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.     

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.     

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.     

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.     

Glimepiride Reduces the Expression of PrPC,Prevents PrPSc Formation and Protects against Prion Mediated Neurotoxicity

Background

A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrP^C) into a disease related, alternatively folded isoform (PrP^Sc). The accumulation of PrP^Sc within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases.

Principal Findings

Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrP^C from the surface of prion-infected neuronal cells. The cell surface is a site where PrP^C molecules may be converted to PrP^Sc and glimepiride treatment reduced PrP^Sc formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82–146. Glimepiride treatment significantly reduce both the amount of PrP82–146 that bound to neurones and PrP82–146 induced activation of cytoplasmic phospholipase A₂ (cPLA₂) and the production of prostaglandin E₂ that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrP^C expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC.

Conclusions

Collectively, these results indicate that glimepiride may be a novel treatment to reduce PrP^Sc formation and neuronal damage in prion diseases.     

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.     

Role of Lipid Rafts and GM1 in the Segregation and Processing of Prion Protein

The prion protein (PrP^C) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrP^C is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrP^C raft association, together with raft lipid composition, appears essential for the conversion of PrP^C into the scrapie isoform PrP^Sc, and the development of prion disease. Controversial findings were reported on the nature of PrP^C-containing rafts, as well as on the distribution of PrP^C between rafts and non-raft membranes. We investigated PrP^C/ganglioside relationships and their influence on PrP^C localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrP^C conformations coexist: in lipid rafts PrP^C is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrP^Sc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrP^C conformation and interaction with lipid bilayers, without modifying PrP^C distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.     

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.     

Clustering of sialylated glycosylphosphatidylinositol anchors mediates PrP-induced activation of cytoplasmic phospholipase A2 and synapse damage

Precisely how the accumulation of PrP^Sc causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrP^Sc oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrP^C, either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A₂ (cPLA₂). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA₂ and synapse damage induced by cross-linked PrP^C. We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA₂ and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrP^C, suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases.     

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.     

Clustering of sialylated glycosylphosphatidylinositol anchors mediates PrP-induced activation of cytoplasmic phospholipase A2 and synapse damage

Precisely how the accumulation of PrP^Sc causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrP^Sc oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrP^C, either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A₂ (cPLA₂). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA₂ and synapse damage induced by cross-linked PrP^C. We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA₂ and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrP^C, suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases.     

Generation of antisera to purified prions in lipid rafts

Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrP^C into alternately folded PrP^Sc. Immunoassays toward pre-clinical detection of infectious PrP^Sc have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrP^Sc selective antibodies. We report a method to purify infectious PrP^Sc from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrP^Sc was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM >20-fold and purified DRM immunogen >40-fold) relative to 1% crude brain homogenate. Purification of PrP^Sc from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrP^Sc antigen was performed using wild-type (wt) and Prnp^0/0 mice, both on Balb/cJ background. A robust immune response against PrP^Sc was observed in all inoculated Prnp^0/0 mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrP^C and PrP^Sc, while binding to other brain-derived protein was not observed. In contrast, the PrP^Sc inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.Key words: prion, scrapie, Prnp0/0 mice, purification methodology, antibody, antisera, lipid-rafts, detergent resistant membranes, neuroscience, immunization, diagnostic     

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

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

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.     

PrP(106-126) does not interact with membranes under physiological conditions

Transmissible spongiform encephalopathies are neurodegenerative diseases characterized by the accumulation of an abnormal isoform of the prion protein PrP^Sc. Its fragment 106-126 has been reported to maintain most of the pathological features of PrP^Sc, and a role in neurodegeneration has been proposed based on the modulation of membrane properties and channel formation. The ability of PrP^Sc to modulate membranes and/or form channels in membranes has not been clearly demonstrated; however, if these processes are important, peptide-membrane interactions would be a key feature in the toxicity of PrP^Sc. In this work, the interaction of PrP(106-126) with model membranes comprising typical lipid identities, as well as more specialized lipids such as phosphatidylserine and GM1 ganglioside, was examined using surface plasmon resonance and fluorescence methodologies. This comprehensive study examines different parameters relevant to characterization of peptide-membrane interactions, including membrane charge, viscosity, lipid composition, pH, and ionic strength. We report that PrP(106-126) has a low affinity for lipid membranes under physiological conditions without evidence of membrane disturbances. Membrane insertion and leakage occur only under conditions in which strong electrostatic interactions operate. These results support the hypothesis that the physiological prion protein PrP^C mediates PrP(106-126) toxic effects in neuronal cells.     

Live imaging of prions reveals nascent PrPSc in cell-surface,raft-associated amyloid strings and webs

Mammalian prions refold host glycosylphosphatidylinositol-anchored PrP^C into β-sheet–rich PrP^Sc. PrP^Sc is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrP^Sc rather than on its truncated PrP27-30 product. We show that N-terminal PrP^Sc epitopes are exposed in their physiological context and visualize, for the first time, PrP^Sc in living cells. PrP^Sc resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrP^Sc amyloids.     

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

During prion infection, the normal, protease-sensitive conformation of prion protein (PrP^C) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP^Sc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP^Sc in prion-infected brain homogenates as an initiating seed to convert PrP^C and trigger the self-propagation of PrP^Sc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP^Sc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP^Sc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP^Sc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.     

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.     

Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds,Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate,Chlorpromazine, and U18666A,in Prion-Infected Mouse Neuroblastoma Cells

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP^Sc) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP^Sc formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP^C) and PrP^Sc in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP^Sc levels without altering intracellular distribution of PrP^Sc. PPS did not change the distribution and levels of PrP^C, whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP^C to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP^Sc formation. In contrast, CPZ and U18666A initiated the redistribution of PrP^Sc from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP^C. The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP^Sc redistribution by CPZ or U18666A partly antagonized PrP^Sc degradation, suggesting that the transfer of PrP^Sc to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP^Sc degradation. This study revealed that precise analysis of the intracellular dynamics of PrP^C and PrP^Sc provides important information for understanding the mechanism of anti-prion agents.     

Lipopolysaccharide induced conversion of recombinant prion protein

The conformational conversion of the cellular prion protein (PrP^C) to the β-rich infectious isoform PrP^Sc is considered a critical and central feature in prion pathology. Although PrP^Sc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrP^C to proteinase K resistant PrP^Sc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrP^C conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrP^C to PrP^Sc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232).