Phospholipase A2 inhibitors or platelet-activating factor antagonists prevent prion replication.

A key feature of prion diseases is the conversion of the cellular prion protein (PrP(C)) into disease-related isoforms (PrP(Sc)), 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 (PrP(res)) in three prion-infected cell lines (ScN2a, SMB, and ScGT1 cells). Daily treatment of these cells with phospholipase A(2) (PLA(2)) inhibitors for 7 days prevented the accumulation of PrP(res). Glucocorticoids with anti-PLA(2) activity also prevented the formation of PrP(res) and reduced the infectivity of SMB cells. Treatment with platelet-activating factor (PAF) antagonists also reduced the PrP(res) content of cells, while the addition of PAF reversed the inhibitory effect of PLA(2) inhibitors on PrP(res) formation. ScGT1 cells treated with PLA(2) 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 PLA(2) inhibitors or PAF antagonists reduced PrP(C) levels suggesting that limiting cellular PrP(C) may restrict prion formation in infected cells. These data indicate a pivotal role for PLA(2) and PAF in controlling PrP(res) formation and identify them as potential therapeutic agents.

Prion diseases, or transmissible spongiform encephalopathies (TSEs), 1 are fatal neurodegenerative disorders that include Kuru, Creutzfeldt-Jakob disease (CJD), and Gerstman-Strä ussler-Scheinker (GSS) disease in man. Central to the pathogenesis of TSEs is the conversion of the host-encoded cellular prion protein (PrP C ) into ␤-sheet-rich disease-related isoforms (PrP Sc ) (1). The formation of PrP Sc is accompanied by changes in biological and biochemical properties such as an increased resistance to proteases (2), the protease-resistant core of PrP Sc designated PrP res . This PrP Sc self-aggregates and forms amyloidgenic fibrils and, in most prion diseases, aggre-gates of PrP Sc 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 PrP Sc fibrils leads to neurodegeneration and the clinical symptoms of prion diseases. Many compounds that interact directly with PrP to prevent PrP Sc formation and/or disrupt preformed PrP Sc aggregates have now been identified; these include large, flat multicyclic compounds and synthetic peptides specifically designed to disrupt the ␤-sheets in PrP Sc (4 -6). However, recent studies demonstrated that the propagation of PrP Sc within prion-infected cells could be reduced following re-routing the trafficking of PrP C following treatment with suramin (7). Other studies have also shown that restricting the supply, or alterations in the trafficking, of PrP C can prevent the formation of PrP Sc (8 -11). In the present study we tested the hypothesis that the trafficking of PrP C within cells, that is vital to PrP Sc formation, is controlled by activation of specific signaling pathways. Previous studies have variously reported that PrP C is associated with activation of the tyrosine kinases Fyn (12), with the cyclic AMP/protein kinase A pathway (13), or with the phospholipase A 2 (PLA 2 )/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 PrP C in non-infected cells, and PrP Sc in scrapie-infected neuroblastoma cell lines (ScN2a, ScGT1, or SMB cells). These studies indicate that activation of PLA 2 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 PrP Sc .

EXPERIMENTAL PROCEDURES
PrP res Production-Scrapie-infected neuroblastoma cells (ScN2a cells; gift from Dr. M. Rogers, University College, Dublin, Ireland) that produce PrP Sc and infectious agent, were grown in Hams F12 medium containing 2 mM glutamine, standard antibiotics (100 units/ml penicillin and 100 g/ml streptomycin) and 2% fetal calf serum. SMB cells (TSE Resource Centre, Institute for Animal Health, Compton, UK), which also produce PrP Sc 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 PrP res , were grown in Optimem supplemented with 2 mM glutamine, 5% fetal calf serum, and standard antibiotics. To measure the effect of drugs on PrP res formation, cells were plated at 1 ϫ 10 5 cells/well in 6-well microtiter plates in the presence or absence of drugs. Cells were then grown with daily changes of media and PrP res 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 ϫ 10 6 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 ϫ 10 6 cell equivalents/ml. Mice under halothane anesthesia were injected intracerebrally with 30 l (7.5 ϫ 10 4 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 PrP res 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 ϫ 10 7 cells/ml. Samples were sonicated at 4°C for 10 min, and cellular debris was removed by centrifugation at 5,000 ϫ g for 1 min. The supernatant was digested with proteinase K at 10 g/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 ϫ g for 4 h at 4°C; the pellet was dissolved in 50 l of Laemmli buffer (Bio-Rad), boiled for 5 min and 20 l 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. PrP res 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 PrP C 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 IgGalkaline 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), AA-COCF 3 , aristolochic acid, bromoenol lactone (BEL), and neomycin sulfate were obtained from Sigma. Cytidine Prostaglandin (PG)E 2 Assay-Analysis of cellular PGE 2 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 PGE 2 in the sample and a fixed amount of labeled PGE 2 for a PGE 2 -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.

PLA 2 Inhibitors Reduce the PrP res Content of Three Prioninfected 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 PrP res content of ScN2a cells. ScN2a cells treated daily for 7 days with one of four different PLA 2 inhibitors (1 M CDP, 1 g/ml aristolochic acid, 1 M BEL, or 1 g/ml AACOCF 3 ) contained significantly less PrP res than did untreated cells. In contrast, the levels of PrP res 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 PLA 2 inhibitors on PrP res production, two other prion-infected neuroblastoma cell lines (SMB and ScGT1 cells) were also treated with these drugs. The PrP res content of SMB or ScGT1 cells, treated with CDP, aristolochic acid, BEL, or AACOCF 3 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).

inhibitors reduced the PrP res content of prion-infected cell lines
To measure the effect of drugs on PrP res formation, ScN2a, SMB, or ScGT1 cells were plated at 1 ϫ 10 5 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 PrP res pg/1 ϫ 10 7 cells Ϯ S.D. of triplicate experiments repeated three times (9 observations a PrP res content of cells significantly less (p Ͻ 0.05) than that of untreated cells.

Signal Transduction and Prion Replication
Corticosteroids Reduce the PrP res Content of Prion-infected Cell Lines-In the present study, ScN2a, SMB, or ScGT1 cells treated with 1 M dexamethasone, 1 M hydrocortisone, or 1 M prednisolone contained undetectable amounts of PrP res , whereas cells treated with 1 M prednisone, an inactive prodrug that is converted to active prednisolone in the liver, did not affect PrP res levels (Table II). In further studies, the inhibitory effects of dexamethasone on PrP res 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 PrP res (data not shown).
The effects of 1 M dexamethasone on ScN2a cells were not immediate as levels of PrP res in treated cells were not significantly different from control cells after 24 h (1009 pg/ml Ϯ 48 in treated cells versus 1112 pg/ml Ϯ 97 in untreated cells, n ϭ 8 independent observations, mean PrP res Ϯ S.D.) but they were significantly reduced after 48 h (561 pg/ml Ϯ 116 versus 1129 pg/ml Ϯ 76, n ϭ 8, p Ͻ 0.05) and further reduced after 72 h (66 pg/ml Ϯ 55 versus 1076 pg/ml Ϯ 77, n ϭ 8, p Ͻ 0.05). We were unable to detect PrP res in cells that had been treated for 4 days or more with 1 M dexamethasone. When ScN2a cells that had been treated with 1 M dexamethasone for 7 days were then grown for a further 12 weeks in drug-free medium these cells remained clear of detectable amounts of PrP res (Ͻ50 pg/ml). Similarly, SMB or ScGT1 that had been treated with 1 M dexamethasone for 7 days remained clear of detectable amounts of PrP res when grown in drug-free medium for 12 weeks.
PLA 2 Inhibitors Reduce PGE 2 Production in Prion-infected Cells-PrP peptides increase PLA 2 activity resulting in the production of PGE 2 (14). In the present study the levels of PGE 2 were significantly raised in prion-infected cells when compared with their non-infected counterparts, which suggests that prion infection activates PLA 2 pathways in neurons. Prion-infected cells treated with 1 M CDP, 1 g/ml AACOCF 3 , or 1 M dexamethasone produced significantly less PGE 2 than did untreated cells showing that drug treatment did indeed inhibit PLA 2 (Fig. 3).
PAF Antagonists Block PrP res Formation-The effects of downstream pathways following PLA 2 activation on the formation of PrP res were investigated. Arachidonic acid, released from membrane phospholipids by PLA 2 , is converted to leucotrienes and prostaglandins by the lipoxygenase (LOX) and COX enzymes respectively. Because the PrP res content of ScN2a, SMB or ScGT1 cells was not affected by treatment with the LOX or COX inhibitors, other factors produced following PLA 2 activation were therefore examined. PAF is generated in neurons by the remodeling pathway following PLA 2 activation (16) and the PrP res 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 PrP res content of ScN2a cells (Fig. 4), and there was a highly significant relationship between the level of PrP res and the concentration of PAF antagonist (p Ͻ 0.05) for all 4 drugs. We noted that at each concentration, PrP res levels were greater in cells treated with gingkolide A compared with gingkolide B (p Ͻ 0.05). Furthermore, the PrP res content of ScN2a cells was reduced to below detectable levels following 7 days of treatment with 2 M PAF antagonists (Ͻ50pg/1 ϫ 10 7 cells). Treatment with the PAF antagonists also caused a dose-dependent reduction in the PrP res content of SMB and ScGT1 cells. Furthermore, ScN2a, SMB, or ScGT1 cells treated with 2 M hexa-PAF, or with 2 M ginkgolide B, for 7 days remained free of detectable PrP res when grown in drug-free medium for a further 12 weeks (data not shown).
PAF Increases PrP res Formation-To compliment the PAF antagonist studies, prion-infected cells were grown in medium containing PAF agonists. The PrP res content of cells treated with PAF agonists (2 M PAF or 2 M 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 PrP res content of all cell types, the increase in ScN2a cells was only 30%. PAF agonist had a greater effect on SMB cells, nearly doubling PrP res content, and, on ScGT1 cells, the PrP res content was increased 2-3-fold.
To determine if the addition of PAF could restore PrP res production to prion-infected cells treated with PLA 2 inhibitors, ScN2a cells were treated with a mixture containing PLA 2 inhibitors (1 M CDP, 1 g/ml AACOCF 3 , or 1 M dexamethasone) and 2 M PAF. The addition of PAF was able to restore PrP res levels in ScN2a cells co-treated with PLA 2 inhibitors or dexamethasone (Fig. 6).
PAF Antagonists Reduce PrP C Levels in Non-infected Cells-Since the production of PrP res is dependent on the presence of PrP C , the effect of PLA 2 inhibitors, PAF antagonists or PAF on PrP C levels in non-infected cells was investigated. The PrP C content of untreated N2a cells (33.2 Ϯ 2.9 ng/1 ϫ 10 7 cells) was significantly higher than that of cells treated with PLA 2 inhibitors (1 M CDP: 12.8 Ϯ 3.2, n ϭ 9, p Ͻ 0.05; 1 g/ml AACOCF 3

Signal Transduction and Prion Replication
ginkgolide B (0.4 Ϯ 0.8, n ϭ 9, p Ͻ 0.05). All PAF antagonists used caused a dose-dependent reduction in the levels of PrP C in N2a cells, and there was a highly significant relationship between PAF antagonist concentration and PrP C levels (p Ͻ 0.05). In addition, the type of ginkgolide used had a significant effect on PrP C levels (p Ͻ 0.05) with cells treated with ginkgolide A containing more PrP C than cells treated with the same concentration of ginkgolide B (Fig. 7). Time course studies showed that PrP C 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 PrP C levels returning to normal within 24 h. Conversely, the PrP C content of N2a cells treated with PAF agonists, 2 M PAF (74.6 Ϯ 4.8, n ϭ 9, p Ͻ 0.05), or 2 M C-PAF (58.9 Ϯ 3.8, n ϭ 9, p Ͻ 0.05) was significantly higher than that of untreated N2a cells. The PrP C formed in N2a cells treated with PAF agonists remained sensitive to digestion with proteinase K. 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 PLA 2 . 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 Ϯ 8 days) (incubation period Ϯ S.D.) was significantly longer than in mice inoculated with lysates from untreated SMB cells (179 Ϯ 6 days; n ϭ 8 mice in each group, p Ͻ 0.05).

DISCUSSION
In the present study we utilized a pharmacological approach to determine the metabolic pathways that underlie the formation of PrP res 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 PLA 2 (aristolochic acid, AACOCF 3 , BEL, and CDP) reduced the PrP res content of prion-infected cells. The concentrations of the PLA 2 inhibitors used were at least 10 times less than the concentration of these drugs that had a toxic effect and treatment with PLA 2 inhibitors did not affect total cellular protein levels. 2 We confirmed that the drugs used inhibited PLA 2 by measuring levels of PGE 2 (a marker of PLA 2 activity). In the present study prion-infected cells treated with CDP, aristolochic acid or AACOCF 3 produced significantly less PGE 2 than untreated cells. It is of interest to note that none of the drugs completely inhibited PLA 2 activity, possibly because there exist several distinct enzymes with PLA 2 activity including cytosolic (cPLA 2 ) and secretory (sPLA 2 ) isozymes (19). Although aristolochic acid and CDP inhibit both cPLA 2 and sPLA 2 , low concentrations of AACOCF 3 or BEL, which are reported to selectively inhibit cPLA 2 (20), inhibited PrP res formation (Table  I) indicating that cPLA 2 may be the isozyme of interest. PLA 2 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 PrP res content, whereas the inactive precursor prednisone had no effect. The effect of dexamethasone was dose-dependent, and PrP res was reduced to below detectable levels at nanomolar concentrations of dexamethasone. A significant effect on PrP res content was not seen until 2 days after the commencement of treatment with dexamethasone, and cells were not clear of PrP res until 4 days after treatment. Nevertheless, ScN2a cells that had been treated with 1 M dexamethasone for 7 days remained free of detectable PrP res 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 PLA 2 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 PrP res content of cells indirectly. Although the activation of PLA 2 is functionally associated with the production of prostaglandins the PrP res 2 C. Bate and A. Williams, unpublished data. content of cells was not affected by treatment with inhibitors of either COX or LOX. The activation of PLA 2 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 cellspecific stimuli (15) and in this study four different PAF antagonists all reduced the PrP res content of ScN2a, ScGT1, or SMB cells. The effects of PAF antagonists were dose-dependent with an IC 50 ϳ50 nM, and at a concentration of 2 M two PAF antagonists (hexa-PAF and ginkgolide B) were able to reduce PrP res to below detectable levels. The finding in the present study that ginkgolide B had a greater effect on PrP res 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 PrP res 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 PrP res content seen in ScGT1 cells than in SMB cells and both showing greater effects than the ScN2a cells. Secondly, the addition of PAF restored PrP res production in dexamethasone or CDP-treated ScN2a cells. Collectively, these results suggest that the effect of dexamethasone or the PLA 2 inhibitors on PrP res formation is mediated via a reduction in PAF formation.
The observation that PrP C is essential for the development of prion diseases (25) suggests that the density and cellular location of PrP C may influence PrP res production. Both the PLA 2 inhibitors and the PAF antagonists reduced cellular PrP C levels indicating that these drugs may prevent the formation of PrP res by limiting the supply of the PrP C substrate. Ginkgolide B, a more potent PAF antagonist than ginkgolide A (24), had a greater effect on PrP C levels in N2a cells than ginkgolide A. In contrast, PAF agonists increased cellular PrP C levels, further indicating the importance of PAF in controlling PrP C expression. The PrP C in PAF treated cells remained sensitive to proteinase K digestion, unlike PrP C species induced in N2a cells treated with proteasome inhibitors (26). The regulation of PrP C expression is poorly understood, previous studies have shown that in neuronal cell lines PrP C expression was increased after treatment with insulin, nerve growth factor, epidermal growth factor, or tumor necrosis factor ␣ (27,28).
There are a number of possible mechanisms for the exact manner by which PAF antagonists could affect PrP res formation. PrP C 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 PrP res (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 PrP res 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 PrP C to PrP Sc is thought to occur after PrP C has reached the plasma membrane and subsequently been re-internalized for degradation (36 -38). These observations raise the possibility that the activation of PLA 2 seen in prion infected cells and the production of PAF may encourage the formation of PrP res by enhancing propitious trafficking and sorting pathways. In some cell lines PAF antagonists prevent endocytosis (39), while in other studies, cPLA 2 inhibitors (AA-COCF 3 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 PrP C (43), these observations suggest that treatment of neurons with PLA 2 inhibitors or PAF antagonists may inhibit PrP res formation by altering the intracellular trafficking of PrP C .
Currently, the development of therapeutic strategies to combat prion disease is largely based on the identification of drugs that bind to and disrupt aggregated PrP Sc . This strategy is based on the belief that PrP Sc is a major, if not the only, component of the infectious agent (44), and that the formation of fibrillar aggregates of PrP Sc leads to neurodegeneration. Thus, it is thought that inhibiting PrP Sc formation, or disrupting pre-formed PrP Sc , will prevent the establishment of disease. The data presented here support the view that PLA 2 and PAF regulate the formation of PrP res , 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 PrP res content at nanomolar concentrations. Both PLA 2 inhibitors and PAF antagonists caused a rapid reduction in the PrP C content of N2a cells. Thus, the effects of PLA 2 inhibitors and PAF antagonists on PrP res formation may result from reducing the supply of PrP C to sites conducive to conversion of PrP C to PrP res . While PrP res formation is undoubtedly a complex process, these observations provide insight into the signaling processes that initiate the formation of PrP res 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 PrP Sc itself.