1 Department of Veterinary Pathology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, UK
2 Department of Pathology and Infectious Diseases, Royal Veterinary College, Hawkshead Lane, North Mymms, Herts AL9 7TA, UK
Correspondence
Clive Bate
c.bate{at}vet.gla.ac.uk
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ABSTRACT |
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INTRODUCTION |
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The cellular mechanisms leading to neuronal death can be studied in tissue-culture systems to determine the functional significance of in vivo observations. Thus, neuronal culture systems developed to investigate interactions between prions and neurons have demonstrated that synthetic peptides derived from the prion protein are neurotoxic (Forloni et al., 1993; Salmona et al., 2003
), provided that the peptides contain substantial
-sheet content (Hope et al., 1996
). One of the important events leading to the degeneration of cultured neurons following the addition of toxic PrP peptides is the activation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid to prostaglandins (PGs) by the cyclo-oxygenases (COXs) (Bate et al., 2004b
). This observation is consistent with observations that levels of PGE2 are raised in brain areas showing neuronal death in murine scrapie (Williams et al., 1994
, 1997b
) and that raised levels of PGE2 are detected in the cerebrospinal fluid of patients with CJD (Minghetti et al., 2000
, 2002
). However, little is known about the processes by which prions or neurotoxic PrP peptides activate PLA2.
Cellular PrPc is required for the process by which PrP peptides induce apoptosis (Brown et al., 1994), suggesting that there are specific interactions between PrP peptides, PrPc, PLA2 and apoptotic pathways. Most PrPc molecules are linked to membranes via a glycosylphosphatidylinositol (GPI) anchor (Stahl et al., 1992
); the presence of the GPI anchor affects the properties of PrPc (Taraboulos et al., 1995
). GPI anchors contain a conserved core that consists of ethanolamine phosphate in an amide linkage to the carboxyl terminus of the protein, three mannose residues, glucosamine and phosphatidylinositol (Mayor & Riezman, 2004
). However, many variations on this core structure are possible and the GPIs isolated from PrPc in hamster brains contain high amounts of galactose, mannose and sialic acid (Stahl et al., 1992
). We therefore investigated the ability of GPIs extracted from both PrPc and PrPSc, obtained from uninfected and infected cells of a murine neuroblastoma cell line, and GPIs extracted from Thy-1 to activate PLA2, as measured by the induction of PGE2. We also examined the ability of GPIs from these sources to induce caspase-3 activity in primary cultures of cortical neurons, as this enzyme is known to be involved in apoptotic cell death. To determine which moiety of the GPI molecules was responsible for the effects observed in each case, the inhibitory effect of some GPI-related molecules on the induction of PGE2 and on the neuronal toxicity of PrP peptides was investigated.
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METHODS |
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Primary neuronal cultures.
Primary cortical neurons were prepared from the brains of mouse embryos as described previously (Bate et al., 2002) and plated in 48-well plates at 2x106 cells per well. Cultures were pretreated with test compounds for 3 h before the addition of PrP peptides. Caspase-3 activity was measured 24 h later by using a fluorometric immunosorbent enzyme assay kit according to the manufacturer's instructions (Roche Diagnostics). For cell-survival assays, microglia [prepared by dissociating the cerebral cortices of newborn mice, as described previously (Bate et al., 2002
)] were added to peptide-treated neurons in the ratio of 1 microglial cell : 10 neurons. After 4 days, microglia were removed by shaking (260 r.p.m for 30 min) and survival of neurons was determined by treating cultures with WST-1.
Prion peptides.
The toxic peptide HuPrP82146, containing aa 82146 of the human prion protein found in GerstmannSträusslerScheinker disease (Salmona et al., 2003), and the synthetic miniprion sPrP106 derived from the murine PrP sequence were synthesized by solid-phase chemistry and purified by reverse-phase HPLC (Bonetto et al., 2002
).
Prion preparations.
PrP molecules resistant to limited protease digestion (10 µg proteinase K ml1 for 1 h at 37 °C) were partially purified by reverse-phase chromatography on a C18 Sep-Pak column (Waters) and quantified by a PrP-specific ELISA, as described previously (Bate et al., 2004a).
Reagents.
Phosphatidylinositol, inositol monophosphate, inositol, inositol-1,4-bisphosphate, inositol-1,4,5-triphosphate, arachidonic acid, platelet-activating factor (PAF), hydrogen peroxide, mannose, glucosamine, galactose, sialic acid (N-acetylneuraminic acid) and staurosporine were obtained from Sigma.
Isolation of GPI anchors.
GPIs were isolated from uninfected N2a cells that were lysed in water, passed through a 26-gauge needle to solubilize cellular debris and centrifuged (10 min at 14 000 g). Insoluble material was suspended in a buffer containing 10 mM Tris/HCl, 100 mM NaCl, 10 mM EDTA, 0·5 % Nonidet P-40, 0·5 % sodium deoxycholate and 2 mM PMSF, pH 7·2. PrPc was immunoprecipitated following the addition of mAb SAF53 (a gift from Professor J. Grassi, CEA, Saclay, France) and protein Gagarose (Sigma). A sample of immunoprecipitated PrPc was retained for analysis by Western blot. The depleted lysate was subsequently incubated with an anti-Thy-1 mAb (Serotech) and protein Gagarose and immunoprecipitated. Precipitates were washed five times with PBS containing 0·02 % Tween 20, suspended in PBS containing 100 µg proteinase K ml1 and digested at 37 °C for 24 h to release GPIs. Insoluble material was collected by centrifugation at 14 000 g and the pellet was washed five times with water. The released GPIs were extracted with water-saturated butan-1-ol, washed with water, split into two and lyophilized. One sample was dissolved in ethanol at 2 µg ml1 and applied to silica gel 60 high-performance TLC (HPTLC) plates (Whatman) for analysis; the other sample was dissolved in tissue-culture medium for bioassays. Controls were prepared by incubating mAb SAF53 in buffer (in the absence of cellular lysates) and protein Gagarose. Control preparations were treated as above.
Isolation of PrPSc-GPI.
GPIs were also isolated from prion-infected ScN2a cells that were lysed in water and treated as described above before solubilization in extraction buffer that did not contain PMSF. As ScN2a cells contain both PrPc and PrPSc, cell lysates were predigested with 10 µg proteinase K ml1 for 1 h at 37 °C to remove PrPc. Digestion was stopped with 5 mM PMSF and protease-resistant PrPSc was immunoprecipitated with mAb SAF53 and protein Gagarose. After extensive washing, immunoprecipitated PrPSc was split into two samples. One sample was retained for Western blot analysis, whilst the other was further digested with 100 µg proteinase K ml1 at 37 °C for 24 h to release the GPIs. GPIs were subsequently extracted with water-saturated butan-1-ol, as described above.
Western blotting.
Samples were dissolved in 50 µl Laemmli buffer (Bio-Rad), boiled and subjected to electrophoresis on a 15 % polyacrylamide gel. Proteins were transferred onto a Hybond-P PVDF membrane (Amersham Biosciences) by semi-dry blotting. Membranes were blocked by using 10 % milk powder in Tris-buffered saline, pH 7·2, containing 0·2 % Tween 20. PrP was detected by incubation with mAb SAF53, followed by a secondary anti-mouse IgG conjugated to peroxidase. Bound antibody was visualized by using an enhanced chemiluminescence kit (Amersham Biosciences).
TLC immunoblotting.
Extracted GPIs were examined by HPTLC on silica gel 60 HPTLC plates by using a mixture of choloroform/methanol/water (10 : 10 : 2·5 by volume). Plates were soaked in 0·1 % poly(isobutylmethacrylate) in hexane, dried and blocked with PBS containing 5 % milk powder. They were probed with 1 µg mAb 5AB3-11 ml1 [which binds to phosphatidylinositol (Bate & Kwiatkowski, 1994)], washed with PBS/Tween and incubated with goat anti-mouse IgG conjugated to peroxidase (Sigma) for 1 h. Bound antibody was washed and visualized by using an enhanced chemiluminescence kit (Amersham Biosciences).
Chemical manipulation of GPIs.
To remove acyl chains from GPIs (deacylation), extracted GPIs (2 µg ml1) were treated with 0·05 M NaOH at 60 °C for 2 h and the reaction mixture was then neutralized. Extracted GPIs (2 µg ml1) were also deaminated by treatment with a mixture containing 0·1 M sodium acetate, pH 3·8, and 0·5 M sodium nitrite (NaNO2) at room temperature for 24 h, after which time the reaction mixture was neutralized.
Statistical analysis.
Results were compared by using one- and two-way analysis of variance techniques as appropriate. Statistical significance was set at the 1 % level.
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RESULTS |
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Inhibition of caspase-3 production in neurons
Caspase-3 activity was measured in primary cortical neurons as an indication of apoptosis. Caspase-3 activity was increased significantly above that of untreated cells following 24 h incubation with 10 µM sPrP106, 10 µM HuPrP82146 or 20 ng PrP-GPI ml1, but not in cells incubated with 20 ng Thy-1-GPI ml1 or 100 µM partial GPIs. Pretreating PrPc-GPI with 1 µg mAb 5AB3-11 ml1 reduced the caspase-3 activity response to GPIs, but caspase-3 activity in neurons pretreated with sPrP106 or HuPrP82146 was not affected (Table 4). Competition experiments showed that caspase-3 activity in response to sPrP106, HuPrP82146 or PrP-GPI was reduced significantly in neurons that had been pretreated with inositol monophosphate or sialic acid, but was not affected by pretreatment with galactose, glucosamine, inositol or mannose.
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DISCUSSION |
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The studies reported here provide indications of the structures required of the GPI for biological activity. Thus, the compounds generated following deamination, which releases phosphatidylinositol from the glycan component of GPI, did not stimulate PGE2 production. Deacylated GPIs failed to run on HPTLC, indicating a hydrophilic compound consistent with the removal of acyl chains. Deacylated GPIs also failed to stimulate PGE2 production, demonstrating that the ester-linked acyl chains on the phosphatidylinositol are required for biological activity. In addition, mAb studies demonstrated that a phosphatidylinositol moiety of GPIs is essential for biological activity. The effects of mAb 5AB3-11 were specific in that it did not affect PGE2 produced by cells treated with arachidonic acid, indicating that mAb 5AB3-11 did not affect COX. Such observations suggest that the biological activity of GPI anchors requires both phosphatidylinositol and another, unspecified glycan component.
Little is known about the process by which prions or PrP peptides activate neuronal PLA2. One possibility is that high concentrations of GPI anchors bind directly to PLA2. In these assays, high concentrations of GPI anchors were required to activate PLA2, perhaps mimicking the high concentration of GPI anchors that occurs following the aggregation of PrPSc molecules or the cross-linkage of PrPc by specific mAbs (Solforosi et al., 2004) or by PrP peptides. The clustering of GPI-anchored proteins is thought to occur in specific membrane microdomains that are known as lipid rafts (Mayor & Riezman, 2004
), which are essential for the activation of PLA2 and the toxicity of prions (Bate et al., 2004a
). The results presented here are consistent with exogenous GPI anchors inserting into membranes and trafficking to lipid rafts, where high concentrations activate PLA2. It is of interest to note that, although the addition of exogenous GPI anchors activated PLA2 and increased caspase-3 activity in neurons, it did not cause neuronal death. It remains to be seen whether prion-induced neuronal death requires additional, non-GPI signals that are inherent in the prion protein structure or whether the lack of neuronal death in response to GPI anchors is simply a concentration effect.
The neurotoxicity of PrP peptides or prion preparations was reduced by pretreatment with some compounds that are common to all GPI anchors. Initial studies showed that whilst phosphatidylinositol, inositol monophosphate and sialic acid reduced the neurotoxicity of sPrP106 and prion preparations, other components of GPI anchors, namely galactose, glucosamine and mannose, had no effect. The presence of a single phosphate on the inositol ring was essential, as inositol alone and inositols containing more than one phosphate did not affect neurotoxicity. Whilst the protective effects of phosphatidylinositol and inositol monophosphate on sPrP106-induced neurotoxicity were evident at micromolar concentrations, the protective effect of sialic acid was only observed at higher concentrations.
The toxicity of prions or PrP peptides involves the activation of neuronal PLA2 and the production of bioactive second messengers, including arachidonic acid and PAF (Bate et al., 2004b). Treatment of neurons with inositol monophosphate or sialic acid did not affect the toxicity of arachidonic acid or PAF, indicating that these compounds prevent the formation, rather than the action, of such neurotoxins. The production of PGE2 that is associated closely with PrP-induced neurotoxicity is a two-stage process that requires the release of arachidonic acid by PLA2 and the conversion of arachidonic acid to PGs by the COX enzymes. The addition of inositol monophosphate or sialic acid reduced PGE2 production in response to HuPrP82146, sPrP106 and PrP-GPIs, but did not affect PGE2 production in response to arachidonic acid, showing that these compounds had no direct effect on COX. In regard to prion-induced toxicity, these observations identify PLA2 as the target of inositol monophosphate and sialic acid, a result that is compatible with previous reports that sialic acid inhibits PLA2 (Yang et al., 1994
).
The presence of inositol monophosphate or sialic acid greatly reduced microglial killing of neurons damaged by sPrP106. Microglia respond to changes in neurons that are induced by PrP peptides (Bate et al., 2002) and our data are consistent with the concept that the presence of inositol monophosphate or sialic acid prevents the PrP-induced neuronal changes that activate microglia. An alternative explanation, i.e. that inositol monophosphate and sialic acid have a direct effect on microglia, was discounted, as these compounds did not affect the production of interleukin 6 from microglia incubated with lipopolysaccharide (unpublished data).
To our knowledge, this is the first report to demonstrate that high concentrations of PrP-GPIs result in activation of PLA2 and neuronal apoptotic pathways (caspase-3). The activity of GPIs was dependent on a phosphatidylinositol moiety, on ester-linked acyl chains and on an unspecified glycan component. There were no obvious physical differences between GPIs isolated from PrPc or PrPSc, nor any significant differences in their biological activity. We propose that the high concentrations of GPIs added here mimic the locally high concentrations of GPIs that occur when PrPc molecules cluster following the addition of PrP peptides, or when the GPI anchors are concentrated following the aggregation of PrPSc molecules. Pretreatment with some partial GPIs, including inositol monophosphate or sialic acid, protected neurons against the toxicity of PrP peptides, sPrP106 and prion preparations. These partial GPIs prevented the activation of PLA2, rather than inhibiting neurotoxins generated following PLA2 activation. Inositol monophosphate and sialic acid also reduced the HuPrP82146- or sPrP106-induced activation of apoptotic pathways in cortical neurons and prevented HuPrP82146- or sPrP106-treated neurons from activating microglia, resulting in increased neuronal survival. The present results are compatible with the hypothesis that inositol monophosphate and sialic acid compete with the complete GPI anchors of PrPc or PrPSc for cellular receptors and prevent PLA2 activation. Whilst neuronal death in response to prions in vivo is undoubtedly a complex process that may include other mechanisms, these observations provide insight into the signalling processes that result in prion-induced neuronal loss.
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ACKNOWLEDGEMENTS |
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Received 15 June 2004;
accepted 30 August 2004.
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