[1] 24w SA4503, a sigma-1 receptor agonist, prevents cultured cortical neurons from oxidative stress-induced cell death via suppression of MAPK pathway activation and glutamate receptor expression

[1] 184w Many studies suggest that antidepressants act as neuroprotective agents in the central nervous system (CNS), though the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of SA4503, which is a sigma-1 receptor agonist and a novel antidepressant candidate, on oxidative stress-induced cell death in cultured cortical neurons. Exposure of the neurons to H 2 O 2 induced cell death, while pretreatment with SA4503 inhibited neuronal cell death. The SA4503-dependent survival effect was reversed by co-application with BD1047 (an antagonist of sigma-1/2 receptors). Previously we found that H 2 O 2 triggers a series of events including over-activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and intracellular Ca 2+ accumulation via voltagegated Ca 2+ channels and ionotropic glutamate receptors, resulting in neuronal cell death (Numakawa et al. (2007) [20]). Importantly, we found in this study that SA4503 reduced the activation of the MAPK/ERK pathway and down-regulated the ionotropic glutamate receptor, GluR1. Taking these findings together, it is possible that SA4503 blocks neuronal cell death via repressing activation of the MAPK/ERK pathway and, consequently, expression levels of glutamate receptors.

[1] 246w We first examined the effect of pretreatment with SA4503 on cell death caused by H 2 O 2 application. SA4503 (0.1 M) was applied to cultured cortical neurons at 4 days in vitro (DIV4). Twenty-four hours later, exposure to H 2 O 2 (50 M) was carried out to induce cell death. Following H 2 O 2 treatment for 12 h, an immunostaining with anti-MAP2 (microtubule-associated protein 2, neuronal marker) antibody was performed. As shown in Fig. 1A, H 2 O 2 induced significant neuronal cell death compared with the control (without SA4503 and H 2 O 2 ). We found that SA4503 significantly reduced the cell death caused by H 2 O 2 (Fig. 1A). We previously determined that the ratio of MAP2-positive cells to the total cells at DIV5 was about 80% in our cultures [14]. To further investigate cell survival, the MTT assay was also conducted. As illustrated, SA4503 decreased cell death by H 2 O 2 at any dose of SA4503 (0.01-10 M, Fig. 1B). The survival effect by SA4503 reached a plateau at 0.1 M (Fig. 1B). Therefore, the following experiments for analyzing cell survival were performed with 0.1 M of SA4503. Next, we examined the possible involvement of sigma-1/2 receptors in the SA4503 effect. Co-pretreatment with SA4503 and BD1047 (1 M, an antagonist of sigma-1/2 receptors) failed to protect cortical neurons from cell death by H 2 O 2 (Fig. 1C), suggesting that SA4503 has survival-promoting effect via sigma-1/2 receptors.

[2] 343w To clarify the mechanisms underlying the SA4503-dependent survival, we examined the change in activation of intracellular signaling. PI3K, phosphatidylinositol 3-kinase, and MAPK/ERK pathways are essential for neuronal survival in the CNS [5,24]. On the other hand, we previously reported that over-activation of ERK1/2 caused by H 2 O 2 is involved in cell death [20]. Thus, to examine the activation of MAPK/ERK pathway to the downstream effects of SA4503, we applied an inhibitor of the MAPK/ERK pathway, U0126, to the cortical cultures. U0126 (10 M, 3 h before H 2 O 2 stimulation) significantly inhibited H 2 O 2 -dependent cell death (Fig. 2A), suggesting that the MAPK/ERK pathway is involved in cell death by H 2 O 2 . No additional or synergistic effect by co-application of U0126 and SA4503 as compared with solo SA4503 or U0126 application was observed (Fig. 2A), suggesting that reduction in the activation of the MAPK/ERK pathway contributes to cell protection by SA4503. We confirmed that a decrease in activation (phosphorylation) of p44/42 MAPK (ERK1/2) after U0126 exposure (3 h) occurred in a dose-dependent manner, though the total expression of ERK1/2 was not changed (Fig. 2B). Next, we examined activation of ERK1/2 after SA4503 exposure. SA4503 treatment for 0.5-3 h reduced levels of pERK1/2 (Fig. 2C). The total ERK1/2 was not changed by SA4503 (Fig. 2C). SA4503 depressed the pERK1/2 levels in a dose-dependent manner (Fig. 2D). We checked the activation of Akt (a component of the PI3K pathway). When the time-or dose-dependency of SA4503 on Akt activation was determined, the activation levels were not influenced by SA4503 (Fig. 2E and F). Total Akt expression was intact after SA4503 application (Fig. 2E and F). Furthermore, both total JNK1/2 (c-JunNH2-terminal kinase1/2, an another member of MAPKs) and pJNK1/2, which regulate apoptosis [9,31], were examined. After the time-or dosedependency of SA4503 was determined, it was revealed that total JNK1/2 and pJNK1/2 were not changed by SA4503 (Fig. 2G and H). These results suggest that SA4503 has a protective effect on cortical neurons via repressing activation of the MAPK/ERK pathway.

[3] 203w We investigated whether ionotropic glutamate receptors are involved in SA4503-dependent neuroprotection as contribution of glutamate function to oxidative stress was reported [17]. Interestingly, SA4503 dramatically decreased expression of GluR1 in a dose-dependent manner (Fig. 3A). Small decreases in the NR2A (not NR2B), and GluR2/3 expression levels after SA4503 exposure were also observed (Fig. 3A). The level of ␤-actin is shown as a control (Fig. 3A). The time-course analysis of SA4503-induced reduction of GluR1 expression was performed. Cultures were treated with SA4503 for 0.5-3 h, and down-regulation of GluR1 expression occurred at all time points (Fig. 3B). ␤-actin levels were intact after SA4503 application (Fig. 3B). Furthermore, to elucidate the possibility that a change in the level of ionotropic glutamate receptors, such as GluR1 AMPA receptors, is associated with SA4503-mediated survival promotion, we tested the effect of CNQX, an antagonist for AMPA receptors. CNQX application (20 min) prevented cortical neurons from cell death by H 2 O 2 . As shown, no additional or synergistic effect of co-application of CNQX and SA4503 as compared with solo drug treatment was confirmed (Fig. 3C), implying that a decrease in AMPA receptor activation is involved in the SA4503-dependent survival against H 2 O 2 -mediated cell death.

[1] 92w The sigma-1 receptor (sig-1R) is a possible target to treat for several brain-related illnesses [6], because sig-1R is putatively involved in synaptic plasticity and neuroprotection in the CNS. Sig-1R has been shown to play a role in critical intracellular processes that regulate Ca 2+ signaling and protein transport. Hayashi and Su demonstrated that the endoplasmic reticulum (ER) protein Sig-1R is a Ca 2+ (via inositol 1,4,5-trisphosphate receptors, IP 3 R)-sensitive and ligand-operated receptor chaperone at the ER membrane [7]. Sig-1R chaperones at the ER-mitochondrion interface regulate Ca 2+ signaling and cell survival.

[2] 106w SA4503 [1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl) piperazine dihydrochloride] is recognized as a selective ligand for sig-1R chaperones [15]. Previous studies suggest that SA4503 positively affects memory by improving memory impairments and increasing cell survival [25,26]. Up-regulation of hippocampal BDNF levels (brain-derived neurotrophic factor), which play a crucial role in synaptic function and neuronal survival [11,21,29,30], have been demonstrated after chronic treatment with SA4503 [8]. Previously, we reported that BDNF rapidly induces release of the neurotransmitter glutamate through PLC␥/IP 3 R/Ca 2+ signaling and that antidepressants, including imipramine and fluvoxamine, enhance the PLC␥/IP 3 R/Ca 2+ system via Sig-1R [19,32]. These studies indicate that Sig-1R has multiple roles in the CNS.

[3] 121w Interestingly, SA4503 shows antidepressant-like, and neuroprotective effects [16,27]. SA4503 decreased the immobility time in the forced swim test in rats, suggesting that SA4503 may have potential antidepressive properties [27]. Moreover, in the same study, SA4503 showed a synergistic effect with imipramine during the forced swim test. Lucas et al. reported further evidence supporting the antidepressant potential of SA 4503 through electrophysiological, morphological and behavioural studies [12]. In addition, Nakazawa et al. showed that SA4503 demonstrates a protective effect on neuronal cultures against the hypoxia/hypoglycemia-induced neurotoxicity [16]. Thus, it is possible that SA4503 is a potential drug for treatment for several brain-related illnesses, including depressive disorder. However, little is known concerning detailed cellular mechanisms underlying the effect of SA4503 in CNS neurons.

[4] 49w Oxidative stress may be associated with various neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis [1,2]. In this study, we examined whether SA4503 demonstrates a preventive effect on cultured cortical neurons from oxidative stress when SA4503 is applied before H 2 O 2 exposure.

[5] 196w Primary cortical cultures were prepared from postnatal 2day-old rats as previously reported [22,23]. The culture medium consisted of 5% fetal bovine serum, 5% heated-inactivated horse serum, 90% of a 1:1 mixture of Dulbecco's modified Eagle's medium, and Ham's F-12 medium. Dissociated cortical neurons were cultured for 4 or 5 days before SA4503 (a gift from M's Science Corporation, Hyogo, Japan) was applied. Twenty-four hours after SA4503 addition, H 2 O 2 (final 50 M) was applied for 12 h. Then, the cell viability was analyzed. To determine the cell viability, we carried out a mitochondrial-dependent conversion of the tetrazolium salt (MTT) assay. The metabolic activity of mitochondria was estimated with the MTT assay as previously reported [20]. BD1047 (1 M, Tocris Cookson Ltd., Avonmouth, UK), an antagonist of sigma-1/2 receptors [13], was applied 20 min before adding SA4503. U0126 (an inhibitor for MEK, an upstream molecule of MAPK/ERK) was purchased from Promega (WI, USA), and used at a final concentration of 10 M. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was purchased from Tocris Bioscience (Bristol, UK). Other reagents were obtained from SIGMA (MO, USA). All animals were treated according to the institutional guidelines for the care and use of animals.

[6] 101w Immunostaining was carried out as reported previously with some modifications [18]. First, cultured cells were fixed in 4% paraformaldehyde at room temperature for 20 min. After three times washes with PBS, the cells were permeabilized, and the nonspecific binding of antibodies was blocked with 10% goat serum, 0.2% Triton X-100 in PBS for 30 min at room temperature. The anti-MAP2 (1:1000, SIGMA) antibody was applied overnight at 4 • C. Following three times washes with PBS, Alexa Fluor 488conjugated anti-mouse IgG (1:200, Invitrogen) was applied as a secondary antibody. Immunoreactivity was monitored with a fluorescence microscope (Axiovert 200, ZEISS, Tokyo, Japan).

[7] 143w Cells were lysed in SDS lysis buffer containing 1% SDS, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA (pH 8.0), 10 mM NaF, 2 mM Na 3 VO 4 , 0.5 mM phenylarsine oxide, and 1 mM phenylmethylsulfonyl fluoride. The protein concentration was quantified using a BCA Protein Assay Kit (PIERCE), and equivalent amounts of total protein were assayed for each immunoblotting. Primary antibodies were used at the following dilutions: anti-Akt (1:1000, Cell Signaling, MA, USA), anti-pAkt (1:1000, Cell Signaling), anti-ERK (1:1000, Cell Signaling), anti-pERK (1:1000, Cell Signaling), anti-pJNK (1:1000, Cell Signaling), anti-JNK (1:1000, Cell Signaling), anti-␤-actin (1:5000, SIGMA), anti-NR2A (1:500, SIGMA), anti-NR2B (1:500, SIGMA), anti-GluR1 (1:1000, CHEMICON, CA, USA), and anti-GluR2/3 (1:500, CHEMICON) antibodies. The intensity of the immunoreactivity was quantified by using Lane & Spot Analyzer software (ATTO Corpora-tion, Tokyo, Japan). The n indicates the number of experiments in separated cultures.

[8] 41w Data shown in this study are presented as mean ± standard deviation (SD). Statistical significance was evaluated using a one-way ANOVA followed by Tukey's test in SPSS ver11 (SPSS Japan, Tokyo, Japan). Probability values less than 5% were considered statistically significant.

[9] 87w In the present study, we found that SA4503, a sig-1R agonist and a novel antidepressant candidate, blocked H 2 O 2 -induced neuronal cell death. SA4503 caused the down-regulation of the MAPK/ERK pathway activation and reduced levels of ionotropic glutamate receptors. U0126, an inhibitor of the MAPK/ERK pathway, and CNQX, an inhibitor of AMPA glutamate receptors, prevented cultured cells from the H 2 O 2 -induced death. Remarkably, after cotreatment with U0126 or CNQX, SA4503 exerted no additional or synergistic survival effects as compared with solo application.

[10] 306w ERK1/2 (p44/p42MAPK) are two isoforms of ERK that belong to the family of MAPKs, including JNK1/2 and the p38 MAP kinase. ERK activation controls various cell responses, including proliferation, survival, and synaptic maturation; indeed, we previously showed that neurotrophin BDNF up-regulated synaptic proteins via the MAPK/ERK pathway [10,14]. Paradoxically, depending on the duration, the magnitude and its subcellular localization, aberrant ERK activation can promote cell death [3]. Persistent activation of ERK contributes to glutamate-induced oxidative toxicity in cortical neurons [28]. Consistently, we previously found that H 2 O 2 induced over-activation of ERK1/2 [20]. Crossthwaite et al. showed MAPK activation in cultured cortical neurons exposed to H 2 O 2 (300 M), however, U0126 did not rescue but slightly enhanced the cell death [4]. In our system, U0126 significantly inhibited H 2 O 2induced cell death, and marked reduction of activated ERK1/2 levels after SA4503 exposure was observed. In contrast, phosphorylation of JNK1/2, which is known as pro-apoptotic molecule [9,31], was not changed by SA4503 exposure. In addition, no additional or synergistic effect of co-application of U0126 and SA4503 on neuroprotection as compared with solo drug application was confirmed. These results suggest that SA4503 exerts the survival effect via repressing activation of the MAPK/ERK pathway. Importantly, though the reduction in activation of ERK1/2 induced by SA4503 or by U0126 was significant, a complete abolishment of basal activated ERK1/2 was not achieved by each drug treatment. Activation of MAPK/ERK signaling beyond the normal threshold may be toxic to cells, while basal activity of this signaling is essential for neuronal survival. Furthermore, there is a possibility that subcellular localization of pERK1/2 is involved in multiple functions of this kinase [3]. It may be valuable to study whether an unknown phosphatase that prevents the activation of ERK1/2 or upstream molecules of ERK1/2, is involved in SA4503-dependent inhibition of pERK1/2.

[11] 167w We previously reported that exposure to H 2 O 2 causes a series of events including ERK1/2 over-activation and an increase in intracellular Ca 2+ via voltage-gated Ca 2+ channels and ionotropic glutamate receptors, ultimately resulting in cell death [20]. Under H 2 O 2 stress, the ERK1/2 signal may work as a death mediator, as U0126 blocks cell death. We recently found that the MAPK/ERK pathway is also involved in maintenance of the expression of ionotropic glutamate receptors [10]. Indeed, in the present study, SA4503 induced the marked down-regulation of glutamate receptors (especially, GluR1). Therefore, we investigated the possibility that a decrease in AMPA receptor activation is required for the SA4503-dependent survival. As expected, CNQX (AMPA receptors antagonist) blocked H 2 O 2 -induced death, and any additional or synergistic effect of co-application of CNQX and SA4503 was not observed. It is possible that SA4503 protects neurons from oxida-tive toxicity via decreasing activation of ERK1/2, which is a critical signaling component for maintenance of GluR1 expression.

[12] 105w In summary, SA4503, a sig-1R agonist, stimulates survivalpromoting effects on cultured cortical neurons. Previously, we found that antidepressants (imipramine, and fluvoxamine) potentiate BDNF-induced intracellular signaling for release of glutamate via stimulation of sig-1R [32]. Recently, up-regulation of BDNF protein in the rat hippocampus by chronic treatment with SA4503 has been reported [8]. Collectively, these results, including our present study, suggest that SA4503 plays various functions in the CNS. In addition to the potential as a novel antidepressant agent, SA4503 may be valuable to study as a therapeutic agent in the treatment of neurodegenerative diseases of the CNS, although further studies concerning intracellular mechanisms are needed.