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Originally published In Press as doi:10.1074/jbc.M404086200 on June 21, 2004

J. Biol. Chem., Vol. 279, Issue 35, 36405-36411, August 27, 2004
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Phospholipase A2 Inhibitors or Platelet-activating Factor Antagonists Prevent Prion Replication*

Clive Bate{ddagger}§, Stuart Reid¶||, and Alun Williams**

From the {ddagger}Department of Veterinary Pathology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, United Kingdom, the Comparative Epidemiology and Informatics, Department of Veterinary Clinical Studies, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, United Kingdom, the ||Department of Statistics and Modeling Sciences, University of Strathclyde, Livingston Tower, Glasgow, G1 1XW United Kingdom, and the **Department of Pathology and Infectious Diseases, Royal Veterinary College, Hawkshead Lane, North Mymms, Herts, AL9 7TA United Kingdom

Received for publication, April 13, 2004 , and in revised form, June 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A key feature of prion diseases is the conversion of the cellular prion protein (PrPC) into disease-related isoforms (PrPSc), the deposition of which is thought to lead to neurodegeneration. In this study a pharmacological approach was used to determine the metabolic pathways involved in the formation of protease-resistant PrP (PrPres) in three prion-infected cell lines (ScN2a, SMB, and ScGT1 cells). Daily treatment of these cells with phospholipase A2 (PLA2) inhibitors for 7 days prevented the accumulation of PrPres. Glucocorticoids with anti-PLA2 activity also prevented the formation of PrPres and reduced the infectivity of SMB cells. Treatment with platelet-activating factor (PAF) antagonists also reduced the PrPres content of cells, while the addition of PAF reversed the inhibitory effect of PLA2 inhibitors on PrPres formation. ScGT1 cells treated with PLA2 inhibitors or PAF antagonists for 7 days remained clear of detectable PrPres when grown in control medium for a further 12 weeks. Treatment of non-infected cells with PLA2 inhibitors or PAF antagonists reduced PrPC levels suggesting that limiting cellular PrPC may restrict prion formation in infected cells. These data indicate a pivotal role for PLA2 and PAF in controlling PrPres formation and identify them as potential therapeutic agents.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prion diseases, or transmissible spongiform encephalopathies (TSEs),1 are fatal neurodegenerative disorders that include Kuru, Creutzfeldt-Jakob disease (CJD), and Gerstman-Sträussler-Scheinker (GSS) disease in man. Central to the pathogenesis of TSEs is the conversion of the host-encoded cellular prion protein (PrPC) into {beta}-sheet-rich disease-related isoforms (PrPSc) (1). The formation of PrPSc is accompanied by changes in biological and biochemical properties such as an increased resistance to proteases (2), the protease-resistant core of PrPSc designated PrPres. This PrPSc self-aggregates and forms amyloidgenic fibrils and, in most prion diseases, aggregates of PrPSc are detected in the diseased brain before neuronal loss is observed (3).

The development of current therapeutic strategies is largely based on the belief that the deposition of amyloidgenic PrPSc fibrils leads to neurodegeneration and the clinical symptoms of prion diseases. Many compounds that interact directly with PrP to prevent PrPSc formation and/or disrupt preformed PrPSc aggregates have now been identified; these include large, flat multicyclic compounds and synthetic peptides specifically designed to disrupt the {beta}-sheets in PrPSc (4–6). However, recent studies demonstrated that the propagation of PrPSc within prion-infected cells could be reduced following re-routing the trafficking of PrPC following treatment with suramin (7). Other studies have also shown that restricting the supply, or alterations in the trafficking, of PrPC can prevent the formation of PrPSc (8–11). In the present study we tested the hypothesis that the trafficking of PrPC within cells, that is vital to PrPSc formation, is controlled by activation of specific signaling pathways. Previous studies have variously reported that PrPC is associated with activation of the tyrosine kinases Fyn (12), with the cyclic AMP/protein kinase A pathway (13), or with the phospholipase A2 (PLA2)/cyclo-oxygenase (COX) pathway (14). Thus, in this study, a pharmacological approach was used to investigate the role of signal transduction mechanisms on levels of PrPC in non-infected cells, and PrPSc in scrapie-infected neuroblastoma cell lines (ScN2a, ScGT1, or SMB cells). These studies indicate that activation of PLA2 and the production of platelet-activating factor (PAF) (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a bioactive phospholipid that is not stored in a preformed state (15) but rapidly synthesized in neurons in response to cell specific stimuli via the remodeling pathway (16), are essential factors in the production of PrPSc.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
PrPres Production—Scrapie-infected neuroblastoma cells (ScN2a cells; gift from Dr. M. Rogers, University College, Dublin, Ireland) that produce PrPSc and infectious agent, were grown in Hams F12 medium containing 2 mM glutamine, standard antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) and 2% fetal calf serum. SMB cells (TSE Resource Centre, Institute for Animal Health, Compton, UK), which also produce PrPSc and infectious agent, were grown in RPMI 1640 medium containing standard antibiotics, 2 mM glutamine and 2% fetal calf serum. ScGT1 cells (supplied by Dr. Sylvain Lehmann, CNRS-IGH, Montpellier, France), an immortalized murine hypothalamic neuronal cell line infected by the scrapie Chandler isolate and that persistently expresses PrPres, were grown in Optimem supplemented with 2 mM glutamine, 5% fetal calf serum, and standard antibiotics. To measure the effect of drugs on PrPres formation, cells were plated at 1 x 105 cells/well in 6-well microtiter plates in the presence or absence of drugs. Cells were then grown with daily changes of media and PrPres production was evaluated after 7 days. Non-infected N2a cells or SMB cells that had been "cured" of infectivity by serial passages in the presence of pentosan polysulphate (PS cells) (17) were used as controls. For time course experiments, ScN2a cells were plated at 5 x 106 cells/well in the presence or absence of dexamethasone and collected after 24, 48, or 72 h. At the end of the treatment, cells were detached and counted to establish cell numbers.

Evaluation of Infectivity—To challenge mice directly, cultured SMB cells were detached and counted, washed twice with phosphate-buffered saline then put through one rapid freeze-thaw cycle. The homogenate was precipitated, washed twice with phosphate-buffered saline, and finally homogenized in sterile 0.9% (w/v) saline at 2.5 x 106 cell equivalents/ml. Mice under halothane anesthesia were injected intracerebrally with 30 µl (7.5 x 104 cell equivalents) of this homogenate. Mice were monitored for clinical signs of scrapie until reaching a predefined clinical end point. All animal work was conducted strictly according to local and national guidelines.

Cell Lysates—Lysates were made from ScN2a, SMB, or ScGT1 cells to evaluate PrPres content. Cells were detached and counted, washed twice in PBS and finally suspended in an extraction buffer containing 10 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate at 1 x 107 cells/ml. Samples were sonicated at 4 °C for 10 min, and cellular debris was removed by centrifugation at 5,000 x g for 1 min. The supernatant was digested with proteinase K at 10 µg/ml for 1 h at 37 °C, digestion was blocked with 5 mM phenylmethylsulfonyl fluoride, and samples were then halved; one half was tested for PrP by an enzyme-linked immunosorbent assay (ELISA; see below) and the other examined by PrP Western blot. This second sample was centrifuged at 50,000 x g for 4 h at 4 °C; the pellet was dissolved in 50 µl of Laemmli buffer (Bio-Rad), boiled for 5 min and 20 µl subjected to electrophoresis on a 15% polyacrylamide gel. Proteins were transferred onto a Hybond-P polyvinylidene difluoride membrane (Amersham Biosciences) by semidry blotting. Membranes were blocked using 10% milk powder in Tris-buffered saline containing 0.2% Tween 20. PrPres was detected by incubation with mAb SAF83 (a gift from J. Grassi, CEA, Saclay, France) for 1 h at room temperature, followed by a secondary anti-mouse IgG conjugated to peroxidase (1 h at room temperature). Detection of bound antibody was visualized using an enhanced chemiluminescence kit (Amersham Biosciences). Lysates were also made from the non-infected N2a cells to evaluate PrPC content. Cells were treated as above except that proteinase K digestion was excluded.

PrP ELISA—PrP in lysates was measured using a PrP-specific ELISA as previously described (18). Briefly, Nunc Maxisorb Immunoplates were coated with antibodies isolated from rabbit antiserum raised to the ovine PrP100-111 peptide conjugated to keyhole limpet hemocyanin (gift from Dr. J. P. M. Langeveld, Central Institute for Animal Disease Control, Lelystad, The Netherlands). Cell lysates were applied, and specific binding was detected by mAb SAF83 (gift from Prof. J. Grassi, CEA, Saclay, France), followed by an anti-mouse IgG-alkaline phosphatase conjugate (Sigma) and an appropriate indicator. Results were calculated by reference to a standard curve of recombinant murine PrP (Prionics, Zurich, Switzerland). The detection limit of this assay is 50 pg/ml.

Drugs—Dexamethasone, prednisolone, prednisone, hydrocortisone, ibuprofen, acetyl salicylic acid, nordihydroguaiaretic acid (NDGA), AACOCF3, aristolochic acid, bromoenol lactone (BEL), and neomycin sulfate were obtained from Sigma. Cytidine-5-diphosphocholine (CDP), caffeic acid, 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine (PAF), and 1-O-alkyl-2-acetyl-sn-glycerol-3-phospho-(N,N,N-trimethyl)-hexanolamine (hexa-PAF) were obtained from Novabiochem (Nottingham, UK). C-PAF, CV-6209, SQ-22536, U73122 [GenBank] , and ethyl-18-OCH3 were obtained from Biomol (Exeter, UK).

Prostaglandin (PG)E2 Assay—Analysis of cellular PGE2 levels was determined in cells by using an enzyme immunoassay kit (Amersham Biosciences) according to the manufacturer's instructions. This assay is based on competition between unlabeled PGE2 in the sample and a fixed amount of labeled PGE2 for a PGE2-specific antibody. The detection limit of this assay is 20 pg/ml.

Statistical Analysis—Comparison of treatment effects were carried out using one and two way analysis of variance techniques as appropriate. Post-hoc comparisons of means were performed as necessary. For all statistical tests, significance was set at the 5% level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PLA2 Inhibitors Reduce the PrPres Content of Three Prion-infected Cell Lines—In an initial screening experiment, the effects of drugs that inhibit some of the common signal transduction pathways were investigated for their effects on the PrPres content of ScN2a cells. ScN2a cells treated daily for 7 days with one of four different PLA2 inhibitors (1 µM CDP, 1 µg/ml aristolochic acid, 1 µM BEL, or 1 µg/ml AACOCF3) contained significantly less PrPres than did untreated cells. In contrast, the levels of PrPres in ScGT1 cells were not significantly affected by treatment with three inhibitors of phospholipase C (Table I and Fig. 1). To confirm the effects of PLA2 inhibitors on PrPres production, two other prion-infected neuroblastoma cell lines (SMB and ScGT1 cells) were also treated with these drugs. The PrPres content of SMB or ScGT1 cells, treated with CDP, aristolochic acid, BEL, or AACOCF3 was also greatly reduced. Even at concentrations 10 times higher than those used in these experiments, the drugs used did not alter cell survival or cell growth (data not shown).


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  		TABLE I
PLA2 inhibitors reduced the PrPres content of prion-infected cell lines

To measure the effect of drugs on PrPres formation, ScN2a, SMB, or ScGT1 cells were plated at 1 x 105 cells/well in 6-well plates. Cells were grown for 7 days, with daily changes of medium, in the presence or absence of the drugs shown. The levels of protease-resistant PrP in cellular lysates were then determined using an ELISA. Values shown are the mean PrPres pg/1 x 107 cells ± S.D. of triplicate experiments repeated three times (9 observations).

 



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  		FIG. 1.
PLA2 inhibitors reduce the PrPres content of ScGT1 cells. ScGT1 cells were grown for 7 days in the presence of control medium (lane 1), 1 µg/ml AACOCF3 (lane 2), 0.5 µg/ml aristolochic acid (lane 3), 1 µM CDP (lane 4), or 10 µM ethyl-18-OCH3 (lane 5). Cellular lysates were digested with 10 µg/ml proteinase K for 1 h at 37 °C and protease-resistant PrP was visualized by immunoblot with mAb SAF83 using enhanced chemiluminescence.

 
Corticosteroids Reduce the PrPres Content of Prion-infected Cell Lines—In the present study, ScN2a, SMB, or ScGT1 cells treated with 1 µM dexamethasone, 1 µM hydrocortisone, or 1 µM prednisolone contained undetectable amounts of PrPres, whereas cells treated with 1 µM prednisone, an inactive prodrug that is converted to active prednisolone in the liver, did not affect PrPres levels (Table II). In further studies, the inhibitory effects of dexamethasone on PrPres content of ScN2a cells was shown to be dose-dependent (Fig. 2). SMB and ScGT1 cells treated with dexamethasone also demonstrated a dose-dependent reduction in PrPres (data not shown).


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  		TABLE II
Glucocorticoids reduce the PrPres content of prion-infected cell lines

The effects of glucocorticoids on PrPres formation were determined by plating ScN2a, SMB or ScGT1 cells at 1 x 105 cells/well in 6 well plates. Cells were grown for 7 days in the presence of the 1 µM glucocorticoids as shown. The levels of protease-resistant PrP in cellular lysates were then determined using an ELISA. Values shown are the mean PrPres pg/1 x 107 cells ± S.D. of quadruplicate experiments repeated three times (12 observations).

 



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  		FIG. 2.
Dexamethasone causes a dose-dependent reduction in the PrPres content of ScN2a cells. To determine the efficacy of dexamethasone, ScN2a cells were grown for 7 days in the presence of different concentrations of dexamethasone as shown. The levels of protease-resistant PrP in cellular lysates were then determined using an ELISA. Values shown are the mean PrPres pg/1 x 107 cells ± S.D. of quadruplicate experiments repeated three times (12 observations).

 
The effects of 1 µM dexamethasone on ScN2a cells were not immediate as levels of PrPres in treated cells were not significantly different from control cells after 24 h (1009 pg/ml ± 48 in treated cells versus 1112 pg/ml ± 97 in untreated cells, n = 8 independent observations, mean PrPres± S.D.) but they were significantly reduced after 48 h (561 pg/ml ± 116 versus 1129 pg/ml ± 76, n = 8, p < 0.05) and further reduced after 72 h (66 pg/ml ± 55 versus 1076 pg/ml ± 77, n = 8, p < 0.05). We were unable to detect PrPres in cells that had been treated for 4 days or more with 1 µM dexamethasone. When ScN2a cells that had been treated with 1 µM dexamethasone for 7 days were then grown for a further 12 weeks in drug-free medium these cells remained clear of detectable amounts of PrPres (<50 pg/ml). Similarly, SMB or ScGT1 that had been treated with 1 µM dexamethasone for 7 days remained clear of detectable amounts of PrPres when grown in drug-free medium for 12 weeks.

PLA2 Inhibitors Reduce PGE2 Production in Prion-infected Cells—PrP peptides increase PLA2 activity resulting in the production of PGE2 (14). In the present study the levels of PGE2 were significantly raised in prion-infected cells when compared with their non-infected counterparts, which suggests that prion infection activates PLA2 pathways in neurons. Prion-infected cells treated with 1 µM CDP, 1 µg/ml AACOCF3, or 1 µM dexamethasone produced significantly less PGE2 than did untreated cells showing that drug treatment did indeed inhibit PLA2 (Fig. 3).



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  		FIG. 3.
PLA2 inhibitors reduced PGE2 production in prion-infected cells. ScN2a cells (shaded bars), SMB cells (open bars), or ScGT1 cells (striped bars) were incubated in control medium (Con), 1 µg/ml AACOCF3 (AA), 1 µM CDP (CDP), or 1 µM dexamethasone (DXM). Levels of PGE2 were measured after 24 h. Values shown are the mean level of PGE2 pg/ml ± S.D. produced during quadruplicate experiments repeated three times (12 observations).

 
PAF Antagonists Block PrPres Formation—The effects of downstream pathways following PLA2 activation on the formation of PrPres were investigated. Arachidonic acid, released from membrane phospholipids by PLA2, is converted to leucotrienes and prostaglandins by the lipoxygenase (LOX) and COX enzymes respectively. Because the PrPres content of ScN2a, SMB or ScGT1 cells was not affected by treatment with the LOX or COX inhibitors, other factors produced following PLA2 activation were therefore examined. PAF is generated in neurons by the remodeling pathway following PLA2 activation (16) and the PrPres content of cells was reduced following treatment with the PAF antagonists hexa-PAF, CV-6209, ginkgolide A, or ginkgolide B (Table III). These PAF antagonists did not affect the survival, or growth rates of prion-infected cells. All four PAF antagonists caused a dose-dependent reduction in the PrPres content of ScN2a cells (Fig. 4), and there was a highly significant relationship between the level of PrPres and the concentration of PAF antagonist (p < 0.05) for all 4 drugs. We noted that at each concentration, PrPres levels were greater in cells treated with gingkolide A compared with gingkolide B (p < 0.05). Furthermore, the PrPres content of ScN2a cells was reduced to below detectable levels following 7 days of treatment with 2 µM PAF antagonists (<50pg/1 x 107 cells). Treatment with the PAF antagonists also caused a dose-dependent reduction in the PrPres content of SMB and ScGT1 cells. Furthermore, ScN2a, SMB, or ScGT1 cells treated with 2 µM hexa-PAF, or with 2 µM ginkgolide B, for 7 days remained free of detectable PrPres when grown in drug-free medium for a further 12 weeks (data not shown).


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  		TABLE III
PAF antagonists prevent PrPres formation in prion-infected cells

ScN2a, SMB, or ScGT1 cells were grown for 7 days, with daily changes of medium, in the presence of control medium, in media supplemented with COX inhibitors (1 µM acetyl salicylic acid (aspirin) or 1 µM ibuprofen), with LOX inhibitors (5 µM NDGA or 5 µM caffeic acid), or with PAF antagonists (1 µM hexa-PAF, 1 µM CV-6209, 1 µM ginkgolide A or 1 µM ginkgolide B). The levels of protease-resistant PrP in cellular lysates were then determined using an ELISA. Values shown are the mean PrPres pg/1 x 107 cells ± S.D. of triplicate experiments repeated three times (9 observations).

 



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  		FIG. 4.
PAF antagonists cause a dose-dependent reduction in the PrPres content of prion-infected cells. ScN2a cells were grown in the presence of different concentrations of PAF antagonists: hexa-PAF (open circles), CV-6209 (closed circles), ginkgolide A (open squares), or ginkgolide B (closed squares) for 7 days. The levels of protease-resistant PrP were subsequently determined in an ELISA. Values shown are the mean PrPres pg/1 x 107 cells ± S.D. of triplicate experiments repeated three times (9 observations).

 
PAF Increases PrPres Formation—To compliment the PAF antagonist studies, prion-infected cells were grown in medium containing PAF agonists. The PrPres content of cells treated with PAF agonists (2 µM PAF or 2 µM C-PAF) were significantly higher than untreated cells (Table IV and Fig. 5). The PAF agonists had no effect on the survival, growth rates, or protein concentration of ScN2a, SMB, or ScGT1 cells. While PAF caused an increase in the PrPres content of all cell types, the increase in ScN2a cells was only 30%. PAF agonist had a greater effect on SMB cells, nearly doubling PrPres content, and, on ScGT1 cells, the PrPres content was increased 2–3-fold. To determine if the addition of PAF could restore PrPres production to prion-infected cells treated with PLA2 inhibitors, ScN2a cells were treated with a mixture containing PLA2 inhibitors (1 µM CDP, 1 µg/ml AACOCF3, or 1 µM dexamethasone) and 2 µM PAF. The addition of PAF was able to restore PrPres levels in ScN2a cells co-treated with PLA2 inhibitors or dexamethasone (Fig. 6).


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  		TABLE IV
PAF agonists increase the PrPres prion-infected cells

Incubating ScN2a, SMB, or ScGT1 cells with PAF agonists showed the effect of PAF agonists on the formation of PrPres. Cells were grown for 7 days, with daily changes of medium, in the presence of control medium, or in media supplemented with PAF agonists (2 µM PAF or 2 µM C-PAF). The levels of protease-resistant PrP in cellular lysates were then determined using an ELISA. Values shown are the mean PrPres pg/ml ± S.D. of triplicate experiments repeated 4 times (n = 12).

 



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  		FIG. 5.
PAF increases the PrPres content of ScN2a and ScGT1 cells. ScN2a or ScGT1 cells were grown for 7 days in the presence of control medium (lanes 3 and 4), 2 µM C-PAF (lanes 2 and 5), or 2 µM PAF (lanes 1 and 6). Protease-resistant PrP was demonstrated by immunoblot with mAb SAF83.

 



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  		FIG. 6.
PAF reverses the inhibition of PrPres formation by PLA2 inhibitors. ScN2a cells were grown in control medium (Con), in 1 µM dexamethasone (DXM), 1 µM CDP, or 1 µg/ml aristolochic acid (AA) in the absence (shaded bars) or presence of 1 µM PAF (open bars). Values shown are the mean PrPres pg/1 x 107 cells ± S.D. of triplicate experiments repeated four times (n = 12).

 
PAF Antagonists Reduce PrPC Levels in Non-infected Cells— Since the production of PrPres is dependent on the presence of PrPC, the effect of PLA2 inhibitors, PAF antagonists or PAF on PrPC levels in non-infected cells was investigated. The PrPC content of untreated N2a cells (33.2 ± 2.9 ng/1 x 107 cells) was significantly higher than that of cells treated with PLA2 inhibitors (1 µM CDP: 12.8 ± 3.2, n = 9, p < 0.05; 1 µg/ml AACOCF3: 2.5 ± 2.1, n = 9, p < 0.05; or 1 µM dexamethasone: 8.4 ± 2.4, n = 9, p < 0.05), or with the PAF antagonists 2 µM hexa-PAF (1.1 ± 1.2, n = 9, p < 0.05), 1 µM CV-6209 (6.8 ± 1.4, n = 9, p < 0.05), 1 µM ginkgolide A (1.5 ± 1.7, n = 9, p < 0.05), or 1 µM ginkgolide B (0.4 ± 0.8, n = 9, p < 0.05). All PAF antagonists used caused a dose-dependent reduction in the levels of PrPC in N2a cells, and there was a highly significant relationship between PAF antagonist concentration and PrPC levels (p < 0.05). In addition, the type of ginkgolide used had a significant effect on PrPC levels (p < 0.05) with cells treated with ginkgolide A containing more PrPC than cells treated with the same concentration of ginkgolide B (Fig. 7). Time course studies showed that PrPC levels were reduced within 24 h and remained low in the presence of any of the PAF antagonists for up to 7 days. However, even after prolonged treatment (7 days), removal of the PAF antagonists resulted in PrPC levels returning to normal within 24 h. Conversely, the PrPC content of N2a cells treated with PAF agonists, 2 µM PAF (74.6 ± 4.8, n = 9, p < 0.05), or 2 µM C-PAF (58.9 ± 3.8, n = 9, p < 0.05) was significantly higher than that of untreated N2a cells. The PrPC formed in N2a cells treated with PAF agonists remained sensitive to digestion with proteinase K.



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  		FIG. 7.
PAF antagonists cause a dose-dependent reduction in the PrPC content of N2a cells. A, N2a cells were grown in the presence of different concentrations of PAF antagonists: hexa-PAF (open squares), CV-6209 (closed squares), ginkgolide A (open circles), or ginkgolide B (closed circles) for 24 h. The levels of PrPC/1 x 107 cells were subsequently determined in an ELISA. Values shown are the mean ± S.D. of quadruplicate experiments repeated two times (8 observations). B, N2a cells were grown in the presence of control medium (lane 1), 2 µM hexa-PAF (lane 2), 1 µM CV-6209 (lane 3), 1 µM ginkgolide B (lane 4), 2 µM PAF (lane 5), or 2 µM PAF (lane 6) after digestion with proteinase K. PrP was visualized by immunoblot with mAb SAF83 using enhanced chemiluminescence.

 
Dexamethasone Reduces the Infectivity of SMB Cells—Dexamethasone is a glucocorticoid widely used in medical practice. It has various mechanisms of action, including inhibition of PLA2. To determine if dexamethasone-treated SMB cells retained infectivity, C57/Bl mice were inoculated via the intracerebral route with homogenates from untreated SMB cells or SMB cells treated for 7 days with 200 nM dexamethasone. The mean incubation period in mice inoculated with lysates from dexamethasone-treated cells (206 ± 8 days) (incubation period ± S.D.) was significantly longer than in mice inoculated with lysates from untreated SMB cells (179 ± 6 days; n = 8 mice in each group, p < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we utilized a pharmacological approach to determine the metabolic pathways that underlie the formation of PrPres in three prion-infected neuroblastoma cell lines (ScN2a, ScGT1, and SMB cells). In a broad screen of compounds we found that 4 different drugs that inhibit PLA2 (aristolochic acid, AACOCF3, BEL, and CDP) reduced the PrPres content of prion-infected cells. The concentrations of the PLA2 inhibitors used were at least 10 times less than the concentration of these drugs that had a toxic effect and treatment with PLA2 inhibitors did not affect total cellular protein levels.2 We confirmed that the drugs used inhibited PLA2 by measuring levels of PGE2 (a marker of PLA2 activity). In the present study prion-infected cells treated with CDP, aristolochic acid or AACOCF3 produced significantly less PGE2 than untreated cells. It is of interest to note that none of the drugs completely inhibited PLA2 activity, possibly because there exist several distinct enzymes with PLA2 activity including cytosolic (cPLA2) and secretory (sPLA2) isozymes (19). Although aristolochic acid and CDP inhibit both cPLA2 and sPLA2, low concentrations of AACOCF3 or BEL, which are reported to selectively inhibit cPLA2 (20), inhibited PrPres formation (Table I) indicating that cPLA2 may be the isozyme of interest.

PLA2 can also be inhibited by the lipocortins, a family of proteins that are produced in response to the glucocorticoids (21). In the present study cells treated with the active glucocorticoids: dexamethasone, hydrocortisone, and prednisolone showed a reduced PrPres content, whereas the inactive precursor prednisone had no effect. The effect of dexamethasone was dose-dependent, and PrPres was reduced to below detectable levels at nanomolar concentrations of dexamethasone. A significant effect on PrPres content was not seen until 2 days after the commencement of treatment with dexamethasone, and cells were not clear of PrPres until 4 days after treatment. Nevertheless, ScN2a cells that had been treated with 1 µM dexamethasone for 7 days remained free of detectable PrPres when grown in drug-free medium for a further 12 weeks. Our in vivo observations showed that SMB cells treated with 200 nM dexamethasone for 7 days contained reduced levels of infectivity. Such observations are consistent with previous reports that transient steroid administration immediately postinfection reduced the susceptibility of mice to scrapie after peripheral challenge (22). However, the use of glucocorticoids in prion diseases should be treated with caution due to the observation that chronic administration of glucocorticoids can itself lead to neuronal atrophy (23).

Since PLA2 and many of its metabolites play important roles in signal transduction, it is possible that altered levels of second messengers could cause the decrease in the PrPres content of cells indirectly. Although the activation of PLA2 is functionally associated with the production of prostaglandins the PrPres content of cells was not affected by treatment with inhibitors of either COX or LOX. The activation of PLA2 also leads to the synthesis of the bioactive phospholipid PAF in neurons via the remodeling pathway (16). PAF is not stored in a preformed state, but rather is rapidly synthesized in response to cell-specific stimuli (15) and in this study four different PAF antagonists all reduced the PrPres content of ScN2a, ScGT1, or SMB cells. The effects of PAF antagonists were dose-dependent with an IC50 ~50 nM, and at a concentration of 2 µM two PAF antagonists (hexa-PAF and ginkgolide B) were able to reduce PrPres to below detectable levels. The finding in the present study that ginkgolide B had a greater effect on PrPres formation than ginkgolide A is consistent with previous reports that ginkgolide B a more potent PAF antagonist than ginkgolide A (24). The role of PAF in prion replication was supported by two further complementary studies. Firstly, the addition of PAF agonists (PAF or C-PAF) increased the production of PrPres in all 3 prion-infected cell lines without affecting total cellular protein concentrations. The magnitude of the effects of the PAF agonists were cell type-dependent, with a greater increase in PrPres content seen in ScGT1 cells than in SMB cells and both showing greater effects than the ScN2a cells. Secondly, the addition of PAF restored PrPres production in dexamethasone or CDP-treated ScN2a cells. Collectively, these results suggest that the effect of dexamethasone or the PLA2 inhibitors on PrPres formation is mediated via a reduction in PAF formation.

The observation that PrPC is essential for the development of prion diseases (25) suggests that the density and cellular location of PrPC may influence PrPres production. Both the PLA2 inhibitors and the PAF antagonists reduced cellular PrPC levels indicating that these drugs may prevent the formation of PrPres by limiting the supply of the PrPC substrate. Ginkgolide B, a more potent PAF antagonist than ginkgolide A (24), had a greater effect on PrPC levels in N2a cells than ginkgolide A. In contrast, PAF agonists increased cellular PrPC levels, further indicating the importance of PAF in controlling PrPC expression. The PrPC in PAF treated cells remained sensitive to proteinase K digestion, unlike PrPC species induced in N2a cells treated with proteasome inhibitors (26). The regulation of PrPC expression is poorly understood, previous studies have shown that in neuronal cell lines PrPC expression was increased after treatment with insulin, nerve growth factor, epidermal growth factor, or tumor necrosis factor {alpha} (27, 28).

There are a number of possible mechanisms for the exact manner by which PAF antagonists could affect PrPres formation. PrPC is found in lipid rafts or caveolae (29), specialized membrane compartments that contain high levels of cholesterol and sphingomyelin (30). Since the formation of these lipid rafts is cholesterol-dependent (31), and drugs that affect cholesterol levels influence the formation of PrPres (8, 18), it is possible that PAF may regulate the composition and hence the function of lipid rafts. In this respect it should be noted that PAF induces sphingomyelinase which itself has been shown to increase the formation of PrPres in ScN2a cells (32). PAF has been demonstrated to increase sterol synthesis (34) and to inhibit cholesterol esterification (33), while PAF antagonists inhibit cholesterol biosynthesis from lanosterol (35). Collectively, these data suggest that PAF may be involved in the maintenance of cholesterol-dependent lipid rafts.

The conversion of PrPC to PrPSc is thought to occur after PrPC has reached the plasma membrane and subsequently been re-internalized for degradation (36–38). These observations raise the possibility that the activation of PLA2 seen in prion infected cells and the production of PAF may encourage the formation of PrPres by enhancing propitious trafficking and sorting pathways. In some cell lines PAF antagonists prevent endocytosis (39), while in other studies, cPLA2 inhibitors (AACOCF3 or BEL) prevent the maintenance of the Golgi network (40), endosome fusion, and endocytosis (41), and modulate the intracellular trafficking of some proteins (42). Together with the observation that the Golgi and the endosomal compartments are involved in the trafficking of a GFP-tagged PrPC (43), these observations suggest that treatment of neurons with PLA2 inhibitors or PAF antagonists may inhibit PrPres formation by altering the intracellular trafficking of PrPC.

Currently, the development of therapeutic strategies to combat prion disease is largely based on the identification of drugs that bind to and disrupt aggregated PrPSc. This strategy is based on the belief that PrPSc is a major, if not the only, component of the infectious agent (44), and that the formation of fibrillar aggregates of PrPSc leads to neurodegeneration. Thus, it is thought that inhibiting PrPSc formation, or disrupting pre-formed PrPSc, will prevent the establishment of disease. The data presented here support the view that PLA2 and PAF regulate the formation of PrPres, and thus presumably the propagation of infectious prions since dexamethasone-treated SMB cells showed reduced levels of infectivity. The effects of the PAF antagonists were dose-dependent and caused a 50% reduction in PrPres content at nanomolar concentrations. Both PLA2 inhibitors and PAF antagonists caused a rapid reduction in the PrPC content of N2a cells. Thus, the effects of PLA2 inhibitors and PAF antagonists on PrPres formation may result from reducing the supply of PrPC to sites conducive to conversion of PrPC to PrPres. While PrPres formation is undoubtedly a complex process, these observations provide insight into the signaling processes that initiate the formation of PrPres and presumably prions. We therefore propose that PAF antagonists may have a role in preventing neurodegeneration in prion diseases when used in combination with drugs targeted at the structure of PrPSc itself.


   FOOTNOTES
 
* This work was supported by the European Commission (Contract QLK3-CT-2001-00283). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 0141-330-2874; Fax: 0141-330-5602; E-mail: c.bate{at}vet.gla.ac.uk.

1 The abbreviations used are: TSE, transmissible spongiform encephalopathies; PAF, platelet-activating factor; CDP, cytidine-5-diphosphocholine; PrP, prion protein; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; PLA2, ase; phospholipase A2; COX, cyclo-oxygenhexa-PAF, 1-O-alkyl-2-acetyl-sn-glycerol-3-phospho-(N,N,N-trimethyl)-hexanolamine; PG, prostaglandin. Back

2 C. Bate and A. Williams, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. R. Veerhuis and Prof. P. Eikelenboom (Vrije Universiteit University Medical Center, Amsterdam, The Netherlands) for helpful discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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