assemble to form small platforms of varying size that "float" within the more liquid disordered phospholipid-rich regions of the PM (17, 18). Rafts may first assemble in the Golgi complex after synthesis of sphingolipids (19), and can be found in other cellular membranes, including those of the endocytic pathway (20, 21).
3.1. Detergent resistant membranes
Cellular membranes, when extracted in non-ionic detergent buffers at low temperatures (e.g. 0-4oC) can be fractionated into detergent soluble and insoluble components. Cellular phospholipids comprise the detergent soluble fraction, whereas the majority of sphingolipids and some cholesterol (i.e. the lipid components of rafts) remain in the insoluble fraction (22); thus leading to the term 'detergent resistant membranes' (DRMs). Extracted DRMs are also characterized by the capacity to 'float' to buoyant fractions when subject to density gradient fractionation. Interestingly, depending on the detergent used for DRM extraction, there can be substantial differences in the protein and lipid content of the isolated raft fraction (23). Rafts are therefore characterized biochemically by their resistance to solubilization in non-ionic detergents at low temperatures, although the end result of DRM extraction does not necessarily reflect the organization of membranes and the protein or lipid association in vivo.
The physical nature of rafts, namely the enrichment of sphingolipids with their high melting temperatures and saturated acyl chains when mixed with cholesterol, facilitates raft association with a specific group of membrane proteins; that is, proteins which prefer an ordered environment such as the cholesterol-rich liquid-ordered membrane phase (24). Glycosylphosphatidylinositol (GPI)-anchored proteins, some transmembrane proteins, and proteins acylated by myristate or palmitate all localize to rafts and can be recovered in DRMs (24-26).
3.2. Cellular rafts
Despite countless studies isolating DRMs from buoyant fractions of fractionated cells, there has been some debate as to whether membrane rafts actually exist in cells. That is, the presence of DRMs in fractionated cells is not absolute proof of in vivo raft domains. However, the decades of biochemical, microscopy, biophysical, cellular biological and proteomics studies have now provided enough evidence such that it is generally agreed that rafts are heterogeneous in nature, may exist as relatively stable or transient domains, and are very small, only 10-200nm in size (27). It also appears that small membrane rafts, upon stimulation or antibody cross-linking, may cluster together to form larger platforms, playing a role in bringing together proteins, for example those that are functionally related, so they can interact on the cell surface (18). Further, it is hypothesized that this clustering is specifically activated through protein-protein or protein-lipid interactions (28). One example of this clustering occurs in caveolae, a subset of rafts characterized as small surface invaginations or dimples which form when the raft associated protein caveolin is brought together by raft clustering, thus allowing polymerization of the caveolin, driving the dimpling process and resulting in endocytosis (18).
Due to the types of proteins identified as associated with DRMs, the formation of membrane rafts has been implicated in numerous cellular functions including signal transduction (29), membrane sorting and cell polarization (30), and cell fusion (31). As mentioned above, specific rafts may play a role in endocytic sorting. Not only does endocytosis occur through the caveolae subset of rafts, but in cells devoid of caveolin and morphological caveolae, there is still raft dependent endocytosis (reviewed in (32)). Further, there is evidence that for some proteins, endocytosis mediated through clathrin-coated pits may also be dependent on lipid-rafts (33-35). Lipid rafts also function in cellular signaling, whereby raft localization and/or clustering concentrates receptors to an environment of ligands, kinases and phosphatases which act in specific signaling cascades. Various proteins and receptors involved in cellular signaling have been localized to lipid rafts, and disruption of rafts, for example through cholesterol depletion or sequestration, impacts on signaling pathways (29). Rafts appear to be particularly important in immune (36) and central nervous system (37) cell signaling and sorting.
4. PRION PROTEIN LOCALIZATION AND TRAFFICKING
Following synthesis, immature PrPC undergoes a number of post-translational modifications, and follows a cellular pathway similar to other cell surface or secreted proteins (38) (see Figure 2). More specifically, following synthesis into the endoplasmic reticulum (ER), PrPC undergoes cleavage by signal peptidase to remove the N-terminal signal peptide, partial N-linked glycosylation with addition of simple sugars at two asparagine (N) residues in the C-terminal half of the protein (39), formation of a single intramolecular disulphide bond (40), and cleavage of the C-terminal signal peptide resulting in attachment of a GPI anchor (41, 42) (see Figure 1). PrPC is then directed to the Golgi where the glycans are modified to more complex oligosaccharides (43). The majority of this mature PrPC continues to the cell surface, retaining its GPI anchor. The specific membrane environment where PrPC resides at the cell surface is likely to be important, not only in the normal biological functions of the protein, but also in the prion disease process, as it may influence any direct interactions of PrPC with the disease associated isoform, PrPSc.
4.1. GPI anchor attachment to lipid rafts
When the C-terminus of a protein is lipidated via phosphoethanolamine attaching to an oligosaccharide-linked phosphatidylinositol lipid, it is said to possess a GPI anchor (44). Early studies of the cellular and disease associated prion proteins determined both to be attached to cellular membranes via a GPI anchor, which was sensitive to cleavage by phosphatidylinositol-specific phospholipase C (PIPLC) (4). A later study identified serine 231 as the site of phosphoethanolamine attachment, which links to a core tetrasaccharide, with the glucosamine component of the core linking to the headgroup of phosphatidylinositol (41) .
Like many other GPI-anchored proteins, PrPC has been shown to associate at steady state levels predominantly with DRMs including lipid rafts and caveolae-like domains (CLD), the latter of which have a lipid composition similar to caveolae but lack the caveolin-1 protein (45-48). Due to the detection of mature PrPC in these DRMs (i.e. after the N-linked glycans have been modified to more complex oligosaccharides), the localization of PrPC to rafts is thought to occur as the protein traverses the Golgi. Additionally, there is evidence that immature PrPC may localize to cholesterol-rich DRMs earlier in the secretory pathway, in the ER (49). The authors suggested that this may have implications for PrPC biosynthesis and protein folding, as depletion of cholesterol slowed the maturation of PrPC and led to an association (as assessed by co-immunoprecipitation) with various ER chaperones (49). However, whether this early association of PrPC with DRMs was exclusively via a GPI-attachment or through another means, for example N-terminal targeting (see below), was not established. Importantly, PIPLC treatment of cultured cells results in the accumulation and detection of PrPC in culture medium with a simultaneous loss of cell associated PrPC (4), indicating that a large proportion of PrPC is localized via its GPI anchor at the cell surface. Further, in some cell lines which express caveolin and have morphological caveolae, PrPC has been detected at the cell surface localized within these raft domains (50-52).
4.2. N-terminal targeting to rafts
Although PrPC associates with lipid rafts through a GPI anchor, a number of cell-free studies demonstrated that PrPC could interact with sphingolipid- and cholesterol-rich model membranes in a GPI-independent manner (53-55), with one study identifying the flexible N-terminus of PrP as crucial to this interaction (54). Subsequent cell based experiments utilizing cells expressing various PrPC constructs mutated such that they were no longer GPI-anchored, confirmed that the N-terminus of PrPC between residues 23-90 contains a raft targeting determinant (56). The far N-terminus of mature mammalian PrPC is highly conserved (10), and contains a positively charged polybasic region between residues 23-28 (-KKRPKP-) immediately following the N-terminal signal peptide (Figure 1). This region is known to bind glycosaminoglycans (GAGs) (57, 58), the long polysaccharide chains covalently linked to the protein core of proteoglycans. Critically, in the context of lipid raft targeting, we determined recently that the major neuronal heparan sulphate proteoglycan, GPI-anchored glypican-1, was involved in the recruitment and stabilization of PrPC in lipid rafts (59).
4.3. PrPC endocytosis
Internalization of PM proteins can occur by distinct mechanisms, broadly described as clathrin-dependent and independent. Endocytosis through clathrin-coated pits, involves the assembly of clathrin, a large oligomeric protein, into lattice structures on the inner surface of the PM, assisting membrane invaginaton to form the coated pits. These 'pits' then have the ability to pinch off and form vesicles, which can traffic to and fuse with other intracellular organelles (60). Generally, this mechanism supports the internalization of transmembrane proteins, which localize in pits through association of their cytoplasmic domain with cytoplasmic clathrin-pit-accessory proteins (61). Somewhat surprisingly then, given the lack of a transmembrane or cytoplasmic domain in the majority of PrPC topological variants, PrPC has been shown to be present at the cell surface in these structures (62-64), and experimental evidence has shown that PrPC does cycle between the plasma membrane and early endosomes via a clathrin coated pit dependent pathway (63-65). Importantly, there is evidence to suggest that for PrPC to be internalized in this classical clathrin-mediated manner it must first translocate out of the lipid raft membrane region, which occurs upon Cu2+ binding to the protein (66). Then this non-raft localized PrPC is rapidly endocytosed through an interaction involving the polybasic region of the N-terminus (65, 66) (Figure 1), with an adaptor protein. One such protein has been identified as the transmembrane low-density lipoprotein receptor-related protein-1 (LRP1) (67, 68), although others may exist.
As previously mentioned, PrPC also localizes within caveolae and CLDs. Further, it is well established that in a clathrin-independent manner, caveolae can be endocytosed, and this has been shown to provide a mechanism for the internalization of PrPC molecules and cycling to late endosomes and lysosomes (51). Previously it has been difficult to reconcile these different modes of PrPC endocytosis, and it has been assumed that the manner of PrPC internalization is largely influenced by the cell type studied. However, evidence that clathrin coated pit and lipid-raft mediated endocytosis are not necessarily mutually exclusive in the internalization of other proteins (33-35), lends to the possibility of a similar scenario for PrPC internalization. Indeed, a recent study found that the capacity for PrPC internalization was reduced with either lipid raft disruption or inhibition of clathrin-mediated endocytosis within a cell line, but was only completely abolished when cells were treated such as to simultaneously disrupt both pathways (69), indicating that even within a single cell there may be different pathways of PrPC internalization. Figure 2 provides a summary of PrPC endocytosis.
Evidence from kinetic studies suggests that PrPC turnover in neuronal cell lines is rapid, with a half-life of approximately five hours (43, 70), and in primary cultures of neuronal and lymphoid cells, turnover is even more rapid (71). PrPC can be degraded through the endosomal-lysosomal pathway (72) and approximately 10% of wild type PrPC is subject to retrograde transport through endoplasmic reticulum associated degradation (ERAD), probably because it has been incorrectly processed, and is degraded via the proteasome (73). PrPC has also been identified in the conditioned media of cultured cells, as well in human cerebrospinal fluid (74), providing evidence of PrPC shedding from the cell surface into the extracellular space. This soluble extracellular PrPC has been characterized as no longer having a GPI anchor, suggesting cleavage either within (by a phospholipase) or immediately adjacent to (by a protease) the GPI moiety (71, 75, 76). Recently, we demonstrated that PrPC was shed from cells through the action of the zinc metalloprotease ADAM10, cleaving the Gly-Arg bond just upstream of the site of GPI attachment (77). Extracellular PrPC has also been found in association with exosomes (78, 79), membrane bound vesicles contained within the 'lumen' of multivesicular bodies (MVB), expelled from the cell with the fusion of the MVB with the plasma membrane (80). As exosomes have a lipid composition, detergent insolubility and buoyant density which is similar to lipid rafts, and contain lipid raft marker proteins (reviewed in (81, 82)), it is possible that PrPC secreted from the cell by this process, is fundamentally still lipid raft associated.
5. LIPID RAFTS AND PrPC FUNCTION
The exact cellular function of the prion protein is presently unknown, although experimental evidence has suggested a number of potential roles. Surprisingly PrPC knockout mice appeared normal, with no overt phenotypic abnormalities (83-85), suggesting that PrPC could be functionally redundant. However, further more comprehensive analysis of PrPC knockout mice and derived cell lines has implicated the cellular prion protein in a variety of biological functions.
5.1. PrPC endoproteolytic processing
Like many structural and functional classes of proteins (86), PrPC undergoes post-translational endoproteolytic cleavage. Endogenous N-terminally truncated PrPC fragments have been identified in cultured cells, and importantly also in vivo. These PrPC species remain glycosylated and GPI-anchored like the full-length protein (87, 88), and because of retention of the GPI anchor are most likely still membrane raft associated. The predominant cleavage, alpha-cleavage, occurs at residues 111 or 112 (human PrPC sequence nomenclature) (87) producing C1. PrPC beta-cleavage occurs around the C-terminus of the octapeptide repeat domain (89, 90), producing C2, which is found at low levels in normal human (87, 88, 90) and cultured cells (89). The corresponding N-terminal fragments, N1 and N2, can also be detected, both in cell lysates and conditioned media (89, 91).
These endoproteolytic cleavages of PrPC may result in membrane-bound and soluble fragments with biological functions distinct from the full-length protein and to each other. Furthermore, the specific subcellular localization of the different PrPC N- and C-terminal species may influence these functions. As already mentioned, the N-terminus of PrPC is required for its translocation out of lipid rafts and internalization via clathrin coated pits, therefore endoproteolytic cleavage to remove the N-terminus is likely to shift its trafficking pathways and mechanisms relative to the full length protein. This may be particularly relevant, given that often such a high percentage of PrPC is N-terminally truncated (92), and that alpha-cleavage has recently been shown to occur before the protein reaches the cell surface, in a late stage of the secretory pathway, independent of PrPC lipid raft localization (93). Conceivably this lack of the important N-terminal polybasic/raft targeting/co-factor binding domain in C1 as it traffics to the cell surface could result in localization to different membrane rafts relative to the full length protein, which may have subsequent implications.
There is some experimental evidence to suggest separate functions for the different PrPC species. Some results indicate that the alpha-cleavage event produces biologically active fragments, with C1 being pro-apoptotic and N1 being protective against staurosporine treatment in a p53-dependent manner, whereas C2 and N2 are inert (94, 95). Conversely, it is known that PrPC beta-cleavage occurs at the cell surface and is carried out by reactive oxygen species (ROS) (96, 97), and neuronal cells expressing PrPC which has been mutated such that it is no longer subject to ROS-mediated beta-cleavage are more sensitive to oxidative stress (97), indicating that this cleavage event is a protective mechanism. In agreement with this, a separate study utilizing synthetic peptides representing the N2 fragment and its different domains found N2 was protective against oxidative stress when it contained an unmodified polybasic motif and was bound to Cu2+ (98). Interestingly, the protective effect appeared to be mediated through a proteoglycan binding partner found within, or at least requiring, intact lipid rafts.
5.2. Cellular signaling
The possibility of PrPC involvement in cellular signaling was raised due to its ubiquitous expression and localization at the cell surface, especially in lipid raft domains which have long been recognized as signaling platforms (99). PrPC is involved in signal transduction in T-lymphocytes, particularly in T-cell activation (100, 101), which is dependent on its lipid raft localization (102). Further, it has been shown that PrPC cross-linking in lymphocytes leads to mitogen-activated protein kinase (MAPK) and consequently extracellular signal regulated kinase 1/2 (ERK1/2) activation (103). Similarly, antibody mediated clustering of cell surface PrPC in neuroectodermal progenitor and differentiated cells, and other neuronal and non-neuronal cells resulted in ERK1/2 activation (104-106), which was shown to occur through nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase stimulation of ROS and in some cells a signaling cascade involving Fyn kinase (105, 106). Interestingly, a recent study highlighted a relationship between PrPC alpha-cleavage with the MAPK/ERK signaling pathway, which was dependent on membrane raft integrity (107). Finally, PrPC has been shown to bind synapsin Ib and Grb2, proteins both involved in neuronal signaling (108).
There are data which indicate that the PrPC linked signaling and neuroprotection functions (see also sections 5.1 and 5.3) may be somewhat concomitant. Stress-inducible protein 1, which binds to PrPC between residues 113-128, stimulated a PrPC-dependent neuroprotective response in the neuroblastic layer of cultured retinal cells, through activation of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase pathway (109, 110). Further, there is the suggestion that the changes observed in signaling proteins in PrPC knockout mice are a compensatory mechanism to maintain cell survival, related to the loss of the antioxidant function of PrPC (111). However, conflicting results, showing antibody mediated cross-linking of PrPC in vivo triggers apoptosis in hippocampal and cerebellar neurons (112), provokes questions as to whether alternative PrPC membrane localizations (for example, in specific raft subsets), and therefore accessibility to different resident binding partners and ligands, can promote different signaling pathways.
5.3. Neuroprotection
As mentioned in section 5.1, PrPC may also have a role as an antioxidant, which appears to be related to the beta-cleavage event and production of the N2 fragment. In addition to the cell based studies already mentioned, in vivo studies have shown similar findings. Mice devoid of PrPC have an increased susceptibility to oxidative stress (111, 113, 114), and in PrPC knockout mice (compared to wild type mice), the peripheral organs as well as the central nervous system showed marked increases in lipid peroxidation and protein oxidation, and decreased catalase activity (114), all indicators of oxidative stress. There is also evidence that the antioxidant and other related protective effects of PrPC are more specific to its neuronal expression (111, 115). The prion protein has been found to behave in a superoxide dismutase (SOD)-like manner, dependent on copper binding to the octarepeat region, specifically during and not subsequent to, protein folding (116). Moreover, cytosolic SOD-1 activity was lower in PrPC-deficient mice compared to wild-type mice, and presumably as a compensatory mechanism, the activities of 'other-SODs' were increased (111). However, there is conflicting evidence that shows PrPC expression levels have no effect on SOD-1 activity in cultured cerebellar neurons and in vivo (117, 118), and that recombinant PrP does not possess any SOD-1 activity (119). Interestingly though, the murine prion protein gene, Prnp, shares a number of features with other mouse SOD genes (120), and is up-regulated as part of a cellular stress response (121, 122). Ultimately, as oxidative damage generated by ROS can be catalyzed by the presence of metals, putative anti-oxidant functions of PrPC may relate to its metal binding affinities, likely influenced by protein conformation and localization within specific lipid environments, and interactions with local binding partners within these environments.
5.4. PrPC and copper
PrPC has two putative copper binding regions, one in the octapeptide repeat sequence (113, 123-126), and another in the neurotoxic and amyloidogenic region between residues 106 to 126 (human PrPC numbering) (127, 128) (see Figure 1), implicating a possible role for PrPC in copper metabolism, homeostasis or transport. As mentioned earlier, it is known that copper binding to PrPC, dependent on an intact octarepeat region, results in lateral movement of PrPC out of lipid rafts (66) and internalization of the protein (129, 130) through a clathrin-dependent route (63, 65). Therefore, PrPC could function to internalize copper, which once liberated is available for use by other cupro-proteins. Alternatively, given the multitude of possible trafficking pathways and cellular environments where it can be found (see Figure 2), PrPC could transport copper directly to or from specific intracellular proteins, cellular compartments or environments. In support of PrPC functioning in copper transport is the finding that copper uptake and PrPC expression levels correlate in mouse cerebellar cells (131). However, there is some evidence that argues against copper transport being a predominant function of PrPC. This includes data indicating that in cell cultures with doxycycline-controlled levels of PrPC expression that PrPC does not traffic copper from the extracellular space into the cell at physiological concentrations (132), and the unaltered levels of ionic copper in the brains of mice with a range of PrPC expression levels (117).
5.5. Other putative PrPC functions
PrPC has also been implicated in a variety of other physiological roles, including an involvement in normal synaptic function and long-term potentiation (133-139), and regulation of normal sleep patterns (111, 140, 141). Importantly we reported that PrPC is involved in the regulation of the beta-secretase BACE-1 which is responsible for cleavage of the Alzheimer's disease (AD) amyloid precursor protein (APP) in the generation of the pathogenic amyloid-beta peptide (142). Raft-association of PrPC was required for its inhibitory action on BACE-1, as neither a transmembrane-anchored construct that is no longer associated with rafts nor a soluble construct lacking any type of anchoring domain was effective. More recently it has been reported that oligomers of the amyloid-beta peptide bind to the charged cluster region (residues 95-110) of cell surface PrPC and affect synaptic plasticity (143) and that expression of PrPC is controlled by the APP intracellular domain, AICD (144). These studies all implicate PrPC in the pathogenesis of AD (reviewed in (145). Although it might not seem possible for a single protein to have so many functions as described above, it may be that the specific biological role of PrPC will differ, and be entirely dependent on, the cell type in which it is expressed, the precise localization within the cell, including the different plasma membrane environments, and/or the ratio or differential expression of any processed variants of the wild-type protein.
6. LIPID RAFTS IN PRION DISEASE
The expression of PrPC is an absolute requirement for development of prion disease and a key event in prion diseases is the conversion of alpha-helical PrPC into the beta-sheet disease-associated PrPSc isoforms. Experimental data supports that the conversion process is facilitated by another protein or proteins or other ancillary factor(s). Some studies have shown that PrP-lipid interactions can influence PrP conformation (53, 146, 147), therefore the PrP lipid environment has the potential to influence the PrPC to PrPSc conversion process. One hypothesis states that potential molecular chaperones which might mediate PrPC unfolding to allow, for example, biologically relevant endogenous cleavage, could have an inadvertent second function in the conversion process (148). There is evidence for the participation of a species-specific macromolecule, posited to be a protein (designated 'protein X') (149-151). There is also evidence to support the involvement of negatively charged macromolecules such as nucleic acids (152-156) or GAGs (157-159). Significantly, our recent finding that reducing levels of the GPI-anchored proteoglycan glypican-1 results in a decrease in the amount of protease resistant PrPSc detected in 22L-scrapie infected mouse neuroblastoma (ScN2a) cells (59) ties together some of this previous evidence. That is, glypican-1 may act as a co-factor in the conversion of PrP, perhaps by bringing together PrPC and PrPSc isoforms as evidenced by the co-immunoprecipitation of glypican-1 with both isoforms (59). Although a similar study utilizing a different cell line (RML-scrapie infected mouse hypothalamic cells, ScGT1-1) observed the opposite effect, with reduction of glypican-1 increasing levels of PrPSc (160), these contrasting results may reflect differences due to cell line PrPC and PrPSc strain specific interactions. This is not an unreasonable assumption due to the known varied expression and glycosylation profiles of PrPC in different cell types (92, 161), along with strain specific PrPres profiles, (162-164) which may reflect slightly different PrPSc conformations (165-168), and evidence that conversion is more favorable when PrPC and the prion strain have similar glycosylation states (169, 170).
6.1. Sites of PrPC to PrPSc conversion
There is much importance in trying to determine the site and mechanism of conversion of PrPC to PrPSc, as inhibition of conversion is an obvious potential therapeutic option. Unlike PrPC, the disease associated PrPSc conformers localize predominantly intracellularly, in late endosomes, multivesicular organelles, lysosomes or lysosome-like structures (171-177), perhaps in lipid raft domains within these organelles (48). Nevertheless, PrPres has been shown to localize at the cell surface (177, 178), and importantly in plasma membrane (45) and endocytic pathway (48) DRMs. The mechanisms of internalization of PrPSc are not well understood, although recent evidence suggests the possibility of endocytosis through an association with clathrin coated pits (177). Due to the varied cellular localization of PrPC and PrPSc / PrPres, there are a number of different possible sites of conversion (summarized in Figure 3).
One plausible site of conversion is at the cell surface. This may be particularly true for cases of transmitted prion disease where a misfolded prion conformer would initially not be produced endogenously, notwithstanding the possibility of internalization of exogenously introduced PrPSc and conversion within a cellular compartment. However, two studies have shown that for conversion and propagation of infectivity to occur, the infectious agent does not necessarily need to be 'free', strongly suggesting initiation of conversion of PrPC at the cell surface (179, 180). The first study utilized infectious prions adsorbed to steel wires as the infectious source in vivo and in vitro, and found infectious titers did not reduce upon multiple uses of the steel wires (179), suggesting that cellular infection required direct contact with the infectious prion, but did not depend on consumption or removal of the infectivity from the wire. Similarly, the second (in vitro) study found that glutaraldehyde fixation of infected cells prior to allowing direct surface contact with recipient cells did not abolish transfer of infection, although it did reduce efficiency (180), suggesting in this cell culture model there may be a trafficking pathway or intracellular compartment where conversion may be more efficient, or perhaps the fixation procedure adversely altered the structure or binding propensities (either to PrPC or other essential co-factors) of the PrPSc on the donor cells.
For conversion and propagation at the cell surface, the specific membrane localization is likely to be important. As previously mentioned, PrPC and PrPres have been localized within DRMs, which presumably represent cellular lipid rafts, although interestingly these two PrP species are not necessarily in the same DRM (48). It remains possible then that PrPC and PrPSc, and catalysts or participants in the conversion process, possibly other GPI-anchored proteins which are enriched in these membranes (such as glypican-1, see above) or other cellular factors which are recruited to rafts by ligand binding, may be brought together by a specific raft clustering event, thus allowing interaction and template directed conversion of PrPC to PrPSc at the cell surface. Further, experimental evidence has shown that lovastatin, a compound known to diminish cholesterol levels and therefore potentially disorder cholesterol-rich domains, inhibited PrPSc formation (62). However, there are some conflicting results which indicate that PrPC GPI-anchored within lipid rafts in vitro is resistant to conversion, with PrPC needing to be free of its GPI anchor, or that there be movement of PrPC and PrPres into adjoining membranes, for efficient conversion (47). Moreover, when rafts were depleted of their sphingolipid component, the conversion rate of PrPC was increased (181), suggesting at least in this model that disruption of rafts increases conversion, i.e. that raft localization protects against conversion. Interestingly, mice expressing GPI-anchorless PrPC (in place of normal GPI-anchored PrPC), which is secreted by cells (182) although appears to be able to associate with DRMs (183), are still susceptible to prion infection. Transgenic mice heterozygous for GPI-anchorless PrPC produce GPI-anchorless PrPres and harbour high titres of prion infectivity (182), whereas mice homozygous for the transgene and therefore expressing higher levels of GPI-anchorless PrPC than the heterozygous mice, develop a unique clinical symptomology and neuropathology after prion infection which is directly related to the GPI-anchorless state of PrPC (184). Furthermore, cultured cells expressing the same GPI-anchorless PrPC permit conversion to GPI-anchorless PrPres in the short-term but do not remain persistently infected and propagating PrPres (185), with the authors suggesting that in vivo perhaps ongoing conversion leading to the distinctive disease is occurring extracellularly. Nevertheless, whichever scenario is considered, these results all underscore the influence of PrPC and PrPSc localization on prion conversion and the pathogenesis of prion disease.
Raft-mediated conversion as described above could also occur intracellularly, as not only do lipid raft domains exist in vesicles of the endocytic pathway, but membrane rafts and raft clustering may be crucial to the endocytosis of PrP conformers. PrPC has been localized to vesicles of the endosomal and lysosomal pathways, and PrPres is known to accumulate in both late endosomes and lysosomes. Therefore, another location of conversion from PrPC to PrPSc is postulated to be shortly after internalization, during an endocytic process. Evidence for this was provided when researchers found that a more acidic pH, similar to that observed in late endosomes or lysosomes, promoted the conformational change of PrPC to a more detergent insoluble (i.e. PrPSc-like) form (186) and changed recombinant PrP from predominantly alpha-helical to beta-sheet (187, 188). In addition, researchers have inhibited PrPres production through lowering the culturing temperature to 18oC and thereby slowing the rate of PrPC endocytosis (189). Similarly, others have shown through a different method of disrupting PrPC traffic to the cell surface and therefore preventing normal passage through the endosomal / lysosomal pathway, that PrPres production after exposure to scrapie was inhibited (72). Finally, through the use of selective inhibitors and siRNA treatments, researchers have found that at least a proportion of conversion occurs not in early or late endosomes, but more specifically in recycling endosomal compartments (190). However, these in vitro data do not completely eliminate the possibility of early stage conversion events, perhaps to intermediate protease sensitive molecules, occurring at the cell surface prior to internalization where the intermediate then acquires protease resistance and propagates further. Nevertheless, overall these data suggest that the conversion process may be most efficient in acidic environments, such as that provided by vesicles of the endosomal / lysosomal pathway, and therefore internalization of the PrP conformers might be crucial for ongoing propagation of prions.
6.2. Cell-to-cell prion transmission
Mechanisms of prion spread, including trafficking and release of disease associated PrP conformers from the cell have been proposed, and are summarized in Figure 4. As eluded to in section 6.1, one way in which the spread of prions may occur is through direct contact between cell surface PrPSc on an infected cell and PrPC on a naive cell, with initiation of conversion at the cell surface, or internalization of the donor cell PrPSc. Linked to its intracellular endosomal or multivesicular body localization is the presence of PrPres extracellularly, packaged in exosomes (78, 79), thereby secreted from the cell, and providing a mechanism of cell-to-cell spread of infectivity. In contrast tunnelling nanotubes (TNTs), actin containing membrane bridges which form between cells as a mode of communication and exchange, and allow movement of molecules along their surface and internally within small vesicles of mostly endosomal or lysosomal origin (191), were speculated to provide a means of prion transmission based on the co-localization of PrPSc with lysosomal marker positive small neuritic vesicles (192). More recently, TNTs were conclusively shown to support the traffic of PrPC as well as exogenous and endogenous PrPSc between various cell types of both neuronal and non-neuronal origin (193). Interestingly this report found that under conditions where exosomal spread of prions was made impossible, efficient transmission of PrPSc (through TNTs) still occurred. Separate to vesicular release of disease associated PrP, approximately 15% of PrPres purified from hamster brain was found to be C-terminally truncated to residue 228 (41). It remains possible that in vivo some of this truncation may arise from cleavage and release of plasma membrane associated PrPres, perhaps through a phospholipase- or protease-like activity, providing another potential mechanism for cell-to-cell transmission. Transmission of infectious prions to recipient cells is also suggested to be possible via a process of "GPI-painting", whereby GPI-anchored PrPSc would transfer from one cell to another by re-inserting into the recipient plasma membrane whilst retaining normal function (47). A GPI-painting model of prion cell-to-cell transmission would therefore require the release of intact GPI-anchored PrPSc from the infected cell or possibly from exosomes, by a mechanism not yet identified. GPI-painting has been shown to occur in vitro (194) and in vivo (195) for proteins unrelated to PrP, and GPI-anchored proteins that re-insert into the plasma membrane can reorganize into membrane microdomains such as rafts (196).
The overall efficiency of cell-to-cell prion spread and therefore prion propagation would conceivably depend on the localization of PrPSc and the efficiency of these different proposed transmission mechanisms, and, similar to the possible sites of conversion and replication, are likely influenced by specific host cell-prion strain combinations.
7. CONCLUSIONS AND PERSPECTIVES
There is a large and increasing body of evidence implicating the membrane microenvironment, particularly cholesterol- and sphingolipid-rich lipid rafts, in both the normal cellular processing and functions of PrPC, as well as its conformational conversion to the disease isoform. This field is not without controversy, due mainly to the limitation of techniques to study rafts in situ. The most widely used technique to study rafts involving their relative resistance to solubilization with detergents is not without its pitfalls but, together with other fractionation and imaging techniques, has clearly shown that rafts, or components therein, are critically involved in the function and conformational conversion of PrPC. Not only because of its GPI anchor but also due to a raft targeting determinant in its N-terminal region, PrPC associates with these membrane microdomains. The recent identification of glypican-1 as targeting PrPC to rafts and facilitating conversion to PrPSc opens up new avenues for prion research and may, in part, explain the therapeutic effects of heparin and other GAG mimetics in prion disease. Further analysis of the molecules within rafts that PrPC and PrPSc interact with will undoubtedly provide additional openings for prion biology researchers.
8. ACKNOWLEDGEMENTS
VL is supported by an Australian Government, National Health and Medical Research Council Training Fellowship #567123. We gratefully acknowledge the Medical Research Council of Great Britain and the Wellcome Trust for supporting our work on prion biology.
1. P. E. Bendheim, H. R. Brown, R. D. Rudelli, L. J. Scala, N. L. Goller, G. Y. Wen, R. J. Kascsak, N. R. Cashman and D. C. Bolton: Nearly ubiquitous tissue distribution of the scrapie agent precursor protein. Neurology 42(1), 149-56 (1992) doi:10.1099/0022-1317-76-10-2583 Abbreviations: AD: Alzheimer's disease, AICD: amyloid precursor protein intracellular domain, APP: amyloid precursor protein, cAMP: cyclic adenosine monophosphate, CLD: caveolae-like domain, DRM: detergent resistant membrane, ER: endoplasmic reticulum, ERAD: endoplasmic reticulum associated degradation, ERK: extracellular signal regulated kinase, GAG: glycosaminoglycan, GPI: glycosylphosphatidylinositol, LRP1: low-density lipoprotein receptor-related protein-1, MAPK: mitogen-activated protein kinase, MVB: multivesicular body, NADPH: nicotinamide adenine dinucleotide phosphate, PIPLC: phosphatidylinositol-specific phospholipase C, PM: plasma membrane, Prnp: prion protein gene, PrP: prion protein, PrPC: prion protein cellular isoform, PrPres: prion protein protease resistant isoform, PrPSc: prion protein misfolded disease associated isoform, ROS: reactive oxygen species, SOD: superoxide dismutase, TNT: tunnelling nanotube
Key Words: Prion, Lipid Raft, Cholesterol, Sphingolipid, Caveolae, Review
Send correspondence to: Nigel M. Hooper, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, LIGHT Laboratories, University of Leeds, Leeds LS2 9JT, UK, Tel: 44 113 343 3163, Fax: 44 113 343 5638, E-mail:n.m.hooper@leeds.ac.uk
2. M. Horiuchi, N. Yamazaki, T. Ikeda, N. Ishiguro and M. Shinagawa: A cellular form of prion protein (PrPC) exists in many non-neuronal tissues of sheep. J Gen Virol 76 (Pt 10), 2583-7 (1995)
3. M. J. Ford, L. J. Burton, R. J. Morris and S. M. Hall: Selective expression of prion protein in peripheral tissues of the adult mouse. Neuroscience 113(1), 177-92 (2002)
doi:10.1016/S0306-4522(02)00155-0
4. N. Stahl, D. R. Borchelt, K. Hsiao and S. B. Prusiner: Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51(2), 229-40 (1987)
doi:10.1016/0092-8674(87)90150-4
5. W. Goldmann: PrP gene and its association with spongiform encephalopathies. Br Med Bull 49(4), 839-59 (1993)
6. P. Mastrangelo and D. Westaway: Biology of the prion gene complex. Biochem Cell Biol 79(5), 613-28 (2001)
doi:10.1139/bcb-79-5-613
7. J. F. Bazan, R. J. Fletterick, M. P. McKinley and S. B. Prusiner: Predicted secondary structure and membrane topology of the scrapie prion protein. Protein Eng 1(2), 125-35 (1987)
doi:10.1093/protein/1.2.125
8. E. Sulkowski: Aromatic palindrome motif in prion proteins. Faseb J 6(6), 2363 (1992)
9. E. Paramithiotis, M. Pinard, T. Lawton, S. LaBoissiere, V. L. Leathers, W. Q. Zou, L. A. Estey, J. Lamontagne, M. T. Lehto, L. H. Kondejewski, G. P. Francoeur, M. Papadopoulos, A. Haghighat, S. J. Spatz, M. Head, R. Will, J. Ironside, K. O'Rourke, Q. Tonelli, H. C. Ledebur, A. Chakrabartty and N. R. Cashman: A prion protein epitope selective for the pathologically misfolded conformation. Nat Med 9(7), 893-9 (2003)
doi:10.1038/nm883
10. F. Wopfner, G. Weidenhofer, R. Schneider, A. von Brunn, S. Gilch, T. F. Schwarz, T. Werner and H. M. Schatzl: Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein. J Mol Biol 289(5), 1163-78 (1999)
doi:10.1006/jmbi.1999.2831
11. S. B. Prusiner, R. Gabizon and M. P. McKinley: On the biology of prions. Acta Neuropathol 72(4), 299-314 (1987)
doi:10.1007/BF00687261
12. K. M. Pan, M. Baldwin, J. Nguyen, M. Gasset, A. Serban, D. Groth, I. Mehlhorn, Z. Huang, R. J. Fletterick, F. E. Cohen and et al.: Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A 90(23), 10962-6 (1993)
doi:10.1073/pnas.90.23.10962
13. H. Bueler, A. Aguzzi, A. Sailer, R. A. Greiner, P. Autenried, M. Aguet and C. Weissmann: Mice devoid of PrP are resistant to scrapie. Cell 73(7), 1339-47 (1993)
doi:10.1016/0092-8674(93)90360-3
14. J. C. Manson, A. R. Clarke, P. A. McBride, I. McConnell and J. Hope: PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology. Neurodegeneration 3(4), 331-40 (1994)
15. S. Sakaguchi, S. Katamine, K. Shigematsu, A. Nakatani, R. Moriuchi, N. Nishida, K. Kurokawa, R. Nakaoke, H. Sato, K. Jishage and et al.: Accumulation of proteinase K-resistant prion protein (PrP) is restricted by the expression level of normal PrP in mice inoculated with a mouse-adapted strain of the Creutzfeldt-Jakob disease agent. J Virol 69(12), 7586-92 (1995)
16. G. K. Radda: The dynamic properties of biological membranes. Philos Trans R Soc Lond B Biol Sci 272(915), 159-71 (1975)
doi:10.1098/rstb.1975.0078
17. H. Hering, C. C. Lin and M. Sheng: Lipid rafts in the maintenance of synapses, dendritic spines, and surface AMPA receptor stability. J Neurosci 23(8), 3262-71 (2003)
18. K. Simons and R. Ehehalt: Cholesterol, lipid rafts, and disease. J Clin Invest 110(5), 597-603 (2002)
19. G. van Meer: Lipid traffic in animal cells. Annu Rev Cell Biol 5, 247-75 (1989)
doi:10.1146/annurev.cb.05.110189.001335
20. J. Gruenberg: The endocytic pathway: a mosaic of domains. Nat Rev Mol Cell Biol 2(10), 721-30 (2001)
doi:10.1038/35096054
21. S. Mayor, S. Sabharanjak and F. R. Maxfield: Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 17(16), 4626-38 (1998)
doi:10.1093/emboj/17.16.4626
22. D. A. Brown and J. K. Rose: Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68(3), 533-44 (1992)
doi:10.1016/0092-8674(92)90189-J
23. S. Schuck, M. Honsho, K. Ekroos, A. Shevchenko and K. Simons: Resistance of cell membranes to different detergents. Proc Natl Acad Sci U S A 100(10), 5795-800 (2003)
doi:10.1073/pnas.0631579100
24. D. A. Brown and E. London: Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 14, 111-36 (1998)
doi:10.1146/annurev.cellbio.14.1.111
25. K. Simons and E. Ikonen: Functional rafts in cell membranes. Nature 387(6633), 569-72 (1997)
doi:10.1038/42408
26. L. J. Pike: The challenge of lipid rafts. J Lipid Res 50 Suppl, S323-8 (2009)
doi:10.1194/jlr.R800040-JLR200
27. L. J. Pike: Rafts defined: a report on the Keystone Symposium on Lipid Rafts and Cell Function. J Lipid Res 47(7), 1597-8 (2006)
doi:10.1194/jlr.E600002-JLR200
28. D. Lingwood and K. Simons: Detergent resistance as a tool in membrane research. Nat Protoc 2(9), 2159-65 (2007)
doi:10.1038/nprot.2007.294
29. K. Simons and D. Toomre: Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1(1), 31-9 (2000)
doi:10.1038/35036052
30. S. Schuck and K. Simons: Polarized sorting in epithelial cells: raft clustering and the biogenesis of the apical membrane. J Cell Sci 117(Pt 25), 5955-64 (2004)
doi:10.1242/jcs.01596
31. C. Salaun, D. J. James and L. H. Chamberlain: Lipid rafts and the regulation of exocytosis. Traffic 5(4), 255-64 (2004)
doi:10.1111/j.1600-0854.2004.0162.x
32. R. G. Parton and A. A. Richards: Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic 4(11), 724-38 (2003)
doi:10.1034/j.1600-0854.2003.00128.x
33. K. Deinhardt, O. Berninghausen, H. J. Willison, C. R. Hopkins and G. Schiavo: Tetanus toxin is internalized by a sequential clathrin-dependent mechanism initiated within lipid microdomains and independent of epsin1. J Cell Biol 174(3), 459-71 (2006)
doi:10.1083/jcb.200508170
34. G. H. Hansen, S. M. Dalskov, C. R. Rasmussen, L. Immerdal, L. L. Niels-Christiansen and E. M. Danielsen: Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism. Biochemistry 44(3), 873-82 (2005)
doi:10.1021/bi047959+
35. R. Rollason, V. Korolchuk, C. Hamilton, P. Schu and G. Banting: Clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif. J Cell Sci 120(Pt 21), 3850-8 (2007)
doi:10.1242/jcs.003343
36. M. Dykstra, A. Cherukuri, H. W. Sohn, S. J. Tzeng and S. K. Pierce: Location is everything: lipid rafts and immune cell signaling. Annu Rev Immunol 21, 457-81 (2003)
doi:10.1146/annurev.immunol.21.120601.141021
37. S. Maekawa, S. Iino and S. Miyata: Molecular characterization of the detergent-insoluble cholesterol-rich membrane microdomain (raft) of the central nervous system. Biochim Biophys Acta 1610(2), 261-70 (2003)
doi:10.1016/S0005-2736(03)00023-3
38. D. A. Harris: Trafficking, turnover and membrane topology of PrP. Br Med Bull 66, 71-85 (2003)
doi:10.1093/bmb/66.1.71
39. V. A. Lawson, S. J. Collins, C. L. Masters and A. F. Hill: Prion protein glycosylation. J Neurochem 93(4), 793-801 (2005)
doi:10.1111/j.1471-4159.2005.03104.x
40. E. Turk, D. B. Teplow, L. E. Hood and S. B. Prusiner: Purification and properties of the cellular and scrapie hamster prion proteins. Eur J Biochem 176(1), 21-30 (1988)
doi:10.1111/j.1432-1033.1988.tb14246.x
41. N. Stahl, M. A. Baldwin, A. L. Burlingame and S. B. Prusiner: Identification of glycoinositol phospholipid linked and truncated forms of the scrapie prion protein. Biochemistry 29(38), 8879-84 (1990)
doi:10.1021/bi00490a001
42. M. A. Ferguson and A. F. Williams: Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu Rev Biochem 57, 285-320 (1988)
doi:10.1146/annurev.bi.57.070188.001441
43. B. Caughey, R. E. Race, D. Ernst, M. J. Buchmeier and B. Chesebro: Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells. J Virol 63(1), 175-81 (1989)
44. M. A. Baldwin: Analysis of glycosylphosphatidylinositol protein anchors: the prion protein. Methods Enzymol 405, 172-87 (2005)
doi:10.1016/S0076-6879(05)05008-1
45. M. Vey, S. Pilkuhn, H. Wille, R. Nixon, S. J. DeArmond, E. J. Smart, R. G. Anderson, A. Taraboulos and S. B. Prusiner: Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains. Proc Natl Acad Sci U S A 93(25), 14945-9 (1996)
doi:10.1073/pnas.93.25.14945
46. A. Gorodinsky and D. A. Harris: Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin. J Cell Biol 129(3), 619-27 (1995)
doi:10.1083/jcb.129.3.619
47. G. S. Baron, K. Wehrly, D. W. Dorward, B. Chesebro and B. Caughey: Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrP(Sc)) into contiguous membranes. Embo J 21(5), 1031-40 (2002)
doi:10.1093/emboj/21.5.1031
48. N. Naslavsky, R. Stein, A. Yanai, G. Friedlander and A. Taraboulos: Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform. J Biol Chem 272(10), 6324-31 (1997) doi:10.1074/jbc.272.10.6324
49. D. Sarnataro, V. Campana, S. Paladino, M. Stornaiuolo, L. Nitsch and C. Zurzolo: PrP(C) association with lipid rafts in the early secretory pathway stabilizes its cellular conformation. Mol Biol Cell 15(9), 4031-42 (2004)
doi:10.1091/mbc.E03-05-0271
50. J. H. Harmey, D. Doyle, V. Brown and M. S. Rogers: The cellular isoform of the prion protein, PrPc, is associated with caveolae in mouse neuroblastoma (N2a) cells. Biochem Biophys Res Commun 210(3), 753-9 (1995)
doi:10.1006/bbrc.1995.1723
51. P. J. Peters, A. Mironov, Jr., D. Peretz, E. van Donselaar, E. Leclerc, S. Erpel, S. J. DeArmond, D. R. Burton, R. A. Williamson, M. Vey and S. B. Prusiner: Trafficking of prion proteins through a caveolae-mediated endosomal pathway. J Cell Biol 162(4), 703-17 (2003)
doi:10.1083/jcb.200304140
52. S. Mouillet-Richard, B. Schneider, E. Pradines, M. Pietri, M. Ermonval, J. Grassi, J. G. Richards, V. Mutel, J. M. Launay and O. Kellermann: Cellular prion protein signaling in serotonergic neuronal cells. Ann N Y Acad Sci 1096, 106-19 (2007)
doi:10.1196/annals.1397.076
53. N. Sanghera and T. J. Pinheiro: Binding of prion protein to lipid membranes and implications for prion conversion. J Mol Biol 315(5), 1241-56 (2002)
doi:10.1006/jmbi.2001.5322
54. G. S. Baron and B. Caughey: Effect of glycosylphosphatidylinositol anchor-dependent and -independent prion protein association with model raft membranes on conversion to the protease-resistant isoform. J Biol Chem 278(17), 14883-92 (2003)
doi:10.1074/jbc.M210840200
55. R. Mahfoud, N. Garmy, M. Maresca, N. Yahi, A. Puigserver and J. Fantini: Identification of a common sphingolipid-binding domain in Alzheimer, prion, and HIV-1 proteins. J Biol Chem 277(13), 11292-6 (2002)
doi:10.1074/jbc.M111679200
56. A. R. Walmsley, F. Zeng and N. M. Hooper: The N-terminal region of the prion protein ectodomain contains a lipid raft targeting determinant. J Biol Chem 278(39), 37241-8 (2003)
doi:10.1074/jbc.M302036200
57. T. Pan, B. S. Wong, T. Liu, R. Li, R. B. Petersen and M. S. Sy: Cell-surface prion protein interacts with glycosaminoglycans. Biochem J 368(Pt 1), 81-90 (2002)
doi:10.1042/BJ20020773
58. R. G. Warner, C. Hundt, S. Weiss and J. E. Turnbull: Identification of the heparan sulfate binding sites in the cellular prion protein. J Biol Chem 277(21), 18421-30 (2002)
doi:10.1074/jbc.M110406200
59. D. R. Taylor, I. J. Whitehouse and N. M. Hooper: Glypican-1 mediates both prion protein lipid raft association and disease isoform formation. PLoS Pathog 5(11), e1000666 (2009)
doi:10.1371/journal.ppat.1000666
60. T. Kirchhausen: Clathrin. Annu Rev Biochem 69, 699-727 (2000)
doi:10.1146/annurev.biochem.69.1.699
61. J. S. Bonifacino and L. M. Traub: Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem 72, 395-447 (2003)
doi:10.1146/annurev.biochem.72.121801.161800
62. A. Taraboulos, M. Scott, A. Semenov, D. Avrahami, L. Laszlo, S. B. Prusiner and D. Avraham: Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform. J Cell Biol 129(1), 121-32 (1995)
doi:10.1083/jcb.129.1.121
63. S. L. Shyng, J. E. Heuser and D. A. Harris: A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J Cell Biol 125(6), 1239-50 (1994)
doi:10.1083/jcb.125.6.1239
64. J. Laine, M. E. Marc, M. S. Sy and H. Axelrad: Cellular and subcellular morphological localization of normal prion protein in rodent cerebellum. Eur J Neurosci 14(1), 47-56 (2001)
doi:10.1046/j.0953-816x.2001.01621.x
65. C. Sunyach, A. Jen, J. Deng, K. T. Fitzgerald, Y. Frobert, J. Grassi, M. W. McCaffrey and R. Morris: The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein. Embo J 22(14), 3591-601 (2003)
doi:10.1093/emboj/cdg344
66. D. R. Taylor, N. T. Watt, W. S. Perera and N. M. Hooper: Assigning functions to distinct regions of the N-terminus of the prion protein that are involved in its copper-stimulated, clathrin-dependent endocytosis. J Cell Sci 118(Pt 21), 5141-53 (2005)
doi:10.1242/jcs.02627
67. C. J. Parkyn, E. G. Vermeulen, R. C. Mootoosamy, C. Sunyach, C. Jacobsen, C. Oxvig, S. Moestrup, Q. Liu, G. Bu, A. Jen and R. J. Morris: LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein. J Cell Sci 121(Pt 6), 773-83 (2008)
doi:10.1242/jcs.021816
68. D. R. Taylor and N. M. Hooper: The low-density lipoprotein receptor-related protein 1 (LRP1) mediates the endocytosis of the cellular prion protein. Biochem J 402(1), 17-23 (2007)
doi:10.1042/BJ20061736
69. D. Sarnataro, A. Caputo, P. Casanova, C. Puri, S. Paladino, S. S. Tivodar, V. Campana, C. Tacchetti and C. Zurzolo: Lipid rafts and clathrin cooperate in the internalization of PrP in epithelial FRT cells. PLoS One 4(6), e5829 (2009)
doi:10.1371/journal.pone.0005829
70. D. R. Borchelt, M. Scott, A. Taraboulos, N. Stahl and S. B. Prusiner: Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J Cell Biol 110(3), 743-52 (1990)
doi:10.1083/jcb.110.3.743
71. P. Parizek, C. Roeckl, J. Weber, E. Flechsig, A. Aguzzi and A. J. Raeber: Similar turnover and shedding of the cellular prion protein in primary lymphoid and neuronal cells. J Biol Chem 276(48), 44627-32 (2001)
doi:10.1074/jbc.M107458200
72. V. Vetrugno, A. Cardinale, I. Filesi, S. Mattei, M. S. Sy, M. Pocchiari and S. Biocca: KDEL-tagged anti-prion intrabodies impair PrP lysosomal degradation and inhibit scrapie infectivity. Biochem Biophys Res Commun 338(4), 1791-7 (2005)
doi:10.1016/j.bbrc.2005.10.146
73. Y. Yedidia, L. Horonchik, S. Tzaban, A. Yanai and A. Taraboulos: Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein. Embo J 20(19), 5383-91 (2001)
doi:10.1093/emboj/20.19.5383
74. F. Tagliavini, F. Prelli, M. Porro, M. Salmona, O. Bugiani and B. Frangione: A soluble form of prion protein in human cerebrospinal fluid: implications for prion-related encephalopathies. Biochem Biophys Res Commun 184(3), 1398-404 (1992)
doi:10.1016/S0006-291X(05)80038-5
75. E. T. Parkin, N. T. Watt, A. J. Turner and N. M. Hooper: Dual mechanisms for shedding of the cellular prion protein. J Biol Chem 279(12), 11170-8 (2004)
doi:10.1074/jbc.M312105200
76. D. R. Borchelt, M. Rogers, N. Stahl, G. Telling and S. B. Prusiner: Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor. Glycobiology 3(4), 319-29 (1993)
doi:10.1093/glycob/3.4.319
77. D. R. Taylor, E. T. Parkin, S. L. Cocklin, J. R. Ault, A. E. Ashcroft, A. J. Turner and N. M. Hooper: Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein. J Biol Chem 284(34), 22590-600 (2009)
doi:10.1074/jbc.M109.032599
78. L. J. Vella, R. A. Sharples, V. A. Lawson, C. L. Masters, R. Cappai and A. F. Hill: Packaging of prions into exosomes is associated with a novel pathway of PrP processing. J Pathol 211(5), 582-90 (2007)
doi:10.1002/path.2145
79. B. Fevrier, D. Vilette, F. Archer, D. Loew, W. Faigle, M. Vidal, H. Laude and G. Raposo: Cells release prions in association with exosomes. Proc Natl Acad Sci U S A 101(26), 9683-8 (2004)
doi:10.1073/pnas.0308413101
80. K. Denzer, M. J. Kleijmeer, H. F. Heijnen, W. Stoorvogel and H. J. Geuze: Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J Cell Sci 113 Pt 19, 3365-74 (2000)
81. N. R. Smalheiser: Exosomal transfer of proteins and RNAs at synapses in the nervous system. Biol Direct 2, 35 (2007)
doi:10.1186/1745-6150-2-35
82. G. van Niel, I. Porto-Carreiro, S. Simoes and G. Raposo: Exosomes: a common pathway for a specialized function. J Biochem 140(1), 13-21 (2006)
doi:10.1093/jb/mvj128
83. H. Bueler, M. Fischer, Y. Lang, H. Bluethmann, H. P. Lipp, S. J. DeArmond, S. B. Prusiner, M. Aguet and C. Weissmann: Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356(6370), 577-82 (1992)
doi:10.1038/356577a0
84. J. C. Manson, A. R. Clarke, M. L. Hooper, L. Aitchison, I. McConnell and J. Hope: 129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal. Mol Neurobiol 8(2-3), 121-7 (1994)
doi:10.1007/BF02780662
85. H. P. Lipp, M. Stagliar-Bozicevic, M. Fischer and D. P. Wolfer: A 2-year longitudinal study of swimming navigation in mice devoid of the prion protein: no evidence for neurological anomalies or spatial learning impairments. Behav Brain Res 95(1), 47-54 (1998)
doi:10.1016/S0166-4328(97)00209-X
86. M. R. Ehlers and J. F. Riordan: Membrane proteins with soluble counterparts: role of proteolysis in the release of transmembrane proteins. Biochemistry 30(42), 10065-74 (1991)
doi:10.1021/bi00106a001
87. S. G. Chen, D. B. Teplow, P. Parchi, J. K. Teller, P. Gambetti and L. Autilio-Gambetti: Truncated forms of the human prion protein in normal brain and in prion diseases. J Biol Chem 270(32), 19173-80. (1995)
doi:10.1074/jbc.270.32.19173
88. T. Pan, R. Li, B. S. Wong, T. Liu, P. Gambetti and M. S. Sy: Heterogeneity of normal prion protein in two- dimensional immunoblot: presence of various glycosylated and truncated forms. J Neurochem 81(5), 1092-101 (2002)
doi:10.1046/j.1471-4159.2002.00909.x
89. A. Mange, F. Beranger, K. Peoc'h, T. Onodera, Y. Frobert and S. Lehmann: Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein. Biol Cell 96(2), 125-32 (2004)
doi:10.1016/j.biolcel.2003.11.007
90. A. Jimenez-Huete, P. M. Lievens, R. Vidal, P. Piccardo, B. Ghetti, F. Tagliavini, B. Frangione and F. Prelli: Endogenous proteolytic cleavage of normal and disease-associated isoforms of the human prion protein in neural and non-neural tissues. Am J Pathol 153(5), 1561-72. (1998)
91. B. Vincent, E. Paitel, Y. Frobert, S. Lehmann, J. Grassi and F. Checler: Phorbol ester-regulated cleavage of normal prion protein in HEK293 human cells and murine neurons. J Biol Chem 275(45), 35612-6 (2000)
doi:10.1074/jbc.M004628200
92. V. Lewis, A. F. Hill, C. L. Haigh, G. M. Klug, C. L. Masters, V. A. Lawson and S. J. Collins: Increased Proportions of C1 Truncated Prion Protein Protect Against Cellular M1000 Prion Infection. J Neuropathol Exp Neurol 68(10), 1125-35 (2009)
doi:10.1097/NEN.0b013e3181b96981
93. A. R. Walmsley, N. T. Watt, D. R. Taylor, W. S. Perera and N. M. Hooper: alpha-cleavage of the prion protein occurs in a late compartment of the secretory pathway and is independent of lipid rafts. Mol Cell Neurosci 40(2), 242-8 (2009)
doi:10.1016/j.mcn.2008.10.012
94. C. Sunyach, M. A. Cisse, C. A. da Costa, B. Vincent and F. Checler: The C-terminal products of cellular prion protein processing, C1 and C2, exert distinct influence on p53-dependent staurosporine-induced caspase-3 activation. J Biol Chem 282(3), 1956-63 (2007)
doi:10.1074/jbc.M609663200
95. M. V. Guillot-Sestier, C. Sunyach, C. Druon, S. Scarzello and F. Checler: The alpha-secretase-derived N-terminal product of cellular prion, N1, displays neuroprotective function in vitro and in vivo. J Biol Chem 284(51), 35973-86 (2009)
doi:10.1074/jbc.M109.051086
96. H. E. McMahon, A. Mange, N. Nishida, C. Creminon, D. Casanova and S. Lehmann: Cleavage of the amino terminus of the prion protein by reactive oxygen species. J Biol Chem 276(3), 2286-91 (2001)
doi:10.1074/jbc.M007243200
97. N. T. Watt, D. R. Taylor, A. Gillott, D. A. Thomas, W. S. Perera and N. M. Hooper: Reactive oxygen species-mediated beta-cleavage of the prion protein in the cellular response to oxidative stress. J Biol Chem 280(43), 35914-21 (2005)
doi:10.1074/jbc.M507327200
98. C. L. Haigh, S. C. Drew, M. P. Boland, C. L. Masters, K. J. Barnham, V. A. Lawson and S. J. Collins: Dominant roles of the polybasic proline motif and copper in the PrP23-89-mediated stress protection response. J Cell Sci 122(Pt 10), 1518-28 (2009)
doi:10.1242/jcs.043604
99. J. A. Allen, R. A. Halverson-Tamboli and M. M. Rasenick: Lipid raft microdomains and neurotransmitter signalling. Nat Rev Neurosci 8(2), 128-40 (2007)
doi:10.1038/nrn2059
100. N. A. Mabbott, K. L. Brown, J. Manson and M. E. Bruce: T-lymphocyte activation and the cellular form of the prion protein. Immunology 92(2), 161-5 (1997)
doi:10.1046/j.1365-2567.1997.00331.x
101. N. R. Cashman, R. Loertscher, J. Nalbantoglu, I. Shaw, R. J. Kascsak, D. C. Bolton and P. E. Bendheim: Cellular isoform of the scrapie agent protein participates in lymphocyte activation. Cell 61(1), 185-92 (1990)
doi:10.1016/0092-8674(90)90225-4
102. B. Hugel, M. C. Martinez, C. Kunzelmann, T. Blattler, A. Aguzzi and J. M. Freyssinet: Modulation of signal transduction through the cellular prion protein is linked to its incorporation in lipid rafts. Cell Mol Life Sci 61(23), 2998-3007 (2004)
doi:10.1007/s00018-004-4318-2
103. C. A. Stuermer, M. F. Langhorst, M. F. Wiechers, D. F. Legler, S. H. Von Hanwehr, A. H. Guse and H. Plattner: PrPc capping in T cells promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads to signal transduction. FASEB J 18(14), 1731-3 (2004)
104. C. Monnet, J. Gavard, R. M. Mege and A. Sobel: Clustering of cellular prion protein induces ERK1/2 and stathmin phosphorylation in GT1-7 neuronal cells. FEBS Lett 576(1-2), 114-8 (2004)
doi:10.1016/j.febslet.2004.08.076
105. S. Mouillet-Richard, M. Ermonval, C. Chebassier, J. L. Laplanche, S. Lehmann, J. M. Launay and O. Kellermann: Signal transduction through prion protein. Science 289(5486), 1925-8 (2000)
doi:10.1126/science.289.5486.1925
106. B. Schneider, V. Mutel, M. Pietri, M. Ermonval, S. Mouillet-Richard and O. Kellermann: NADPH oxidase and extracellular regulated kinases 1/2 are targets of prion protein signaling in neuronal and nonneuronal cells. Proc Natl Acad Sci U S A 100(23), 13326-31 (2003)
doi:10.1073/pnas.2235648100
107. C. L. Haigh, V. A. Lewis, L. J. Vella, C. L. Masters, A. F. Hill, V. A. Lawson and S. J. Collins: PrPC-related signal transduction is influenced by copper, membrane integrity and the alpha cleavage site. Cell Res 19(9), 1062-78 (2009)
doi:10.1038/cr.2009.86
108. C. Spielhaupter and H. M. Schatzl: PrPC directly interacts with proteins involved in signaling pathways. J Biol Chem 276(48), 44604-12 (2001)
doi:10.1074/jbc.M103289200
109. L. B. Chiarini, A. R. Freitas, S. M. Zanata, R. R. Brentani, V. R. Martins and R. Linden: Cellular prion protein transduces neuroprotective signals. Embo J 21(13), 3317-26 (2002)
doi:10.1093/emboj/cdf324
110. S. M. Zanata, M. H. Lopes, A. F. Mercadante, G. N. Hajj, L. B. Chiarini, R. Nomizo, A. R. Freitas, A. L. Cabral, K. S. Lee, M. A. Juliano, E. de Oliveira, S. G. Jachieri, A. Burlingame, L. Huang, R. Linden, R. R. Brentani and V. R. Martins: Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection. EMBO J 21(13), 3307-16 (2002)
doi:10.1093/emboj/cdf325
111. D. R. Brown, R. S. Nicholas and L. Canevari: Lack of prion protein expression results in a neuronal phenotype sensitive to stress. J Neurosci Res 67(2), 211-24 (2002)
doi:10.1002/jnr.10118
112. L. Solforosi, J. R. Criado, D. B. McGavern, S. Wirz, M. Sanchez-Alavez, S. Sugama, L. A. DeGiorgio, B. T. Volpe, E. Wiseman, G. Abalos, E. Masliah, D. Gilden, M. B. Oldstone, B. Conti and R. A. Williamson: Cross-linking cellular prion protein triggers neuronal apoptosis in vivo. Science 303(5663), 1514-6 (2004)
doi:10.1126/science.1094273
113. D. R. Brown, K. Qin, J. W. Herms, A. Madlung, J. Manson, R. Strome, P. E. Fraser, T. Kruck, A. von Bohlen, W. Schulz-Schaeffer, A. Giese, D. Westaway and H. Kretzschmar: The cellular prion protein binds copper in vivo. Nature 390(6661), 684-7 (1997)
doi:10.1038/37733
114. F. Klamt, F. Dal-Pizzol, M. J. Conte da Frota, R. Walz, M. E. Andrades, E. G. da Silva, R. R. Brentani, I. Izquierdo and J. C. Fonseca Moreira: Imbalance of antioxidant defense in mice lacking cellular prion protein. Free Radic Biol Med 30(10), 1137-44 (2001)
doi:10.1016/S0891-5849(01)00512-3
115. K. J. Lee, A. Panzera, D. Rogawski, L. E. Greene and E. Eisenberg: Cellular prion protein (PrPC) protects neuronal cells from the effect of huntingtin aggregation. J Cell Sci 120(Pt 15), 2663-71 (2007)
doi:10.1242/jcs.004598
116. D. R. Brown, B. S. Wong, F. Hafiz, C. Clive, S. J. Haswell and I. M. Jones: Normal prion protein has an activity like that of superoxide dismutase. Biochem J 344 Pt 1, 1-5 (1999)
doi:10.1042/0264-6021:3440001
117. D. J. Waggoner, B. Drisaldi, T. B. Bartnikas, R. L. Casareno, J. R. Prohaska, J. D. Gitlin and D. A. Harris: Brain copper content and cuproenzyme activity do not vary with prion protein expression level. J Biol Chem 275(11), 7455-8 (2000)
doi:10.1074/jbc.275.11.7455
118. G. Hutter, F. L. Heppner and A. Aguzzi: No superoxide dismutase activity of cellular prion protein in vivo. Biol Chem 384(9), 1279-85 (2003)
doi:10.1515/BC.2003.142
119. S. Jones, M. Batchelor, D. Bhelt, A. R. Clarke, J. Collinge and G. S. Jackson: Recombinant prion protein does not possess SOD-1 activity. Biochem J 392(Pt 2), 309-12 (2005)
120. M. Premzl, M. Delbridge, J. E. Gready, P. Wilson, M. Johnson, J. Davis, E. Kuczek and J. A. Marshall Graves: The prion protein gene: identifying regulatory signals using marsupial sequence. Gene 349, 121-34 (2005)
doi:10.1016/j.gene.2004.11.049
121. V. Wang, T. C. Chuang, Y. D. Hsu, W. Y. Chou and M. C. Kao: Nitric oxide induces prion protein via MEK and p38 MAPK signaling. Biochem Biophys Res Commun 333(1), 95-100 (2005)
doi:10.1016/j.bbrc.2005.05.091
122. W. C. Shyu, S. Z. Lin, K. Saeki, A. Kubosaki, Y. Matsumoto, T. Onodera, M. F. Chiang, P. Thajeb and H. Li: Hyperbaric oxygen enhances the expression of prion protein and heat shock protein 70 in a mouse neuroblastoma cell line. Cell Mol Neurobiol 24(2), 257-68 (2004)
doi:10.1023/B:CEMN.0000018620.41913.d2
123. M. P. Hornshaw, J. R. McDermott and J. M. Candy: Copper binding to the N-terminal tandem repeat regions of mammalian and avian prion protein. Biochem Biophys Res Commun 207(2), 621-9 (1995)
doi:10.1006/bbrc.1995.1233
124. M. P. Hornshaw, J. R. McDermott, J. M. Candy and J. H. Lakey: Copper binding to the N-terminal tandem repeat region of mammalian and avian prion protein: structural studies using synthetic peptides. Biochem Biophys Res Commun 214(3), 993-9 (1995)
doi:10.1006/bbrc.1995.2384
125. C. S. Burns, E. Aronoff-Spencer, C. M. Dunham, P. Lario, N. I. Avdievich, W. E. Antholine, M. M. Olmstead, A. Vrielink, G. J. Gerfen, J. Peisach, W. G. Scott and G. L. Millhauser: Molecular features of the copper binding sites in the octarepeat domain of the prion protein. Biochemistry 41(12), 3991-4001. (2002)
doi:10.1021/bi011922x
126. A. P. Garnett and J. H. Viles: Copper binding to the octarepeats of the prion protein. Affinity, specificity, folding, and cooperativity: insights from circular dichroism. J Biol Chem 278(9), 6795-802 (2003)
doi:10.1074/jbc.M209280200
127. M. F. Jobling, X. Huang, L. R. Stewart, K. J. Barnham, C. Curtain, I. Volitakis, M. Perugini, A. R. White, R. A. Cherny, C. L. Masters, C. J. Barrow, S. J. Collins, A. I. Bush and R. Cappai: Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126. Biochemistry 40(27), 8073-84 (2001)
doi:10.1021/bi0029088
128. G. S. Jackson, I. Murray, L. L. Hosszu, N. Gibbs, J. P. Waltho, A. R. Clarke and J. Collinge: Location and properties of metal-binding sites on the human prion protein. Proc Natl Acad Sci U S A 98(15), 8531-5 (2001)
doi:10.1073/pnas.151038498
129. P. C. Pauly and D. A. Harris: Copper stimulates endocytosis of the prion protein. J Biol Chem 273(50), 33107-10 (1998)
doi:10.1074/jbc.273.50.33107
130. K. S. Lee, A. C. Magalhaes, S. M. Zanata, R. R. Brentani, V. R. Martins and M. A. Prado: Internalization of mammalian fluorescent cellular prion protein and N-terminal deletion mutants in living cells. J Neurochem 79(1), 79-87 (2001)
doi:10.1046/j.1471-4159.2001.00529.x
131. D. R. Brown: Prion protein expression aids cellular uptake and veratridine-induced release of copper. J Neurosci Res 58(5), 717-25 (1999)
doi:10.1002/(SICI)1097-4547(19991201)58:5<717::AID-JNR13>3.0.CO;2-S
132. W. Rachidi, D. Vilette, P. Guiraud, M. Arlotto, J. Riondel, H. Laude, S. Lehmann and A. Favier: Expression of prion protein increases cellular copper binding and antioxidant enzyme activities but not copper delivery. J Biol Chem 278(11), 9064-72 (2003)
doi:10.1074/jbc.M211830200
133. J. W. Herms, T. Tings, S. Dunker and H. A. Kretzschmar: Prion protein affects Ca2+-activated K+ currents in cerebellar purkinje cells. Neurobiol Dis 8(2), 324-30 (2001)
doi:10.1006/nbdi.2000.0369
134. S. B. Colling, J. Collinge and J. G. Jefferys: Hippocampal slices from prion protein null mice: disrupted Ca(2+)-activated K+ currents. Neurosci Lett 209(1), 49-52 (1996)
doi:10.1016/0304-3940(96)12596-9
135. J. Collinge, M. A. Whittington, K. C. Sidle, C. J. Smith, M. S. Palmer, A. R. Clarke and J. G. Jefferys: Prion protein is necessary for normal synaptic function. Nature 370(6487), 295-7 (1994)
doi:10.1038/370295a0
136. G. R. Mallucci, S. Ratte, E. A. Asante, J. Linehan, I. Gowland, J. G. Jefferys and J. Collinge: Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration. Embo J 21(3), 202-10 (2002)
doi:10.1093/emboj/21.3.202
137. J. R. Criado, M. Sanchez-Alavez, B. Conti, J. L. Giacchino, D. N. Wills, S. J. Henriksen, R. Race, J. C. Manson, B. Chesebro and M. B. Oldstone: Mice devoid of prion protein have cognitive deficits that are rescued by reconstitution of PrP in neurons. Neurobiol Dis 19(1-2), 255-65 (2005)
doi:10.1016/j.nbd.2005.01.001
138. A. S. Coitinho, R. Roesler, V. R. Martins, R. R. Brentani and I. Izquierdo: Cellular prion protein ablation impairs behavior as a function of age. Neuroreport 14(10), 1375-9 (2003)
doi:10.1097/00001756-200307180-00019
139. J. C. Manson, J. Hope, A. R. Clarke, A. Johnston, C. Black and N. MacLeod: PrP gene dosage and long term potentiation. Neurodegeneration 4(1), 113-4 (1995)
140. I. Tobler, T. Deboer and M. Fischer: Sleep and sleep regulation in normal and prion protein-deficient mice. J Neurosci 17(5), 1869-79 (1997)
141. I. Tobler, S. E. Gaus, T. Deboer, P. Achermann, M. Fischer, T. Rulicke, M. Moser, B. Oesch, P. A. McBride and J. C. Manson: Altered circadian activity rhythms and sleep in mice devoid of prion protein. Nature 380(6575), 639-42 (1996)
doi:10.1038/380639a0
142. E. T. Parkin, N. T. Watt, I. Hussain, E. A. Eckman, C. B. Eckman, J. C. Manson, H. N. Baybutt, A. J. Turner and N. M. Hooper: Cellular prion protein regulates beta-secretase cleavage of the Alzheimer's amyloid precursor protein. Proc Natl Acad Sci U S A 104(26), 11062-7 (2007)
doi:10.1073/pnas.0609621104
143. J. Lauren, D. A. Gimbel, H. B. Nygaard, J. W. Gilbert and S. M. Strittmatter: Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature 457(7233), 1128-32 (2009)
doi:10.1038/nature07761
144. B. Vincent, C. Sunyach, H. D. Orzechowski, P. St George-Hyslop and F. Checler: p53-Dependent transcriptional control of cellular prion by presenilins. J Neurosci 29(20), 6752-60 (2009)
doi:10.1523/JNEUROSCI.0789-09.2009
145. K. A. Kellett and N. M. Hooper: Prion protein and Alzheimer disease. Prion 3(4), 190-4 (2009)
146. J. Kazlauskaite, N. Sanghera, I. Sylvester, C. Venien-Bryan and T. J. Pinheiro: Structural changes of the prion protein in lipid membranes leading to aggregation and fibrillization. Biochemistry 42(11), 3295-304 (2003)
doi:10.1021/bi026872q
147. F. Wang, F. Yang, Y. Hu, X. Wang, C. Jin and J. Ma: Lipid interaction converts prion protein to a PrPSc-like proteinase K-resistant conformation under physiological conditions. Biochemistry 46(23), 7045-53 (2007)
doi:10.1021/bi700299h
148. N. S. Hachiya, M. Imagawa and K. Kaneko: The possible role of protein X, a putative auxiliary factor in pathological prion replication, in regulating a physiological endoproteolytic cleavage of cellular prion protein. Med Hypotheses 68(3), 670-3 (2007)
doi:10.1016/j.mehy.2006.07.038
149. K. Kaneko, L. Zulianello, M. Scott, C. M. Cooper, A. C. Wallace, T. L. James, F. E. Cohen and S. B. Prusiner: Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proc Natl Acad Sci U S A 94(19), 10069-74. (1997)
doi:10.1073/pnas.94.19.10069
150. G. C. Telling, M. Scott, J. Mastrianni, R. Gabizon, M. Torchia, F. E. Cohen, S. J. DeArmond and S. B. Prusiner: Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 83(1), 79-90 (1995)
doi:10.1016/0092-8674(95)90236-8
151. F. Yehiely, P. Bamborough, M. Da Costa, B. J. Perry, G. Thinakaran, F. E. Cohen, G. A. Carlson and S. B. Prusiner: Identification of candidate proteins binding to prion protein. Neurobiol Dis 3(4), 339-55 (1997)
doi:10.1006/nbdi.1997.0130
152. M. P. Gomes, T. A. Millen, P. S. Ferreira, E. S. N. L. Cunha, T. C. Vieira, M. S. Almeida, J. L. Silva and Y. Cordeiro: Prion protein complexed to N2a cellular RNAs through its N-terminal domain forms aggregates and is toxic to murine neuroblastoma cells. J Biol Chem (2008)
153. N. R. Deleault, R. W. Lucassen and S. Supattapone: RNA molecules stimulate prion protein conversion. Nature 425(6959), 717-20 (2003)
doi:10.1038/nature01979
154. V. Adler, B. Zeiler, V. Kryukov, R. Kascsak, R. Rubenstein and A. Grossman: Small, highly structured RNAs participate in the conversion of human recombinant PrP(Sen) to PrP(Res) in vitro. J Mol Biol 332(1), 47-57 (2003)
doi:10.1016/S0022-2836(03)00919-7
155. J. C. Geoghegan, P. A. Valdes, N. R. Orem, N. R. Deleault, R. A. Williamson, B. T. Harris and S. Supattapone: Selective incorporation of polyanionic molecules into hamster prions. J Biol Chem 282(50), 36341-53 (2007)
doi:10.1074/jbc.M704447200
156. M. Liu, S. Yu, J. Yang, X. Yin and D. Zhao: RNA and CuCl2 induced conformational changes of the recombinant ovine prion protein. Mol Cell Biochem 294(1-2), 197-203 (2007)
doi:10.1007/s11010-006-9260-1
157. O. Schonberger, L. Horonchik, R. Gabizon, D. Papy-Garcia, D. Barritault and A. Taraboulos: Novel heparan mimetics potently inhibit the scrapie prion protein and its endocytosis. Biochem Biophys Res Commun 312(2), 473-9 (2003)
doi:10.1016/j.bbrc.2003.10.150
158. O. Ben-Zaken, S. Tzaban, Y. Tal, L. Horonchik, J. D. Esko, I. Vlodavsky and A. Taraboulos: Cellular heparan sulfate participates in the metabolism of prions. J Biol Chem 278(41), 40041-9 (2003)
doi:10.1074/jbc.M301152200
159. T. Mayer-Sonnenfeld, M. Zeigler, M. Halimi, Y. Dayan, C. Herzog, C. I. Lasmezas and R. Gabizon: The metabolism of glycosaminoglycans is impaired in prion diseases. Neurobiol Dis 20(3), 738-43 (2005)
doi:10.1016/j.nbd.2005.05.009
160. K. Lofgren, F. Cheng, L. A. Fransson, K. Bedecs and K. Mani: Involvement of glypican-1 autoprocessing in scrapie infection. Eur J Neurosci 28(5), 964-72 (2008)
doi:10.1111/j.1460-9568.2008.06386.x
161. C. Monnet, V. Marthiens, H. Enslen, Y. Frobert, A. Sobel and R. M. Mege: Heterogeneity and regulation of cellular prion protein glycoforms in neuronal cell lines. Eur J Neurosci 18(3), 542-8 (2003)
doi:10.1046/j.1460-9568.2003.02777.x
162. R. A. Bessen and R. F. Marsh: Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent. J Virol 66(4), 2096-101 (1992)
163. R. A. Bessen and R. F. Marsh: Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol 68(12), 7859-68 (1994)
164. R. A. Somerville, A. Chong, O. U. Mulqueen, C. R. Birkett, S. C. Wood and J. Hope: Biochemical typing of scrapie strains. Nature 386(6625), 564 (1997)
doi:10.1038/386564a0
165. R. A. Bessen, D. A. Kocisko, G. J. Raymond, S. Nandan, P. T. Lansbury and B. Caughey: Non-genetic propagation of strain-specific properties of scrapie prion protein. Nature 375(6533), 698-700 (1995)
doi:10.1038/375698a0
166. D. Peretz, R. A. Williamson, G. Legname, Y. Matsunaga, J. Vergara, D. R. Burton, S. J. DeArmond, S. B. Prusiner and M. R. Scott: A change in the conformation of prions accompanies the emergence of a new prion strain. Neuron 34(6), 921-32 (2002)
doi:10.1016/S0896-6273(02)00726-2
167. G. C. Telling, P. Parchi, S. J. DeArmond, P. Cortelli, P. Montagna, R. Gabizon, J. Mastrianni, E. Lugaresi, P. Gambetti and S. B. Prusiner: Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity. Science 274(5295), 2079-82 (1996)
doi:10.1126/science.274.5295.2079
168. B. Caughey, G. J. Raymond and R. A. Bessen: Strain-dependent differences in beta-sheet conformations of abnormal prion protein. J Biol Chem 273(48), 32230-5 (1998)
doi:10.1074/jbc.273.48.32230
169. K. A. Nishina, N. R. Deleault, S. P. Mahal, I. Baskakov, T. Luhrs, R. Riek and S. Supattapone: The stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro. Biochemistry 45(47), 14129-39 (2006)
doi:10.1021/bi061526k
170. N. L. Tuzi, E. Cancellotti, H. Baybutt, L. Blackford, B. Bradford, C. Plinston, A. Coghill, P. Hart, P. Piccardo, R. M. Barron and J. C. Manson: Host PrP Glycosylation: A Major Factor Determining the Outcome of Prion Infection. In: PLoS Biol. (2008)
171. L. Laszlo, J. Lowe, T. Self, N. Kenward, M. Landon, T. McBride, C. Farquhar, I. McConnell, J. Brown, J. Hope and et al.: Lysosomes as key organelles in the pathogenesis of prion encephalopathies. J Pathol 166(4), 333-41 (1992)
doi:10.1002/path.1711660404
172. B. Caughey, G. J. Raymond, D. Ernst and R. E. Race: N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. J Virol 65(12), 6597-603 (1991)
173. M. P. McKinley, A. Taraboulos, L. Kenaga, D. Serban, A. Stieber, S. J. DeArmond, S. B. Prusiner and N. Gonatas: Ultrastructural localization of scrapie prion proteins in cytoplasmic vesicles of infected cultured cells. Lab Invest 65(6), 622-30 (1991)
174. J. E. Arnold, C. Tipler, L. Laszlo, J. Hope, M. Landon and R. J. Mayer: The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain. J Pathol 176(4), 403-11 (1995)
doi:10.1002/path.1711760412
175. V. Grigoriev, F. Escaig-Haye, N. Streichenberger, N. Kopp, J. Langeveld, P. Brown and J. G. Fournier: Submicroscopic immunodetection of PrP in the brain of a patient with a new-variant of Creutzfeldt-Jakob disease. Neurosci Lett 264(1-3), 57-60 (1999)
doi:10.1016/S0304-3940(99)00146-9
176. F. Pimpinelli, S. Lehmann and I. Maridonneau-Parini: The scrapie prion protein is present in flotillin-1-positive vesicles in central- but not peripheral-derived neuronal cell lines. Eur J Neurosci 21(8), 2063-72 (2005)
doi:10.1111/j.1460-9568.2005.04049.x
177. N. M. Veith, H. Plattner, C. A. Stuermer, W. J. Schulz-Schaeffer and A. Burkle: Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy. Eur J Cell Biol 88(1), 45-63 (2009)
doi:10.1016/j.ejcb.2008.08.001
178. M. Kristiansen, P. Deriziotis, D. E. Dimcheff, G. S. Jackson, H. Ovaa, H. Naumann, A. R. Clarke, F. W. van Leeuwen, V. Menendez-Benito, N. P. Dantuma, J. L. Portis, J. Collinge and S. J. Tabrizi: Disease-associated prion protein oligomers inhibit the 26S proteasome. Mol Cell 26(2), 175-88 (2007)
doi:10.1016/j.molcel.2007.04.001
179. E. Flechsig, I. Hegyi, M. Enari, P. Schwarz, J. Collinge and C. Weissmann: Transmission of scrapie by steel-surface-bound prions. Mol Med 7(10), 679-84 (2001)
180. N. Kanu, Y. Imokawa, D. N. Drechsel, R. A. Williamson, C. R. Birkett, C. J. Bostock and J. P. Brockes: Transfer of scrapie prion infectivity by cell contact in culture. Curr Biol 12(7), 523-30 (2002)
doi:10.1016/S0960-9822(02)00722-4
181. N. Naslavsky, H. Shmeeda, G. Friedlander, A. Yanai, A. H. Futerman, Y. Barenholz and A. Taraboulos: Sphingolipid depletion increases formation of the scrapie prion protein in neuroblastoma cells infected with prions. J Biol Chem 274(30), 20763-71 (1999)
doi:10.1074/jbc.274.30.20763
182. B. Chesebro, M. Trifilo, R. Race, K. Meade-White, C. Teng, R. LaCasse, L. Raymond, C. Favara, G. Baron, S. Priola, B. Caughey, E. Masliah and M. Oldstone: Anchorless prion protein results in infectious amyloid disease without clinical scrapie. Science 308(5727), 1435-9 (2005)
doi:10.1126/science.1110837
183. V. Campana, A. Caputo, D. Sarnataro, S. Paladino, S. Tivodar and C. Zurzolo: Characterization of the properties and trafficking of an anchorless form of the prion protein. J Biol Chem 282(31), 22747-56 (2007)
doi:10.1074/jbc.M701468200
184. B. Chesebro, B. Race, K. Meade-White, R. Lacasse, R. Race, M. Klingeborn, J. Striebel, D. Dorward, G. McGovern and M. Jeffrey: Fatal transmissible amyloid encephalopathy: a new type of prion disease associated with lack of prion protein membrane anchoring. PLoS Pathog 6(3), e1000800 (2010)
doi:10.1371/journal.ppat.1000800
185. K. L. McNally, A. E. Ward and S. A. Priola: Cells expressing anchorless prion protein are resistant to scrapie infection. J Virol 83(9), 4469-75 (2009)
doi:10.1128/JVI.02412-08
186. W. Q. Zou and N. R. Cashman: Acidic pH and detergents enhance in vitro conversion of human brain PrPC to a PrPSc-like form. J Biol Chem 277(46), 43942-7 (2002)
doi:10.1074/jbc.M203611200
187. S. Hornemann and R. Glockshuber: A scrapie-like unfolding intermediate of the prion protein domain PrP(121-231) induced by acidic pH. Proc Natl Acad Sci U S A 95(11), 6010-4 (1998)
doi:10.1073/pnas.95.11.6010
188. W. Swietnicki, R. Petersen, P. Gambetti and W. K. Surewicz: pH-dependent stability and conformation of the recombinant human prion protein PrP(90-231). J Biol Chem 272(44), 27517-20 (1997)
doi:10.1074/jbc.272.44.27517
189. D. R. Borchelt, A. Taraboulos and S. B. Prusiner: Evidence for synthesis of scrapie prion proteins in the endocytic pathway. J Biol Chem 267(23), 16188-99 (1992)
190. Z. Marijanovic, A. Caputo, V. Campana and C. Zurzolo: Identification of an intracellular site of prion conversion. PLoS Pathog 5(5), e1000426 (2009)
doi:10.1371/journal.ppat.1000426
191. S. Gurke, J. F. Barroso and H. H. Gerdes: The art of cellular communication: tunneling nanotubes bridge the divide. Histochem Cell Biol 129(5), 539-50 (2008)
doi:10.1007/s00418-008-0412-0
192. A. C. Magalhaes, G. S. Baron, K. S. Lee, O. Steele-Mortimer, D. Dorward, M. A. Prado and B. Caughey: Uptake and neuritic transport of scrapie prion protein coincident with infection of neuronal cells. J Neurosci 25(21), 5207-16 (2005)
doi:10.1523/JNEUROSCI.0653-05.2005
193. K. Gousset, E. Schiff, C. Langevin, Z. Marijanovic, A. Caputo, D. T. Browman, N. Chenouard, F. de Chaumont, A. Martino, J. Enninga, J. C. Olivo-Marin, D. Mannel and C. Zurzolo: Prions hijack tunnelling nanotubes for intercellular spread. Nat Cell Biol 11(3), 328-36 (2009)
doi:10.1038/ncb1841
194. D. F. Legler, M. A. Doucey, P. Schneider, L. Chapatte, F. C. Bender and C. Bron: Differential insertion of GPI-anchored GFPs into lipid rafts of live cells. Faseb J 19(1), 73-5 (2005)
195. D. L. Kooyman, G. W. Byrne, S. McClellan, D. Nielsen, M. Tone, H. Waldmann, T. M. Coffman, K. R. McCurry, J. L. Platt and J. S. Logan: In vivo transfer of GPI-linked complement restriction factors from erythrocytes to the endothelium. Science 269(5220), 89-92 (1995)
doi:10.1126/science.7541557
196. D. R. Premkumar, Y. Fukuoka, D. Sevlever, E. Brunschwig, T. L. Rosenberry, M. L. Tykocinski and M. E. Medof: Properties of exogenously added GPI-anchored proteins following their incorporation into cells. J Cell Biochem 82(2), 234-45 (2001)
doi:10.1002/jcb.1154