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15-Deoxy-delta12,14-prostaglandin J2, a neuroprotectant or a neurotoxicant?
Toxicology 216 (2005) 232–243
15-Deoxy-delta12,14-prostaglandin J2, a neuroprotectant or a neurotoxicant? Seong-Ho Koh a,1 , Boo Jung a,1 , Chi Won Song b , Youngchul Kim a , Yong Soon Kim b , Seung Hyun Kim a,∗ a
b
Department of Neurology, Institute of Biomedical Science, College of Medicine, Hanyang University, #17 Haengdang-dong, Seongdong-ku, Seoul 133-791, Republic of Korea Department of General Toxicology, National Institute of Toxicological Research, KFDA, Seoul, Republic of Korea Received 1 July 2005; received in revised form 9 August 2005; accepted 12 August 2005 Available online 26 September 2005
Abstract 15-Deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) is a potent ligand for peroxisome proliferators-activated receptor ␥ (PPAR␥). However, its various effects independent of PPAR␥ have recently been observed. The effect of 15d-PGJ2 on neuronal cells is still controversial. We investigated its effect on neuronal cells (N18D3 cells). When N18D3 cells were treated with 15d-PGJ2, the viability was not changed up to 8 M, but decreased at higher than 8 M. The expressions of survival signals, such as p85a phosphatidylinositol 3-kinase, phospho-Akt, and phospho-glycogen synthase kinase-3 beta (Ser-9), slightly increased up to 8 M, however, decreased at higher than 8 M. The levels of free radicals and membrane lipid peroxidation and the expression of c-Jun N-terminal Kinase increased in a dose-dependent manner, especially at higher than 8 M. However, the expressions of death signals, such as cytosolic cytochrome c, activated caspase-3, and cleaved poly(ADP-ribose) polymerase, decreased up to 8 M, however, increased at higher than 8 M. In the study to evaluate whether low dose of 15d-PGJ2, up to 8 M, had protective effect on oxidative stress-injured N18D3 cells, compared to the cells treated with only 100 M H2 O2 , the pretreatment with 8 M 15d-PGJ2 increased the viability and the expressions of the survival signals, but decreased them of the death signals. These results indicate that 15d-PGJ2 could be a neuroprotectant or a neurotoxicant, depending on its concentration. Therefore, some specific optimum dose of 15d-PGJ2 may be a new potential therapeutic candidate for oxidative stress-injury model of neurodegenerative diseases. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: 15d-PGJ2; Neuron; JNK; PI3-K; Akt
1. Introduction 15-Deoxy-delta12,14-prostagladin J2 (15d-PGJ2) is a natural ligand activating peroxisome proliferators∗
Corresponding author. Tel.: +82 2 2290 8371; fax: +82 2 2296 8370. E-mail address: [email protected] (S.H. Kim). 1 Both authors (S.-H. Koh, B. Jung) equally contributed to this journal.
activated receptor ␥ (PPAR␥), a nuclear hormone receptor/transcription factor that regulates adipocyte differentiation and fatty acid homeostasis (Tontonoz et al., 1994; Ichiki et al., 2004). It is well known that 15d-PGJ2-activated PPAR␥ forms a heterodimer with retinoid X receptor, binds to a specific DNA sequence, PPAR response element (PPRE), and activates target gene transcription (Schoonjans et al., 1997). However, its various effects independent of PPAR␥, including induction of cyclooxygenase-2 expression and activa-
0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2005.08.015
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tion of the mitogen-activated protein kinase kinase (MEK)/extracellular signals-regulated kinase (ERK) pathway, have recently been reported (Li et al., 2004a; Hashimoto et al., 2004). And, in vascular smooth muscle, 15d-PGJ2-induced DNA synthesis was also proven to be partially dependent on phosphatidylinositol 3-kinase (PI3-K) (Takeda et al., 2001). Recently, PI3-K/Akt signaling pathway has been shown to be related with pathogenic mechanism of neurodegenerative diseases (Cantley, 2002; Cantrell, 2001; Doble and Woodgett, 2003; Kirschenbaum et al., 2001; Kulich and Chu, 2001; Pap and Cooper, 1998; Pei et al., 2002; Sang et al., 2001; Xia and Hyman, 2002; Xu et al., 2005; Zhao et al., 2002), and oxidative stress neuronal injury (Kim et al., 2005; Koh et al., 2003, 2004, 2005). Phosphorylated Akt activated by PI3-K plays very important role in the neuronal cell survival by both enhancing the expression of anti-apoptotic proteins and inhibiting the activity of proapoptotic ones (Cantley, 2002; Cantrell, 2001; Doble and Woodgett, 2003; Kirschenbaum et al., 2001; Koh et al., 2003, 2004, 2005; Pap and Cooper, 1998; Sang et al., 2001). GSK-3 regulates the function of metabolic, structural, and signaling proteins and is regulated by PI3-K/Akt activity. Furthermore, evidence indicates that GSK-3 is associated with Alzheimer’s disease by participating in phosphorylation of the microtubulebinding protein tau and contributing to the formation of neurofibrillary tangles and the interaction with presenilin (Doble and Woodgett, 2003; Kirschenbaum et al., 2001; Sang et al., 2001). Activated GSK-3 is inactivated through phosphorylation by pAkt. Phosphorylation of GSK-3 is important in the inhibition of oxidative stress-induced apoptosis, because unphosphorylated GSK-3, activated form which activates the mitochondrial death pathway, results in the damage of mitochondria. Increase of cytoplasmic cytochrome c released from damaged mitochondria activates caspase9 and caspase-3, accompanied by PARP cleavage, and then results in apoptosis (Cantley, 2002; Cantrell, 2001; Koh et al., 2003, 2004, 2005; Pap and Cooper, 1998). There have been several reports about effects of PGJ2 on neuronal cells (Aoun et al., 2003; Heneka et al., 1999; Li et al., 2004a,b; Rohn et al., 2001; Yagami et al., 2003; Zhuang et al., 2003). Nevertheless, its precise effects on neuronal cells have not yet been clearly established. 15d-PGJ2 was described to have neuroprotective effect in some reports (Aoun et al., 2003; Heneka et al., 1999; Zhuang et al., 2003), but to have neurotoxic effect in others (Li et al., 2004a,b; Rohn et al., 2001). Considering that various studies on the effects of
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15d-PGJ2 on neuronal cells had been focused on specific concentration and shown different effects (Aoun et al., 2003; Li et al., 2004a), the precise concentration–response relationships should be clearly examined. This study was designed to evaluate the dosedependent different roles of PGJ2 on neuronal cells (N18D3 cells). The levels of free radicals and membrane lipid peroxidation, and the cellular signals, such as phosphatidylinositol 3-kinase (PI3-K), Akt, glycogen synthase kinase-3 beta (GSK-3), c-Jun N-terminal Kinase (JNK), phospho-JNK, cytosolic cytochrome c, caspase3, and poly(ADP-ribose) polymerase (PARP) were investigated in PGJ2-treated N18D3 cells with/without hydrogen peroxide treatment. 2. Materials and methods 2.1. Cell culture, induction of differentiation, and treatment N18D3 cells, hybrid neuron line obtained by fusion of dorsal root ganglion neurons isolated from 4-week-old Balb/C mouse with the mouse neuroblastoma N18TG2 cells, were maintained in logarithmic-phase growth on poly-l-lysine (Sigma)-precoated 100-mm dish (Corning) in Minium Essential Media (GIBCO BRL, Grand Island, NY, USA) containing 10% heat-inactivated donor horse serum, 5% fetal bovine serum (FBS), 100 units/ml penicillin, 100 g/ml streptomycin, 4 g/l glucose, 2.2 g/l NaHCO3 , and 10 ml/l l-glutamine. After cells were neuronally differentiated with treatment of nerve growth factor, the cells were maintained in humidified incubators with 5% CO2 and 95% air at 37 ◦ C. Cells were grown to 50% confluence, and then harvested in Ca2+ /Mg2+ -free Hank’s balanced salt solution containing 1 mM EDTA. N18D3 cells were plated at a density of 106 cells/100-mm poly-l-lysineprecoated dish (Kim et al., 2005; Koh et al., 2004; Park et al., 2000; Sanfeliu et al., 1999). To examine the effect of 15d-PGJ2 (Fig. 1) on the viability of neuronal cells, 15d-PGJ2 (Cayman Ann Arbor, MI, USA) was dissolved in ethanol, the final concentration of ethanol never exceeding 0.1% when added to cells as ethanol solution (Satoh et al., 1999). N18D3 cells were treated for 6 h (Aoun et al., 2003) with several concentrations of 15d-PGJ2 [0 (only
Fig. 1. Structure of 15-deoxy-delta12,14-prostaglandin J2 (15dPGJ2).
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vehicle, control), 2, 4, 8, 16, and 32 M] and were washed more than three times with phosphate buffered saline (PBS). Cell viability was measured after 24 h by using the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Trypan blue stain (Carmichael et al., 1987; McCarthy and Evan, 1998). To evaluate the effect of PGJ2 on cellular signals, including PI3-K/Akt, GSK-3, JNK, cytosolic cytochrome c, caspase-3, and PARP, N18D3 cells were treated with several concentrations of 15d-PGJ2. And to evaluate its protective effect on oxidative stress-injury, N18D3 cells were pretreated with different concentrations of 15d-PGJ2 (0, 2, 4, or 8 M) for 6 h, washed carefully several times with PBS, and then treated with 100 M H2 O2 for 30 min. Subsequently, the cells were washed carefully, and the cell viability was measured after 24 h. To exclude the possibility of direct hydroxyl radical scavenging effect of residual 15d-PGJ2 on wells, washing steps were repeated several times prior to the addition of H2 O2 . In addition, the levels of hydrogen peroxide were measured using luminol-dependent chemiluminescence which is formed by the reaction between H2 O2 and luminol catalyzed by horseradish peroxidase (Wilhelm et al., 1995), and the amounts of reactive hydroxyl radicals (OH• ) from H2 O2 were indirectly measured by quantifying 2,5- and 2,3-dihydroxybenzoic acid (DHBA) generated by salicylate (Wako Pure Chemical Industries Ltd., Osaka, Japan) hydroxylation using HPLC (Kaneko et al., 2000; Teismann and Ferger, 2000). 2,5- and 2,3-DHBA used as standard were purchased from Sigma Chemical (St. Louis, MO, USA). After washing more than three times, the levels of H2 O2 and the amounts of reactive hydroxyl radicals were found to be not statistically significantly different between the group treated with 100 M H2 O2 only and the group treated with 100 M H2 O2 and 15d-PGJ2. Harvested cells after 30 min of H2 O2 treatment were immediately used for PI3-K/Akt, GSK3, and JNK immunoblotting, and cytochrome c, caspase-3, and PARP immunoblotting after 6 h (Fahn and Cohen, 1992; Mausuda et al., 2002; McCarthy and Evan, 1998; McCord, 1994; Molina-Holgade et al., 2002; Mudher et al., 2001). To examine the role of PI3-K/Akt in the 15d-PGJ2 mediated survival protection of N18D3 cells, we designed an experiment on N18D3 cells with PI3-K inhibitor, LY294002 (Sigma, St. Louis, MO, USA). N18D3 cells were divided into four groups, such as control (group 1), 100 M H2 O2 (group 2), 100 M H2 O2 + 8 M 15d-PGJ2 (group 3), and 50 M LY294002 + 100 M H2 O2 + 8 M 15d-PGJ2 (group 4) groups. LY294002 was treated at group 4 before 1 h of 15dPGJ2 treatment. 2.2. MTT assay and Trypan blue staining to evaluate cell survival MTT is absorbed into cell, and formazan is then formed by the action of mitochondrial succinate dehydrogenase. Accumulation of formazan reflects the activity of mitochondria directly and the cell viability indirectly. Thus, cells were plated at a density of 1 × 104 cells/well in the 96-well plates, cultured,
differentiated, and treated according to the methods described above. A total 50 l of 2 mg/ml MTT (Sigma) was added after 200 l of medium was added into each well. An aliquot (220 l) of solution was removed from each well, and 150 l of dimethyl sulfoxide was then added to each well. Optical density (OD) at 540 nm was evaluated on the ELISA plate reader after precipitate in the well was dissolved by a microplate mixer for 10 min. All results were calibrated for OD measured in the same conditioned well without cell culture (Koh et al., 2003). For Trypan blue staining, 10 l of Trypan blue solution were incubated with 10 l of cells collected from each plate for 2 min, and unstained live cells were counted on a hemacytometer (Koh et al., 2003). 2.3. DAPI staining to evaluate apoptosis DAPI staining was performed to evaluate apoptosis. The procedures for DAPI staining were as follows: N18D3 cells were treated (1) without any of H2 O2 and 15d-PGJ2 (control), (2) with only 8 M 15d-PGJ2 for 6 h, (3) with only 32 M 15d-PGJ2 for 6 h, (4) with only 100 M H2 O2 for 30 min, and (5) with 100 M H2 O2 for 30 min after pretreatment with 8 M 15d-PGJ2 for 6 h. Subsequently, the differently treated cells were further incubated in the 96-well plates for 24 h after washing, harvested in PBS, and then centrifuged at 261 × g for 2 min, and 4% neutral buffered formalin (100 l) was added to the cell pellet for fixation. A 50 l aliquot of the cell suspension was applied to a glass slide and dried at room temperature. The fixed cells were then washed in PBS, air dried, and stained with the DNA-specific fluorochrome 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) for 20 min. The adhered cells were rinsed with PBS, air dried, and mounted with 90% glycerol. The slides were observed under an Olympus fluorescence microscope. Percent of apoptotic cells, which coincided with morphological criteria of apoptosis, such as nuclear condensation and segmentation, was calculated based on the total number of cells. 2.4. Determination of free radical production and oxidative damage induced by 15d-PGJ2 itself To measure free radical production by 15d-PGJ2 itself, neuronally differentiated N18D3 cells treated with only 0 (control), 2, 4, 8, 16, and 32 M 15d-PGJ2 for 30 min were incubated with fluorescent probe 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) (Molecular Probes Inc., Eugene, OR, USA), which freely crosses the cell membranes and is hydrolysed by cellular esterases to 2 ,7 -dichlorodihydrofluorescein (DCFH2 ). DSFH2 is a non-fluorescent molecule, however, it is oxidized to the fluorescent 2 ,7 -dichlorofluorescein (DCF) in the presence of peroxides. Therefore, accumulation of DCF in the cells is indicated by an increment in fluorescence at 525 nm, when the sample is excited at 488 nm by a Microplate Fluorescence Reader (FL600) (D.I. Biotech Ltd., Seoul, Korea) (Tammariello et al., 2000).
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To measure oxidative damage by 15d-PGJ2 itself, lipid peroxidation was evaluated by measuring the thiobarbituric acidreacting substances (TBARS) (Sigma Chemical) in N18D3 cells treated with only 0 (control), 2, 4, 8, 16, and 32 M 15dPGJ2 for 1 h. Briefly, the cells were washed and harvested with ice-cold 50 mM phosphate buffer, pH 7.4, and then 500 l of 1% thiobarbituric acid and 500 l of 8N HCl were added to the sample. The samples were boiled for 20 min and subsequently cooled with tap water. 1-Butanol (1.5 ml) was then added to the samples, and the mixture was shaken for 2 min. After centrifugation at 2000 × g for 10 min, the fluorescence intensity at 550 nm (excitation) and 534 nm (emission) in the butanol phase was measured by a Microplate Fluorescence Reader (FL600) (Ohkawa et al., 1979). Incubation time used in these studies (30 min for measuring free radical production and 1 h for measuring lipid peroxidation) was chosen, because maximal effects were shown at these time points. 2.5. Western blot analyses For Western blotting, we treated N18D3 cells: (1) without any of H2 O2 and 15d-PGJ2, (2) with 2, 4, 8, 16, or 32 M 15d-PGJ2 for 6 h, (3) with only 100 M H2 O2 for 30 min, and (4) with 100 M H2 O2 for 30 min after pretreatment with 8 M 15d-PGJ2 for 6 h. PI3-K, Akt, GSK3, JNK, phospho-JNK, cytosolic cytochrome c, caspase-3, and PARP were assayed by Western blot analyses. Briefly, 5 × 106 cells were washed twice in cold PBS and incubated for 10 min on ice in a lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 100 g/ml phenylmethylsulfonylfluoride (PMSF), 50 l/ml aprotinin, 1% Igapel 630, 100 mM NaF, 0.5% sodium deoxy choate, 0.5 mM EDTA, and 0.1 mM EGTA]. Cell lysates were centrifuged at 17,000 × g to evaluate cleaved and non-cleaved forms of PARP and caspase-3, phosphorylated and non-phosphorylated form of JNK1, GSK-3 and Akt, and p85a PI3-K. And, to evaluate released cytochrome c, cells after washing were suspended in sucrose-supplemented cell extract buffer (SCEB, 300 mM sucrose, 10 mM HEPES at pH 7.4, 50 mM KCl, 5 mM EGTA, 5 mM MgCl2 , 1 mM DTT, 10 M cytochalasin B, and 1 mM PMSF), left on ice for 30 min, and then homogenized by 50 strokes in an ice-cold Dounce homogenizer. Unbroken cells and nuclei were pelleted by centrifugation at 2000 × g for 10 min. Mitochondria were collected by centrifugation of the resulting supernatant at 13,000 × g for 10 min. The postmitochondrial fraction, namely the supernatant in the above step, was used for immunoblotting for cytochrome c. Protein concentrations of cell lysates and postmitochondrial fraction were determined by Bio-Rad protein assay kit. An equal amount (20 g) of protein from each sample was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were blocked with 5% skim milk and sequentially incubated with specific antibodies for PARP (1:500, Pharmin-
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gen, San Diego, CA, USA), caspase-3 (1:1000, Santa Cruz Biotech, Delaware, CA, USA), cleaved caspase-3 (Asp 175) (1:1000, Cell Signaling, Beverly, MA, USA), cytochrome c (1:500, Santa Cruz Biotech), JNK1, phospho-JNK1 (1:1000, Santa Cruz Biotech), GSK-3 (1:1000, Santa Cruz Biotech), phospho-GSK-3 (Ser-9) (1:1000, Santa Cruz Biotech), Akt (1:1000, Cell Signaling), phospho-Akt (1:1000, Cell Signaling), and p85a PI3-K (1:1000, Sigma). The membranes were washed with Tris buffered saline containing 0.05% Tween20 (TBST), and then processed using an HRP-conjugated anti-rabbit antibody or anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA) followed by ECL detection (Amersham Pharmacia Biotech) (Juin et al., 1999; Koh et al., 2003). The same membrane was used to measure the each ratio by reprobing: for example, the membrane used for evaluation of pAkt was reused to evaluate Akt through reprobing procedure. The results of Western blots were quantified with image analyzer (Bio-Rad, Quantity One-4,2,0). 2.6. Statistical analysis All data were presented as means ± S.E.M. from five or more independent experiments. To determine the overall significance, ANOVA was performed. Statistical comparisons between different treatment groups were done by Duncan’s Multiple Range Test. Differences with p-values of less than 0.05 were considered statistically significant.
3. Results 3.1. Effects of 15d-PGJ2 on N18D3 cells, depending on its concentration To evaluate the viability of N18D3 cells after their exposure to 15d-PGJ2, N18D3 cells were treated with different concentrations of 15d-PGJ2 [0 (control group), 2, 4, 8, 16, and 32 M] for 6 h. After washing and 24 h of further incubation, cell viability was measured by using the MTT assay and Trypan blue stain. As shown in Fig. 2A, there was no significant change in viability up to the dose of 8 M 15d-PGJ2, however, the viabilities at higher than 8 M concentrations were significantly decreased in a concentration-dependent pattern (p < 0.035). And, to evaluate the neuroprotective effects of low dose 15d-PGJ2 on oxidative stress-injured neuronal cells, N18D3 cells were pretreated with several concentrations of 15d-PGJ2 (0, 2, 4, and 8 M) in the medium for 6 h. The cells were washed and then exposed to 100 M H2 O2 for 30 min. The pretreatment of the cells with 15d-PGJ2 significantly increased the cell viability, and the increase was concentration-dependent (p < 0.049) (Fig. 2B). DAPI staining revealed that the treatment with 32 M 15d-PGJ2 significantly increased
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4 M 15d-PGJ2, compared with the control (Fig. 4B) (p < 0.05). 3.3. Effect of 15d-PGJ2 on the expression of p85a PI3-K, phosphorylation of JNK, Akt and GSK-3β, cytochrome c release, caspase-3 activation, and PARP cleavage
Fig. 2. Measurement of viability of N18D3 cells by MTT assay and Trypan blue stain. The data are represented as means (percent of control) ± S.E.M. Each treatment group was compared with the other groups using Duncan’s Multiple Range Test.
the percent of apoptotic cells, compared with the control and the treatment with 8 M 15d-PGJ2 (p < 0.021), however, the pretreatment with 8 M 15d-PGJ2 before 100 M H2 O2 treatment significantly reduced it, compared with the treatment of only 100 M H2 O2 (p < 0.014) (Fig. 3). 3.2. Oxidant effects of 15d-PGJ2 To appreciate the oxidant effects of 15d-PGJ2 itself, the amounts of free radicals in N18D3 cells treated with only 0 (control), 2, 4, 8, 16, and 32 M 15d-PGJ2 for 30 min were measured by using the DCFH-DA (Fig. 4A). Although the levels of free radicals were generally increased as the 15dPGJ2 concentration was increased, they were significantly increased in the cells treated with higher than 4 M 15d-PGJ2, compared with the control (p < 0.05). Furthermore, the levels of thiobarbituric acid-reacting substances (TBARS), which are products of membrane lipid peroxidation, were also significantly increased in the cells treated with higher than
The effects of 15d-PGJ2 on p85a PI3-K, phosphorylation of JNK, Akt and GSK-3, cytochrome c release, caspase-3 activation, and PARP cleavage were determined by immunoblotting homogenates of N18D3 cells treated under several conditions. Compared with the control, the immunoreactivities (IRs) of p85a PI3-K, phospho-Akt, and phospho-GSK3 (Ser-9) were slightly increased up to 8 M 15dPGJ2 (p < 0.041), but they were significantly decreased at higher than 8 M 15d-PGJ2 (p < 0.01) (Fig. 5A–F). And, compared with the cells treated with only 100 M H2 O2 , they were significantly increased in the cells pretreated with 8 M 15d-PGJ2 for 6 h before 100 M H2 O2 treatment (p < 0.025). In case of JNK, the IR of JNK1 and phospho-JNK1 was increased in a dosedependent manner, especially at higher than 8 M 15dPGJ2 (p < 0.037), and this finding is similar to previously reported result (Li et al., 2004a; Hashimoto et al., 2004). However, the IR of JNK1 and phospho-JNK1 of the cells pretreated with 8 M 15d-PGJ2 for 6 h before 100 M H2 O2 treatment was significantly lower than that of the cells only treated with 100 M H2 O2 (p < 0.009) (Fig. 5G and H). There was a slight reduction in the IRs of released cytochrome c, activated caspase-3, and cleaved PARP up to 8 M 15d-PGJ2 (p < 0.039), but they were significantly increased at higher than 8 M (p < 0.01), compared with the control group (Fig. 6). And, compared with the cells treated only with 100 M H2 O2 , the pretreatment of 8 M 15d-PGJ2 reduced those IRs (p < 0.014). 3.4. The inhibition of PI3-K/Akt partially blocks the effect of 15d-PGJ2 To examine the role of PI3-K/Akt in the 15d-PGJ2 mediated protection of N18D3 cells, we performed an experiment on N18D3 cells with PI3-K inhibitor. N18D3 cells were divided into four groups, such as control (group 1), 100 M H2 O2 only (group 2), 100 M H2 O2 + 8 M 15D-PGJ2 (group 3), and 50 M PI3-K inhibitor + 100 M H2 O2 + 8 M 15D-PGJ2 (group 4) groups. Comparing group 4 with group 3 (Fig. 7), cell viability was decreased about 12.5% in group 4.
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Fig. 3. Detection of apoptosis in N18D3 cells by DAPI staining. Treated and untreated cells were stained with DAPI and examined for condensation and fragmentation. (A) Normal cells have a round or an ovoid shape. (B) Cells treated with only 100 M H2 O2 show condensation and fragmentation of the nuclei, suggesting that they are undergoing apoptosis-like cell death. (C) Alteration of percent of apoptosis according to several conditions.
4. Discussion This study demonstrated that 15d-PGJ2 could have different effects on neuronal cells, depending on its concentrations: It could have neuroprotective effect at lower concentrations (up to 8 M) or neurotoxic effect at higher concentrations (higher than 8 M) in N18D3 cells (Fig. 2). In DAPI staining to evaluate apoptosis (Fig. 3), the treatment with 32 M 15d-PGJ2 treatment significantly increased the percent of apoptotic cells, compared with the control and the treatment with 8 M. However, the pretreatment with 8 M 15d-PGJ2 before inflicting oxidative stress rather decreased apoptotic cells. The levels of free radicals and membrane lipid peroxidation were dose-dependently increased according to the concentrations of 15d-PGJ2 (Fig. 4), although the simultaneous treatment of a direct scavenger or antioxidant, such as epigallocatechin gallate, could reduce free radical formation induced by the treatment with 15dPGJ2 (data not shown). In the study to evaluate the change of cellular signals (Figs. 5 and 6), lower con-
centration (8 M) of PGJ2 increased survival signals, such as PI3-K, phospho-Akt, and phospho-GSK-3, but decreased death signals, such as cytosolic cytochrome c, activated caspase-3, and cleaved PARP. On the contrary, its higher concentration (32 M) reduced the survival signals, but increased the death signals. Differently from other signals, JNK, proapoptotic signal related with the MEK/ERK pathway (Hashimoto et al., 2004; Li et al., 2004a,b; Liu and Lin, 2005; Zhao et al., 2004), was continuously increased in a dose-dependent manner, although increase of JNK was particularly significant at higher than 8 M, and this pattern was similar to the change pattern of free radicals and membrane lipid peroxidation. In the test to examine the role of PI3-K/Akt in the PGJ2-mediated survival protection of N18D3 cells, cell viability was decreased about 12.5% in 50 M PI3-K inhibitor + 100 M H2 O2 + 8 M PGJ2 group compared with 100 M H2 O2 + 8 M PGJ2 group. This decrement of cell viability might be caused by the inhibition of PI3K. Therefore, the effects of PGJ2 seen here were partially dependent on the activation of PI3-K.
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Fig. 4. Measurement of free radicals production and oxidative damage by treatment of 15d-PGJ2. The data are represented as means ± S.E.M. Each treatment group was compared with the other groups using Duncan’s Multiple Range Test. (A) N18D3 cells were treated with several concentrations of 15d-PGJ2 for 30 min to measure free radicals generated in the presence of 15d-PGJ2 itself by using the fluorescent probe DCFH-DA (see Section 2). (B) To measure oxidative damage by free radicals due to 15d-PGJ2 treatment for 1 h, membrane lipid peroxidation was measured as TBARS levels (see Section 2).
Prostaglandins are a family of oxygenated metabolites of arachidonic acid. They have a diverse range of actions depending on the PG type and cell target, and PGJ2 is one of them. Effects of PGJ2 on several kinds of cells, including cancer cells, vascular smooth muscles, and astrocyte (Chen et al., 2005; Giri et al., 2004; Hashimoto et al., 2004; Ichiki et al., 2004; Takeda et al., 2001; Zhao et al., 2004), have been being investigated. Although there are several reports about its effects on neuronal cells (Aoun et al., 2003; Heneka et al., 1999; Li et al., 2004a,b; Rohn et al., 2001; Yagami et al., 2003; Zhuang et al., 2003), the precise effects of 15d-PGJ2 on neuronal cells have not yet been clearly established. In the reports that PGJ2 was neurotoxic (Li et al., 2004a,b; Rohn et al., 2001), as the mechanism of neurotoxic effect of 15d-PGJ2, it has been suggested that 15d-PGJ2 enhances COX-2 expression (Li et al., 2004a), inhibits the ubiquitin hydrolase UCH-L1 and elicits ubiquitinprotein aggregation without proteasome inhibition (Li et al., 2004b), and induces apoptosis (Rohn et al., 2001). In the reports that PGJ2 had neuroprotective effect (Aoun et al., 2003; Heneka et al., 1999; Zhuang et al., 2003),
while, as the neuroprotective mechanism of 15d-PGJ2, it has been suggested that it controls heme oxygenase expression (Zhuang et al., 2003), prevents glutamateinduced cytotoxicity (Aoun et al., 2003), and inhibits inducible nitric oxide synthase (Heneka et al., 1999). The precise effects and mechanisms of 15d-PGJ2 are still controversial. Interrelationship between free radical level depending on 15d-PGJ2 concentration and neuronal cell survival has not yet been reported, although it has been well known that 15d-PGJ2 can induce free radical formation (Chen et al., 2005; Kondo et al., 2001). Furthermore, the effect of 15d-PGJ2 on the PI3K/Akt pathway, which has been being emphasized to be very important in neuronal cell survival (Cantley, 2002; Cantrell, 2001; Doble and Woodgett, 2003; Kirschenbaum et al., 2001; Pap and Cooper, 1998; Sang et al., 2001), has not yet been investigated in neuronal cells. It is novel to find that 15d-PGJ2, depending on its concentration, could play different roles as a neuroprotectant or a neurotoxicant. These findings may support the clue on the controversy about the effect on neuronal cells in the previous studies; Aoun et al. (2003) suggested that 15d-PGJ2 had neuroprotective effect against glutamate induced cytotoxicity in retinal ganglion cells, but Li et al. (2004a) suggested that it acted as a neurotoxicant on neuronal cells by enhancing COX-2 expression. Considering the results of two studies, 15d-PGJ2 acted as a neuroprotectant in lower concentrations from 1 to 5 M (Aoun et al., 2003), but as a neurotoxicant in higher concentration from 10 M (Li et al., 2004a). These previous findings are in accord with our present findings. These different effects of 15d-PGJ2 on neuronal cells, depending on its concentration, were achieved by differently acting cellular signals. 15d-PGJ2 at low concentration increased the expression of the survival signals, such as PI3-K and the phosphorylated Akt and GSK-3 (Ser-9), and decreased that of the death signals, such as cytosolic cytochrome c, activated caspase-3, and cleaved PARP, especially when severe oxidative stress, such as hydrogen peroxide, was inflicted, while, high concentration 15d-PGJ2 decreased the expression of the survival signals and increased that of the death signals. These different responses might be related with cellular tolerability. Namely, under the threshold level which cells can tolerate, stress might increase the expression of survival signals and decrease that of death signals, and then might keep cells alive. However, over the level, it can induce neuronal cell death by modifying cellular proteins, lipids and nucleic acids (Czapski et al., 2004; Kim et al., 2005; Koh et al., 2005).
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Fig. 6. Immunoreactivity (IR) of cytosolic cytochrome c, caspase-3, and PARP in N18D3 cells. Quantitative data of N18D3 cells are expressed as arbitrary unit of the simultaneously assayed control group’s value. Each treatment group was compared with the other groups using Duncan’s Multiple Range Test. Representative ECL radiograph of immunoblot, demonstrating that the IRs of cytosolic cytochrome c (A), activated caspase-3 (C) and cleaved PARP (E), and quantitative data expressing the IRs of cytosolic cytochrome c (B), activated caspase-3 (D), and cleaved PARP (F) (when each of them is normalized to actin immunostaining) in the control group, in only 15d-PGJ2 treated groups, in the only 100 M H2 O2 treated group, and in the 8 M 15d-PGJ2 pretreated group before the treatment with 100 M H2 O2 .
Fig. 5. Immunoreactivity (IR) of p85a PI3-K, phospho-Akt, phospho-GSK-3 (Ser-9), JNK1, and phospho-JNK1 in N18D3 cells. Quantitative data of N18D3 cells are expressed as arbitrary unit of the simultaneously assayed control group’s value. Each treatment group was compared with the other groups using Duncan’s Multiple Range Test. Representative ECL radiograph of immunoblot, demonstrating that the IRs of p85a PI3-K (A), phospho-Akt (C), phospho-GSK-3 (Ser-9) (E), and phospho-JNK1 (G) and quantitative data expressing the IRs of p85a PI3-K (normalized to actin immunostaining) (B), phospho-Akt (normalized to Akt immunostaining) (D), phospho-GSK-3 (Ser-9) (normalized to GSK-3 immunostaining) (F), and JNK1 and phospho-JNK1 (normalized to actin immunostaining) (H) in the control group, in only 15d-PGJ2 treated groups, in the only 100 M H2 O2 treated group, and in the 8 M 15d-PGJ2 pretreated group before the treatment with 100 M H2 O2 .
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Fig. 7. The role of PI3-K/Akt in the 15d-PGJ2 mediated survival protection of N18D3 cells. N18D3 cells were divided into 4 groups, such as control (group 1), 100 M H2 O2 only (group 2), 100 M H2 O2 + 8 M 15d-PGJ2 (group 3), and 50 M PI3K inhibitor, LY294002 (Sigma) + 100 M H2 O2 + 8 M 15d-PGJ2 (group 4) groups.
N18D3 cells could tolerate up to the threshold level of free radicals produced by 15d-PGJ2. However, they begin to die at over threshold level of free radicals caused by oxidative stress. The fact that pretreatment with 8 M 15d-PGJ2 protected N18D3 cells against oxidative stress by reducing the decrement of the survival signals caused by the stress suggests that free radicals below the threshold level might have preconditioning effects by stimulating survival signals. Our present results together with previous reports (Kim et al., 2005; Levites et al., 2003; Weinreb et al., 2003) suggest that low concentration of antioxidants, such as 15d-PGJ2, induces anti-apoptotic patterns in gene and protein expressions and high concentration induces proapoptotic patterns, again emphasizing the fact that the level of free radicals produced by antioxidants has a very important role in inducing different effects of antioxidants. These suggestions were also indirectly proven by the findings that, although JNK, free radicals and membrane lipid peroxidation, which reflect stress given to cells, were even slightly increased in low concentration of 15d-PGJ2, the viability was not decreased by increasing survival signals and decreasing death signals. Describing in detail, although low concentration of 15d-PGJ2 even induced oxidative stress, based on the results of the studies on the alteration of the levels of free radicals and membrane lipid peroxidation depending on its concentration, neuronal cells could tolerate it enough by increasing survival signals, including PI3-K, phospho-Akt, and phospho-GSK-3 (Ser-9), and decreasing death signals, including cytosolic cytochrome c, activated caspase-3, and cleaved PARP, based on the change of cellular signals. However, oxidative stress induced by high con-
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centration of 15d-PGJ2 was over the threshold level. Therefore, survival signals were decreased and death signals were increased, and neuronal cells began to die. There are some limitations in this study which included: (1) the dose of 15d-PGJ2 in vivo could not physiologically reach the M level, (2) it was not confirmed whether the observed response of cells to free radicals is a general phenomenon or is specific to N18D3 cells, and (3) DCFH for measuring reactive oxygen species might generate free radicals and TBARS for measuring membrane lipid peroxidation might be nonspecific (Rota et al., 1999a,b). Therefore, further studies are needed to evaluate the neuroprotective effect of 15dPGJ2 in vivo and to evaluate whether the response of cells to free radicals is general phenomenon. And, precise and more appropriate measurement of PGJ2 induced ROS using spin-trap should be performed (Rota et al., 1999a,b). In conclusion, the results of this study showed that 15d-PGJ2 could act as a neuroprotectant or a neurotoxicant, depending on its concentration, that it, especially at some specific concentrations, had neuroprotective effects on oxidative stress-injured N18D3 cells, by increasing p85a PI3-K, phospho-Akt, and phospho-GSK-3 (Ser-9) and decreasing cytosolic cytochrome c, activated caspase-3, and cleaved PARP, and that the level of free radicals produced by 15d-PGJ2, might have a very important role in deciding the fate of cells. These results suggest that low concentration of 15d-PGJ2 could be a potential candidate of therapeutic or modulating agents for neurodegenerative and other diseases influenced by oxidative damage. Acknowledgements The authors would like to thank Prof. Woon K. Paik, Hanyang University, and anonymous reviewer for careful review of this manuscript. N18D3 cells were generous gift from Prof. Seung U. Kim (Brain Disease Research Center, Ajou University School of Medicine, Suwon, South Korea and Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, Canada). References Aoun, P., Simpkins, J.W., Agarwal, N., 2003. Role of PPAR-gamma ligands in neuroprotection against glutamate-induced cytotoxicity in retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 44, 2999–3004. Cantley, L.C., 2002. The phosphoinositide 3-kinase pathway. Science 296, 1655–1657.
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15-Deoxy-delta12,14-prostaglandin J2, a neuroprotectant or a neurotoxicant? Seong-Ho Koh a,1 , Boo Jung a,1 , Chi Won Song b , Youngchul Kim a , Yong Soon Kim b , Seung Hyun Kim a,∗ a
b
Department of Neurology, Institute of Biomedical Science, College of Medicine, Hanyang University, #17 Haengdang-dong, Seongdong-ku, Seoul 133-791, Republic of Korea Department of General Toxicology, National Institute of Toxicological Research, KFDA, Seoul, Republic of Korea Received 1 July 2005; received in revised form 9 August 2005; accepted 12 August 2005 Available online 26 September 2005
Abstract 15-Deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) is a potent ligand for peroxisome proliferators-activated receptor ␥ (PPAR␥). However, its various effects independent of PPAR␥ have recently been observed. The effect of 15d-PGJ2 on neuronal cells is still controversial. We investigated its effect on neuronal cells (N18D3 cells). When N18D3 cells were treated with 15d-PGJ2, the viability was not changed up to 8 M, but decreased at higher than 8 M. The expressions of survival signals, such as p85a phosphatidylinositol 3-kinase, phospho-Akt, and phospho-glycogen synthase kinase-3 beta (Ser-9), slightly increased up to 8 M, however, decreased at higher than 8 M. The levels of free radicals and membrane lipid peroxidation and the expression of c-Jun N-terminal Kinase increased in a dose-dependent manner, especially at higher than 8 M. However, the expressions of death signals, such as cytosolic cytochrome c, activated caspase-3, and cleaved poly(ADP-ribose) polymerase, decreased up to 8 M, however, increased at higher than 8 M. In the study to evaluate whether low dose of 15d-PGJ2, up to 8 M, had protective effect on oxidative stress-injured N18D3 cells, compared to the cells treated with only 100 M H2 O2 , the pretreatment with 8 M 15d-PGJ2 increased the viability and the expressions of the survival signals, but decreased them of the death signals. These results indicate that 15d-PGJ2 could be a neuroprotectant or a neurotoxicant, depending on its concentration. Therefore, some specific optimum dose of 15d-PGJ2 may be a new potential therapeutic candidate for oxidative stress-injury model of neurodegenerative diseases. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: 15d-PGJ2; Neuron; JNK; PI3-K; Akt
1. Introduction 15-Deoxy-delta12,14-prostagladin J2 (15d-PGJ2) is a natural ligand activating peroxisome proliferators∗
Corresponding author. Tel.: +82 2 2290 8371; fax: +82 2 2296 8370. E-mail address: [email protected] (S.H. Kim). 1 Both authors (S.-H. Koh, B. Jung) equally contributed to this journal.
activated receptor ␥ (PPAR␥), a nuclear hormone receptor/transcription factor that regulates adipocyte differentiation and fatty acid homeostasis (Tontonoz et al., 1994; Ichiki et al., 2004). It is well known that 15d-PGJ2-activated PPAR␥ forms a heterodimer with retinoid X receptor, binds to a specific DNA sequence, PPAR response element (PPRE), and activates target gene transcription (Schoonjans et al., 1997). However, its various effects independent of PPAR␥, including induction of cyclooxygenase-2 expression and activa-
0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2005.08.015
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tion of the mitogen-activated protein kinase kinase (MEK)/extracellular signals-regulated kinase (ERK) pathway, have recently been reported (Li et al., 2004a; Hashimoto et al., 2004). And, in vascular smooth muscle, 15d-PGJ2-induced DNA synthesis was also proven to be partially dependent on phosphatidylinositol 3-kinase (PI3-K) (Takeda et al., 2001). Recently, PI3-K/Akt signaling pathway has been shown to be related with pathogenic mechanism of neurodegenerative diseases (Cantley, 2002; Cantrell, 2001; Doble and Woodgett, 2003; Kirschenbaum et al., 2001; Kulich and Chu, 2001; Pap and Cooper, 1998; Pei et al., 2002; Sang et al., 2001; Xia and Hyman, 2002; Xu et al., 2005; Zhao et al., 2002), and oxidative stress neuronal injury (Kim et al., 2005; Koh et al., 2003, 2004, 2005). Phosphorylated Akt activated by PI3-K plays very important role in the neuronal cell survival by both enhancing the expression of anti-apoptotic proteins and inhibiting the activity of proapoptotic ones (Cantley, 2002; Cantrell, 2001; Doble and Woodgett, 2003; Kirschenbaum et al., 2001; Koh et al., 2003, 2004, 2005; Pap and Cooper, 1998; Sang et al., 2001). GSK-3 regulates the function of metabolic, structural, and signaling proteins and is regulated by PI3-K/Akt activity. Furthermore, evidence indicates that GSK-3 is associated with Alzheimer’s disease by participating in phosphorylation of the microtubulebinding protein tau and contributing to the formation of neurofibrillary tangles and the interaction with presenilin (Doble and Woodgett, 2003; Kirschenbaum et al., 2001; Sang et al., 2001). Activated GSK-3 is inactivated through phosphorylation by pAkt. Phosphorylation of GSK-3 is important in the inhibition of oxidative stress-induced apoptosis, because unphosphorylated GSK-3, activated form which activates the mitochondrial death pathway, results in the damage of mitochondria. Increase of cytoplasmic cytochrome c released from damaged mitochondria activates caspase9 and caspase-3, accompanied by PARP cleavage, and then results in apoptosis (Cantley, 2002; Cantrell, 2001; Koh et al., 2003, 2004, 2005; Pap and Cooper, 1998). There have been several reports about effects of PGJ2 on neuronal cells (Aoun et al., 2003; Heneka et al., 1999; Li et al., 2004a,b; Rohn et al., 2001; Yagami et al., 2003; Zhuang et al., 2003). Nevertheless, its precise effects on neuronal cells have not yet been clearly established. 15d-PGJ2 was described to have neuroprotective effect in some reports (Aoun et al., 2003; Heneka et al., 1999; Zhuang et al., 2003), but to have neurotoxic effect in others (Li et al., 2004a,b; Rohn et al., 2001). Considering that various studies on the effects of
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15d-PGJ2 on neuronal cells had been focused on specific concentration and shown different effects (Aoun et al., 2003; Li et al., 2004a), the precise concentration–response relationships should be clearly examined. This study was designed to evaluate the dosedependent different roles of PGJ2 on neuronal cells (N18D3 cells). The levels of free radicals and membrane lipid peroxidation, and the cellular signals, such as phosphatidylinositol 3-kinase (PI3-K), Akt, glycogen synthase kinase-3 beta (GSK-3), c-Jun N-terminal Kinase (JNK), phospho-JNK, cytosolic cytochrome c, caspase3, and poly(ADP-ribose) polymerase (PARP) were investigated in PGJ2-treated N18D3 cells with/without hydrogen peroxide treatment. 2. Materials and methods 2.1. Cell culture, induction of differentiation, and treatment N18D3 cells, hybrid neuron line obtained by fusion of dorsal root ganglion neurons isolated from 4-week-old Balb/C mouse with the mouse neuroblastoma N18TG2 cells, were maintained in logarithmic-phase growth on poly-l-lysine (Sigma)-precoated 100-mm dish (Corning) in Minium Essential Media (GIBCO BRL, Grand Island, NY, USA) containing 10% heat-inactivated donor horse serum, 5% fetal bovine serum (FBS), 100 units/ml penicillin, 100 g/ml streptomycin, 4 g/l glucose, 2.2 g/l NaHCO3 , and 10 ml/l l-glutamine. After cells were neuronally differentiated with treatment of nerve growth factor, the cells were maintained in humidified incubators with 5% CO2 and 95% air at 37 ◦ C. Cells were grown to 50% confluence, and then harvested in Ca2+ /Mg2+ -free Hank’s balanced salt solution containing 1 mM EDTA. N18D3 cells were plated at a density of 106 cells/100-mm poly-l-lysineprecoated dish (Kim et al., 2005; Koh et al., 2004; Park et al., 2000; Sanfeliu et al., 1999). To examine the effect of 15d-PGJ2 (Fig. 1) on the viability of neuronal cells, 15d-PGJ2 (Cayman Ann Arbor, MI, USA) was dissolved in ethanol, the final concentration of ethanol never exceeding 0.1% when added to cells as ethanol solution (Satoh et al., 1999). N18D3 cells were treated for 6 h (Aoun et al., 2003) with several concentrations of 15d-PGJ2 [0 (only
Fig. 1. Structure of 15-deoxy-delta12,14-prostaglandin J2 (15dPGJ2).
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vehicle, control), 2, 4, 8, 16, and 32 M] and were washed more than three times with phosphate buffered saline (PBS). Cell viability was measured after 24 h by using the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Trypan blue stain (Carmichael et al., 1987; McCarthy and Evan, 1998). To evaluate the effect of PGJ2 on cellular signals, including PI3-K/Akt, GSK-3, JNK, cytosolic cytochrome c, caspase-3, and PARP, N18D3 cells were treated with several concentrations of 15d-PGJ2. And to evaluate its protective effect on oxidative stress-injury, N18D3 cells were pretreated with different concentrations of 15d-PGJ2 (0, 2, 4, or 8 M) for 6 h, washed carefully several times with PBS, and then treated with 100 M H2 O2 for 30 min. Subsequently, the cells were washed carefully, and the cell viability was measured after 24 h. To exclude the possibility of direct hydroxyl radical scavenging effect of residual 15d-PGJ2 on wells, washing steps were repeated several times prior to the addition of H2 O2 . In addition, the levels of hydrogen peroxide were measured using luminol-dependent chemiluminescence which is formed by the reaction between H2 O2 and luminol catalyzed by horseradish peroxidase (Wilhelm et al., 1995), and the amounts of reactive hydroxyl radicals (OH• ) from H2 O2 were indirectly measured by quantifying 2,5- and 2,3-dihydroxybenzoic acid (DHBA) generated by salicylate (Wako Pure Chemical Industries Ltd., Osaka, Japan) hydroxylation using HPLC (Kaneko et al., 2000; Teismann and Ferger, 2000). 2,5- and 2,3-DHBA used as standard were purchased from Sigma Chemical (St. Louis, MO, USA). After washing more than three times, the levels of H2 O2 and the amounts of reactive hydroxyl radicals were found to be not statistically significantly different between the group treated with 100 M H2 O2 only and the group treated with 100 M H2 O2 and 15d-PGJ2. Harvested cells after 30 min of H2 O2 treatment were immediately used for PI3-K/Akt, GSK3, and JNK immunoblotting, and cytochrome c, caspase-3, and PARP immunoblotting after 6 h (Fahn and Cohen, 1992; Mausuda et al., 2002; McCarthy and Evan, 1998; McCord, 1994; Molina-Holgade et al., 2002; Mudher et al., 2001). To examine the role of PI3-K/Akt in the 15d-PGJ2 mediated survival protection of N18D3 cells, we designed an experiment on N18D3 cells with PI3-K inhibitor, LY294002 (Sigma, St. Louis, MO, USA). N18D3 cells were divided into four groups, such as control (group 1), 100 M H2 O2 (group 2), 100 M H2 O2 + 8 M 15d-PGJ2 (group 3), and 50 M LY294002 + 100 M H2 O2 + 8 M 15d-PGJ2 (group 4) groups. LY294002 was treated at group 4 before 1 h of 15dPGJ2 treatment. 2.2. MTT assay and Trypan blue staining to evaluate cell survival MTT is absorbed into cell, and formazan is then formed by the action of mitochondrial succinate dehydrogenase. Accumulation of formazan reflects the activity of mitochondria directly and the cell viability indirectly. Thus, cells were plated at a density of 1 × 104 cells/well in the 96-well plates, cultured,
differentiated, and treated according to the methods described above. A total 50 l of 2 mg/ml MTT (Sigma) was added after 200 l of medium was added into each well. An aliquot (220 l) of solution was removed from each well, and 150 l of dimethyl sulfoxide was then added to each well. Optical density (OD) at 540 nm was evaluated on the ELISA plate reader after precipitate in the well was dissolved by a microplate mixer for 10 min. All results were calibrated for OD measured in the same conditioned well without cell culture (Koh et al., 2003). For Trypan blue staining, 10 l of Trypan blue solution were incubated with 10 l of cells collected from each plate for 2 min, and unstained live cells were counted on a hemacytometer (Koh et al., 2003). 2.3. DAPI staining to evaluate apoptosis DAPI staining was performed to evaluate apoptosis. The procedures for DAPI staining were as follows: N18D3 cells were treated (1) without any of H2 O2 and 15d-PGJ2 (control), (2) with only 8 M 15d-PGJ2 for 6 h, (3) with only 32 M 15d-PGJ2 for 6 h, (4) with only 100 M H2 O2 for 30 min, and (5) with 100 M H2 O2 for 30 min after pretreatment with 8 M 15d-PGJ2 for 6 h. Subsequently, the differently treated cells were further incubated in the 96-well plates for 24 h after washing, harvested in PBS, and then centrifuged at 261 × g for 2 min, and 4% neutral buffered formalin (100 l) was added to the cell pellet for fixation. A 50 l aliquot of the cell suspension was applied to a glass slide and dried at room temperature. The fixed cells were then washed in PBS, air dried, and stained with the DNA-specific fluorochrome 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) for 20 min. The adhered cells were rinsed with PBS, air dried, and mounted with 90% glycerol. The slides were observed under an Olympus fluorescence microscope. Percent of apoptotic cells, which coincided with morphological criteria of apoptosis, such as nuclear condensation and segmentation, was calculated based on the total number of cells. 2.4. Determination of free radical production and oxidative damage induced by 15d-PGJ2 itself To measure free radical production by 15d-PGJ2 itself, neuronally differentiated N18D3 cells treated with only 0 (control), 2, 4, 8, 16, and 32 M 15d-PGJ2 for 30 min were incubated with fluorescent probe 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) (Molecular Probes Inc., Eugene, OR, USA), which freely crosses the cell membranes and is hydrolysed by cellular esterases to 2 ,7 -dichlorodihydrofluorescein (DCFH2 ). DSFH2 is a non-fluorescent molecule, however, it is oxidized to the fluorescent 2 ,7 -dichlorofluorescein (DCF) in the presence of peroxides. Therefore, accumulation of DCF in the cells is indicated by an increment in fluorescence at 525 nm, when the sample is excited at 488 nm by a Microplate Fluorescence Reader (FL600) (D.I. Biotech Ltd., Seoul, Korea) (Tammariello et al., 2000).
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To measure oxidative damage by 15d-PGJ2 itself, lipid peroxidation was evaluated by measuring the thiobarbituric acidreacting substances (TBARS) (Sigma Chemical) in N18D3 cells treated with only 0 (control), 2, 4, 8, 16, and 32 M 15dPGJ2 for 1 h. Briefly, the cells were washed and harvested with ice-cold 50 mM phosphate buffer, pH 7.4, and then 500 l of 1% thiobarbituric acid and 500 l of 8N HCl were added to the sample. The samples were boiled for 20 min and subsequently cooled with tap water. 1-Butanol (1.5 ml) was then added to the samples, and the mixture was shaken for 2 min. After centrifugation at 2000 × g for 10 min, the fluorescence intensity at 550 nm (excitation) and 534 nm (emission) in the butanol phase was measured by a Microplate Fluorescence Reader (FL600) (Ohkawa et al., 1979). Incubation time used in these studies (30 min for measuring free radical production and 1 h for measuring lipid peroxidation) was chosen, because maximal effects were shown at these time points. 2.5. Western blot analyses For Western blotting, we treated N18D3 cells: (1) without any of H2 O2 and 15d-PGJ2, (2) with 2, 4, 8, 16, or 32 M 15d-PGJ2 for 6 h, (3) with only 100 M H2 O2 for 30 min, and (4) with 100 M H2 O2 for 30 min after pretreatment with 8 M 15d-PGJ2 for 6 h. PI3-K, Akt, GSK3, JNK, phospho-JNK, cytosolic cytochrome c, caspase-3, and PARP were assayed by Western blot analyses. Briefly, 5 × 106 cells were washed twice in cold PBS and incubated for 10 min on ice in a lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 100 g/ml phenylmethylsulfonylfluoride (PMSF), 50 l/ml aprotinin, 1% Igapel 630, 100 mM NaF, 0.5% sodium deoxy choate, 0.5 mM EDTA, and 0.1 mM EGTA]. Cell lysates were centrifuged at 17,000 × g to evaluate cleaved and non-cleaved forms of PARP and caspase-3, phosphorylated and non-phosphorylated form of JNK1, GSK-3 and Akt, and p85a PI3-K. And, to evaluate released cytochrome c, cells after washing were suspended in sucrose-supplemented cell extract buffer (SCEB, 300 mM sucrose, 10 mM HEPES at pH 7.4, 50 mM KCl, 5 mM EGTA, 5 mM MgCl2 , 1 mM DTT, 10 M cytochalasin B, and 1 mM PMSF), left on ice for 30 min, and then homogenized by 50 strokes in an ice-cold Dounce homogenizer. Unbroken cells and nuclei were pelleted by centrifugation at 2000 × g for 10 min. Mitochondria were collected by centrifugation of the resulting supernatant at 13,000 × g for 10 min. The postmitochondrial fraction, namely the supernatant in the above step, was used for immunoblotting for cytochrome c. Protein concentrations of cell lysates and postmitochondrial fraction were determined by Bio-Rad protein assay kit. An equal amount (20 g) of protein from each sample was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were blocked with 5% skim milk and sequentially incubated with specific antibodies for PARP (1:500, Pharmin-
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gen, San Diego, CA, USA), caspase-3 (1:1000, Santa Cruz Biotech, Delaware, CA, USA), cleaved caspase-3 (Asp 175) (1:1000, Cell Signaling, Beverly, MA, USA), cytochrome c (1:500, Santa Cruz Biotech), JNK1, phospho-JNK1 (1:1000, Santa Cruz Biotech), GSK-3 (1:1000, Santa Cruz Biotech), phospho-GSK-3 (Ser-9) (1:1000, Santa Cruz Biotech), Akt (1:1000, Cell Signaling), phospho-Akt (1:1000, Cell Signaling), and p85a PI3-K (1:1000, Sigma). The membranes were washed with Tris buffered saline containing 0.05% Tween20 (TBST), and then processed using an HRP-conjugated anti-rabbit antibody or anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA) followed by ECL detection (Amersham Pharmacia Biotech) (Juin et al., 1999; Koh et al., 2003). The same membrane was used to measure the each ratio by reprobing: for example, the membrane used for evaluation of pAkt was reused to evaluate Akt through reprobing procedure. The results of Western blots were quantified with image analyzer (Bio-Rad, Quantity One-4,2,0). 2.6. Statistical analysis All data were presented as means ± S.E.M. from five or more independent experiments. To determine the overall significance, ANOVA was performed. Statistical comparisons between different treatment groups were done by Duncan’s Multiple Range Test. Differences with p-values of less than 0.05 were considered statistically significant.
3. Results 3.1. Effects of 15d-PGJ2 on N18D3 cells, depending on its concentration To evaluate the viability of N18D3 cells after their exposure to 15d-PGJ2, N18D3 cells were treated with different concentrations of 15d-PGJ2 [0 (control group), 2, 4, 8, 16, and 32 M] for 6 h. After washing and 24 h of further incubation, cell viability was measured by using the MTT assay and Trypan blue stain. As shown in Fig. 2A, there was no significant change in viability up to the dose of 8 M 15d-PGJ2, however, the viabilities at higher than 8 M concentrations were significantly decreased in a concentration-dependent pattern (p < 0.035). And, to evaluate the neuroprotective effects of low dose 15d-PGJ2 on oxidative stress-injured neuronal cells, N18D3 cells were pretreated with several concentrations of 15d-PGJ2 (0, 2, 4, and 8 M) in the medium for 6 h. The cells were washed and then exposed to 100 M H2 O2 for 30 min. The pretreatment of the cells with 15d-PGJ2 significantly increased the cell viability, and the increase was concentration-dependent (p < 0.049) (Fig. 2B). DAPI staining revealed that the treatment with 32 M 15d-PGJ2 significantly increased
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4 M 15d-PGJ2, compared with the control (Fig. 4B) (p < 0.05). 3.3. Effect of 15d-PGJ2 on the expression of p85a PI3-K, phosphorylation of JNK, Akt and GSK-3β, cytochrome c release, caspase-3 activation, and PARP cleavage
Fig. 2. Measurement of viability of N18D3 cells by MTT assay and Trypan blue stain. The data are represented as means (percent of control) ± S.E.M. Each treatment group was compared with the other groups using Duncan’s Multiple Range Test.
the percent of apoptotic cells, compared with the control and the treatment with 8 M 15d-PGJ2 (p < 0.021), however, the pretreatment with 8 M 15d-PGJ2 before 100 M H2 O2 treatment significantly reduced it, compared with the treatment of only 100 M H2 O2 (p < 0.014) (Fig. 3). 3.2. Oxidant effects of 15d-PGJ2 To appreciate the oxidant effects of 15d-PGJ2 itself, the amounts of free radicals in N18D3 cells treated with only 0 (control), 2, 4, 8, 16, and 32 M 15d-PGJ2 for 30 min were measured by using the DCFH-DA (Fig. 4A). Although the levels of free radicals were generally increased as the 15dPGJ2 concentration was increased, they were significantly increased in the cells treated with higher than 4 M 15d-PGJ2, compared with the control (p < 0.05). Furthermore, the levels of thiobarbituric acid-reacting substances (TBARS), which are products of membrane lipid peroxidation, were also significantly increased in the cells treated with higher than
The effects of 15d-PGJ2 on p85a PI3-K, phosphorylation of JNK, Akt and GSK-3, cytochrome c release, caspase-3 activation, and PARP cleavage were determined by immunoblotting homogenates of N18D3 cells treated under several conditions. Compared with the control, the immunoreactivities (IRs) of p85a PI3-K, phospho-Akt, and phospho-GSK3 (Ser-9) were slightly increased up to 8 M 15dPGJ2 (p < 0.041), but they were significantly decreased at higher than 8 M 15d-PGJ2 (p < 0.01) (Fig. 5A–F). And, compared with the cells treated with only 100 M H2 O2 , they were significantly increased in the cells pretreated with 8 M 15d-PGJ2 for 6 h before 100 M H2 O2 treatment (p < 0.025). In case of JNK, the IR of JNK1 and phospho-JNK1 was increased in a dosedependent manner, especially at higher than 8 M 15dPGJ2 (p < 0.037), and this finding is similar to previously reported result (Li et al., 2004a; Hashimoto et al., 2004). However, the IR of JNK1 and phospho-JNK1 of the cells pretreated with 8 M 15d-PGJ2 for 6 h before 100 M H2 O2 treatment was significantly lower than that of the cells only treated with 100 M H2 O2 (p < 0.009) (Fig. 5G and H). There was a slight reduction in the IRs of released cytochrome c, activated caspase-3, and cleaved PARP up to 8 M 15d-PGJ2 (p < 0.039), but they were significantly increased at higher than 8 M (p < 0.01), compared with the control group (Fig. 6). And, compared with the cells treated only with 100 M H2 O2 , the pretreatment of 8 M 15d-PGJ2 reduced those IRs (p < 0.014). 3.4. The inhibition of PI3-K/Akt partially blocks the effect of 15d-PGJ2 To examine the role of PI3-K/Akt in the 15d-PGJ2 mediated protection of N18D3 cells, we performed an experiment on N18D3 cells with PI3-K inhibitor. N18D3 cells were divided into four groups, such as control (group 1), 100 M H2 O2 only (group 2), 100 M H2 O2 + 8 M 15D-PGJ2 (group 3), and 50 M PI3-K inhibitor + 100 M H2 O2 + 8 M 15D-PGJ2 (group 4) groups. Comparing group 4 with group 3 (Fig. 7), cell viability was decreased about 12.5% in group 4.
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Fig. 3. Detection of apoptosis in N18D3 cells by DAPI staining. Treated and untreated cells were stained with DAPI and examined for condensation and fragmentation. (A) Normal cells have a round or an ovoid shape. (B) Cells treated with only 100 M H2 O2 show condensation and fragmentation of the nuclei, suggesting that they are undergoing apoptosis-like cell death. (C) Alteration of percent of apoptosis according to several conditions.
4. Discussion This study demonstrated that 15d-PGJ2 could have different effects on neuronal cells, depending on its concentrations: It could have neuroprotective effect at lower concentrations (up to 8 M) or neurotoxic effect at higher concentrations (higher than 8 M) in N18D3 cells (Fig. 2). In DAPI staining to evaluate apoptosis (Fig. 3), the treatment with 32 M 15d-PGJ2 treatment significantly increased the percent of apoptotic cells, compared with the control and the treatment with 8 M. However, the pretreatment with 8 M 15d-PGJ2 before inflicting oxidative stress rather decreased apoptotic cells. The levels of free radicals and membrane lipid peroxidation were dose-dependently increased according to the concentrations of 15d-PGJ2 (Fig. 4), although the simultaneous treatment of a direct scavenger or antioxidant, such as epigallocatechin gallate, could reduce free radical formation induced by the treatment with 15dPGJ2 (data not shown). In the study to evaluate the change of cellular signals (Figs. 5 and 6), lower con-
centration (8 M) of PGJ2 increased survival signals, such as PI3-K, phospho-Akt, and phospho-GSK-3, but decreased death signals, such as cytosolic cytochrome c, activated caspase-3, and cleaved PARP. On the contrary, its higher concentration (32 M) reduced the survival signals, but increased the death signals. Differently from other signals, JNK, proapoptotic signal related with the MEK/ERK pathway (Hashimoto et al., 2004; Li et al., 2004a,b; Liu and Lin, 2005; Zhao et al., 2004), was continuously increased in a dose-dependent manner, although increase of JNK was particularly significant at higher than 8 M, and this pattern was similar to the change pattern of free radicals and membrane lipid peroxidation. In the test to examine the role of PI3-K/Akt in the PGJ2-mediated survival protection of N18D3 cells, cell viability was decreased about 12.5% in 50 M PI3-K inhibitor + 100 M H2 O2 + 8 M PGJ2 group compared with 100 M H2 O2 + 8 M PGJ2 group. This decrement of cell viability might be caused by the inhibition of PI3K. Therefore, the effects of PGJ2 seen here were partially dependent on the activation of PI3-K.
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Fig. 4. Measurement of free radicals production and oxidative damage by treatment of 15d-PGJ2. The data are represented as means ± S.E.M. Each treatment group was compared with the other groups using Duncan’s Multiple Range Test. (A) N18D3 cells were treated with several concentrations of 15d-PGJ2 for 30 min to measure free radicals generated in the presence of 15d-PGJ2 itself by using the fluorescent probe DCFH-DA (see Section 2). (B) To measure oxidative damage by free radicals due to 15d-PGJ2 treatment for 1 h, membrane lipid peroxidation was measured as TBARS levels (see Section 2).
Prostaglandins are a family of oxygenated metabolites of arachidonic acid. They have a diverse range of actions depending on the PG type and cell target, and PGJ2 is one of them. Effects of PGJ2 on several kinds of cells, including cancer cells, vascular smooth muscles, and astrocyte (Chen et al., 2005; Giri et al., 2004; Hashimoto et al., 2004; Ichiki et al., 2004; Takeda et al., 2001; Zhao et al., 2004), have been being investigated. Although there are several reports about its effects on neuronal cells (Aoun et al., 2003; Heneka et al., 1999; Li et al., 2004a,b; Rohn et al., 2001; Yagami et al., 2003; Zhuang et al., 2003), the precise effects of 15d-PGJ2 on neuronal cells have not yet been clearly established. In the reports that PGJ2 was neurotoxic (Li et al., 2004a,b; Rohn et al., 2001), as the mechanism of neurotoxic effect of 15d-PGJ2, it has been suggested that 15d-PGJ2 enhances COX-2 expression (Li et al., 2004a), inhibits the ubiquitin hydrolase UCH-L1 and elicits ubiquitinprotein aggregation without proteasome inhibition (Li et al., 2004b), and induces apoptosis (Rohn et al., 2001). In the reports that PGJ2 had neuroprotective effect (Aoun et al., 2003; Heneka et al., 1999; Zhuang et al., 2003),
while, as the neuroprotective mechanism of 15d-PGJ2, it has been suggested that it controls heme oxygenase expression (Zhuang et al., 2003), prevents glutamateinduced cytotoxicity (Aoun et al., 2003), and inhibits inducible nitric oxide synthase (Heneka et al., 1999). The precise effects and mechanisms of 15d-PGJ2 are still controversial. Interrelationship between free radical level depending on 15d-PGJ2 concentration and neuronal cell survival has not yet been reported, although it has been well known that 15d-PGJ2 can induce free radical formation (Chen et al., 2005; Kondo et al., 2001). Furthermore, the effect of 15d-PGJ2 on the PI3K/Akt pathway, which has been being emphasized to be very important in neuronal cell survival (Cantley, 2002; Cantrell, 2001; Doble and Woodgett, 2003; Kirschenbaum et al., 2001; Pap and Cooper, 1998; Sang et al., 2001), has not yet been investigated in neuronal cells. It is novel to find that 15d-PGJ2, depending on its concentration, could play different roles as a neuroprotectant or a neurotoxicant. These findings may support the clue on the controversy about the effect on neuronal cells in the previous studies; Aoun et al. (2003) suggested that 15d-PGJ2 had neuroprotective effect against glutamate induced cytotoxicity in retinal ganglion cells, but Li et al. (2004a) suggested that it acted as a neurotoxicant on neuronal cells by enhancing COX-2 expression. Considering the results of two studies, 15d-PGJ2 acted as a neuroprotectant in lower concentrations from 1 to 5 M (Aoun et al., 2003), but as a neurotoxicant in higher concentration from 10 M (Li et al., 2004a). These previous findings are in accord with our present findings. These different effects of 15d-PGJ2 on neuronal cells, depending on its concentration, were achieved by differently acting cellular signals. 15d-PGJ2 at low concentration increased the expression of the survival signals, such as PI3-K and the phosphorylated Akt and GSK-3 (Ser-9), and decreased that of the death signals, such as cytosolic cytochrome c, activated caspase-3, and cleaved PARP, especially when severe oxidative stress, such as hydrogen peroxide, was inflicted, while, high concentration 15d-PGJ2 decreased the expression of the survival signals and increased that of the death signals. These different responses might be related with cellular tolerability. Namely, under the threshold level which cells can tolerate, stress might increase the expression of survival signals and decrease that of death signals, and then might keep cells alive. However, over the level, it can induce neuronal cell death by modifying cellular proteins, lipids and nucleic acids (Czapski et al., 2004; Kim et al., 2005; Koh et al., 2005).
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Fig. 6. Immunoreactivity (IR) of cytosolic cytochrome c, caspase-3, and PARP in N18D3 cells. Quantitative data of N18D3 cells are expressed as arbitrary unit of the simultaneously assayed control group’s value. Each treatment group was compared with the other groups using Duncan’s Multiple Range Test. Representative ECL radiograph of immunoblot, demonstrating that the IRs of cytosolic cytochrome c (A), activated caspase-3 (C) and cleaved PARP (E), and quantitative data expressing the IRs of cytosolic cytochrome c (B), activated caspase-3 (D), and cleaved PARP (F) (when each of them is normalized to actin immunostaining) in the control group, in only 15d-PGJ2 treated groups, in the only 100 M H2 O2 treated group, and in the 8 M 15d-PGJ2 pretreated group before the treatment with 100 M H2 O2 .
Fig. 5. Immunoreactivity (IR) of p85a PI3-K, phospho-Akt, phospho-GSK-3 (Ser-9), JNK1, and phospho-JNK1 in N18D3 cells. Quantitative data of N18D3 cells are expressed as arbitrary unit of the simultaneously assayed control group’s value. Each treatment group was compared with the other groups using Duncan’s Multiple Range Test. Representative ECL radiograph of immunoblot, demonstrating that the IRs of p85a PI3-K (A), phospho-Akt (C), phospho-GSK-3 (Ser-9) (E), and phospho-JNK1 (G) and quantitative data expressing the IRs of p85a PI3-K (normalized to actin immunostaining) (B), phospho-Akt (normalized to Akt immunostaining) (D), phospho-GSK-3 (Ser-9) (normalized to GSK-3 immunostaining) (F), and JNK1 and phospho-JNK1 (normalized to actin immunostaining) (H) in the control group, in only 15d-PGJ2 treated groups, in the only 100 M H2 O2 treated group, and in the 8 M 15d-PGJ2 pretreated group before the treatment with 100 M H2 O2 .
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Fig. 7. The role of PI3-K/Akt in the 15d-PGJ2 mediated survival protection of N18D3 cells. N18D3 cells were divided into 4 groups, such as control (group 1), 100 M H2 O2 only (group 2), 100 M H2 O2 + 8 M 15d-PGJ2 (group 3), and 50 M PI3K inhibitor, LY294002 (Sigma) + 100 M H2 O2 + 8 M 15d-PGJ2 (group 4) groups.
N18D3 cells could tolerate up to the threshold level of free radicals produced by 15d-PGJ2. However, they begin to die at over threshold level of free radicals caused by oxidative stress. The fact that pretreatment with 8 M 15d-PGJ2 protected N18D3 cells against oxidative stress by reducing the decrement of the survival signals caused by the stress suggests that free radicals below the threshold level might have preconditioning effects by stimulating survival signals. Our present results together with previous reports (Kim et al., 2005; Levites et al., 2003; Weinreb et al., 2003) suggest that low concentration of antioxidants, such as 15d-PGJ2, induces anti-apoptotic patterns in gene and protein expressions and high concentration induces proapoptotic patterns, again emphasizing the fact that the level of free radicals produced by antioxidants has a very important role in inducing different effects of antioxidants. These suggestions were also indirectly proven by the findings that, although JNK, free radicals and membrane lipid peroxidation, which reflect stress given to cells, were even slightly increased in low concentration of 15d-PGJ2, the viability was not decreased by increasing survival signals and decreasing death signals. Describing in detail, although low concentration of 15d-PGJ2 even induced oxidative stress, based on the results of the studies on the alteration of the levels of free radicals and membrane lipid peroxidation depending on its concentration, neuronal cells could tolerate it enough by increasing survival signals, including PI3-K, phospho-Akt, and phospho-GSK-3 (Ser-9), and decreasing death signals, including cytosolic cytochrome c, activated caspase-3, and cleaved PARP, based on the change of cellular signals. However, oxidative stress induced by high con-
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centration of 15d-PGJ2 was over the threshold level. Therefore, survival signals were decreased and death signals were increased, and neuronal cells began to die. There are some limitations in this study which included: (1) the dose of 15d-PGJ2 in vivo could not physiologically reach the M level, (2) it was not confirmed whether the observed response of cells to free radicals is a general phenomenon or is specific to N18D3 cells, and (3) DCFH for measuring reactive oxygen species might generate free radicals and TBARS for measuring membrane lipid peroxidation might be nonspecific (Rota et al., 1999a,b). Therefore, further studies are needed to evaluate the neuroprotective effect of 15dPGJ2 in vivo and to evaluate whether the response of cells to free radicals is general phenomenon. And, precise and more appropriate measurement of PGJ2 induced ROS using spin-trap should be performed (Rota et al., 1999a,b). In conclusion, the results of this study showed that 15d-PGJ2 could act as a neuroprotectant or a neurotoxicant, depending on its concentration, that it, especially at some specific concentrations, had neuroprotective effects on oxidative stress-injured N18D3 cells, by increasing p85a PI3-K, phospho-Akt, and phospho-GSK-3 (Ser-9) and decreasing cytosolic cytochrome c, activated caspase-3, and cleaved PARP, and that the level of free radicals produced by 15d-PGJ2, might have a very important role in deciding the fate of cells. These results suggest that low concentration of 15d-PGJ2 could be a potential candidate of therapeutic or modulating agents for neurodegenerative and other diseases influenced by oxidative damage. Acknowledgements The authors would like to thank Prof. Woon K. Paik, Hanyang University, and anonymous reviewer for careful review of this manuscript. N18D3 cells were generous gift from Prof. Seung U. Kim (Brain Disease Research Center, Ajou University School of Medicine, Suwon, South Korea and Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, Canada). References Aoun, P., Simpkins, J.W., Agarwal, N., 2003. Role of PPAR-gamma ligands in neuroprotection against glutamate-induced cytotoxicity in retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 44, 2999–3004. Cantley, L.C., 2002. The phosphoinositide 3-kinase pathway. Science 296, 1655–1657.
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