European Journal of Pharmacology 727 (2014) 167–173
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Interaction of new antidepressants with sigma-1 receptor chaperones and their potentiation of neurite outgrowth in PC12 cells Tamaki Ishima, Yuko Fujita, Kenji Hashimoto n Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, 1-8-1 Inohana, Chiba 260-8670, Japan
art ic l e i nf o
a b s t r a c t s
Article history: Received 17 December 2013 Received in revised form 28 January 2014 Accepted 29 January 2014 Available online 5 February 2014
The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [3H] ( þ)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine 4sertraline4fluoxetine 4escitalopram 4 citalopram4paroxetine 4duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect. & 2014 Elsevier B.V. All rights reserved.
Keywords: Antidepressant Mirtazapine Neurite outgrowth Selective serotonin reuptake inhibitor Serotonin and noradrenaline reuptake inhibitor Sigma-1 receptor chaperone
1. Introduction Selective serotonin reuptake inhibitors (SSRIs) and serotonin and noradrenaline reuptake inhibitors (SNRIs) are used as therapeutic drugs for a number of neuropsychiatric diseases, including major depression, obsessive–compulsive disorder and social anxiety disorder (Millan, 2006; Owens, 2004). Although these SSRIs and SNRIs increase serotonin and/or noradrenaline levels throughout the central nervous system (CNS), through blockade of serotonin and/or noradrenaline transporters, it is well known that their pharmacological actions are very heterogenous (Goodnick and Goldstein, 1998a, 1998b; Hosenbocus and Chahal, 2011; Nemeroff and Owens, 2004; Stahl, 1998). Mirtazapine is a noradrenergic and specific serotonergic antidepressant (NaSSA), differentiating from other antidepressants (Benjamin and Doraiswamy, 2011; Croom et al., 2009; Watanabe et al., 2011). Interestingly, it is likely that mirtazapine has a faster
n
Corresponding author. Tel.: þ 81 43 226 2517; fax: þ81 43 226 2561. E-mail address: [email protected] (K. Hashimoto).
http://dx.doi.org/10.1016/j.ejphar.2014.01.064 0014-2999 & 2014 Elsevier B.V. All rights reserved.
onset of action compared with SSRIs and SNRIs, during the acutephase treatment of major depression (Nagao et al., 2013; Watanabe et al., 2011). The sigma-1 receptor is a novel endoplasmic reticulum (ER) chaperone, which regulates a variety of cellular functions, such as inositol 1,4,5-triphosphate receptor-mediated Ca2 þ signaling, ion channel firing, protein kinase location/activation, cellular redox, neurotransmitter release, inflammation, cellular differentiation, neuronal survival and synaptogenesis (Hayashi and Su, 2007; Su et al., 2010). Accumulating evidence suggests that the sigma-1 receptor chaperone plays a role in cognition, and the pathophysiology of a number of neuropsychiatric diseases, including major depression, schizophrenia and addiction (Bermack and Debonnel, 2005; Hashimoto, 2009a, 2009b, 2013; Hashimoto and Furuse, 2012; Hashimoto and Ishiwata, 2006; Hayashi and Su, 2004, 2007, 2008, 2010; Hayashi et al., 2011; Hindmarch and Hashimoto, 2010; Ishikawa and Hashimoto, 2010; Katz et al., 2011; Kourrich et al., 2012; Maurice et al., 2001; Niitsu et al., 2012; Su et al., 2010). In 1996, we reported that some SSRIs such as fluvoxamine, sertraline, fluoxetine and citalopram possess high to moderate affinity for
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sigma-1 receptors in the rat brain (Narita et al., 1996). Subsequent to this, we reported that fluvoxamine was capable of improving phencyclidine-treated cognitive deficits via the sigma-1 receptor chaperone (Hashimoto et al., 2007). These findings implicate the sigma-1 receptor chaperone in the mechanistic action of some SSRIs, such as fluvoxamine (Hashimoto et al., 2007; Hashimoto and Furuse, 2012; Ishikawa et al., 2007). After publication of the paper from Narita et al. (1996), a number of new antidepressants have been developed and used in clinical practice. The purpose of this study is to examine whether these new antidepressants, such as escitalopram, SNRIs and mirtazapine, interact with the sigma-1 receptor chaperone. First, we examined the effects of SSRIs namely fluvoxamine, paroxetine, sertraline, citalopram and escitalopram, and SNRIs, such as duloxetine, venlafaxine and milnacipran, and mirtazapine, on [3H](þ)-pentazocine binding to the sigma-1 receptor chaperone. Since, sigma-1 receptor agonists, including fluvoxamine, fluoxetine, donepezil, and ifenprodil, could potentiate nerve growth factor (NGF)-induced neurite outgrowth, in PC12 cells (Ishima et al., 2008; Ishima and Hashimoto, 2012; Nishimura et al., 2008; Takebayashi et al., 2002), we also examined the effects of these drugs on NGF-induced neurite outgrowth in these cells.
2. Materials and methods 2.1. Drugs Drugs were obtained from the following sources: fluvoxamine from Solvay Seiyaku K.K., Tokyo, Japan and paroxetine, sertraline, fluoxetine, citalopram, escitalopram, duloxetine, venlafaxine, milnacipran, and mirtazapine from Wako Chemical Co., Ltd., Tokyo, Japan. NE-100 was synthesized in our laboratory. WAY-100635 was purchased from Sigma-Aldrich, St Louis, MO, USA. Other chemicals were purchased commercially. Drugs were dissolved at 10 mM in dimethyl sulfoxide (DMSO), and then diluted in either binding buffer or cell culture medium. NE-100 was dissolved in distilled water. 2.2. [3H]( þ)Pentazocine binding to rat brain membranes Binding assays for the sigma-1 receptor chaperone were performed according to a previous method (Narita et al., 1996), with a slight modification. Male Sprague–Dawley rats (250–300 g; Oriental Yeast Co., Ltd., Tokyo, Japan) were killed by decapitation, and their brains were removed rapidly. The brains were homogenized in 20 volumes of ice-cold 50 mM Tris-HCI (pH 8.0 at 25 1C), using a Polytron homogenizer set at 5, for 30 s. The homogenate was centrifuged at 48,000g for 10 m (4 1C). The resulting pellet was resuspended in buffer and recentrifuged. This procedure was repeated twice. The final pellet was suspended in 20 volumes of 50 mM Tris-HCI (pH 8.0 at 25 1C). For assays measuring sigma-1 receptor binding, aliquots of crude membranes (approximately 350–400 pg protein) were incubated with [3H] (þ )-pentazocine (a specific radioligand for sigma-1 receptor chaperones, 1.25–40 nM for Scatchard analysis and 5.0 nM for pharmacological inhibition: 1.254 TBq/mmol, GE Healthcare, Tokyo, Japan) and 50 mM Tris–HCl (pH 8.0 at 25 1C), in a final volume of 0.5 ml, for 2 h at 27 1C. After the addition of 4 ml of icecold buffer, the membranes were filtered rapidly, using a Brandell 24-channel cell harvester (Biochemical Research Laboratories, Gaithersburg, MD, USA), through Whatman GF/B filters pretreated with 0.5% polyethyleneimine, for at least 2 h. The filters were washed three times with 4 ml of ice-cold buffer. The radioactivity trapped by the filters was measured using a liquid scintillation counter (Tri-Carb 2800 TR, PerkinElmer Japan, Tokyo, Japan). Nonspecific binding was estimated in the presence of 10 μM NE-100 (a
selective sigma-1 receptor antagonist) (Okuyama and Nakazato, 1996). Protein concentrations were measured colorimetrically in a Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA, USA), using bovine serum albumin (BSA) as the standard. The dissociation constant (Kd) and maximal binding (Bmax) values from saturation-binding and the IC50 values from binding displacement by each drug were determined using GraphPad Prism (GraphPad Software, San Diego, CA, USA). The Ki values were calculated from the IC50 values, using the formula of Chung and Prusoff, Ki ¼ IC50/(1 þ[L]/Kd) (Cheng and Prusoff, 1973), where IC50 is the concentration that resulted in 50% inhibition of specific binding, [L] is the concentration of radioligand used and Kd the dissociation constant. 2.3. Effects of antidepressants on NGF-induced neurite outgrowth PC12 cells (RIKEN Cell Bank, Tsukuba, Japan) were cultured at 37 1C, in 5% CO2 with Dulbecco's modified Eagle's medium (DMEM), supplemented with 5% heat-inactivated fetal bovine serum (FBS), 10% heat-inactivated horse serum and 1% penicillin. The medium was changed two to three times a week. PC12 cells were plated onto 24-well tissue culture plates coated with poly-Dlysine/laminin. Cells were plated at relatively low density (0.25 104 cells/cm2) in DMEM containing 0.5% FBS with 1% penicillin and streptomycin. Media containing minimal levels of serum (0.5% FBS) were used, since it is known that serum contains steroid hormones that bind to sigma-1 receptors (Ishima et al., 2008; Ishima and Hashimoto, 2012; Nishimura et al., 2008). As reported previously (Ishima et al., 2008, 2012; Ishima and Hashimoto, 2012; Nishimura et al., 2008), 2.5 ng/ml of NGF was used to study the effects of antidepressant drugs on NGF-induced neurite outgrowth. Twenty-four hours after plating, media were replaced with DMEM containing 0.5% FBS along plus 1% penicillin and streptomycin with NGF (2.5 ng/ml), with or without added antidepressant drugs. Five days after incubation with NGF (2.5 ng/ ml) alone or supplemented with antidepressant drugs, morphometric analysis was performed on digitized images of live cells taken under phase-contrast illumination, with an inverted microscope linked to a camera. Images of three fields per well were taken, with an average of 100 cells per field. Differentiated cells were counted by visual examination of the field; only cells showing at least one neurite with a length equal to the cell body diameter were counted and expressed as a percentage of the total cells in the field. The scoring was performed in a blinded manner. 2.4. Statistical analysis Data are the mean 7standard error of the mean (S.E.M.). Statistical analysis was performed using the software package SPSS (SPSS 12.0J, Tokyo, Japan). The data were analyzed by oneway analysis of variance (ANOVA) or two-way ANOVA. When appropriate, post-hoc comparisons were performed using the Bonferroni/Dunn test. Significance for results was set at Po 0.05.
3. Results 3.1. Effects of antidepressant drugs on [3H]( þ)pentazocine binding For saturation-binding isotherms, six grade-diluted concentrations of [3H]( þ)-pentazocine (1.25–40 nM) were used. The specific binding of [3H]( þ)-pentazocine to rat brain membranes was saturable. Scatchard analysis of specific binding revealed an apparent Kd of 13.1 72.18 nM and a Bmax of 858.0 750.8 fmol/mg protein (n ¼ 3).
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Next, we examined the pharmacological inhibition of specific [3H] (þ )-pentazocine (5.0 nM) binding to rat brain membranes. Fluvoxamine, sertraline, fluoxetine, escitalopram, citalopram, paroxetine and duloxetine were found to displace [3H](þ )-pentazocine binding at rat brain membranes (Table 1). The order of potency for sigma-1 receptor chaperone binding drugs was as follows: fluvoxamine (Ki ¼ 17.0 nM)4sertraline (Ki ¼31.6 nM)4fluoxetine (Ki ¼191.2 nM) 4escitalopram (Ki ¼ 288.3 nM)4citalopram (Ki ¼403.8 nM)4paroxetine (Ki ¼2041 nM)4duloxetine (Ki ¼3533 nM) (Table 1). The Ki values of venlafaxine, milnacipran, and mirtazapine for [3H](þ )-pentazocine binding were greater than 10,000 nM (Table 1). 3.2. Effects of antidepressant drugs on NGF-induced neurite outgrowth In this assay, antidepressant drugs were tested at two concentrations (1.0 and 10 μM) in PC12 cells. At these concentrations, fluvoxamine and fluoxetine significantly increased the proportion of cells with NGF (2.5 ng/ml) induced neurite outgrowth in PC12 Table 1 Affinity of antidepressant drugs for the sigma-1 receptor chaperone in rat brain. Drugs
Ki (nM)
Fluvoxamine Sertraline Fluoxetine Escitalopram Citalopram Paroxetine Duoxetine Venlafaxine Milnacipran Mirtazapine
17.0 7 1.4 31.6 7 2.9 191.2 7 11.6 288.3 747.0 403.8 712.0 20417 144 35337 325 4 10,000 4 10,000 4 10,000
Assays were carried out under conditions described in the Materials and methods section. The specified concentrations of drug were incubated with rat brain membranes and 5 nM [3H](þ )-pentazocine. Non-specific binding was estimated in the presence of NE-100 (10 μM). Values are the mean7 S.E.M. of three experiments performed in duplicate.
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cells (Fig. 1). At the lower concentration (1.0 μM), escitalopram significantly increased the number of cells with NGF (2.5 ng/ml) induced neurite outgrowth (Fig. 1). Escitalopram (10 μM) and citalopram (1.0 and 10 μM) promoted slight increases in the number of cells with NGF-induced neurite outgrowth, but these differences were not statistically significant (Fig. 1). In contrast, sertraline and paroxetine produced no effect, consistent with a previous report (Nishimura et al., 2008). The higher concentration (10 μM) of sertraline significantly decreased the number of PC12 cells with NGF-induced neurite outgrowth, suggestive of cellular toxicity (Fig. 1) and consistent with a previous report (Nishimura et al., 2008). None of the SNRIs tested altered the number of cells with NGFinduced neurite outgrowth (Fig. 2). In contrast, the higher concentration (10 μM) of mirtazapine significantly increased the number of PC12 cells with neurite outgrowth induced by NGF (2.5 ng/ml), although the potency of mirtazapine was weaker than that of fluvoxamine at the same concentration (Fig. 2). To investigate the role of sigma-1 receptor chaperones, we examined the effects of NE-100 on the potentiation of NGF-induced neurite outgrowth by fluvoxamine, fluoxetine, escitalopram, and mirtazapine. Two-way ANOVA analysis revealed significant interactions [fluvoxamine NE-100: F(1, 20)¼21.68, Po0.001], [fluoxetine NE-100: F(1, 92)¼21.31, Po0.001], and [escitalopram NE-100: F (1, 20)¼23.63, Po0.001]. Co-administration of NE-100 (1.0 μM) significantly blocked potentiation by fluvoxamine (10 μM), fluoxetine (10 μM) and escitalopram (1.0 μM) (Fig. 3). In contrast, two-way ANOVA analysis revealed no differences in interaction [mirtazapine NE-100: F(1, 44)¼ 0.078, P¼0.781]. NE-100 had no effect on potentiation of NGF-induced neurite outgrowth by mirtazapine (10 μM) (Fig. 3D), suggesting that the sigma-1 receptor chaperone may not be involved in the mechanisms driving neurite outgrowth in this drug. Addition of NE-100 (1.0 μM) alone did not alter NGF-induced neurite outgrowth in PC12 cells (Fig. 3). To investigate the pharmacology of sertraline at sigma-1 receptor chaperones, we examined its effect on the potentiation of NGFinduced neurite outgrowth by fluvoxamine and fluoxetine. Twoway ANOVA analysis revealed significant interactions [fluvoxamine sertraline: F(1, 20)¼ 8.01, P¼0.01] and [fluoxetine sertraline: F (1, 20)¼13.41, P¼0.002]. Co-administration of sertraline (1.0 μM)
Fig. 1. Effects of SSRIs (fluvoxamine, sertraline, fluoxetine, paroxetine, escitalopram, citalopram and paroxetine) on NGF-induced neurite outgrowth in PC12 cells. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean7 S.E.M. The number of columns indicates the number of assays. nnn P o 0.001 compared with the control group.
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Fig. 2. Effects of fluvoxamine, SNRIs (duloxetine, venlafaxine, and milnacipran), and mirtazapine, on NGF-induced neurite outgrowth in PC12 cells. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean 7S.E.M. The number of columns indicates the number of assays. nnnP o0.001 compared with the control group.
Fig. 3. Effects of NE-100 on the potentiation of NGF-induced neurite outgrowth by fluvoxamine, fluoxetine, escitalopram and mirtazapine. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean 7 S.E.M. The number of columns indicates the number of assays. nnnPo 0.001 compared with the antidepressant drug alone group.
significantly blocked potentiation by fluvoxamine (1.0 μM) and fluoxetine (1.0 μM) (Fig. 4), suggesting that sertraline might be an antagonist at the sigma-1 receptor chaperone. To investigate the role of 5-hydroxytryptamine 5-HT1A receptor in the mechanisms of mirtazapine, we examined the effect of the selective 5-HT1A receptor antagonist WAY-100635 (10 μM) on the
potentiation of NGF-induced neurite outgrowth by mirtazapine (10 μM). Two-way ANOVA analysis revealed a significant interaction [mirtazapine WAY-100635: F(1, 44) ¼17.26, P o0.001]. Co-administration of WAY-100635 (10 μM) significantly blocked potentiation by mirtazapine (10 μM) (Fig. 5), suggesting the role of 5-HT1A receptor in the mechanisms of action of mirtazapine.
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Fig. 4. Effects of sertraline on the potentiation of NGF-induced neurite outgrowth by fluvoxamine or fluoxetine. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean 7S.E.M. The number of columns indicates the number of assays. nnnPo 0.001 compared with the antidepressant drug alone group.
Fig. 5. Effect of 5-HT1A receptor antagonist WAY-100635 on the potentiation of NGF-induced neurite outgrowth by mirtazapine. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean 7 S.E.M. The number of columns indicates the number of assays. nnnPo 0.001 compared with the antidepressant drug alone group.
Fig. 6 denotes representative photomicrographs showing the effects of fluvoxamine, fluoxetine and mirtazapine on PC12 cells. All these drugs increased the number of cells with neurite outgrowth.
4. Discussion In this study, we found that some SSRIs, including fluvoxamine, sertraline, fluoxetine, escitalopram and citalopram, had high to moderate affinity at sigma-1 receptor chaperones in the rat brain. The Ki values of fluvoxamine, sertraline, fluoxetine and citalopram were similar to those recorded in a previous report (Narita et al., 1996). In contrast, paroxetine, all tested SNRIs (duloxetine, venlafaxine, and milnacipran), and mirtazapine showed weak affinity at sigma-1 receptor chaperones. Furthermore, we found that the SSRIs, fluvoxamine, fluoxetine, escitalopram, could potentiate NGF-induced neurite outgrowth in PC12 cells, and that these effects could be blocked by NE-100, suggesting that these drugs function as agonists at sigma-1 receptor chaperones. This is the first paper demonstrating that escitalopram, a new SSRI, is capable of potentiating NGF-induced neurite outgrowth in a cell assay. Here, we detected no agonist activity for citalopram in cell assays, although it is reported that citalopram
affects fetal thalamic axon responsiveness to netrin-1, via sigma-1 receptors (Bonnin et al., 2012). These findings imply that for the SSRIs, fluvoxamine, fluoxetine and escitalopram, potentiation of NGFinduced neurite outgrowth in PC12 cells is mediated through agonism at sigma-1 receptors. As mentioned in Introduction, previous reports suggest that sigma1 receptor agonists, such as fluvoxamine, fluoxetine, donepezil, and ifenprodil, potentiate NGF-induced neurite outgrowth in PC12 cells, and that these effects can be blocked by NE-100, an antagonist at the sigma-1 receptor chaperone (Ishima et al., 2008; Ishima and Hashimoto, 2012; Nishimura et al., 2008; Takebayashi et al., 2002). Thus, it is likely that a drug's ability to exert an agonist or antagonist effect at the sigma-1 receptor chaperone determines its ability to promote NGF-induced neurite outgrowth in PC12 cells, and therefore its usefulness as a pharmacological agent. Unlike fluvoxamine, sartraline, which has a high affinity for sigma-1 receptor chaperones, did not alter NGF-induced neurite outgrowth, consistent with a previous report (Nishimura et al., 2008). The reasons underlying this discrepancy between these two SSRIs are currently unclear. One possibility may involve the difference in pharmacological action, namely whether the drugs exert opposing effects at sigma-1 receptor chaperones. In this study, we found that sertraline antagonized the potentiation of NGF-induced neurite outgrowth by fluvoxamine and fluoxetine, indicating that sertraline might act as an antagonist at sigma-1 receptor chaperones. Interestingly, phencyclidine-induced cognitive deficits in mice are significantly improved, by subsequent subchronic (14 days) doses of fluvoxamine, but not of sertraline, indicating that sigma-1 receptor agonism forms a part of fluvoxamine's mechanism of action (Hashimoto et al., 2007; Ishima et al., 2009). Taken together, these findings suggest that fluvoxamine and sertraline may function as an agonist and antagonist respectively, at sigma-1 receptor chaperones (Hashimoto, 2009a; Ishima et al., 2009), although a further study is needed to confirm this action. In this study, we found that mirtazapine potentiated NGFinduced neurite outgrowth in PC12 cells, although its effect could not be antagonized by NE-100. This is the first paper demonstrating that mirtazapine can potentiate NGF-induced neurite outgrowth in a cell culture assay. Mirtazapine antagonizes noradrenergic α2 autoreceptors and noradrenergic α2 receptors, resulting in enhanced release of noradrenaline from noradrenergic terminals, and increased serotonin release from serotonergic terminals, respectively (Croom et al., 2009). Thus, it seems that the net effect of mirtazapine is to increase noradrenergic and serotonergic transmission, notably 5-HT1A receptor-mediated transmission (Croom et al., 2009). Previously, we reported that the selective 5-HT1A receptor agonist, 8-hydroxy-2-dipropylaminotetraline (8-OH-DPAT), could potentiate NGF-induced neurite outgrowth in PC12 cells
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Fig. 6. Representative photomicrographs of PC12 cells. (A) Control (NGF (2.5 ng/ml) alone), (B) NGFþ fluvoxamine (10 μM), (C) NGFþ fluoxetine (10 μM) and (D) NGFþmirtazapine (10 μM). These drugs increased the number of cells with neurite outgrowth. Bar¼ 50 μm
(Ishima et al., 2012). In the present study, we found that the selective 5-HT1A receptor antagonist WAY-100635 significantly antagonized the effect of mirtazapine on neurite outgrowth, suggesting the role of the 5-HT1A receptor in the mechanisms of mirtazapine on neurite outgrowth. The Meta-analyses of new generation antidepressants (MANGA) study reported that mirtazapine was significantly more efficacious than duloxetine (Cipriani et al., 2009). Furthermore, a recent meta-analysis showed that augmentation therapy with mirtazapine improved overall and negative symptoms in patients with schizophrenia (Kishi and Iwata, 2014). It is therefore noteworthy that mirtazapine potentiates neurite outgrowth, since at the cellular level, this type of neuronal plasticity can underlie the therapeutic effect of drugs targeting the CNS (Lieberman et al., 2008; Lu and Dwyer, 2005; Molteni et al., 2009; Williams and Dwyer, 2009). In conclusion, our results suggest that sigma-1 receptor agonists, fluvoxamine, fluoxetine and escitalopram, potentiate NGFinduced neurite outgrowth in PC12 cells, implying that the sigma1 receptor chaperone plays a role in the pharmacological effects of these SSRIs. In contrast, mirtazapine is able to potentiate NGFinduced neurite outgrowth in PC12 cells, via 5-HT1A receptor.
Acknowledgments This study was supported by a grant from Grants-in-Aid for Scientific Research on Innovative Areas of The Ministry of Education, Culture, Sports, Science and Technology, Japan (to K.H., Grant number: 24116006). Dr. Hashimoto has served as a scientific consultant to Astellas and Taisho, and he also received the research grant support from Abbvie, Dainippon Sumitomo, Otsuka, and Taisho over
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Interaction of new antidepressants with sigma-1 receptor chaperones and their potentiation of neurite outgrowth in PC12 cells Tamaki Ishima, Yuko Fujita, Kenji Hashimoto n Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, 1-8-1 Inohana, Chiba 260-8670, Japan
art ic l e i nf o
a b s t r a c t s
Article history: Received 17 December 2013 Received in revised form 28 January 2014 Accepted 29 January 2014 Available online 5 February 2014
The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [3H] ( þ)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine 4sertraline4fluoxetine 4escitalopram 4 citalopram4paroxetine 4duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect. & 2014 Elsevier B.V. All rights reserved.
Keywords: Antidepressant Mirtazapine Neurite outgrowth Selective serotonin reuptake inhibitor Serotonin and noradrenaline reuptake inhibitor Sigma-1 receptor chaperone
1. Introduction Selective serotonin reuptake inhibitors (SSRIs) and serotonin and noradrenaline reuptake inhibitors (SNRIs) are used as therapeutic drugs for a number of neuropsychiatric diseases, including major depression, obsessive–compulsive disorder and social anxiety disorder (Millan, 2006; Owens, 2004). Although these SSRIs and SNRIs increase serotonin and/or noradrenaline levels throughout the central nervous system (CNS), through blockade of serotonin and/or noradrenaline transporters, it is well known that their pharmacological actions are very heterogenous (Goodnick and Goldstein, 1998a, 1998b; Hosenbocus and Chahal, 2011; Nemeroff and Owens, 2004; Stahl, 1998). Mirtazapine is a noradrenergic and specific serotonergic antidepressant (NaSSA), differentiating from other antidepressants (Benjamin and Doraiswamy, 2011; Croom et al., 2009; Watanabe et al., 2011). Interestingly, it is likely that mirtazapine has a faster
n
Corresponding author. Tel.: þ 81 43 226 2517; fax: þ81 43 226 2561. E-mail address: [email protected] (K. Hashimoto).
http://dx.doi.org/10.1016/j.ejphar.2014.01.064 0014-2999 & 2014 Elsevier B.V. All rights reserved.
onset of action compared with SSRIs and SNRIs, during the acutephase treatment of major depression (Nagao et al., 2013; Watanabe et al., 2011). The sigma-1 receptor is a novel endoplasmic reticulum (ER) chaperone, which regulates a variety of cellular functions, such as inositol 1,4,5-triphosphate receptor-mediated Ca2 þ signaling, ion channel firing, protein kinase location/activation, cellular redox, neurotransmitter release, inflammation, cellular differentiation, neuronal survival and synaptogenesis (Hayashi and Su, 2007; Su et al., 2010). Accumulating evidence suggests that the sigma-1 receptor chaperone plays a role in cognition, and the pathophysiology of a number of neuropsychiatric diseases, including major depression, schizophrenia and addiction (Bermack and Debonnel, 2005; Hashimoto, 2009a, 2009b, 2013; Hashimoto and Furuse, 2012; Hashimoto and Ishiwata, 2006; Hayashi and Su, 2004, 2007, 2008, 2010; Hayashi et al., 2011; Hindmarch and Hashimoto, 2010; Ishikawa and Hashimoto, 2010; Katz et al., 2011; Kourrich et al., 2012; Maurice et al., 2001; Niitsu et al., 2012; Su et al., 2010). In 1996, we reported that some SSRIs such as fluvoxamine, sertraline, fluoxetine and citalopram possess high to moderate affinity for
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sigma-1 receptors in the rat brain (Narita et al., 1996). Subsequent to this, we reported that fluvoxamine was capable of improving phencyclidine-treated cognitive deficits via the sigma-1 receptor chaperone (Hashimoto et al., 2007). These findings implicate the sigma-1 receptor chaperone in the mechanistic action of some SSRIs, such as fluvoxamine (Hashimoto et al., 2007; Hashimoto and Furuse, 2012; Ishikawa et al., 2007). After publication of the paper from Narita et al. (1996), a number of new antidepressants have been developed and used in clinical practice. The purpose of this study is to examine whether these new antidepressants, such as escitalopram, SNRIs and mirtazapine, interact with the sigma-1 receptor chaperone. First, we examined the effects of SSRIs namely fluvoxamine, paroxetine, sertraline, citalopram and escitalopram, and SNRIs, such as duloxetine, venlafaxine and milnacipran, and mirtazapine, on [3H](þ)-pentazocine binding to the sigma-1 receptor chaperone. Since, sigma-1 receptor agonists, including fluvoxamine, fluoxetine, donepezil, and ifenprodil, could potentiate nerve growth factor (NGF)-induced neurite outgrowth, in PC12 cells (Ishima et al., 2008; Ishima and Hashimoto, 2012; Nishimura et al., 2008; Takebayashi et al., 2002), we also examined the effects of these drugs on NGF-induced neurite outgrowth in these cells.
2. Materials and methods 2.1. Drugs Drugs were obtained from the following sources: fluvoxamine from Solvay Seiyaku K.K., Tokyo, Japan and paroxetine, sertraline, fluoxetine, citalopram, escitalopram, duloxetine, venlafaxine, milnacipran, and mirtazapine from Wako Chemical Co., Ltd., Tokyo, Japan. NE-100 was synthesized in our laboratory. WAY-100635 was purchased from Sigma-Aldrich, St Louis, MO, USA. Other chemicals were purchased commercially. Drugs were dissolved at 10 mM in dimethyl sulfoxide (DMSO), and then diluted in either binding buffer or cell culture medium. NE-100 was dissolved in distilled water. 2.2. [3H]( þ)Pentazocine binding to rat brain membranes Binding assays for the sigma-1 receptor chaperone were performed according to a previous method (Narita et al., 1996), with a slight modification. Male Sprague–Dawley rats (250–300 g; Oriental Yeast Co., Ltd., Tokyo, Japan) were killed by decapitation, and their brains were removed rapidly. The brains were homogenized in 20 volumes of ice-cold 50 mM Tris-HCI (pH 8.0 at 25 1C), using a Polytron homogenizer set at 5, for 30 s. The homogenate was centrifuged at 48,000g for 10 m (4 1C). The resulting pellet was resuspended in buffer and recentrifuged. This procedure was repeated twice. The final pellet was suspended in 20 volumes of 50 mM Tris-HCI (pH 8.0 at 25 1C). For assays measuring sigma-1 receptor binding, aliquots of crude membranes (approximately 350–400 pg protein) were incubated with [3H] (þ )-pentazocine (a specific radioligand for sigma-1 receptor chaperones, 1.25–40 nM for Scatchard analysis and 5.0 nM for pharmacological inhibition: 1.254 TBq/mmol, GE Healthcare, Tokyo, Japan) and 50 mM Tris–HCl (pH 8.0 at 25 1C), in a final volume of 0.5 ml, for 2 h at 27 1C. After the addition of 4 ml of icecold buffer, the membranes were filtered rapidly, using a Brandell 24-channel cell harvester (Biochemical Research Laboratories, Gaithersburg, MD, USA), through Whatman GF/B filters pretreated with 0.5% polyethyleneimine, for at least 2 h. The filters were washed three times with 4 ml of ice-cold buffer. The radioactivity trapped by the filters was measured using a liquid scintillation counter (Tri-Carb 2800 TR, PerkinElmer Japan, Tokyo, Japan). Nonspecific binding was estimated in the presence of 10 μM NE-100 (a
selective sigma-1 receptor antagonist) (Okuyama and Nakazato, 1996). Protein concentrations were measured colorimetrically in a Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA, USA), using bovine serum albumin (BSA) as the standard. The dissociation constant (Kd) and maximal binding (Bmax) values from saturation-binding and the IC50 values from binding displacement by each drug were determined using GraphPad Prism (GraphPad Software, San Diego, CA, USA). The Ki values were calculated from the IC50 values, using the formula of Chung and Prusoff, Ki ¼ IC50/(1 þ[L]/Kd) (Cheng and Prusoff, 1973), where IC50 is the concentration that resulted in 50% inhibition of specific binding, [L] is the concentration of radioligand used and Kd the dissociation constant. 2.3. Effects of antidepressants on NGF-induced neurite outgrowth PC12 cells (RIKEN Cell Bank, Tsukuba, Japan) were cultured at 37 1C, in 5% CO2 with Dulbecco's modified Eagle's medium (DMEM), supplemented with 5% heat-inactivated fetal bovine serum (FBS), 10% heat-inactivated horse serum and 1% penicillin. The medium was changed two to three times a week. PC12 cells were plated onto 24-well tissue culture plates coated with poly-Dlysine/laminin. Cells were plated at relatively low density (0.25 104 cells/cm2) in DMEM containing 0.5% FBS with 1% penicillin and streptomycin. Media containing minimal levels of serum (0.5% FBS) were used, since it is known that serum contains steroid hormones that bind to sigma-1 receptors (Ishima et al., 2008; Ishima and Hashimoto, 2012; Nishimura et al., 2008). As reported previously (Ishima et al., 2008, 2012; Ishima and Hashimoto, 2012; Nishimura et al., 2008), 2.5 ng/ml of NGF was used to study the effects of antidepressant drugs on NGF-induced neurite outgrowth. Twenty-four hours after plating, media were replaced with DMEM containing 0.5% FBS along plus 1% penicillin and streptomycin with NGF (2.5 ng/ml), with or without added antidepressant drugs. Five days after incubation with NGF (2.5 ng/ ml) alone or supplemented with antidepressant drugs, morphometric analysis was performed on digitized images of live cells taken under phase-contrast illumination, with an inverted microscope linked to a camera. Images of three fields per well were taken, with an average of 100 cells per field. Differentiated cells were counted by visual examination of the field; only cells showing at least one neurite with a length equal to the cell body diameter were counted and expressed as a percentage of the total cells in the field. The scoring was performed in a blinded manner. 2.4. Statistical analysis Data are the mean 7standard error of the mean (S.E.M.). Statistical analysis was performed using the software package SPSS (SPSS 12.0J, Tokyo, Japan). The data were analyzed by oneway analysis of variance (ANOVA) or two-way ANOVA. When appropriate, post-hoc comparisons were performed using the Bonferroni/Dunn test. Significance for results was set at Po 0.05.
3. Results 3.1. Effects of antidepressant drugs on [3H]( þ)pentazocine binding For saturation-binding isotherms, six grade-diluted concentrations of [3H]( þ)-pentazocine (1.25–40 nM) were used. The specific binding of [3H]( þ)-pentazocine to rat brain membranes was saturable. Scatchard analysis of specific binding revealed an apparent Kd of 13.1 72.18 nM and a Bmax of 858.0 750.8 fmol/mg protein (n ¼ 3).
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Next, we examined the pharmacological inhibition of specific [3H] (þ )-pentazocine (5.0 nM) binding to rat brain membranes. Fluvoxamine, sertraline, fluoxetine, escitalopram, citalopram, paroxetine and duloxetine were found to displace [3H](þ )-pentazocine binding at rat brain membranes (Table 1). The order of potency for sigma-1 receptor chaperone binding drugs was as follows: fluvoxamine (Ki ¼ 17.0 nM)4sertraline (Ki ¼31.6 nM)4fluoxetine (Ki ¼191.2 nM) 4escitalopram (Ki ¼ 288.3 nM)4citalopram (Ki ¼403.8 nM)4paroxetine (Ki ¼2041 nM)4duloxetine (Ki ¼3533 nM) (Table 1). The Ki values of venlafaxine, milnacipran, and mirtazapine for [3H](þ )-pentazocine binding were greater than 10,000 nM (Table 1). 3.2. Effects of antidepressant drugs on NGF-induced neurite outgrowth In this assay, antidepressant drugs were tested at two concentrations (1.0 and 10 μM) in PC12 cells. At these concentrations, fluvoxamine and fluoxetine significantly increased the proportion of cells with NGF (2.5 ng/ml) induced neurite outgrowth in PC12 Table 1 Affinity of antidepressant drugs for the sigma-1 receptor chaperone in rat brain. Drugs
Ki (nM)
Fluvoxamine Sertraline Fluoxetine Escitalopram Citalopram Paroxetine Duoxetine Venlafaxine Milnacipran Mirtazapine
17.0 7 1.4 31.6 7 2.9 191.2 7 11.6 288.3 747.0 403.8 712.0 20417 144 35337 325 4 10,000 4 10,000 4 10,000
Assays were carried out under conditions described in the Materials and methods section. The specified concentrations of drug were incubated with rat brain membranes and 5 nM [3H](þ )-pentazocine. Non-specific binding was estimated in the presence of NE-100 (10 μM). Values are the mean7 S.E.M. of three experiments performed in duplicate.
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cells (Fig. 1). At the lower concentration (1.0 μM), escitalopram significantly increased the number of cells with NGF (2.5 ng/ml) induced neurite outgrowth (Fig. 1). Escitalopram (10 μM) and citalopram (1.0 and 10 μM) promoted slight increases in the number of cells with NGF-induced neurite outgrowth, but these differences were not statistically significant (Fig. 1). In contrast, sertraline and paroxetine produced no effect, consistent with a previous report (Nishimura et al., 2008). The higher concentration (10 μM) of sertraline significantly decreased the number of PC12 cells with NGF-induced neurite outgrowth, suggestive of cellular toxicity (Fig. 1) and consistent with a previous report (Nishimura et al., 2008). None of the SNRIs tested altered the number of cells with NGFinduced neurite outgrowth (Fig. 2). In contrast, the higher concentration (10 μM) of mirtazapine significantly increased the number of PC12 cells with neurite outgrowth induced by NGF (2.5 ng/ml), although the potency of mirtazapine was weaker than that of fluvoxamine at the same concentration (Fig. 2). To investigate the role of sigma-1 receptor chaperones, we examined the effects of NE-100 on the potentiation of NGF-induced neurite outgrowth by fluvoxamine, fluoxetine, escitalopram, and mirtazapine. Two-way ANOVA analysis revealed significant interactions [fluvoxamine NE-100: F(1, 20)¼21.68, Po0.001], [fluoxetine NE-100: F(1, 92)¼21.31, Po0.001], and [escitalopram NE-100: F (1, 20)¼23.63, Po0.001]. Co-administration of NE-100 (1.0 μM) significantly blocked potentiation by fluvoxamine (10 μM), fluoxetine (10 μM) and escitalopram (1.0 μM) (Fig. 3). In contrast, two-way ANOVA analysis revealed no differences in interaction [mirtazapine NE-100: F(1, 44)¼ 0.078, P¼0.781]. NE-100 had no effect on potentiation of NGF-induced neurite outgrowth by mirtazapine (10 μM) (Fig. 3D), suggesting that the sigma-1 receptor chaperone may not be involved in the mechanisms driving neurite outgrowth in this drug. Addition of NE-100 (1.0 μM) alone did not alter NGF-induced neurite outgrowth in PC12 cells (Fig. 3). To investigate the pharmacology of sertraline at sigma-1 receptor chaperones, we examined its effect on the potentiation of NGFinduced neurite outgrowth by fluvoxamine and fluoxetine. Twoway ANOVA analysis revealed significant interactions [fluvoxamine sertraline: F(1, 20)¼ 8.01, P¼0.01] and [fluoxetine sertraline: F (1, 20)¼13.41, P¼0.002]. Co-administration of sertraline (1.0 μM)
Fig. 1. Effects of SSRIs (fluvoxamine, sertraline, fluoxetine, paroxetine, escitalopram, citalopram and paroxetine) on NGF-induced neurite outgrowth in PC12 cells. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean7 S.E.M. The number of columns indicates the number of assays. nnn P o 0.001 compared with the control group.
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Fig. 2. Effects of fluvoxamine, SNRIs (duloxetine, venlafaxine, and milnacipran), and mirtazapine, on NGF-induced neurite outgrowth in PC12 cells. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean 7S.E.M. The number of columns indicates the number of assays. nnnP o0.001 compared with the control group.
Fig. 3. Effects of NE-100 on the potentiation of NGF-induced neurite outgrowth by fluvoxamine, fluoxetine, escitalopram and mirtazapine. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean 7 S.E.M. The number of columns indicates the number of assays. nnnPo 0.001 compared with the antidepressant drug alone group.
significantly blocked potentiation by fluvoxamine (1.0 μM) and fluoxetine (1.0 μM) (Fig. 4), suggesting that sertraline might be an antagonist at the sigma-1 receptor chaperone. To investigate the role of 5-hydroxytryptamine 5-HT1A receptor in the mechanisms of mirtazapine, we examined the effect of the selective 5-HT1A receptor antagonist WAY-100635 (10 μM) on the
potentiation of NGF-induced neurite outgrowth by mirtazapine (10 μM). Two-way ANOVA analysis revealed a significant interaction [mirtazapine WAY-100635: F(1, 44) ¼17.26, P o0.001]. Co-administration of WAY-100635 (10 μM) significantly blocked potentiation by mirtazapine (10 μM) (Fig. 5), suggesting the role of 5-HT1A receptor in the mechanisms of action of mirtazapine.
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Fig. 4. Effects of sertraline on the potentiation of NGF-induced neurite outgrowth by fluvoxamine or fluoxetine. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean 7S.E.M. The number of columns indicates the number of assays. nnnPo 0.001 compared with the antidepressant drug alone group.
Fig. 5. Effect of 5-HT1A receptor antagonist WAY-100635 on the potentiation of NGF-induced neurite outgrowth by mirtazapine. The numbers in parentheses denote the concentration (μM) of drug used. The data show the mean 7 S.E.M. The number of columns indicates the number of assays. nnnPo 0.001 compared with the antidepressant drug alone group.
Fig. 6 denotes representative photomicrographs showing the effects of fluvoxamine, fluoxetine and mirtazapine on PC12 cells. All these drugs increased the number of cells with neurite outgrowth.
4. Discussion In this study, we found that some SSRIs, including fluvoxamine, sertraline, fluoxetine, escitalopram and citalopram, had high to moderate affinity at sigma-1 receptor chaperones in the rat brain. The Ki values of fluvoxamine, sertraline, fluoxetine and citalopram were similar to those recorded in a previous report (Narita et al., 1996). In contrast, paroxetine, all tested SNRIs (duloxetine, venlafaxine, and milnacipran), and mirtazapine showed weak affinity at sigma-1 receptor chaperones. Furthermore, we found that the SSRIs, fluvoxamine, fluoxetine, escitalopram, could potentiate NGF-induced neurite outgrowth in PC12 cells, and that these effects could be blocked by NE-100, suggesting that these drugs function as agonists at sigma-1 receptor chaperones. This is the first paper demonstrating that escitalopram, a new SSRI, is capable of potentiating NGF-induced neurite outgrowth in a cell assay. Here, we detected no agonist activity for citalopram in cell assays, although it is reported that citalopram
affects fetal thalamic axon responsiveness to netrin-1, via sigma-1 receptors (Bonnin et al., 2012). These findings imply that for the SSRIs, fluvoxamine, fluoxetine and escitalopram, potentiation of NGFinduced neurite outgrowth in PC12 cells is mediated through agonism at sigma-1 receptors. As mentioned in Introduction, previous reports suggest that sigma1 receptor agonists, such as fluvoxamine, fluoxetine, donepezil, and ifenprodil, potentiate NGF-induced neurite outgrowth in PC12 cells, and that these effects can be blocked by NE-100, an antagonist at the sigma-1 receptor chaperone (Ishima et al., 2008; Ishima and Hashimoto, 2012; Nishimura et al., 2008; Takebayashi et al., 2002). Thus, it is likely that a drug's ability to exert an agonist or antagonist effect at the sigma-1 receptor chaperone determines its ability to promote NGF-induced neurite outgrowth in PC12 cells, and therefore its usefulness as a pharmacological agent. Unlike fluvoxamine, sartraline, which has a high affinity for sigma-1 receptor chaperones, did not alter NGF-induced neurite outgrowth, consistent with a previous report (Nishimura et al., 2008). The reasons underlying this discrepancy between these two SSRIs are currently unclear. One possibility may involve the difference in pharmacological action, namely whether the drugs exert opposing effects at sigma-1 receptor chaperones. In this study, we found that sertraline antagonized the potentiation of NGF-induced neurite outgrowth by fluvoxamine and fluoxetine, indicating that sertraline might act as an antagonist at sigma-1 receptor chaperones. Interestingly, phencyclidine-induced cognitive deficits in mice are significantly improved, by subsequent subchronic (14 days) doses of fluvoxamine, but not of sertraline, indicating that sigma-1 receptor agonism forms a part of fluvoxamine's mechanism of action (Hashimoto et al., 2007; Ishima et al., 2009). Taken together, these findings suggest that fluvoxamine and sertraline may function as an agonist and antagonist respectively, at sigma-1 receptor chaperones (Hashimoto, 2009a; Ishima et al., 2009), although a further study is needed to confirm this action. In this study, we found that mirtazapine potentiated NGFinduced neurite outgrowth in PC12 cells, although its effect could not be antagonized by NE-100. This is the first paper demonstrating that mirtazapine can potentiate NGF-induced neurite outgrowth in a cell culture assay. Mirtazapine antagonizes noradrenergic α2 autoreceptors and noradrenergic α2 receptors, resulting in enhanced release of noradrenaline from noradrenergic terminals, and increased serotonin release from serotonergic terminals, respectively (Croom et al., 2009). Thus, it seems that the net effect of mirtazapine is to increase noradrenergic and serotonergic transmission, notably 5-HT1A receptor-mediated transmission (Croom et al., 2009). Previously, we reported that the selective 5-HT1A receptor agonist, 8-hydroxy-2-dipropylaminotetraline (8-OH-DPAT), could potentiate NGF-induced neurite outgrowth in PC12 cells
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Fig. 6. Representative photomicrographs of PC12 cells. (A) Control (NGF (2.5 ng/ml) alone), (B) NGFþ fluvoxamine (10 μM), (C) NGFþ fluoxetine (10 μM) and (D) NGFþmirtazapine (10 μM). These drugs increased the number of cells with neurite outgrowth. Bar¼ 50 μm
(Ishima et al., 2012). In the present study, we found that the selective 5-HT1A receptor antagonist WAY-100635 significantly antagonized the effect of mirtazapine on neurite outgrowth, suggesting the role of the 5-HT1A receptor in the mechanisms of mirtazapine on neurite outgrowth. The Meta-analyses of new generation antidepressants (MANGA) study reported that mirtazapine was significantly more efficacious than duloxetine (Cipriani et al., 2009). Furthermore, a recent meta-analysis showed that augmentation therapy with mirtazapine improved overall and negative symptoms in patients with schizophrenia (Kishi and Iwata, 2014). It is therefore noteworthy that mirtazapine potentiates neurite outgrowth, since at the cellular level, this type of neuronal plasticity can underlie the therapeutic effect of drugs targeting the CNS (Lieberman et al., 2008; Lu and Dwyer, 2005; Molteni et al., 2009; Williams and Dwyer, 2009). In conclusion, our results suggest that sigma-1 receptor agonists, fluvoxamine, fluoxetine and escitalopram, potentiate NGFinduced neurite outgrowth in PC12 cells, implying that the sigma1 receptor chaperone plays a role in the pharmacological effects of these SSRIs. In contrast, mirtazapine is able to potentiate NGFinduced neurite outgrowth in PC12 cells, via 5-HT1A receptor.
Acknowledgments This study was supported by a grant from Grants-in-Aid for Scientific Research on Innovative Areas of The Ministry of Education, Culture, Sports, Science and Technology, Japan (to K.H., Grant number: 24116006). Dr. Hashimoto has served as a scientific consultant to Astellas and Taisho, and he also received the research grant support from Abbvie, Dainippon Sumitomo, Otsuka, and Taisho over
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