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Originally published In Press as doi:10.1074/jbc.M313061200 on January 29, 2004

J. Biol. Chem., Vol. 279, Issue 15, 14983-14990, April 9, 2004
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Squalestatin Cures Prion-infected Neurons and Protects Against Prion Neurotoxicity*

Clive Bate{ddagger}§, Mario Salmona¶, Luisa Diomede¶, and Alun Williams||

From the {ddagger}Institute of Comparative Medicine, Department of Veterinary Pathology, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, United Kingdom, the Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche, Mario Negri, Via Eritrea 62, 20157 Milano, Italy, and the ||Department of Pathology and Infectious Diseases, Royal Veterinary College, Hawkshead Lane, North Mymms, Herts AL9 7TA, United Kingdom

Received for publication, December 1, 2003 , and in revised form, January 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A key feature of prion diseases is the conversion of the normal, cellular prion protein (PrPC) into {beta}-sheet-rich disease-related isoforms (PrPSc), the deposition of which is thought to lead to neurodegeneration. In the present study, the squalene synthase inhibitor squalestatin reduced the cholesterol content of cells and prevented the accumulation of PrPSc in three prion-infected cell lines (ScN2a, SMB, and ScGT1 cells). ScN2a cells treated with squalestatin were also protected against microglia-mediated killing. Treatment of neurons with squalestatin resulted in a redistribution of PrPC away from Triton X-100 insoluble lipid rafts. These effects of squalestatin were dose-dependent, were evident at nanomolar concentrations, and were partially reversed by cholesterol. In addition, uninfected neurons treated with squalestatin became resistant to the otherwise toxic effect of PrP peptides, a synthetic miniprion (sPrP106) or partially purified prion preparations. The protective effect of squalestatin, which was reversed by the addition of water-soluble cholesterol, correlated with a reduction in prostaglandin E2 production that is associated with neuronal injury in prion disease. These studies indicate a pivotal role for cholesterol-sensitive processes in controlling PrPSc formation, and in the activation of signaling pathways associated with PrP-induced neuronal death.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prion diseases, or Transmissible Spongiform Encephalopathies (TSEs), are fatal neurodegenerative disorders that include Kuru, Creutzfeldt-Jakob disease (CJD), and Gerstman-Straussler-Scheinker (GSS) syndrome in man. These diseases are associated with the deposition of aggregates of disease-related isoforms (PrPSc) of a host-encoded protein (PrPC) within the central nervous system (1). During disease, a portion of the {alpha}-helix and random coil structure in PrPC is refolded into a {beta}-pleated sheet in PrPSc (2), a conformational change that renders PrPSc poorly soluble in water and resistant to protease digestion, the resultant protease-resistant PrP being designated PrPres (3). Consequently, aggregates of PrPSc accumulate around neurons in affected brain areas (4), a process which is thought to lead to neuronal dysfunction and death, and subsequently the clinical symptoms of infection.

The observation that cellular PrPC is essential for the development of prion diseases (5–7) suggests that the density and cellular location of PrPC in neurons may influence the production of PrPSc. PrPC is found in lipid rafts or caveolae-like domains (CLDs)1 (8), specialized membrane compartments that contain high levels of cholesterol and sphingomyelin (9). The formation of these lipid rafts is cholesterol-dependent (10), and drugs that affect cholesterol levels have been shown to influence the formation of PrPSc. Thus, the formation of PrPSc in prion-infected neuroblastoma cells (ScN2a cells) was reduced when cells were treated with lovastatin (11). Lovastatin is a competitive inhibitor of 3-hydroxy-3-methylglutaryl-co-enzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol production (12) and HMG-CoA reductase inhibitors are useful clinical tools used to treat hypercholesterolemia (13) or ischemic heart disease (14–15) in man. However, although HMG-CoA reductase inhibitors reduce cholesterol levels, they exhibit many other effects, for example, they also inhibit the production of isoprenoid precursors (16). Recent studies suggest that many of the effects of HMG-CoA reductase inhibitors are independent of their effects on cholesterol metabolism (17–19). In the present study we investigated the effects of squalestatin, a specific inhibitor of squalene synthase that inhibits cholesterol production without affecting the production of non-sterol products (20) (Fig. 1), on the production of PrPres in three prion-infected cell lines, and on the levels and distribution of PrPC in non-infected cells.



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  		FIG. 1.
The metabolic pathway leading to cholesterol production. The biochemical pathway that leads to the production of cholesterol indicating the sites of action of the HMG-CoA reductase inhibitor (lovastatin) and the squalene synthase inhibitor (squalestatin) is shown (17).

 
Although the accumulation of aggregated PrPSc in the CNS is a distinguishing feature of prion diseases (21), the precise mechanisms by which PrPSc deposition leads to neuronal damage remain to be fully determined. The processes of neuronal loss have been examined by incubating neuronal cells with either prion preparations (22) or with peptides derived from the protease-resistant core of PrPSc (23). In the present study we used two peptides derived from the human PrP protein, HuPrP(82–146), a synthetic equivalent of a PrPSc fragment present in the brains of patients with GSS (24) and the truncated, neurotoxic version HuPrP-(106–126) (23), and a synthetic miniprion (sPrP106) (25) to investigate if cholesterol-sensitive microdomains are required for the processes by which prions kill infected neurons. We also examined the effects of cholesterol manipulation on neuronal prostaglandin (PG)E2 production since levels of neuronal PGE2 are closely associated with PrP-induced neuronal death (26).

Microglial activation frequently co-localizes with the accumulation of PrPSc in the CNS (4). In vitro, microglia secrete cytokines in response to PrP peptides (27) or when incubated with prion-infected neurons (28). Moreover, the addition of microglia to neurons treated with prions or PrP peptides results in a significant further reduction in neuronal survival, which is greater than that seen on exposure of neurons to PrP peptides only (29, 30). These observations suggest a significant role of microglia in neuronal loss during prion diseases and support interpretations drawn from time course studies of experimental prion disease in rodent models (1, 30). Thus, we investigated the effect of squalestatin on a second model of prion-induced neurodegeneration that involves the interaction between microglia and prion-infected neuroblastoma cells or PrP-damaged neurons.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
PrPres Production—Scrapie-infected neuroblastoma cells (ScN2a cells) were a gift of Dr. M. Rogers (University College, Dublin, Ireland). These cells produce PrPSc and infectious agent, and were grown in Hams F12 medium containing 2 mM glutamine, standard antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), 2% fetal calf serum (FCS) and 200 nM retinoic acid. 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, 200 nM retinoic acid, and 2% FCS. ScGT1 cells are an immortalized murine hypothalamic neuronal cell line infected by the scrapie Chandler strain and persistently express PrPres and were supplied by Dr. Sylvain Lehmann (CNRS-IGH, Montpellier, France) and were grown in optimem supplemented with 2 mM glutamine, 200 nM retinoic acid, 5% FCS, and standard antibiotics. To measure the effect of squalestatin on the PrPres formation, ScN2a, SMB cells or ScGT1 cells were plated at 1 x 105 cells/well in 6-well microtiter plates in the presence or absence of squalestatin alone (0–100 nM) (GlaxoSmithKline, Stevenage, Herts, UK), 100 nM squalestatin plus 1–100 µg/ml cholesterol (either free cholesterol or water-soluble cholesterol complexed to methyl-{beta}-cyclodextrin)(Sigma), or 100 nM squalestatin plus 500 µM mevalonate (Sigma). Cells were then grown with daily changes of media and PrPres production was evaluated after 8 days. In some studies, ScN2a cells were grown in the serum-free neurobasal medium (NBM) (Invitrogen, Paisley, UK) supplemented with 2 mM glutamine and B27 components. Non-infected N2a cells or SMB cells that had been "cured" of infectivity by serial passages in the presence of pentosan polysulphate (PS cells) were used as controls. At the end of the treatment, cells were detached and counted to establish cell numbers. Washed cells were first lysed in distilled water and the insoluble pellet collected. The pellet was 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 and digested with 10 µg/ml proteinase K for 1 h at 37 °C. Digestion was stopped with 5 mM phenylmethanesulfonyl fluoride (PMSF) and the remaining supernatant was split in two. The first sample was tested in a PrP-specific enzyme-linked immunosorbent assay (ELISA). The 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). Samples were boiled for 5 min and 20 µl of each sample was 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 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 by the 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.

Solubility of PrPc in Triton X-100 —To determine the effect of squalestatin on the distribution of PrPC, uninfected murine N2a neuroblastoma cells were maintained in RPMI 1640 supplemented with 2 mM glutamine, standard antibiotics, and 2% FCS in the presence or absence of 100 nM squalestatin. After 3 days cells were lysed on ice at 1 x 107 cells/ml in either (a) an extraction buffer containing 10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and 5 mM PMSF or (b) a buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.8, and 5 mM PMSF. Soluble material was collected after centrifugation at 10,000 x g. The insoluble pellet was dissolved in extraction buffer (a) at 1 x 107 cells/ml, and all three supernatants were tested in a PrP ELISA.

Animals—129/Ola mice were housed at constant temperature (21 ± 1 °C) and relative humidity (60 ± 10%) and supplied ad libitum with water and a standard diet. Procedures involving animals and their care were conducted in accordance with national and international regulations (Animals (Scientific Procedures) Act, 1986, UK, and EEC Council Directive 86609, OJ L358, 1, 12 December 1987).

Primary Neuronal Cultures—Primary cortical neurons were prepared from embryonic day 15.5 mice as previously described (28). After 2 days, medium was changed to NBM containing B27 components, 2 mM glutamine and 5 µM cytosine arabinoside to prevent the proliferation of astroglia. Mature cultures were treated with 5 mM L-leucine methylester to reduce the number of contaminating microglia before further use (31). For experiments, cultures were preincubated for 3 h in media containing 100 nM squalestatin alone or a mixture of 100 nM squalestatin and 1–100 µg/ml water-soluble cholesterol before the addition of peptides (HuPrP-(82–146), HuPrP-(106–126), sPrP106, or a prion extract. After another 3 h, microglia were added to neuronal cultures in the ratio 1 microglia to 10 neurons. After 4 days, cultures were shaken and washed to remove non-adherent microglia, and neuronal viability was determined using the 3-[4,5 dimethylthiazol-2yl]-2,5 diphenyltetrazolium bromide (MTT) method.

Microglia—Microglia cultures were prepared from newborn 129/Ola mice as previously described (28). Isolated microglia was added to ScN2a cells or treated neuronal cells in the ratio of 1 microglia to 10 neuronal cells. In other studies microglia were added to 96-well flat-bottomed plates at 1 x 105 cells/well and allowed to adhere overnight. Microglia were subsequently treated with 100 nM squalestatin or a vehicle control for 3 h before the addition of 10 ng/ml lipopolysaccharide. After a further 24 h supernatants were collected and tested for IL-6.

Neurotoxicity of PrP Peptides or Prion Preparations—The human neuroblastoma SH-SY5Y cell line (European Collection of Cell Cultures) was grown in RPMI 1640 medium supplemented with 2 mM glutamine, standard antibiotics and 2% FCS. Cells were plated at 3 x 104 cells/well into 96-well microtiter plates and allowed to adhere overnight. The following day, cells were pretreated for 3 h either with 0–100 nM squalestatin alone or with 100 nM squalestatin plus 1–100 µg/ml of water-soluble cholesterol or with 100 nM squalestatin plus 500 µM of mevalonate, before the addition of peptides, sPrP106 or a diluted prion extract. Squalestatin stock solutions were made in di-methyl sulfoxide (Me2SO) and diluted appropriately. Cell viability was determined 24 h later using the MTT method (32). Optical density was measured at 595 nm, and results calculated by reference to untreated cells. PGE2 levels were also determined at this time point.

Microglia-mediated Toxicity—To investigate the effect of squalestatin on microglia-mediated toxicity, ScN2a cells were plated at 1 x 105 cells/well in 24-well plates and allowed to adhere overnight. Cells were cultured in the presence or absence of 100 nM squalestatin alone, or in a mixture of 100 nM squalestatin and 1–100 µg/ml water-soluble cholesterol for a further 24 h after which microglia were added in the ratio 1 microglia to 10 ScN2a cells. The survival of ScN2a cells was measured after cells had been co-cultured for 24 h using the MTT method. N2a neuroblastoma cells were used as non-infected controls.

PrP Peptides—For studies using human neuroblastoma (SH-SY5Y) cells, peptides containing amino acids corresponding to residues 106–126 of the human prion protein (HuPrP-(106–126)) (23) and a peptide consisting of the same amino acids in a scrambled order (HuPrP-(106–126scrambled)) were synthesized by solid-phase chemistry and purified by reverse-phase HPLC. A longer peptide containing amino acids corresponding to residues 82–146 of the human prion protein (HuPrP-(82–146)) (24) was also used, as was a control peptide containing amino acid residues 82–146 in a scrambled sequence (HuPrP-(82–146scrambled)). A synthetic miniprion (sPrP106) derived from the murine PrP sequence was used for studies on murine cortical neurons (25).

Prion Extract Preparations—Partially purified prion extracts were obtained from ScGT1 cells to reduce the number of potential contaminants that may be present in prion preparations from infected brain material. Washed cells were 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 and digested with 10 µg/ml of proteinase K for 1 h at 37 °C. Digestion was stopped with 5 mM PMSF and the supernatant was loaded onto a prewashed C18 Sep-Pak column (Waters, Elstree, UK). Bound material was eluted under a gradient of n-propanol in water (5–100%), fractions containing PrP were pooled (samples tested by a PrP ELISA), washed three times in distilled water and resuspended in 0.01 M pH 7.4 phosphate-buffered saline at 100 ng/ml. Control preparations from non-infected N2a cells were produced by lysis in extraction buffer containing 5 mM PMSF. The supernatant was loaded on to a prewashed C18 Sep-Pak column and eluted under a gradient of n-propanol in water. Fractions containing PrP were pooled, washed three times in distilled water, and resuspended in phosphate-buffered saline at 100 ng/ml.

PrP ELISA—Briefly, treated lysates were added to a Nunc Maxisorb Immunoplates precoated with diluted rabbit antiserum raised to a PrP peptides (ovine PrP-(27–52) and ovine PrP-(100–111)) conjugated to keyhole limpet hemocyanin (a gift from by Dr. J. P. M. Langeveld, Pepscan Systems, Lelystad, The Netherlands). Samples were applied and visualized using mAb SAF83 (gift from Prof. J. Grassi (CEA, Saclay, France), followed by an anti-mouse IgG-alkaline phosphatase conjugate (Sigma) and an appropriate indicator. Plates were read at 450 nM, and results were calculated by reference to a standard curve of recombinant murine PrP (Prionics, Zurich, Switzerland).

PGE2 Assay—Analysis of cellular PGE2 levels was determined in SH-SY5Y 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.

IL-6 ELISA—Levels of interleukin (IL)-6 in cultures containing microglia were assayed in a sandwich ELISA (R&D Systems, Abingdon, UK) as previously described (28). Plates were read at 450 nm, and results were calculated by reference to a standard curve of recombinant murine IL-6. The detection limit of this assay is 20 pg/ml.

Cholesterol and Protein Content—Cellular cholesterol and protein content were determined on 1 x 107 SH-SY5Y cells, or ScN2a cells grown in either Hams F12 medium containing 2% FCS or in serum-free NBM, before and after exposure to 100 nM squalestatin for 3–24 h. At the end of incubation, cells were detached by trypsinization, washed twice with phosphate-buffered saline, and pelleted (550 x g for 10 min). Total lipids were extracted according to Folch et al. (33), and total cholesterol was determined using an enzymatic assay kit (Roche Applied Science). Cellular protein concentration was determined on cell lysates using the Bio-Rad protein assay.

Statistical Analysis—Comparison of treatment effects was 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
 
Squalestatin Reduces PrPres Formation in Prion-infected Cells—Previous studies had indicated that ScN2a cells contain protease-resistant PrP (PrPres) and infectious agent (34). Here, we investigated the effect of squalestatin, an inhibitor of squalene synthase that inhibits the production of cholesterol (20), on ScN2a cells. As shown in Fig. 2, the accumulation of PrPres in ScN2a cells was significantly reduced by squalestatin in a dose-dependent manner. The amounts of PrPres were reduced to below detectable levels in ScN2a cells treated with 100 nM squalestatin (Fig. 2 and Table I). This effect was observed in both lipid-depleted NBM medium and in Hams F12 medium containing 2% FCS that contained extracellular cholesterol. The effect of squalestatin on PrPres production was accompanied by a decrease in the cellular cholesterol levels. ScN2a cells grown in medium containing 2% FCS and treated for 24 h with 100 nM squalestatin contained significantly less cholesterol (0.262 ± 0.040 µg of cholesterol/mg protein, mean ± S.D.) than did untreated ScN2a cells (0.509 ± 0.041, n = 6, p < 0.05). The cholesterol content of ScN2a cells grown in NBM (0.484 ± 0.033) was greatly reduced by treatment with 100 nM squalestatin (0.181 ± 0.025, n = 6, p < 0.05). There was no significant difference between the cholesterol content of ScN2a cells grown in serum-free medium and ScN2a cells grown in 2% FCS. To investigate whether the protective effect of squalestatin was mediated by sterol derivatives, the PrPres formation was evaluated in ScN2a cells treated for 24 h with 100 nM squalestatin alone or combined with 1–100 µg/ml of water-soluble cholesterol, or with 500 µM of mevalonate. As reported in Table I, mevalonate did not affect the content of PrPres in squalestatin-treated cells. In contrast, the addition of water-soluble cholesterol partially reversed the effect of squalestatin on PrPres production although it was not fully restored even with the higher dose of 100 µg/ml of cholesterol (Table I).



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  		FIG. 2.
Squalestatin reduces PrPres in ScN2a cells. A, scrapie-infected neuroblastoma cells (ScN2a) were grown in Hams F12 medium containing 2% FCS (open bars) or in the serum-free NBM (shaded bars). To measure the effect of squalestatin on the PrPres formation, cells were plated at 1 x 105 cells/well in 6-well microtiter plates treated with vehicle (Me2SO) or with different concentrations of squalestatin. The levels of protease-resistant PrPres were then determined in an ELISA as described under "Experimental Procedures." Values shown are the mean ± S.D. of triplicate experiments repeated three times (nine observations). B, ScN2a cells were grown in NBM containing different concentrations of squalestatin as shown. Protease-resistant PrP was demonstrated by immunoblot with mAb SAF83.

 


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  		TABLE I
Effects of squalestatin, mevalonate, and cholesterol on PrPres production ScN2a cells were treated with 100 nM squalestatin alone or in combination with 500 µM mevalonate or 1–100 µg/ml water-soluble cholesterol. The amounts of PrPres were determined as described. Each value is the mean PrPres/1 x 107 cells ± S.D. from triplicate experiments repeated three times (9 observations).

 
To confirm the effects of squalestatin on PrPres production, two other prion-infected neuroblastoma cell lines (SMB or ScGT1 cells) was treated with 100 nM squalestatin. The PrPres content of SMB cells treated with a vehicle control (7,405 ± 512 PrPres pg/ml) was significantly higher than that of SMB cells treated with 100 nM squalestatin (< 50 pg/ml, n = 8, p < 0.05). Similar results were observed with ScGT1 cells, in that the PrPres content of ScGT1 cells treated with a vehicle control (10,014 ± 845 PrPres pg/ml) was significantly higher than that of ScGT1 cells treated with 100 nM squalestatin (< 50 pg/ml, n = 8, p < 0.05).

Effect of Squalestatin on the Solubility of PrPc in Triton X-100 —Previous studies showed that PrPC to preferentially locate in cholesterol-rich lipid rafts which are insoluble in non-ionic detergents such as Triton X-100 (11). To determine the effect of squalestatin on the levels and distribution of PrPC, N2a neuroblastoma cells were grown in the presence or absence of 100 nM squalestatin. There was no significant difference in total levels of PrPC between untreated and squalestatin-treated cells. However, while the majority of PrPC in untreated cells was Triton X-100 insoluble, in squalestatin-treated cells most of the PrPC was Triton X-100 soluble (Fig. 3A). PrPC in squalestatin-treated cells remained sensitive to protease digestion (Fig. 3B).



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  		FIG. 3.
Treatment with squalestatin changes the distribution of PrPc in N2a cells. A, non-infected N2a cells were grown in control medium (untreated) or with 100 nM squalestatin (SQ treated) for 72 h. Cells were subsequently lysed in either an extraction buffer or in a buffer containing 1% Triton X-100 at the equivalent of 1 x 107 cells/ml. The amounts of PrPC in whole cellular lyastes (shaded bars), soluble in 1% Triton X-100 (open bars) or insoluble in Triton X-100 (striped bars) were determined in an ELISA. Values shown are the mean level of PrPC/1 x 107 cells ± S.D. of triplicate experiments repeated three times (9 observations). B, N2a cells were grown in the presence (+) or absence (-) of 100 nM squalestatin (SQ). Cellular lysates were untreated (PK-) or digested with proteinase K (10 µg/ml for1hat37 °C) (PK+). PrP was demonstrated by immunoblot with mAb SAF83.

 
Squalestatin Protects Neurons Against PrP Peptides—The effect of squalestatin on the sensitivity of SH-SY5Y cells to PrP peptides was examined. Cells treated for 3 h with 100 nM squalestatin contained significantly less cholesterol than did untreated ones (0.394 ± 0.024 µg of cholesterol/mg of protein for untreated cells and 0.172 ± 0.033 µg of cholesterol/mg of protein for treated cells, n = 6, p < 0.05). The survival of squalestatin-treated SH-SY5Y cells incubated with 40 µM HuPrP-(106–126), 10 µM HuPrP-(82–146) or 10 µM sPrP106 was significantly greater than that of vehicle treated SH-SY5Y cells incubated with these peptides (Fig. 4A). This protective effect of squalestatin was dose-dependent with an IC50 ~1 nM when tested against 10 µM HuPrP(82–146) (Fig. 4B).



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  		FIG. 4.
Pretreatment of SH-SY5Y cells with squalestatin reduces the neurotoxicity of PrP peptides. A, SH-SY5Y cells were pretreated for 3 h with vehicle (Me2SO) (black bars) or with 100 nM squalestatin (open bars). Cells were then incubated for 24 h with 40 µM HuPrP-(106–126) (106–126), 10 µM HuPrP-(82–146) (82–146), or 10 µM sPrP106 and cell viability was evaluated by MTT. B, SH-SY5Y cells were pretreated for 3 h with vehicle (Me2SO) (open bar) or with different concentrations of squalestatin (black bars). Cells were then incubated for 24 h with 10 µM HuPrP-(82–146), and the cell viability was evaluated by MTT. Each bar represents mean cell survival ± S.D. of triplicate experiments repeated four times (12 observations).

 
Previous studies have shown that the neuronal loss is observed after the addition of partially purified prion preparations (22, 28). In the present study the addition of 10 ng/ml PrP extracted from three different prion preparations significantly reduced the survival of untreated SH-SY5Y cells by 50%, whereas the survival of SH-SY5Y cells was not affected by the addition of 10 ng/ml PrP extracted from uninfected cells (Table II). Although these prion preparations were not pure, the neurotoxicity of these preparations was thought to be due to PrPres since these preparations were not toxic for neurons from PrP-deficient mice, and that the toxicity of these preparations was selectively removed following immunoprecipitation with an anti-PrP antibody (data not shown). The survival of cells pretreated with 100 nM squalestatin, before the addition of the prion extracts, was significantly higher than that of untreated SH-SY5Y cells incubated with the same preparations (Table II). In addition, squalestatin also protected murine primary cortical neurons (non-transformed cells) against the toxicity of prion preparations (data not shown).


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  		TABLE II
Squalestatin-treated cells are resistant to the toxic effects of prion preparations SH-SY5Y cells were pretreated for 3 h with a vehicle control (Me2SO) or 100 nM squalestatin, and then incubated with 10 ng/ml PrP extracted from prion-infected cells (prion extracts), or 10 ng/ml PrP extracted from non-infected cells (control extracts). Cell viability was evaluated after 24 hours using MTT. Each value is the mean cell survival ± S.D. from triplicate experiments repeated three times (9 observations).

 
To determine whether squalestatin-treated cells were completely resistant to PrP peptides, different amounts of HuPrP-(82–146) were added to SH-SY5Y cells treated with 100 nM squalestatin. The addition of HuPrP-(82–146) caused a dose-dependent reduction in the survival of untreated cells, whereas even at high concentrations, HuPrP-(82–146) did not kill cells treated with 100 nM squalestatin (Fig. 5). Further experiments were performed to determine if the addition of cholesterol to squalestatin-treated SH-SY5Y cells would restore their sensitivity to the neurotoxic effect of PrP peptides or to prions. As shown in Fig. 6, the presence of 10 µg/ml of water-soluble cholesterol reversed the protective effect of 100 nM squalestatin on the toxicity induced by 40 µM HuPrP-(106–126), 10 µM HuPrP-(82–146) or by a prion extract in SH-SY5Y cells.



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  		FIG. 5.
Squalestatin-treated SH-SY5Y cells are resistant to HuPrP-(82–146). SH-SY5Y cells were pretreated for 3 h with vehicle (Me2SO) (black circles) or with 100 nM squalestatin (open circles) and then incubated for 24 h with different concentrations of HuPrP-(82–146). Cell viability was evaluated by the MTT method. Each point represents the mean cell survival ± S.D. of triplicate experiments repeated twice (6 observations).

 



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  		FIG. 6.
The neuroprotective effect of squalestatin is reversed by the addition of water-soluble cholesterol. SH-SY5Y cells were pretreated for 3 h with vehicle (Me2SO) (black bars), with 100 nM squalestatin (white bars) or with a mixture of 100 nM squalestatin and 10 µg/ml water-soluble cholesterol (striped bars) and then incubated for 24 h with a prion extract (prion), with 40 µM HuPrP-(106–126) (106–126), or with 10 µM HuPrP-(82–146) (82–146). Cell viability was evaluated by the MTT method. Each point represents mean cell survival ± S.D. of triplicate experiments repeated twice (6 observations).

 
Reduced PGE2 Production in Squalestatin-treated Cells— The cyclo-oxygenases (COX) are enzymes that convert arachidonic acid to PGs. The observations that the levels of neuronal PGE2 are raised in response to PrP peptides, and that COX-1 inhibitors protect neurons against PrP peptides, suggest that activated COX is involved in neuronal toxicity (35, 26). Thus, in this study, the effects of squalestatin on PGE2 levels from SH-SY5Y cells incubated with PrP peptides or a prion extract were examined. We were unable to detect PGE2 in untreated SH-SY5Y cells, squalestatin-treated cells, or cells incubated with scrambled peptides. Cells exposed to either 40 µM HuPrP-(106–126) to 10 µM HuPrP-(82–146), 10 µM sPrP106, or to a prion extract produced significant amounts of PGE2. SH-SY5Y cells pretreated with 100 nM squalestatin produced significantly less PGE2 when subsequently incubated with these peptides or a prion extract (Table III). The effect of squalestatin was reversed by the addition of 10 µg/ml of water-soluble cholesterol.


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  		TABLE III
Effect of squalestatin on the production of PGE2 in SH-SY5Y cells Cells were pretreated for 3 h with a vehicle control (Me2SO) or 100 nM squalestatin alone or combined with 10 µg/ml water-soluble cholesterol before the addition of peptides (10 µM HuPrP-(82–146), 40 µM HuPrP-(106–126)), 10 µM sPrP106, or a prion extract. PGE2 production was evaluated after 24 hours as shown. Results are the mean levels of PGE2 ± S.D. from quadruplicate experiments repeated twice (8 observations).

 
Squalestatin Protects ScN2a Cells Against Microglia-mediated Killing—The effect of squalestatin on interactions between ScN2a cells and microglia was also examined. As shown in Fig. 7A, the survival of ScN2a cells co-cultured with microglia (ratio of 1 microglia to 10 ScN2a cells) was significantly lower than those of non-infected N2a cells co-cultured with microglia, consistent with previous studies (28). The pretreatment of ScN2a cells with 100 nM squalestatin completely protected them from microglia-mediated toxicity, increasing cell viability from 30 to 100%. This protective effect could be fully reversed by the addition of 10 µg/ml of water-soluble cholesterol.



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  		FIG. 7.
Squalestatin protects prion-damaged neurons against microglia. A, ScN2a cells (black bars) or N2a cells (white bars) were pretreated with vehicle (Veh), with 100 nM squalestatin (SQ), or with 100 nM squalestatin and 10 µg/ml water-soluble cholesterol (SQ+Ch) for 24 h before the addition of microglia. Cell viability was evaluated after a further 24 h. Each point represents mean cell survival ± S.D. of triplicate experiments repeated four times (12 observations). B, primary cortical neurons were preincubated for 3 h with vehicle (Me2SO) (black bars) or with 100 nM squalestatin (white bars) or with 100 nM squalestatin and 10 µg/ml water-soluble cholesterol (striped bars) before the addition of a prion extract (Prion), 40 µM HuPrP-(106–126) (106–126), 10 µM HuPrP-(82–146) (82–146), or 10 µM sPrP106. After a further 3 h microglia were added to neuronal cultures in the ratio 1 microglia to 10 neurons, and cell viability was determined 4 days later using the MTT method. Each point represents the mean cell survival ± S.D. of triplicate experiments repeated four times (12 observations).

 
The effect of the squalestatin on interactions between PrP-damaged, non-transformed neurons, and microglia was also studied. Thus, primary cortical neurons were treated with 100 nM squalestatin before the addition of 40 µM HuPrP-(106–126), 10 µM HuPrP-(82–146), 10 µM sPrP106 or a prion extract, and microglia then added 3 h later in the ratio of 1 microglia to 10 neurons. The survival of neurons in co-cultures containing squalestatin and PrP peptides or a prion extract, were significantly higher than the survival of untreated co-cultures exposed to these peptides or prion preparations (Fig. 7B). To determine whether squalestatin had a direct effect on microglia, isolated microglia cultures were pretreated with 100 nM squalestatin before the addition of 10 ng/ml LPS. There was no significant difference in the amounts of IL-6 produced by squalestatin-treated microglia (4205 pg/ml ± 221, n = 6) and vehicle-treated microglia (4286 pg/ml ± 161).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we examined the effect of squalestatin on cellular cholesterol levels, on the accumulation of PrPres in three prion-infected cell lines, and on two models of PrP-induced neurotoxicity. Squalestatin reduced the accumulation of PrPres in ScN2a, SMB, and ScGT1 cells. This effect of squalestatin on PrPres production was dose-dependent with an IC50 ~5 nM, while even 50 µM squalestatin was not toxic for ScN2a cells. None of the effects of squalestatin were reversed by the addition of mevalonate (previously shown to reverse the effects of HMG-CoA reductase inhibitors such as lovastatin), but were reversed by cholesterol indicating that cholesterol depletion was responsible for the effects of squalestatin. Cholesterol homeostasis in mammalian cells is governed by both cholesterol synthesis and by the influx and efflux of cholesterol from the surrounding medium (36). Recent studies have shown that the statins reduce cerebral cholesterol levels (37) indicating that cholesterol production is important in maintaining neuronal cholesterol levels.

Several observations suggest that the maintenance of cholesterol levels in these neuronal cells is largely influenced by the synthesis of cholesterol rather than by the uptake of cholesterol from the surrounding medium. Firstly, the effects of squalestatin were observed in medium containing FCS (and hence cholesterol), and the addition of 100 µg/ml of normal cholesterol did not affect the neuroprotective effect of squalestatin, or the effect of squalestatin on PrPres formation (data not shown). In contrast, low concentrations of water-soluble cholesterol (~10 µg/ml) were enough to reverse the neuroprotective effects of squalestatin, restore PrPres production and restore cellular cholesterol levels. Water-soluble cholesterol is complexed to methyl-{beta}-cyclodextrin that facilitates the insertion of cholesterol into cell membranes while normal cholesterol requires specific uptake mechanisms. Finally, drugs that prevented cellular uptake of cholesterol did not affect either PrPres formation or the neurotoxicity of PrP peptides.2

Previous studies suggest that the concentration and cellular location of PrPC may be critical factors in the production of PrPSc (6, 7) since PrPSc formation is thought to be dependent on the supply of PrPC to an intracellular environment that facilitates the conversion of PrPC to PrPSc (38). Although cells treated with squalestatin contained significantly less cholesterol than untreated cells, the amounts of PrPC in untreated and squalestatin treated cells were not significantly different and, unlike treatment with the cholesterol-binding drug filipin (39), squalestatin did not cause the release of PrPC into culture medium (data not shown). In the present study squalestatin treatment resulted in the dispersion of PrPC into Triton X-100 soluble fractions, an observation compatible with previous studies that showed that cholesterol-depleted cells no longer contain typical caveolae (40). PrPC molecules are normally located in lipid rafts (8) and dispersion from such sites following squalestatin treatment may affect the normal cellular trafficking of PrPC. The golgi and the endosomal compartments are involved in the trafficking of a GFP-tagged PrPC (41) and in hippocampal neurons, the trafficking of cholera toxin (whose receptor is known to reside in lipid rafts) from endosomes to the golgi apparatus is blocked by cholesterol depletion (42). While the current results are suggestive, it is not known whether the trafficking of PrPC is affected in squalestatin-treated cells so as to prevent PrPC interacting with cellular components required for conversion to PrPSc.

The loss of neurons in response to prions or PrP peptides is thought to be an apoptotic event (43) requiring receptor binding and the activation of specific metabolic pathways. The neurotrophin p75 receptor, located in CLDs (44), is thought to act as a receptor for PrP peptides and is involved in the neuronal damage of HuPrP-(106–126) (45). Thus, the protective effect of squalestatin may be mediated by a destabilizing effect on neurotrophin p75. Since many of the molecules involved in cellular signaling are found in cholesterol-sensitive CLDs (46) the possibility that cholesterol depletion may affect the generation of second messengers in response to PrP peptides, or to aggregated PrPSc, was considered. For example, the components of the cyclic adenosine monophosphate (cAMP) signaling pathway are found in CLDs (47) and a recent report has linked cAMP with PrPC and neuroprotection (48). The observation that arachidonic acid (49) and the COX enzymes (50) are found in CLDs is also of interest. Levels of PGE2 (a measure of COX activation) are increased in brain areas showing neuronal death in murine scrapie (51), and raised levels of PGE2 are detected in the cerebrospinal fluid of patients with CJD (52–53). Moreover, the addition of PrP peptides to SH-SY5Y cells increased PGE2 production, and neurons treated with COX inhibitors were resistant to the toxicity of PrP peptides (26). Taken together these results suggest that PrP peptides stimulate the production of neurotoxic prostaglandins. The present studies are compatible with the hypothesis that COX activation by PrP peptides or prion extracts occurs within CLDs as PGE2 production was greatly reduced in squalestatin-treated neurons.

A significant role for microglia in the neuropathogenesis of prion disease is suggested by the close association between the accumulation of prion fibrils and the activation of microglia in scrapie-infected mice (1, 30). Moreover, in vitro studies showed that microglia killed prion-damaged neurons (29) and selectively kill ScN2a cells, but not non-infected N2a cells (28). The presence of squalestatin in co-cultures containing microglia and prion-infected ScN2a cells resulted in a greatly increased neuronal survival. Since squalestatin had no effect on isolated microglia we concluded that the effects of squalestatin are predominantly on ScN2a cells. The addition of PrP peptides to primary cortical neurons induced a time-dependent changes that stimulate microglia to kill these cells (54). The protective effect of squalestatin against microglia-mediated killing is compatible with the hypothesis that these PrP-induced changes in neurons are sterol-dependent. The nature(s) of the changes expressed by ScN2a cells or PrP-damaged neurons that activate microglia are currently under further investigation.

Although previous studies showed that the formation of PrPSc was reduced in lovastatin-treated ScN2a cells (11), the authors concluded that the statins were not compatible with use in man, partly due to the high concentrations of lovastatin required, and partly due to the fact that results were only seen in lipid-depleted medium. The concentration of squalestatin required to affect PrPres formation in three different prion-infected cell lines was shown to be less than 10 nM; considerably lower than that reported for lovastatin (0.3 µM) (11). Moreover, the action of squalestatin was not affected by the cholesterol in the in medium and was only partially restored with high concentrations of water-soluble cholesterol. In contrast, the ability of 100 nM lovastatin to reduce PrPSc production was reversed with 1 µg/ml water-soluble cholesterol (data not shown) suggesting that statins and squalestatin affect different cholesterol pools. The different effects displayed by statins and squalestatin depended on their different pharmacological target (HMG-CoA reductase versus squalene synthase; see Fig. 1) and the effects of these on cholesterol distribution. Time course studies showed that squalestatin produced a prolonged reduction in cholesterol levels (significant reduction after 24 h) that was not observed in lovastatin-treated cells. The greater potency of squalestatin suggests that it may be a more promising candidate than the HMG-CoA reductase inhibitors for use in vivo.

In summary, an understanding of the processes by which prions accumulate in neurons, and how this process leads to neuronal loss, may aid the development of therapies to delay the progression of prion diseases. In the present study, squalestatin had a profound effect on three major aspects of the neuropathogenesis of prion disease. Squalestatin-treated ScN2a, SMB, or ScGT1 cells were cured of PrPres, ScN2a cells were protected against killing by microglia and neurons treated with squalestatin were resistant to the neurotoxicity of PrP peptides. Furthermore, the observation that squalestatin had protective effects at low concentrations, suggest that the use of this drug to treat prion diseases should be considered.


   FOOTNOTES
 
* This work was supported by the European Commission (Contracts BMH4-CT98-6011 and 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.: 44-0-141-330-2874; Fax: 44-0-141-330-5602; E-mail: c.bate{at}vet.gla.ac.uk.

1 The abbreviations used are: CLD, caveolae-like domains; FCS, fetal calf serum; PMSF, phenylmethylsulfonyl fluoride; IL, interleukin; GFP, green fluorescent protein; MTT, 3-[4,5 dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide; Me2SO, dimethyl sulfoxide; HMG-coA, 3-hydroxy-3-methylglutaryl-co-enzyme A; PG, prostaglandin; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; HPLC, high performance liquid chromatography. Back

2 C. Bate, M. Salmona, L. Diomede, and A. Williams, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. R. Veerhuis and Prof. P. Eikelenboom (Free University, Amsterdam, The Netherlands) for helpful discussions. The ScN2a and N2a cells were a kind gift of Dr. M. Rogers (University College,Dublin, Ireland). SMB and SMB(PS) cells were obtained from the TSE Resource Centre, Institute for Animal Health, Compton, UK, and ScGT1 cells were obtained from Dr. S. Lehmann (Montpellier, France).



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C. Bate, S. Reid, and A. Williams
Phospholipase A2 Inhibitors or Platelet-activating Factor Antagonists Prevent Prion Replication
J. Biol. Chem., August 27, 2004; 279(35): 36405 - 36411.
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