Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

In prion diseases, the cellular form of the prion protein, PrP^C, undergoes a conformational conversion to the infectious isoform, PrP^Sc. PrP^C associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI) anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs). We show that heparin displaces PrP^C from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP^C. We then utilised a transmembrane-anchored form of PrP (PrP-TM), which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP^C to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP^C from rafts, promoting its endocytosis. Glypican-1 and PrP^C colocalised on the cell surface and both PrP^C and PrP^Sc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP^Sc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP^C on the β-secretase cleavage of the Alzheimer''s amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP^C and PrP^Sc in lipid rafts.     

Cellular factors implicated in prion replication

Prions are the unconventional infectious agents responsible for prion diseases, which are composed mainly by the misfolded prion protein (PrP^Sc) that replicates by converting the host associated cellular prion protein (PrP^C). Several lines of evidence suggest that other cellular components participate in prion conversion, however, the identity or even the chemical nature of such factors are entirely unknown. In this article we study the conversion factor activity by complementation of a PMCA procedure employing purified PrP^C and PrP^Sc. Our results show that the conversion factor is present in all major organs of diverse mammalian species, and is predominantly located in the lipid raft fraction of the cytoplasmic membrane. On the other hand, it is not present in the lower organisms tested (yeast, bacteria and flies). Surprisingly, treatments that eliminate the major classes of chemical molecules do not affect conversion activity, suggesting that various different compounds may act as conversion factor in vitro. This conclusion is further supported by experiments showing that addition of various classes of molecules have a small, but detectable effect on enhancing prion replication in vitro. More research is needed to elucidate the identity of these factors, their detailed mechanism of action and whether or not they are essential component of the infectious particle.     

Prion Infection Impairs Cholesterol Metabolism in Neuronal Cells

Conversion of prion protein (PrP^C) into a pathological isoform (PrP^Sc) during prion infection occurs in lipid rafts and is dependent on cholesterol. Here, we show that prion infection increases the abundance of cholesterol transporter, ATP-binding cassette transporter type A1 (ATP-binding cassette transporter type A1), but reduces cholesterol efflux from neuronal cells leading to the accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was confirmed in prion-infected mice. Mechanistically, conversion of PrP^C to the pathological isoform led to PrP^Sc accumulation in rafts, displacement of ABCA1 from rafts and the cell surface, and enhanced internalization of ABCA1. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression with liver X receptor agonist or overexpression of heterologous ABCA1 reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, which plays an important role in the pathogenesis of prion infection in neuronal cells.     

Mechanism of aggregation and membrane interactions of mammalian prion protein

The cellular prion protein (PrP^C), which is present ubiquitously in all mammalian neurons, is normally found to be linked to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. The conformational conversion of PrP^C into misfolded and aggregated forms is associated with transmissible neurodegenerative diseases known as prion diseases. The importance of different misfolded conformations in prion diseases, and the mechanism by which prion aggregates induce neurotoxicity remain poorly understood. Multiple studies have been shown that the toxicity of misfolded prion protein is directly correlated with its ability to interact with and perturb membranes. This review describes the current progress toward understanding prion protein misfolding and aggregation, as well as the interaction of prion protein aggregates with lipid membrane.     

Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signaling and Prion Formation

The prion diseases occur following the conversion of the cellular prion protein (PrP^C) into disease-related isoforms (PrP^Sc). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP^C in prion formation was examined using a cell painting technique. PrP^Sc formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP^C. In contrast, PrP^C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP^C) was not converted to PrP^Sc. Furthermore, the presence of desialylated PrP^C inhibited the production of PrP^Sc within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP^C contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP^C. Desialylated PrP^C was less sensitive to cholesterol depletion than PrP^C and was not released from cells by treatment with glimepiride. The presence of desialylated PrP^C in neurons caused the dissociation of cytoplasmic phospholipase A₂ from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A₂. These findings show that the sialic acid moiety of the GPI attached to PrP^C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP^Sc formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases.     

Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrP^C) to the infectious scrapie form (PrP^Sc). However, the mechanism that exogenous PrP^Sc infects cells and where pathologic conversion of PrP^C to the PrP^Sc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrP^C to the pathologic PrP^Sc form in N2a cells exposed to strain RML PrP^Sc infected brain homogenates, suggesting that a critical determinant of PrP^C conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP^Sc infects cells by lipid raft dependent, macropinocytosis.     

Identification of an Intracellular Site of Prion Conversion

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP^C), denoted PrP^Sc, which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrP^C into PrP^Sc and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrP^C and PrP^Sc and measured PrP^Sc levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.     

The GPI-anchoring of PrP

The cellular prion protein (PrP^C) is an N-glycosylated GPI-anchored protein usually present in lipid rafts with numerous putative functions. When it changes its conformation to a pathological isoform (then referred to as PrP^Sc), it is an essential part of the prion, the agent causing fatal and transmissible neurodegenerative prion diseases. There is growing evidence that toxicity and neuronal damage on the one hand and propagation/infectivity on the other hand are two distinct processes of the disease and that the GPI-anchor attachment of PrP^C and PrP^Sc plays an important role in protein localization and in neurotoxicity. Here we review how the signal sequence of the GPI-anchor matters in PrP^C localization, how an altered cellular localization of PrP^C or differences in GPI-anchor composition can affect prion infection, and we discuss through which mechanisms changes on the anchorage of PrP^C can modify the disease process.     

Glycosylphosphatidylinositol Anchor Analogues Sequester Cholesterol and Reduce Prion Formation

A hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP^C where C is cellular) into an alternatively folded, disease-related isoform (PrP^Sc, where Sc is scrapie), the accumulation of which is associated with synapse degeneration and ultimately neuronal death. The formation of PrP^Sc is dependent upon the presence of PrP^C in specific, cholesterol-sensitive membrane microdomains, commonly called lipid rafts. PrP^C is targeted to these lipid rafts because it is attached to membranes via a glycosylphosphatidylinositol anchor. Here, we show that treatment of prion-infected neuronal cell lines (ScN2a, ScGT1, or SMB cells) with synthetic glycosylphosphatidylinositol analogues, glucosamine-phosphatidylinositol (glucosamine-PI) or glucosamine 2-O-methyl inositol octadecyl phosphate, reduced the PrP^Sc content of these cells in a dose-dependent manner. In addition, ScGT1 cells treated with glucosamine-PI did not transmit infection following intracerebral injection to mice. Treatment with glucosamine-PI increased the cholesterol content of ScGT1 cell membranes and reduced activation of cytoplasmic phospholipase A₂ (PLA₂), consistent with the hypothesis that the composition of cell membranes affects key PLA₂-dependent signaling pathways involved in PrP^Sc formation. The effect of glucosamine-PI on PrP^Sc formation was also reversed by the addition of platelet-activating factor. Glucosamine-PI caused the displacement of PrP^C from lipid rafts and reduced expression of PrP^C at the cell surface, putative sites for PrP^Sc formation. We propose that treatment with glucosamine-PI modifies local micro-environments that control PrP^C expression and activation of PLA₂ and subsequently inhibits PrP^Sc formation.     

Insight into early events in the aggregation of the prion protein on lipid membranes

The key molecular event underlying prion diseases is the conversion of the monomeric and α-helical cellular form of the prion protein (PrP^C) to the disease-associated state, which is aggregated and rich in β-sheet (PrP^Sc). The molecular details associated with the conversion of PrP^C into PrP^Sc are not fully understood. The prion protein is attached to the cell membrane via a GPI lipid anchor and evidence suggests that the lipid environment plays an important role in prion conversion and propagation. We have previously shown that the interaction of the prion protein with anionic lipid membranes induces β-sheet structure and promotes prion aggregation, whereas zwitterionic membranes stabilize the α-helical form of the protein. Here, we report on the interaction of recombinant sheep prion protein with planar lipid membranes in real-time, using dual polarization interferometry (DPI). Using this technique, the simultaneous evaluation of multiple physical properties of PrP layers on membranes was achieved. The deposition of prion on membranes of POPC and POPC/POPS mixtures was studied. The properties of the resulting protein layers were found to depend on the lipid composition of the membranes. Denser and thicker protein deposits formed on lipid membranes containing POPS compared to those formed on POPC. DPI thus provides a further insight on the organization of PrP at the surface of lipid membranes.     

Does the tail wag the dog? How the structure of a glycosylphosphatidylinositol anchor affects prion formation

There is increasing interest in the role of the glycosylphosphatidylinositol (GPI) anchor attached to the cellular prion protein (PrP^C). Since GPI anchors can alter protein targeting, trafficking and cell signaling, our recent study examined how the structure of the GPI anchor affected prion formation. PrP^C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP^C) was not converted to PrP^Sc in prion-infected neuronal cell lines and in scrapie-infected primary cortical neurons. In uninfected neurons desialylated PrP^C was associated with greater concentrations of gangliosides and cholesterol than PrP^C. In addition, the targeting of desialylated PrP^C to lipid rafts showed greater resistance to cholesterol depletion than PrP^C. The presence of desialylated PrP^C caused the dissociation of cytoplasmic phospholipase A₂ (cPLA₂) from PrP-containing lipid rafts, reduced the activation of cPLA₂ and inhibited PrP^Sc production. We conclude that the sialic acid moiety of the GPI attached to PrP^C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP^Sc formation.     

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

Background

The cellular prion protein (PrP^C) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrP^Sc). Although endocytosis appears to be required for this conversion, the mechanism of PrP^C internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved.

Methodology/Principal Findings

We have investigated the mechanism of PrP^C endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrP^C internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrP^C endocytosis. PrP^C internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrP^C co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrP^C can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization.

Conclusions/Significance

These findings are of particular interest if we consider that the internalization route/s undertaken by PrP^C can be crucial for the ability of different prion strains to infect and to replicate in different cell lines.     

The Cellular Prion Protein Interacts with the Tissue Non-Specific Alkaline Phosphatase in Membrane Microdomains of Bioaminergic Neuronal Cells

Background

The cellular prion protein, PrP^C, is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP^C in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP^C acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP^C, we have described a neuronal specificity pointing to a role of PrP^C in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11^5-HT) or noradrenergic (1C11^NE) derivatives.

Methodology/Principal Findings

The neuronal specificity of PrP^C signaling prompted us to search for PrP^C partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP^C with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP). This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11^5-HT and 1C11^NE cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11^5-HT and 1C11^NE bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP.

Conclusion/Significance

The identification of a novel PrP^C partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP^C and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP^C-laminin interplay. The partnership between TNAP and PrP^C in neuronal cells may provide new clues as to the neurospecificity of PrP^C function.     

Real-time visualization of prion transport in single live cells using quantum dots

Prion diseases are fatal neurodegenerative disorders resulting from structural conversion of the cellular isoform of PrP^C to the infectious scrapie isoform PrP^Sc. It is believed that such structural alteration may occur within the internalization pathway. However, there is no direct evidence to support this hypothesis. Employing quantum dots (QDs) as a probe, we have recorded a real-time movie demonstrating the process of prion internalization in a living cell for the first time. The entire internalization process can be divided into four discrete but connected stages. In addition, using methyl-beta-cyclodextrin to disrupt cell membrane cholesterol, we show that lipid rafts play an important role in locating cellular PrP^C to the cell membrane and in initiating PrP^C endocytosis.     

Identifying critical sites of PrPc-PrPSc interaction in prion-infected cells by dominant-negative inhibition

A direct physical interaction of the prion protein isoforms is a key element in prion conversion. Which sites interact first and which parts of PrP^c are converted subsequently is presently not known in detail. We hypothesized that structural changes induced by PrP^Sc interaction occur in more than one interface and subsequently propagate within the PrP^C substrate, like epicenters of structural changes. To identify potential interfaces we created a series of systematically-designed mutant PrPs and tested them in prion-infected cells for dominant-negative inhibition (DNI) effects. This showed that mutant PrPs with deletions in the region between first and second α-helix are involved in PrP-PrP interaction and conversion of PrP^C into PrP^Sc. Although some PrPs did not reach the plasma membrane, they had access to the locales of prion conversion and PrP^Sc recycling using autophagy pathways. Using other series of mutant PrPs we already have identified additional sites which constitute potential interaction interfaces. Our approach has the potential to characterize PrP-PrP interaction sites in the context of prion-infected cells. Besides providing further insights into the molecular mechanisms of prion conversion, this data may help to further elucidate how prion strain diversity is maintained.     

Novel amplification mechanism of prions through disrupting sortilin-mediated trafficking

Conformational conversion of the cellular prion protein, PrP^C, into the abnormally folded isoform of prion protein, PrP^Sc, which leads to marked accumulation of PrP^Sc in brains, is a key pathogenic event in prion diseases, a group of fatal neurodegenerative disorders caused by prions. However, the exact mechanism of PrP^Sc accumulation in prion-infected neurons remains unknown. We recently reported a novel cellular mechanism to support PrP^Sc accumulation in prion-infected neurons, in which PrP^Sc itself promotes its accumulation by evading the cellular inhibitory mechanism, which is newly identified in our recent study. We showed that the VPS10P sorting receptor sortilin negatively regulates PrP^Sc accumulation in prion-infected neurons, by interacting with PrP^C and PrP^Sc and trafficking them to lysosomes for degradation. However, PrP^Sc stimulated lysosomal degradation of sortilin, disrupting the sortilin-mediated degradation of PrP^C and PrP^Sc and eventually evoking further accumulation of PrP^Sc in prion-infected neurons. These findings suggest a positive feedback amplification mechanism for PrP^Sc accumulation in prion-infected neurons.     

The cellular prion protein with a monoacylated glycosylphosphatidylinositol anchor modifies cell membranes,inhibits cell signaling and reduces prion formation

The prion diseases occur following the conversion of the cellular prion protein (PrP^C) into a disease-related isoform (PrP^Sc). In this study a cell painting technique was used to examine the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP^C in prion formation. The introduction of PrP^C to infected neuronal cells increased the cholesterol content of cell membranes, increased activation of cytoplasmic phospholipase A₂ (cPLA₂) and increased PrP^Sc formation. In contrast, PrP^C with a monoacylated GPI anchor did not alter the amount of cholesterol in cell membranes, was not found within lipid rafts and did not activate cPLA₂. Although monoacylated PrP^C remains within cells for longer than native PrP^C it was not converted to PrP^Sc. Moreover, the presence of monoacylated PrP^C displaced cPLA₂ from PrP^Sc-containing lipid rafts, reducing the activation of cPLA₂ and PrP^Sc formation. We conclude that acylation of the GPI anchor attached to PrP^C modifies the local membrane microenvironments that control some cell signaling pathways, the trafficking of PrP^C and PrP^Sc formation. In addition, such observations raise the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases.Key words: cholesterol, glycosylphosphatidylinositol, lipid rafts, membranes, phospholipase A₂, prion, trafficA key event in the prion diseases is the conversion of a normal host protein (PrP^C) into a disease-associated isoform (PrP^Sc).¹ Although the presence of PrP^C is essential for prion formation,^2,3 not all cells that express PrP^C are permissive for PrP^Sc replication. The reasons why some cells that express PrP^C do not replicate PrP^Sc are not fully understood. Reports that the targeting of PrP^C to specific membranes is required for efficient PrP^Sc formation⁴ indicate that the factors that affect the cellular targeting and intracellular trafficking of PrP^C are critical in determining PrP^Sc replication.Our study examined the effects of the glycosylphosphatidylinositol (GPI) anchor that links the majority of PrP^C molecules to cell membranes⁵ on PrP^Sc formation. Originally GPI anchors were seen as a simple method of attaching proteins to cell membranes. However, there is increasing interest in the role of GPI anchors in complex biological functions including the regulation of membrane composition, cell signaling and protein trafficking.⁶ To examine the role of the GPI anchor PrP^C preparations were digested with phosphatidylinositol-phospholipase C (PI-PLC) (deacylated PrP^C) or phospholipase A₂ (PLA₂) (monoacylated PrP^C) (Fig. 1) and isolated by reverse phase chromatography. These digestions, coupled with a cell painting technique, allowed us to examine modifications of the GPI anchor that could not be achieved by genetic manipulation methods. Controversy surrounds the role of the GPI anchor in PrP^Sc formation; the seminal observation that transgenic mice producing anchorless PrP^C produced large amounts of extracellular PrP^Sc,⁷ suggests that the GPI has little effect upon PrP^Sc replication. In contrast, a recent study showed that cells that produce anchorless PrP^C were not permissive to PrP^Sc formation⁸ and in our study deacylated PrP^C did not affect PrP^Sc production. Although at first glance these results appear contradictory, they may be explained by reference to the site of conversion of PrP^C to PrP^Sc. Clearly anchorless PrP^C can be converted to PrP^Sc in a process that occurs within the extracellular milieu. However, as anchorless PrP^C is rapidly secreted from cells⁷ it has little contact with cell-associated PrP^Sc. Similarly we found that deacylated PrP^C was fully soluble and did not readily associate with cells.Open in a separate windowFigure 1Phospholipase digestion of PrP^C affects the acylation of the GPI anchor. Cartoon showing the putative GPI anchor attached to PrP^C, monoacylated PrP^C and deacylated PrP^C. Glycan residues shown include inositol (Inos), mannose (Man), sialic acid (SA), galactose (Gal), N-acetyl galactosamine (GalNAc) and glucosamine (GlcN) as well as phosphate (P).Native PrP^C is rapidly transferred to cells⁹ and we showed that the addition of PrP^C caused a dose-dependent increase in the PrP^Sc content of all prion-infected cell lines tested. We used this cell painting technique to introduce monoacylated PrP^C to recipient cells. Our paper describes three major observations; firstly that monoacylated PrP^C behaves differently to native PrP^C with regards to cellular distribution, intracellular trafficking and cell signaling; secondly, that monoacylated PrP^C was not converted to PrP^Sc and thirdly, that monoacylated PrP^C inhibited the conversion of endogenous PrP^C to PrP^Sc.The presence of GPI anchors targets proteins including PrP^C and PrP^Sc to specialized membrane micro-domains that are commonly called lipid rafts.^10,11 Lipid rafts are patches of membranes that are highly enriched in cholesterol and sphingolipids and which are operationally defined by their insolubility in cold non-ionic detergents and floatation as low density membranes on sucrose density gradients. The importance of lipid rafts in prion diseases is based upon studies showing that treatment with cholesterol synthesis inhibitors reduced cellular cholesterol and the formation of PrP^Sc.¹² Since the cholesterol content of cell membranes is critical for the formation of lipid rafts¹³ it is assumed that the integrity of these lipid rafts is necessary for efficient PrP^Sc formation. The presence of GPI-anchored proteins is thought to help lipid rafts form as the saturated fatty acids that are contained within GPI anchors facilitate the solubilisation of cholesterol in the membrane¹⁴ and the glycan component protect cholesterol from water.¹⁵ Thus the nature and number of the acyl chains contained within GPI anchors are factors that affect lipid raft formation (Fig. 2).Open in a separate windowFigure 2Acylation of PrP^C affects the underlying cell membrane. Cartoon showing the proposed membranes surrounding native PrP^C and monoacylated PrP^C, including cholesterol (), lyso-phospholipids (), saturated phospholipids () and unsaturated phospholipids (). Monoacylated PrP^C is not directed to lipid rafts and the membrane surrounding contains less cholesterol and more unsaturated phospholipids.We observed that the addition of native PrP^C significantly increased the amount of cholesterol in cell membranes. This result was unexpected as the amount of cholesterol in cell membranes is tightly controlled by an esterification and hydrolysis cycle which releases cholesterol from stored cholesterol esters.¹⁶ The PrP^C-induced increase in cholesterol was accompanied by a reduction in cholesterol esters suggesting that it was derived from the hydrolysis of cholesterol esters. Pharmacological inhibition of cholesterol ester hydrolysis not only blocked the PrP^C-induced increase in cholesterol and the reduction in cholesterol esters, but also reduced the PrP^C-induced increase in PrP^Sc formation. Collectively, these results indicate that cells respond to the introduction of PrP^C by the hydrolysis of cholesterol esters; which provides cholesterol to stabilize PrP^C within the lipid rafts that are necessary to facilitate PrP^Sc formation.The differences in the cellular distribution of PrP^C and monoacylated PrP^C were examined using neurons from Prnp knockout mice. While PrP^C was targeted to lipid rafts, monoacylated PrP^C was found predominantly within the normal (non-raft) cell membranes. Unlike native PrP^C, monoacylated PrP^C did not affect the cholesterol content of cell membranes; an observation that highlights the critical role of the presence of two saturated fatty acids contained within the GPI anchor to sequester cholesterol and precipitate the formation of lipid rafts.¹⁴ Critically, monoacylated PrP^C was unable to solubilize cholesterol or precipitate lipid raft formation and was consequently found outside lipid rafts (Fig. 2).Since many raft-associated proteins including PrP^C traffic within cells via specific pathways,¹⁷ we argued that monoacylated PrP^C might undergo alternative trafficking pathways to those utilized by native PrP^C. Our observations, that greater amounts of monoacylated PrP^C than native PrP^C were expressed at the cell surface, and that while most of the native PrP^C was removed from these cells within 24 h, monoacylated PrP^C remained in neurons for longer, are indicative of altered intracellular trafficking. It is possible that the cellular location and/or pathway(s) used by monoacylated PrP^C may be physically segregated from PrP^Sc. This hypothesis would explain our observation that the addition of monoacylated PrP^C to prion-infected cell lines did not increase PrP^Sc formation indicating that it was not converted to PrP^Sc.Since monoacylated phospholipids exist only transiently within cell membranes, they are rapidly reacylated by esterases,¹⁸ we wondered whether the monoacylated PrP^C could also be reacylated to form the native, diacylated PrP^C. We found no evidence that the monoacylated PrP^C added to Prnp knockout neurons was reacylated suggesting that the enzymes involved in reacylation of membrane phospholipids do not recognize phosphatidylinositol when it is incorporated as part of the GPI anchor. In addition we were unable to detect monoacylated PrP^C occurring naturally within Prnp wild-type neurons.While these theories explain why monoacylated PrP^C was not readily converted to PrP^Sc; a more refined hypothesis is required to explain why monoacylated PrP^C reduced PrP^Sc production. One possibility is that monoacylated PrP^C is converted to monoacylated PrP^Sc which in turn acts as an inefficient template for PrP^C to PrP^Sc conversion.¹⁹ It is also possible that monoacylated PrP^C competes with endogenous PrP^C for specific partner proteins involved in endocytosis. The depletion of these partner proteins could consequently alter the trafficking of endogenous PrP^C and hence PrP^Sc formation. In our paper we explored the idea that the binding of monoacylated PrP^C to PrP^Sc modifies the lipid rafts that are involved in PrP^Sc formation. Both the composition and function of lipid rafts is dynamic and controlled by an induced fit model.²⁰ Since monoacylated PrP^C does not sequester cholesterol, the membrane surrounding a complex between PrP^Sc and monoacylated PrP^C might be expected to contain less cholesterol than membranes formed following the interaction between PrP^Sc and PrP^C (Fig. 3). Thus, the binding of monoacylated PrP^C to PrP^Sc may reduce the cholesterol content of local membranes to a level below that required for the conversion of PrP^C to PrP^Sc. This hypothesis is consistent with observations that formation of PrP^Sc was affected by the lipid composition of membranes²¹ and that lipids were essential co-factors in prion formation.²²Open in a separate windowFigure 3Monoacylated PrP^C affects the capture of cPLA₂ in PrP^Sc-containing lipid rafts. (A) Cartoon showing the proposed membranes surrounding PrP^Sc and PrP^C including the capture of cPLA₂ in lipid rafts that are dense in cholesterol () and saturated phospholipids (). (B) Cartoon showing the proposed interactions between PrP^Sc and monoacylated PrP^C which reduces the solubility of membranes to cholesterol, increases the concentration of unsaturated phospholipids () and prevents the capture of cPLA₂ into PrP^Sc-containing lipid rafts.Our studies raise the question “why are lipid rafts important in PrP^Sc formation?” Lipid rafts are enriched with signaling molecules and can act as domains in which the GPI anchors attached to PrP^C can interact with cell signaling pathways.²³ Although PrP^C has been reported to interact with many cell signaling pathways we concentrated upon its effects on the activation of cPLA₂, based upon studies showing that the activation of cPLA₂ correlates strongly with the amounts of cholesterol and PrP^Sc,²⁴ and that the inhibition of cPLA₂ reduces PrP^Sc formation.²⁵ These observations underpin the hypothesis that it is the clustering of GPI anchors attached to PrP proteins that leads to the activation of cPLA₂. This hypothesis was tested by incubating Prnp knockout neurons with PrP^C or monoacylated PrP^C and then adding the anti-PrP mAb 4F2. We found that the cross-linkage of PrP^C by mAb 4F2 caused the activation of cPLA₂, whereas the cross-linkage of monoacylated PrP^C by mAb 4F2 had no significant affect. The activation of cPLA₂ is associated with multiple phosphorylation events and the translocation of cPLA₂ to specific membranes.²⁶ In scrapie-infected GT1 (ScGT1) cells most of the cPLA₂ was found within lipid rafts consistent with reports that the activation of cPLA₂ is dependent upon cholesterol-sensitive lipid rafts.²⁷ More specifically, immunoprecipitation studies showed that cPLA₂ was targeted to PrP^Sc-containing lipid rafts.²⁴ Collectively, these observations suggest that PrP^C binds to PrP^Sc in cholesterol-dense lipid rafts, where it activates the cPLA₂ that facilitates the conversion of PrP^C to PrP^Sc (Fig. 3).We found that the presence of monoacylated PrP^C reduced the activation of cPLA₂ within prion-infected cells. As cPLA₂ can be activated by multiple different stimuli we sought to determine whether the inhibitory effect of monoacylated PrP^C was stimulus specific. We reported that monoacylated PrP^C did not affect the activation of cPLA₂ by a phospholipase A₂-activating peptide indicating that monoacylated PrP^C did not have a direct inhibitory effect upon cPLA₂. Rather we found that the addition of monoacylated PrP^C to prion-infected cells caused the dissociation of some cPLA₂ from PrP^Sc-containing lipid rafts. The targeting of cPLA₂ to membranes containing their endogenous substrates can regulate cell signaling, including for the formation of second messengers such as platelet-activating factor that facilitate PrP^Sc formation.²⁵ We propose that the binding of monoacylated PrP^C to PrP^Sc changed the composition of the underlying membrane so that it no longer captured and activated cPLA₂ (Fig. 3).In conclusion our study showed that the addition of monoacylated PrP^C modified cell membranes thus reducing the activation of cPLA₂ and PrP^Sc formation in prion-infected cells. We propose that the 2 acyl chains attached to the GPI anchor is a critical factor that facilitates the conversion to PrP^C to PrP^Sc within cell membranes and that the presence of monoacylated PrP^C disrupts lipid raft micro-domains that are essential for efficient PrP^Sc formation. Moreover, these results raise the possibility that targeting the GPI anchor attached to PrP^C may reveal novel therapeutics/treatments for prion diseases.     

Interaction of Peptide Aptamers with Prion Protein Central Domain Promotes α-Cleavage of PrP<Superscript>C</Superscript>

Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis or treatment exists. Prions are composed of the misfolded isoform PrP^Sc of the cellular prion protein PrP^C. Expression of PrP^C is a prerequisite for prion infection, and conformational conversion of PrP^C is induced upon its direct interaction with PrP^Sc. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrP^C as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrP^Sc replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrP^C α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrP^C at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aβ oligomers characteristic for Alzheimer’s disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrP^C and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.     

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

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

Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC

The mammalian prions replicate by converting cellular prion protein (PrP^C) into pathogenic conformational isoform (PrP^Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP^Sc on conversion of PrP^C in vitro using PrP^Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP^Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP^Sc. The tight correlation between conversion potency of small oligomers of human sPrP^Sc observed in vitro and duration of the disease suggests that sPrP^Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.