LPA1 Mediates Antidepressant-Induced ERK1/2 Signaling and Protection from Oxidative Stress in Glial Cells S

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Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/09/07/jpet.116.236455.DC1

1521-0103/359/2/340–353$25.00 http://dx.doi.org/10.1124/jpet.116.236455 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 359:340–353, November 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

LPA1 Mediates Antidepressant-Induced ERK1/2 Signaling and Protection from Oxidative Stress in Glial Cells s

Maria C. Olianas, Simona Dedoni, and Pierluigi Onali Laboratory of Cellular and Molecular Pharmacology, Section of Neurosciences and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy (M.C.O., S.D., P.O.) Received July 5, 2016; accepted September 6, 2016

ABSTRACT Downloaded from Antidepressants have been shown to affect glial cell functions abrogated by cell treatment with pertussis toxin and by and intracellular signaling through mechanisms that are still not the inhibition of fibroblast growth factor (FGF) receptor (FGF-R) completely understood. In the present study, we provide and platelet-derived growth factor receptor (PDGF-R) tyrosine evidence that in glial cells the lysophosphatidic acid (LPA) kinases. Both Ki16425 and AM966 suppressed antidepressant- receptor LPA1 mediates antidepressant-induced growth fac- induced phosphorylation of FGF-R. Moreover, blockade of tor receptor transactivation, ERK1/2 signaling, and protection LPA1 or inhibition of FGF-R and PDGF-R activities prevented b from oxidative stress. Thus, in C6 glioma cells and rat cortical antidepressant-stimulated Akt and GSK-3 phosphorylations. jpet.aspetjournals.org astrocytes, ERK1/2 activation induced by either amitriptyline or Mianserin protected C6 glioma cells and astrocytes from apopto- mianserin was antagonized by Ki16425 and VPC 12249 (S), tic cell death induced by H2O2, as indicated by increased cell which block LPA1 and LPA3 receptors, and by AM966, which viability, decreased expression of cleaved caspase 3, reduced selectively blocks LPA1. Cell depletion of LPA1 with siRNA cleavage of poly-ADP ribose polymerase and inhibition of DNA treatment markedly reduced antidepressant- and LPA-induced fragmentation. The protective effects of mianserin were antag- ERK1/2 phosphorylation. LPA1 blockade prevented antidepressant- onized by AM966. These data indicate that LPA1 constitutes a induced phosphorylation of the transcription factors CREB and novel molecular target of the regulatory actions of tricyclic and

Elk-1. Antidepressants and LPA signaling to ERK1/2 was tetracyclic antidepressants in glial cells. at ASPET Journals on September 27, 2021

Introduction glutamate transporter GLT1 (Reagan et al., 2004), and the calcium-binding protein S100b (Manev et al., 2001). Many A growing body of evidence indicates that glial cells of these changes have been proposed to contribute to constitute a major cellular target of antidepressants (Czeh the therapeutic efficacy of antidepressants (Czeh and and Di Benedetto, 2013; Sanacora and Banasr, 2013). Animal Di Benedetto, 2013; Sanacora and Banasr, 2013). Further- studies have shown that repeated in vivo administration of more, a number of in vitro studies have documented that antidepressants upregulates the hippocampal expression of antidepressants can act directly on glial cells. In primary glial fibrillary acidic protein (GFAP), a major intermediate cultures of astrocytes and in rat C6 glioma cells, which retain filament protein of astrocytes, and prevents or reverses the several morphologic and neurochemical properties of astro- decrease in hippocampal GFAP induced by chronic stress cytes (Pfeiffer et al., 1970; Bissell et al., 1974), distinct classes (Sillaber et al., 2008; Liu et al., 2009). Animal treatment with of antidepressants have been shown to induce the activation of antidepressants has also been demonstrated to affect the the extracellular signal-regulated kinases 1 and 2 (ERK1/2) brain expression of other specific astroglial proteins, including the proapoptotic protein Ndrg2 (Nichols, 2003), the gap signaling pathway (Khawaja et al., 2004; Li et al., 2008), which junction protein Connexin 43 (Fatemi et al., 2008), the plays a critical role in controlling cell differentiation and survival (Roux and Blenis, 2004), and is a putative key regulator of motivational and affective behavior (Duric et al., This work was supported by Regione Autonoma della Sardegna, Italy, L.R. 2010). In glial cells, antidepressant-induced ERK1/2 activa- n.7/2007 [CRP10810/2012] and Fondazione Banco di Sardegna, Italy [Prot. tion has been reported to mediate the phosphorylation/ U993.2014/AI.875.MGB] to P.O. dx.doi.org/10.1124/jpet.116.236455. activation of the transcription factor cAMP response s This article has supplemental material available at jpet.aspetjournals.org element–binding protein (CREB), and the expression and

ABBREVIATIONS: ANOVA, analysis of variance; CREB, cAMP response element–binding protein; DAPI, 49,6-diamidino-2phenylindole dihydrochloride; EGF, epidermal growth factor; Emax, maximal effect; ERK1/2, extracellular signal-regulated protein kinase 1/2; FCS, fetal calf serum; FGF, basic fibroblast growth factor; FGF-R, FGF receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; GPCR, G protein-coupled receptor; H2O2, hydrogen peroxide; 5-HT, 5-hydroxytryptamine; LPA, lysophosphatidic acid; MTT, 3-4(4,5- dimethyl-thiazol-2yl)-2,5-diphenyl-tetrazolium bromide; NBFI, norbinaltorphimine; PARP, poly (ADP-ribose) polymerase; PBS, phosphate-buffered saline; PDGF-BB, platelet-derived growth factor-BB; PDGF-R, PDGF receptor b; PI3K, phosphoinositide 3-kinase; PMSF, phenylmethylsulphonyl fluoride; P/S, penicillin/streptomycin; PTX, pertussis toxin; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase–dUTP nick- end labeling.

340 Antidepressant Signaling through LPA1 in Glial Cells 341 release of the neurotrophic factors brain-derived neurotrophic (Bristol, UK). Recombinant mouse basic FGF (FGF-2) was from ProSpec- factor and glial-derived neurotrophic factor (Mercier et al., Tany TechnoGene Ltd. (Ness Ziona, Israel). 2004; Hisaoka et al., 2007). Cell Culture. C6 rat glioma cells (European Collection of Cell ’ A major issue in understanding how antidepressants directly Cultures, Porton Down, Wiltshire, UK) were grown in Ham s F-12 affect glial cell activity is the identification of the mechanisms medium containing 2 mM L-glutamine, 10% fetal calf serum (FCS), and 0.5% penicillin/streptomycin (P/S) in a humidified 95% air and 5% by which these drugs can trigger intracellular signaling. Pre- CO2 at 37°C. vious studies have reported that in glial cells distinct classes of Primary cultures of astrocytes were prepared from the cerebral antidepressants alter the subcellular localization of heterotri- cortex of newborn (1-day-old) Sprague-Dawley rats. Experiments were meric G proteins (Donati and Rasenick, 2005), change the performed in accordance with the European Communities Council expression of b-arrestin 2 (Golan et al., 2010), and activate Directive (86/609EEC) and the Principles of Laboratory Animal Care different receptor systems, including FGF-R (Hisaoka et al., in Italy. After removal of meninges, the brain tissue was minced into 2011), 5HT2B receptors (Li et al., 2008), and k-opioid receptors small pieces and incubated in cell dissociation buffer (Invitrogen/Life (Onali et al., 2010; Olianas et al., 2012). These studies suggest Technologies, Monza, Italy) containing 0.2% trypsin for 20 minutes at that antidepressants, besides inhibiting monoamine trans- 37°C. Soyabean trypsin inhibitor and Dnase I (Sigma-Aldrich) were porters in nerve terminals, possess the ability to affect different then added, and the incubation was continued for 5 minutes. Cells were dissociated by aspiration through fire-polished Pasteur pipettes, signaling molecules in glial cells. collected by centrifugation, and resuspended in Dulbecco’s modified The bioactive phospholipid lysophosphatidic acid (LPA), which ’ ’

Eagle s medium-Ham s F-12 medium containing 10% FCS and 0.5% Downloaded from acts as endogenous agonist at six distinct G protein–coupled P/S. Cells were seeded into 75-cm2 flasks and incubated at 37°C in an receptors, termed LPA1–6 (Yung et al., 2015), is known to exert a atmosphere with 5% CO2. After incubation for 2 weeks, astrocytes variety of effects on astrocytes, including the regulation of cell were collected by shaking the cultures at 350 rpm overnight at 37°C morphology, mitogenesis, expression of neurotrophic genes, and (McCarthy and DeVellis, 1980). Astrocytes were grown in Dulbecco’s production of neuronal-differentiating factors (Ramakers and modified Eagle’s medium-Ham’s F-12 medium supplemented with Moolenaar, 1998; Tabuchi et al., 2000; Spohr et al., 2008; Sato 10% FCS and 0.5% P/S at 37°C in an atmosphere with 5% CO2. et al., 2011). We have recently observed that in CHO-K1 Cultured cells displayed a flat and polygonal morphology, typical of jpet.aspetjournals.org fibroblasts different antidepressants induce insulin-like growth type I astrocytes (Raff et al., 1983). Immunofluorescence analysis indicated that the cells were .95% positive by immunofluorescence factor-1 receptor transactivation, ERK1/2 signaling, and en- staining with an anti-GFAP antibody (Sigma-Aldrich). Cells were hanced cell proliferation through the LPA1 receptor (Olianas used 3–4 days after replating. et al., 2015). This observation indicated that LPA1 participates in Small Interfering RNA Transfection. C6 glioma cells were the cellular actions of antidepressants and prompted us to transfected with either control small interfering RNA (siRNA)-A or investigate whether this receptor system could be involved in mouse LPA1 siRNA (Santa Cruz Biotechnology) using Lipofectamine the regulation of glial cell activity by these drugs. RNAiMAX (Invitrogen/Life Technologies) as a transfection reagent. at ASPET Journals on September 27, 2021 In the present study, we show that in C6 glioma cells and Cells were incubated with siRNA duplexes (75 nM) for 12 hours at primary cultures of rat cortical astrocytes LPA1 mediates the 37°C and analyzed 48 hours after transfection. To determine trans- growth factor receptor transactivation and stimulation of fection efficiency, parallel samples were treated with fluorescein- ERK1/2 signaling by the tricyclic antidepressant amitriptyline conjugated control siRNA-A (Santa Cruz Biotechnology). An efficiency ∼ and the tetracyclic antidepressant mianserin. Moreover, we show of 60% was obtained in three separate experiments. Preparation of Crude Plasma Membrane Fraction. C6 gli- that mianserin protects glial cells from oxidative stress–induced oma cells grown to confluency in 100-mm Petri dishes were washed, apoptosis and that this effect is dependent on LPA1. scraped into ice-cold phosphate-buffered saline (PBS), and centrifuged at 1000 rpm at 4°C. Cells were washed twice with PBS, and the final 2 Materials and Methods pellet was stored at 80°C for 24 hours. The frozen cells were thawed and resuspended in ice-cold 10 mM HEPES/NaOH (pH 7.40) contain- Materials. Amitriptyline hydrochloride, nortriptyline hydrochlo- ing 0.1 mM EDTA, 4 mM sodium pyrophosphate, 2 mM sodium ride, imipramine hydrochloride, desipramine hydrochloride, orthovanadate, 10 mM sodium fluoride, 0.1% phosphatase inhibitor mianserin hydrochloride, mirtazapine hydrochloride, clomipramine cocktail 3, 1% protease inhibitor cocktail, and 1 mM phenylmethyl- hydrochloride, and Bordetella pertussis toxin (PTX). Recombinant sulphonyl fluoride (HEPES/EDTA buffer). Cells were lysed by using a human platelet-derived growth factor-BB (PDGF-BB), 3-4(4,5- glass/glass Dounce tissue grinder (pestle A), and the homogenate was dimethyl-thiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT), centrifuged for 10 minutes at 500g at 4°C. The supernatant was 49,6-diamidino-2-phenylindole dihydrochloride (DAPI), and 5- collected and centrifuged at 48,000g for 10 minutes at 4°C. The hydroxytryptamine hydrochloride (5-HT) were from Sigma-Aldrich supernatant was discarded, and the pellet was resuspended in ice-cold (St. Louis, MO). Recombinant human epidermal growth factor (EGF) HEPES/EDTA buffer and centrifuged at 48,000g for 10 minutes at was from Cell Signaling Technology (Danvers, MA). Ki16425 (3-(4-[4- 4°C. The final pellet was resuspended in ice-cold buffer containing ([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl) PBS, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate, 2 mM EDTA, propanoic acid), PD173074 (N-[2-[[4-(diethylamino)butyl]amino]-6- 2 mM EGTA, 4 mM sodium pyrophosphate, 2 mM sodium orthovana- (3,5-dimethoxyphenyl)pyrido[2,3-day]pyrimidin-7-yl]-N-(1,1-dimethy- date, 10 mM sodium fluoride, 20 nM okadaic acid, 0.1% phosphatase lurea), and norbinaltorphimine (NBFI) were obtained from Santa Cruz inhibitor cocktail 3, 1% protease inhibitor cocktail, and 1 mM phenyl- Biotechnology (Dallas, TX). AM966 (49-{4-[(R)-1-(2-chloro-phenyl)- methylsulphonyl fluoride (radioimmunoprecipitation assay buffer) ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4yl)-acetic acid supplemented with 0.1% Triton X-100 and sonicated for 5 seconds was from Chem Scene (Monmouth Junction, NJ). VPC12249 (S) ((S)- before Western blot analysis. phosphoric acid mono-[3-(4-benzyloxy-phenyl)-2-octadec-9-enoylamino- Western Blot Analysis. Cells were serum starved for 24 hours, propyl] ester ammonium salt) was from Avanti Polar Lipids Inc. incubated in fresh serum-free medium for 1 hour at 37°C, and then (Alabaster, AL). 1-Oleoyl-LPA was from either Santa Cruz Biotechnology treated with the test agents, as specified in the text. Cell extracts were or Sigma-Aldrich. Tyrphostin AG1296 was from Merck Millipore (Darm- prepared by scraping the cells in ice-cold radioimmunoprecipitation stadt, Germany). H2L5186303 ((Z,Z)-4-49-[1,3-phenylenebis(oxy-4,1- assay buffer followed by sonication for 5 seconds. Aliquots of cell phenyleneimino)]bis-4-oxo-2-butenoic acid) was from Tocris Bioscience extracts were taken for protein determination by using a protein assay 342 Olianas et al.

(Bio-Rad, Hercules, CA). Cell proteins were separated by SDS-PAGE Cell Viability Analysis. Cell viability of C6 glioma cells was and then electrophoretically transferred to either polyvinylidene determined by fluorescence-based analysis using the Muse Cell Count difluoride or nitrocellulose membranes. Membranes were incubated and Viability kit provided by Millipore (Temecula, CA). Briefly, overnight at 4°C with one of the following primary antibodies: anti- serum-starved cells were exposed to the experimental agents for the phospho-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187) (RA 15002) indicated times in serum-free medium, detached by trypsin/EDTA (1:15,000) (Neuromics, Northfield, MN); anti-ERK1/2 (#9102) treatment, washed in medium containing 10% FCS and incubated (1:1000), anti-phospho-CREB (Ser133) (catalog #9191) (1:2000), anti- with the assay reagent solutions for 5–15 minutes, as specified by the CREB (catalog #9197) (1:1000), anti-phospho-ElK-1(Ser383) (catalog manufacturer protocol. Cells were then analyzed by using the Muse #9181) (1:1000), anti-phospho-Akt (Thr308) (catalog #2965), anti- Cell Analyzer (Millipore). phospho-glycogen synthase kinase-3b (GSK-3b)(Ser9)(catalog Cell viability of rat astrocytes was performed by MTT assay in a #5558) (1:1000), anti-phospho-FGF receptor (Tyr653/654) (catalog 96-well plate. Serum-starved astrocytes were treated with the exper- #3476) (1:1000), anti-phospho-PDGF receptor (PDGF-R) b (Tyr751) imental agents, as specified in the text. After washing with PBS, the (catalog #3166) (1:1000), anti–PDGF-R b (catalog #3175) (1:1000), anti- cells were incubated for 3 hours at 37°C with MTT (0.5 mg/ml), cleaved poly-(ADP ribose) polymerase (PARP) (Asp214) (catalog #9625) dissolved in PBS, and filtered through a 0.20-mm Millipore filter. The (1:1000), anti-PARP (catalog #9542) (1:1000), and anti-pan-cadherin blue formazan product was solubilized by the addition of 10% SDS in (1:2000) (catalog #28E12) (all from Cell Signaling Technology); anti-Akt 10 mM HCl. Absorbance was measured on a Wallach Victor microplate reader (PerkinElmer, Waltham, MA) using a reference wavelength of (sc-8312) (1:1000), anti-GSK-3b (sc-9166) (1:1000), and anti-LPA1 630 nm and a test wavelength of 530 nm. (sc-515665) (1:2000) (all from Santa Cruz Biotechnology); anti-LPA2

Experiments were conducted to establish the concentrations of Downloaded from (ALR-032) (1:500) and anti-LPA3 (ALR-033) (1:500) (all from Alomone Labs, Jerusalem, Israel); anti-FGF receptor 1 (bs-0230R) (1:1000) (Bioss, freshly prepared hydrogen peroxide (H2O2) and the incubation times Inc., Woburn, MA); anti-actin (A2066) (1:2000) (Sigma-Aldrich); and that produced no more than 50% cell death in both C6 glioma cells and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog rat cortical astrocytes. Statistical Analysis. Results are reported as the mean 6 SEM of #247002g) (1:5000) (Synaptic Systems, Gottingen, Germany). After N independent experiments. Concentration-response curves were incubation with horseradish peroxidase–conjugated secondary anti- analyzed by the program GraphPad Prism (GraphPad Software, San bodies, immunoreactive bands were detected by using ECL Plus and ECL

Diego, CA), which yielded the drug concentration producing half- jpet.aspetjournals.org Hyperfilm (Amersham). The size of immunoreactive bands was de- maximal stimulation (EC ) or half-maximal inhibition (IC ), and termined by using molecular weight standards detected with an ECL 50 50 maximal effect (E ). Values of experimental groups were routinely suitable antibody (Santa Cruz Biotechnology) and Sharp Mass V Plus max expressed as a percentage or as the fold stimulation of control, which prestained protein molecular weight marker (EuroClone, Milan, Italy). was included in each independent experiment. The control was set as Band densities were determined by densitometric analysis using Image 100 or 1, with a variance obtained by expressing each control value as Scanner III (GE Healthcare, Milan, Italy) and NIH ImageJ software a ratio or percentage of the mean of the raw values of the control group. (National Institutes of Health, Bethesda, MA). The optical density of Statistical analysis was performed by either unpaired Student’s t test phosphoproteins was normalized to the density of the corresponding total or one-way analysis of variance (ANOVA) followed by Newman-Keuls at ASPET Journals on September 27, 2021 protein in the same samples. For the analysis of cleaved PARP, the post hoc test as appropriate. A P value of ,0.05 was considered to be formation of the cleaved protein was normalized to the level of the statistically significant. uncleaved form determined in the same samples. For the other proteins, the densitometric values were normalized to the levels of either actin or GAPDH. Results Immunofluorescence Analysis. Cells plated on poly-L-lysine– precoated glass coverslips were serum starved and treated with the Stimulation of ERK1/2 Phosphorylation by Amitrip- test compounds at 37°C, as specified in the text. After fixation with 4% tyline, Mianserin, and LPA in C6 Glioma Cells. Expo- paraformaldehyde and permeabilization with 0.2% Triton X-100, the sure of C6 glioma cells to either amitriptyline (15 mM) or cells were blocked with 3% bovine serum albumin and 1% normal goat mianserin (5 mM) for different periods of time showed that both serum, and incubated overnight at 4°C with either anti-phospho- drugs induced a rapid increase of ERK1/2 dual phosphoryla- CREB (Ser133) antibody (1:1000) (catalog #06-519; Upstate Biotech- tion, which is an index of the kinase activation, without – nology Inc., Lake Placid, NY) or anti cleaved caspase 3 (Asp175) changing total ERK1/2 levels. In the presence of these drugs, (1:200) (catalog #9664; Cell Signaling Technology). Control samples phospho-ERK1/2 levels increased about 4-fold (N 5 4) at were incubated in the absence of primary antibodies. After rinsing, the cells were incubated with Alexa Fluor 488–conjugated goat IgG 10 minutes and then declined, remaining above basal level for (Invitrogen/Molecular Probes). Cell nuclei were stained with 0.1 mg/ml at least 3 hours (Fig. 1, A and B). Under the same experimental DAPI. Images were captured at constant camera settings and conditions, ERK1/2 phosphorylation induced by LPA (100 nM) analyzed using the program Cell P (Olympus Soft Imaging Solutions, showed a similar kinetic profile, being high at 10 minutes and Hamburg, Germany), as previously described (Olianas et al., 2008). No slowly decreasing at later time points (Fig. 1C). labeling was detected in samples treated without primary antibodies. The stimulations of ERK1/2 phosphorylation elicited by Four separate culture preparations were analyzed by an investigator amitriptyline and mianserin were concentration dependent, who was unaware of the treatment. with mean EC50 values of 2.1 6 0.5 and 1.2 6 0.3 mM, Terminal Deoxynucleotidyl Transferase–dUTP Nick-End respectively, and Emax values corresponding to 4.2 6 0.7- Labeling Assay. Cultured astrocytes grown on glass coverslips were and 4.7 6 0.8-fold increases in basal values (N 5 5, P , treated with the test compounds at 37°C, as specified in the text. In 0.001), respectively (Fig. 1, D and E). LPA also displayed a situ terminal deoxynucleotidyl transferase–mediated digoxigenin- concentration-dependent stimulation of ERK1/2 with an deoxyuridine nick-end labeling (TUNEL) assay was performed using 6 6 the DeadEnd fluorimetric TUNEL system (Promega, Madison, WI), EC50 of 260 30 nM and a mean 9.4 1.0-fold increase in 5 , according to the manufacturer instructions. Cell nuclei were stained Emax values over basal values (N 5, P 0.001) (Fig. 1F). with DAPI. Images were captured over randomly selected fields and LPA1 Antagonists Inhibit ERK1/2 Phosphorylation analyzed with Cell P software, as described for immunofluorescence Induced by Amitriptyline and Mianserin. As shown in assays. Three separate culture preparations were analyzed by an Fig. 2A, preincubation of C6 glioma cells with either 1 mM investigator who was unaware of the treatment. Ki16425, which antagonizes LPA1 and LPA3 and has no Antidepressant Signaling through LPA1 in Glial Cells 343 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 1. Time- and concentration-dependent stimulation of ERK1/2 phosphorylation by amitriptyline, mianserin, and LPA in C6 glioma cells. Cells were serum starved for 24 hours and then exposed to amitriptyline (15 mM) (A), mianserin (5 mM) (B), or LPA (100 nM) (C) for the indicated periods of time. The levels of phospho-ERK1/2 and total ERK1/2 were measured in cell extracts by Western blot. Densitometric ratios are expressed as the fold change in stimulation with respect to 0 time (vehicle-treated cells) and are the mean 6 SEM of four experiments. *P , 0.05, ***P , 0.001 vs. 0 time by ANOVA followed by Newman-Keuls post hoc test. (D–F) Serum-starved cells were treated for 10 minutes with the indicated concentrations of amitriptyline, mianserin, and LPA. Values are expressed as the fold change in stimulation with respect to controls (vehicle-treated cells) and are the mean 6 SEM of five experiments. activity at the other LPA receptors (Ohta et al., 2003; cell pretreatment with Ki16425, AM966, or VPC12249 (S) Yanagida et al., 2009; Jiang et al., 2013), or 1 mM AM966, (Fig. 2, F–H). which preferentially blocks LPA1 (Swaney et al., 2010), Concentration-response curves performed in C6 glioma cells completely prevented ERK1/2 stimulation induced by indicated that AM966 and Ki16425 completely suppressed LPA 100 nM LPA. Ki16425 (100 nM) and AM966 (100 nM) reduced (1 mM)–induced ERK1/2 phosphorylation with IC50 values of the stimulatory effect elicited by amitriptyline (15 mM) by 84 6 13.8 6 1.2 and 63.5 6 9 nM, respectively (Fig. 3, A and B). The 2% and 74 6 3%, respectively (Fig. 2B), and that induced by stimulatory effect of amitriptyline (15 mM) was antagonized by mianserin (5 mM) by 80 6 3 and 76 6 4%, respectively (Fig. AM966 and Ki16425, with IC50 values of 1.0 6 0.2 and 2.8 6 0.5 2C). Cell treatment with the LPA analog VPC12249 (S) nM, respectively, and maximal inhibitory effects of 83% and 76%, (10 mM), which blocks LPA1 and LPA3 (Heise et al., 2001), respectively (Fig. 3, C and D). Similarly, the stimulatory effect of reduced amitriptyline- and mianserin-stimulated ERK1/2 mianserin (5 mM) was antagonized by either AM966 (IC50 5 1.6 6 phosphorylation by 68 6 4% and 70 6 3%, respectively (Fig. 0.3 nM) or Ki16425 (IC50 5 3.2 6 0.4 nM) with curves that 2D). At the concentrations used, none of the antagonists reached a plateau at approximately 80% inhibition (Fig. 3, E and affected the phosphorylation state of ERK1/2 per se. F). The compound H2L5186303, which displays selectivity for Short-term exposure of rat cortical astrocytes to LPA (1 mM) LPA2 over LPA1 and LPA3 (Fells et al., 2008), had no effect on increased ERK1/2 phosphorylation by approximately 2.3-fold LPA (1 mM) stimulation of ERK1/2 at concentrations up to 1 mM (P , 0.001, N 5 10) (Fig. 2E). Cell pretreatment with AM966 (Fig. 3G). This antagonist also had no effect on the stimulatory (1 mM) completely blocked the stimulatory effect of LPA, responses elicited by either amitriptyline or mianserin (Fig. 3H). whereas Ki16425 (1 mM) curtailed the response by 80% (P , We have previously reported that in rat C6 glioma cells, 0.01) (Fig. 2E). In these cells, amitriptyline (15 mM) and amitriptyline and mianserin stimulated ERK1/2 phosphory- mianserin (5 mM) stimulated ERK1/2 phosphorylation by lation by activating endogenous k-opioid receptors (Onali about 2-fold (P , 0.001, N 5 12). The stimulatory effect et al., 2010; Olianas et al., 2012). To examine whether the elicited by both antidepressants was significantly reduced by incomplete blockade observed with AM966 and Ki16425 was 344 Olianas et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 2. Blockade of antidepressant- and LPA-induced ERK1/2 phosphorylation by LPA receptor antagonists. (A) C6 glioma cells were preincubated for 10 minutes with vehicle, AM966 (1 mM) (AM), or Ki16425 (1 mM) (Ki), and then exposed to either vehicle or LPA (100 nM) for 10 minutes. Values are expressed as the fold change in stimulation with respect to control (vehicle + vehicle) and are the mean 6 SEM of three experiments. (B) C6 glioma cells were preincubated for 10 minutes with vehicle, Ki16425 (100 nM), or AM966 (100 nM), and then exposed to either vehicle or amitriptyline (15 mM) (Amitript) for 10 minutes. Values are the mean 6 SEM of five experiments. (C) C6 glioma cells were preincubated as indicated in (B) and then exposed to either vehicle or mianserin (5 mM) (Mians) for 10 minutes. Values are the mean 6 SEM of five experiments. (D) C6 glioma cells were preincubated for 10 minutes with either vehicle or 10 mM VPC12249 (S), and then exposed to either vehicle, 15 mM amitriptyline, or 5 mM mianserin for 10 minutes. Values are the mean 6 SEM of three experiments. (E) Astrocytes were preincubated with the LPA receptor antagonists, as indicated in (A), and then exposed to either vehicle or 1 mM LPA for 15 minutes. Values are the mean 6 SEM of five experiments. (F–H) Astrocytes were preincubated for 10 minutes with vehicle, 100 nM Ki16425, 100 nM AM966, or 10 mM VPC12249, and then exposed to vehicle, 15 mM amitriptyline, or 5 mM mianserin for 15 minutes. Values are the mean 6 SEM of four experiments. ***P , 0.001 vs. control (vehicle + vehicle); #P , 0.05, ##P , 0.01, ###P , 0.001 vs. the corresponding sample treated with vehicle by ANOVA followed by Newman-Keuls post hoc test.

due to the concomitant stimulation of k-opioid receptors, cells directed against the extracellular domain of LPA1 detected were cotreated with the k-opioid receptor antagonist NBFI. the presence of a prominent immunoreactive band of ∼41 kDa, As shown in Supplemental Fig. 1, the combination of NBFI which corresponds to the molecular mass of LPA1 (Fig. 4A). (100 nM) and Ki16425 (1 mM) completely blocked the Immunoblot analysis using antibodies against the extracellu- stimulatory effect of amitriptyline (15 mM) and mianserin lar domains of LPA2 and LPA3 also showed the presence of (5 mM). immunoreactive bands corresponding to LPA2 (37 kDa) and LPA1 Knockdown Inhibits Antidepressant-Induced LPA3 (∼41 kDa) (Fig. 4A). A similar pattern of receptor ERK1/2 Phosphorylation in C6 Glioma Cells. Western expression was observed when a crude plasma membrane blot analysis of C6 glioma cell lysates with an antibody preparation was examined (Fig. 4A). Antidepressant Signaling through LPA1 in Glial Cells 345 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 3. Concentration-dependent effects of LPA receptor antagonists on antidepressant- and LPA-induced ERK1/2 phosphorylation in C6 glioma cells. Cells were preincubated with either vehicle or the indicated concentrations of AM966 or Ki16425 for 10 minutes, and then exposed to vehicle or 1 mM LPA (A, B); 15 mM amitriptyline (Amitript) (C, D); or 5 mM mianserin (Mians) (E, F) for 10 minutes. Values are expressed as percentages of phospho- ERK1/2 stimulation and are the mean 6 SEM of four separate experiments. (G) Cells were preincubated for 10 minutes with either vehicle or the indicated concentrations of H2L5186303 (H2L5), and then exposed to either vehicle or 1 mM LPA for 10 minutes. Values are the mean 6 SEM of three experiments. (H) Cells were preincubated with either vehicle or 1 mM H2L5186303 (H2L5), and then exposed to vehicle, 15 mM amitriptyline, or 5 mM mianserin for 10 minutes. Values are the mean 6 SEM of three experiments. ***P , 0.001 vs. control (vehicle + vehicle).

Treatment of C6 glioma cells with LPA1-siRNA reduced Ki16425 and AM966 Block the Phosphorylation of CREB the expression levels of LPA1 by 60 6 5% (P , 0.001), andElK-1InducedbyAmitriptylineandMianserin.The compared with control siRNA-treated cells, but failed to transcription factor CREB is activated by phosphorylation at significantly affect the expression levels of either LPA2 or Ser133 in response to growth factors and a variety of LPA3 (Fig. 4B). extracellular stimuli acting through different protein kinase In cells depleted of LPA1, ERK1/2 phosphorylation elicited pathways, including ERK1/2 (Lonze and Ginty, 2002). Im- by either amitriptyline (15 mM) or mianserin (5 mM) was munofluorescence analysis showed that exposure of C6 reduced by 82 6 3% and 80 6 4%, respectively, compared with glioma cells to amitriptyline (15 mM) for 30 minutes induced the responses observed in control siRNA-treated cells (Fig. a significant increase in the number of cell nuclei expressing 4C). Similarly, LPA1-siRNA treatment reduced the stimula- phospho-Ser133-CREB (Fig. 5A). The increase was blocked tions induced by imipramine (15 mM) and LPA (50 nM) by 70 6 by pretreatment with Ki16425 (100 nM). Western blot anal- 2% and 74 6 5%, respectively (Fig. 4D). ysis showed that in both C6 glioma cells and rat cortical 346 Olianas et al. Downloaded from jpet.aspetjournals.org

Fig. 4. LPA1 knockdown attenuates antidepressant- and LPA-induced ERK1/2 phosphorylation in C6 glioma cells. (A) Whole-cell lysates and crude plasma membranes were analyzed for LPA1, LPA2, and LPA3 expression by Western blot. GAPDH and pan-cadherin (plasma membrane marker) were used as loading controls. Data are representative of three separate experiments. (B) Cells were treated with either control siRNA or LPA1 siRNA, and were analyzed for LPA1, LPA2, and LPA3 expression 48 hours after transfection. Each lane was loaded with a sample from three separate transfections. Values of LPA1–3 immunoreactivities normalized to the levels of GAPDH are reported as a percentage of controls (control siRNA-treated cells) and are the mean 6 SEM of three experiments. (C, D) C6 glioma cells treated with siRNAs, as indicated in (B) ,were exposed for 10 minutes to vehicle, at ASPET Journals on September 27, 2021 amitriptyline (Amitript) (15 mM), and mianserin (Mians) (5 mM) (C); and imipramine (Imipr) (15 mM) or LPA (50 nM) (D). Values are the mean 6 SEM of three experiments. ***P , 0.001 vs. control; #P , 0.05, ###P , 0.001 vs. the corresponding sample in the control siRNA-treated group by ANOVA followed by Newman-Keuls post hoc test. astrocytes, amitriptyline (15 mM) and mianserin (5 mM) Antidepressant-Induced ERK1/2 Phosphorylation In- increased phospho-CREB levels, and this response was volves PDGF-R and FGF-R Transactivation. Like other antagonized by AM966 (100 nM) (Fig. 5, B and C). G protein–coupled receptors, LPA receptors have been shown Elk-1 is a transcription factor that is directly phosphory- to stimulate ERK1/2 via transactivation of growth factor lated at Ser383 and Ser389 by ERK1/2 (Besnard et al., 2011). receptor tyrosine kinases, particularly EGF receptor (Wetzker As shown in Fig. 5D, the exposure of C6 glioma cells to either and Bohmer, 2003). Exposure of C6 glioma cells to 50 ng/ml of amitriptyline (15 mM) or mianserin (5 mM) induced a EGF for 5 minutes failed to affect ERK1/2 phosphorylation significant increase in the levels of phospho-Ser383-Elk-1, (results not shown), indicating the absence of functional EGF and this response was abrogated by preincubation with receptor coupled to ERK1/2. On the other hand, in these cells AM966 (100 nM). FGF-R has been identified as a site of convergence between PTX Prevents Antidepressant-Induced ERK1/2 Phos- growth factor and Gi/o-coupled receptor signaling (Belcheva phorylation. In a number of cell types, LPA1 has been et al., 2002). We found that ERK1/2 phosphorylation elicited shown to signal by coupling to distinct families of hetero- by LPA, amitriptyline, or mianserin was significantly reduced trimeric G proteins, including Gi/o,Gq/11,andG12/13 (Yung by pretreatment of C6 glioma cells with the FGF-R tyrosine et al., 2015). Exposure of C6 glioma cells to PTX (100 ng/ml), kinase inhibitor PD173074 at a concentration (100 nM) that which selectively uncouples Gi/o from receptors, caused an completely blocked the stimulatory response to FGF-2 almost complete suppression of ERK1/2 phosphorylation (10 ng/ml) (Fig. 7, A and B). Exposure to PD173074 (100 nM) induced by amitriptyline (20 mM), imipramine (20 mM), completely blocked ERK1/2 phosphorylation induced by am- desipramine (20 mM), nortriptyline (20 mM), mianserin itriptyline and mianserin in rat cortical astrocytes (Fig. 7C). (10 mM), and mirtazapine (15 mM) (Fig. 6, A–C). PTX Short-term treatment of C6 glioma cells with amitriptyline blocked LPA (100 nM)–induced stimulation of ERK1/2 (15 mM) and mianserin (5 mM) elicited a significant increase in phosphorylation (Fig. 6D), but failed to affect the stimula- FGF-R phosphorylation at Tyr653/654 (Fig. 7, D and E), which is tory effect elicited by either FGF-2 (10 ng/ml) or 5-HT critical for the receptor kinase activity and signaling (Mohammadi (10 mM)(Fig.6,DandE).ExposuretoPTXalsoprevented et al., 1996). Pretreatment with either AM966 or Ki16425 blocked ERK1/2 phosphorylation induced by either mianserin antidepressant-induced FGF-R phosphorylation (Fig. 7, D and E). (5 mM) or amitriptyline (15 mM) in rat cortical astrocytes In addition to FGF-2, PDGF has been shown to act on C6 (Fig. 6F). glioma cells (Zhang et al., 1992; Goya et al., 1996). As shown in Antidepressant Signaling through LPA1 in Glial Cells 347

for phosphatidylinositol 3-kinase (PI3K) (Panayotou et al., 1992). Like PDGF-BB, LPA (100 nM), clomipramine (10 mM), mianserin (5 mM), and amitriptyline (15 mM) induced a significant increase in PDGF-R b phosphorylation, although by an extent smaller than that elicited by PDGF-BB (Fig. 7G). Cell treatment with the PDGF-R tyrosine kinase inhibitor tyrphostin AG1296 (10 mM) abrogated ERK1/2 phosphorylation induced by PDGF-BB (3 ng /ml), but had no effect on the stimulation induced by FGF-2 (3 ng/ml), indicating that the inhibitor displayed receptor specificity (Fig. 7H). Pretreat- ment with tyrphostin AG1296 (10 mM) reduced ERK1/2 phosphorylation induced by either amitriptyline or mianserin (Fig. 7I). Stimulation of Akt Signaling by Antidepressants through LPA1 and Growth Factor Receptor Activity. Activation of the PI3K/Akt pathway is a main mechanism by which growth factor receptors induce prosurvival signaling (Hennessy et al., 2005). Plasma membrane 39-phosphoinosi- Downloaded from tides generated by PI3K allow the recruitment and the activation of Akt through phosphorylation at Thr308 by phosphatidylinositol-dependent protein kinase 1. As shown in Fig. 8A, in C6 glioma cells LPA (1mM), mirtazapine (15 mM), imipramine (10 mM), mianserin (5 mM), and amitriptyline (15 mM) caused a significant increase of Akt phosphorylation jpet.aspetjournals.org at Thr308. Cell pretreatment with either AG1296 (10 mM) or PD173074 (100 nM) inhibited amitriptyline- and mianserin- stimulated Akt phosphorylation (Fig. 8, B and C). Pretreatment with AM966 (100 nM) completely blocked the stimulation of Akt phosphorylation elicited by either amitriptyline or mianserin (Fig. 8D). Moreover, AM966 prevented the antidepressant-

induced phosphorylation of GSK-3b at Ser9, a main down- at ASPET Journals on September 27, 2021 stream target of Akt (Hennessy et al., 2005) (Fig. 8E). Mianserin Protects Glial Cells from Oxidative Stress–Induced Apoptosis through LPA1. Exposure of primary astrocytes to H2O2 has been reported to cause apoptotic cell death (Juknat et al., 2005). Immunofluorescence analysis showed that 24 hours of incubation of C6 glioma cells and rat cortical astrocytes with 300 and 100 mMH2O2, respectively, enhanced the percentage of cells positive for a cleaved active form of caspase 3, a marker of apoptosis (Fig. 9, A and B). The deleterious effects of H2O2 were markedly reduced in cells pretreated for 24 hours with mianserin (5 mM). Western blot analysis of C6 glioma cell extracts indicated that Fig. 5. Blockade of LPA1 prevents the phosphorylation of CREB and ElK-1 m induced by amitriptyline (Amitript) and mianserin (Mians). (A) C6 glioma prolonged exposure to 300 MH2O2 caused a significant cells were serum starved for 24 hours, pretreated with either vehicle or increase in the levels of cleaved PARP, a substrate of activated 100 nM Ki16425 for 10 minutes, and then were exposed to either vehicle or caspase 3, and this effect was significantly reduced in cells m 15 M amitriptyline for 30 minutes. Phospho-CREB immunofluorescence pretreated with mianserin (5 mM) (Fig. 9C). The addition of (green color) was measured in nuclei identified by DAPI staining (blue color). Scale bar, 50 mm. Values of quantitative analysis of phospho-CREB AM966 (100 nM) failed to affect H2O2-induced stimulation of immunofluorescence are expressed as the percentage of positive nuclei and PARP cleavage, but completely prevented the inhibitory effect are the mean 6 SEM of three experiments. ***P , 0.001 vs. control; ###P , of mianserin. Similarly, analysis of C6 glioma cell viability 0.001 vs. the respective value with no antagonist by ANOVA followed by m Newman-Keuls post hoc test. C6 glioma cells (B) and rat astrocytes (C) were showed that mianserin (5 M) reduced the cell death induced preincubated with either vehicle or AM966 (100 nM) for 10 minutes and then by H2O2 and that the protective effect of mianserin was exposed for 30 minutes to either 15 mM amitriptyline or 5 mM mianserin. Cell attenuated in cells cotreated with AM966 (100 nM) (Fig. 9D). extracts were then analyzed for phospho-CREB (pCREB) and total CREB by In cortical astrocytes, prolonged exposure to H O (100 mM) Western blot. Values are the mean 6 SEM of four experiments. (D) C6 glioma 2 2 cells were treated as indicated in (B), and cell extracts were analyzed for enhanced the number of cell nuclei displaying DNA fragmen- phospho-Elk-1 (pElk-1) and total Elk-1. Values are the mean 6 SEM of three tation, as assessed by the TUNEL assay (Fig. 9E). Pretreat- experiments. **P , 0.01, ***P , 0.001 vs. control; #P , 0.05, ###P , 0.001 by ment with mianserin (5 mM) reduced this apoptotic marker to ANOVA followed by Newman-Keuls post hoc test. control levels, and this effect was prevented by AM966 (100 nM). Exposure to H2O2 (200 mM) reduced astrocyte Fig. 7F, PDGF-BB (10 ng/ml) induced a marked autophos- viability by 40% (P , 0.001), and this effect was attenuated phorylation of PDGF-R b at Tyr751, which is located in the by pretreatment with mianserin (5 mM) (Fig. 9F). This pro- kinase insert region of the receptor and acts as a docking site tective effect was significantly reduced by AM966 (100 nM). 348 Olianas et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 6. PTX-sensitive G proteins transduce antidepressant-induced ERK1/2 phosphorylation. C6 glioma cells were incubated in serum-free medium containing either vehicle or 100 ng/ml PTX for 24 hours, and then treated for 10 minutes with vehicle, 20 mM amitriptyline (Amitript), and 20 mM imipramine (Imipr) (A); 20 mM desipramine (Desipr) and 20 mM nortriptyline (Nortript) (B); or either 10 mM mianserin (Mians) or 15 mM mirtazapine (Mirtaz) (C). In (D), vehicle- and PTX-treated cells were exposed for 10 minutes to either 100 nM LPA or 10 ng/ml FGF-2. Values are the mean 6 SEM of four experiments. (E) Serum-starved C6 glioma cells preincubated with either vehicle or PTX, as indicated in (A), were exposed for 10 minutes to 5 mM mianserin and for 5 minutes to 10 mM 5HT. Values are the mean 6 SEM of three experiments. (F) Rat cortical astrocytes were preincubated with either vehicle or PTX, as indicated in (A), and then exposed to 5 mM mianserin or 15 mM amitriptyline for 15 minutes. Values are the mean 6 SEM of four experiments. ***P , 0.001 vs. control (vehicle); ###P , 0.001 by ANOVA followed by Newman-Keuls post hoc test.

Discussion behaved as partial agonists. It is noteworthy that their EC50 values (2.1 and 1.2 mM, respectively) are within the concentration A large number of studies have demonstrated that antide- range reached by these drugs in the brain after therapeutic doses pressants affect intracellular signaling and biologic activity of (Glotzbach and Preskorn, 1982; Kurata and Kurachi, 1989). glial cells, but the molecular mechanisms mediating these ERK1/2 phosphorylation elicited by either amitriptyline or effects are still not completely understood. In the present mianserin was antagonized with nanomolar potencies by study, we provide the first evidence that in C6 glioma cells and structurally different LPA1 antagonists, including AM966, rat cortical astrocytes, the activation of G -coupled LPA is i/o 1 which selectively inhibits LPA1 over LPA2–5 (Swaney et al., involved in the stimulation of ERK1/2 signaling by tricyclic and 2010), and Ki16425, which blocks LPA1 and LPA3 and has no tetracyclic antidepressants. Moreover, the study shows that activity at the other LPA receptors (Ohta et al., 2003; Yanagida mianserin inhibits oxidative stress–induced apoptosis of glial et al., 2009; Jiang et al., 2013). VPC12249 (S), an LPA analog cells and that this protective effect involves LPA1 activation. that is a dual LPA1/LPA3 antagonist (Heise et al., 2001), was In C6 glioma cells, amitriptyline and mianserin induced a also effective in antagonizing ERK1/2 phosphorylation induced rapid and persistent increase of ERK1/2 phosphorylation with a by the two antidepressants. Conversely, in C6 glioma cells the kinetic profile similar to that displayed by LPA. Analysis of compound H2L5186303, which blocks LPA2 about 3000 and concentration-response curves showed that LPA stimulated 100 times more potently than LPA1 and LPA3, respectively ERK1/2phosphorylationwithanEC50 of 260 nM, which agrees (Fells et al., 2008), had no effect on ERK1/2 phosphorylation with the potency previously observed in CHO-K1 cells expressing elicited by amitriptyline, mianserin, or LPA over a wide endogenous LPA1 (81 nM) and HEK 293 cells transfected with concentration range. Moreover, in these cells depletion of human LPA1 cDNA (140 nM) (Olianas et al., 2015). Amitriptyline LPA1 by siRNA treatment led to a significant reduction of and mianserin were less potent and efficacious than LPA in antidepressant- and LPA-induced ERK1/2 activation. Collec- stimulating ERK1/2 phosphorylation, suggesting that they tively, these data indicate that the activation of LPA1 plays a Antidepressant Signaling through LPA1 in Glial Cells 349 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 7. Involvement of growth factor receptor transactivation in antidepressant- and LPA-induced ERK1/2 phosphorylation. (A, B) Serum-starved C6 glioma cells were pretreated with either vehicle or the FGF-R inhibitor PD173074 (100 nM) for 2 hours, and then exposed for 10 minutes to 15 mM amitriptyline (Amitript), 10 ng/ml FGF-2, 5 mM mianserin (Mians), or 50 nM LPA. Values are the mean 6 SEM of four experiments. (C) Rat cortical astrocytes were pretreated with either vehicle or PD173074 as indicated in (A) and then exposed for 15 minutes to either 15 mM amitriptyline or 5 mM mianserin. Values are the mean 6 SEM of four experiments. ***P , 0.001 vs. control; #P , 0.05, ###P , 0.001 vs. the corresponding value in the vehicle-pretreated group by ANOVA followed by Newman-Keuls post hoc test. (D, E) C6 glioma cells were preincubated with vehicle, 100 nM AM966, or 100 nM Ki16425 for 10 minutes, and then exposed for 5 minutes to either 15 mM amitriptyline or 5 mM mianserin, respectively. Cell extracts were analyzed for the levels of phospho-Tyr653/654-FGF-R (p-FGF-R) and total FGF-R. Values are the mean 6 SEM of four experiments. ***P , 0.001 vs. control; ##P , 0.01, ###P , 0.001 by ANOVA followed by Newman-Keuls post hoc test. C6 glioma cells were treated for 5 minutes with either vehicle and 10 ng/ml PDGF-BB (F); or vehicle, 100 nM LPA, 10 mM clomipramine (Clomipr), 5 mM mianserin, and 15 mM amitriptyline (G). Cell extracts were analyzed for the levels of phospho-Tyr751-PDGF-R b (p-PDGF-R) and total PDGF-R b (PDGF-R) by Western blot. Values are the mean 6 SEM of four experiments. (H, I) C6 glioma cells were preincubated for 1 hour with either vehicle or 10 mM tyrphostin AG1296 (AG1296), and then exposed for 10 minutes to 3 ng/ml PDGF-BB, 3 ng/ml FGF-2, 15 mM amitriptyline, or 5 mM mianserin. Values are the mean 6 SEM of four experiments. *P , 0.05, ***P , 0.001 vs. control, ###P , 0.001 vs. the corresponding value in the vehicle-treated group by either ANOVA followed by Newman-Keuls post hoc test (G, I) or Student’s t test (G). 350 Olianas et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 8. Antidepressants induce Akt phosphorylation through LPA1 and growth factor receptor activity. (A) C6 glioma cells were exposed for 10 minutes to vehicle, 1 mM LPA, 15 mM mirtazapine (Mirtaz), 10 mM imipramine (Imipr), 5 mM mianserin (Mians), and 15 mM amitriptyline (Amitript). Cell extracts were then analyzed for phospho-Thr308-Akt (pAkt) and total Akt by Western blot. Values are the mean 6 SEM of four experiments. C6 glioma cells were pretreated with either vehicle and 10 mM tyrphostin AG1296 for 1 hour (B), or vehicle and 100 nM PD173074 for 2 hours (C). Thereafter, cells were exposed for 10 minutes to vehicle, 15 mM amitriptyline, or 5 mM mianserin. Values are the mean 6 SEM of four experiments. (D, E) C6 glioma cells were preincubated with either vehicle or AM966 (100 nM) for 10 minutes, and then exposed for 10 minutes to either 15 mM amitriptyline or 5 mM mianserin. Cells extracts were analyzed for phospho-Akt and total Akt (D) and for phospho-Ser9-GSK-3b (pGSK3b) and total GSK-3b (E). Values are the mean 6 SEM of four experiments. ***P , 0.001 vs. control; #P , 0.05, ###P , 0.001 vs. the corresponding sample treated with vehicle by ANOVA followed by Newman-Keuls post hoc test. major role in ERK1/2 activation by amitriptyline and mianserin Cell treatment with either AM966 or Ki16425 prevented the in both C6 glioma cells and rat cortical astrocytes. stimulation of CREB and Elk-1 phosphorylation elicited by It is important to note that, in addition to LPA1, Western amitriptyline and mianserin. Transcriptional activation of blot analysis detected the presence of LPA2 and LPA3, which CREB via phosphorylation at Ser133 is known to trigger the display a high sequence homology to LPA1 (Yung et al., 2015), expression of a large array of genes regulating neuronal in both whole-cell lysates and crude plasma membrane development, synaptic plasticity, learning, and memory preparations of C6 glioma cells. The finding that LPA (Lonze and Ginty, 2002). In glial cells, CREB activation has been stimulated ERK1/2 exclusively via LPA1 suggests the possi- linked not only to the expression of growth factors, but also to cell bility, which remains to be investigated, that in these cells differentiation, proliferation, and survival (Sato-Bigbee et al., LPA2 and LPA3 are coupled to ERK1/2 less efficiently than 1999; Kim et al., 2007; Daniel et al., 2014; Pardo et al., 2016). Elk-1 LPA1. With regard to the stimulatory effects of amitriptyline is a transcription factor that upon phosphorylation by ERK1/2 and mianserin, the selective involvement of LPA1 is consistent translocates to the nucleus, where it regulates immediate early with previous observations that in assays of receptor-b gene expression, neuronal differentiation, and cytoskeleton arrestin interaction both antidepressants failed to show dynamics by binding to the serum response element (Besnard agonist activity in CHO-K1 cells overexpressing either LPA2 et al., 2011). There is evidence that, like CREB, Elk-1 activation or LPA3 (Olianas et al., 2015). induces growth factor expression and antiapoptotic effects (Shin The present study also provides evidence that in C6 glioma et al., 2009; Demir et al., 2011). Thus, the present study suggests cells and primary astrocytes LPA1 mediates antidepressant- that LPA1 may play a role in mediating antidepressant-induced induced signaling events occurring downstream of ERK1/2. prosurvival signaling via CREB and Elk-1 activation. Antidepressant Signaling through LPA1 in Glial Cells 351 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 9. Mianserin (Mians) protects glial cells from oxidative stress–induced apoptosis through LPA1. C6 glioma cells (A) and rat cortical astrocytes (B) were pretreated for 24 hours in serum-free medium with either vehicle or 5 mM mianserin. Thereafter, C6 glioma cells and astrocytes were exposed for 24 hours to 300 and 100 mMH2O2, respectively. Cells positive for cleaved caspase 3 immunofluorescence (green color) were identified, and their number was expressed as a percentage of the total number of cells present in each field examined. Cell nuclei are stained in blue with DAPI. Values are the mean 6 SEM of four independent experiments. Scale bar, 50 mm. (C) C6 glioma cells were pretreated for 10 minutes with either vehicle or 100 nM AM966, and then exposed for 24 hours in serum-free medium to either vehicle or 5 mM mianserin. Cells were then incubated in the absence and in the presence of 300 mMH2O2 for 24 hours. Cell extracts were analyzed for cleaved PARP (cleav PARP) and PARP by Western blot. Values are the mean 6 SEM of four experiments. (D) C6 glioma cells were treated as indicated in (C), and cell viability was determined by cytofluorimetric analysis using the Muse Cell Count and Viability kit. Values are expressed as a percentage of control (vehicle + vehicle) and are the mean 6 SEM of four experiments. (E) Astrocytes were pretreated as indicated in (B) and then incubated for 24 hours with either vehicle or 100 mMH2O2. The number of nuclei displaying DNA fragmentation by in situ TUNEL assay (green color) was determined in each field and reported as the percentage of total nuclei (stained in blue with DAPI). Values are the mean 6 SEM of four experiments. (F) Serum-starved rat astrocytes were pretreated for 10 minutes with either vehicle or 100 nM AM966, and then exposed for 18 hours to either vehicle or 5 mM mianserin. Cells were then incubated in the absence and in the presence of freshly prepared 200 mMH2O2 for 3 hours. Cell viability was determined by the MTT assay. Values are expressed as a percentage of control (vehicle + vehicle) and are the mean 6 SEM of four experiments. ***P , 0.001 vs. control (vehicle); #P , 0.05, ###P , 0.001 by ANOVA followed by Newman-Keuls post hoc test.

In agreement with our previous studies (Olianas et al., ERK1/2 phosphorylation by 5HT. Although this observation 2012), in C6 glioma cells and primary astrocytes ERK1/2 is in line with the finding that astrocytes express 5HT2 stimulation elicited by mianserin and other antidepres- receptors coupled to ERK1/2 via PTX-insensitive G proteins sants, including amitriptyline, imipramine, desipramine, of the Gq/11 type (Li et al., 2008), it also indicates that in C6 and mirtazapine, was suppressed by cell treatment with glioma cells 5HT receptors are unlikely to participate in PTX, implying the involvement of the Gi/o family of G ERK1/2 stimulation by the tricyclic and tetracyclic antide- proteins. This finding is also consistent with the observation pressants examined. that in different cell types LPA1 induces ERK1/2 activity in a In C6 glioma cells, the signaling cascade linking PTX-sensitive manner (Ishii et al., 2004) and further antidepressant-induced LPA1 activation to stimulation of supports the participation of Gi/o-coupled LPA1 in the ERK1/2andAktsignalingappearedtoinvolvethetrans- antidepressant actions in glial cells. PTX treatment failed activation of both FGF-R and PDGF-R. Thus, the phos- to prevent ERK1/2 phosphorylation by FGF-2, indicating phorylation of ERK1/2 and Akt induced by amitriptyline that this response was independent of Gi/o activation. More- and mianserin was diminished by either PD173074 or over, under similar experimental conditions, treatment of tyrphostin AG1296, used at a concentration that selec- C6 glioma cells with PTX did not prevent the stimulation of tively inhibited the tyrosine kinase activity of FGF-R and 352 Olianas et al.

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LPA1 mediates antidepressant-induced ERK1/2 signalling and protection from

oxidative stress in glial cells

Maria C. Olianas, Simona Dedoni, Pierluigi Onali

The Journal of Pharmacology and Experimental Therapeutics

Supplemental Figure 1. Blockade of amitriptyline- and mianserin-stimulated ERK1/2 phosphorylation by the combination of Ki16425 and NBFI. C6 glioma cells were preincubated for 10 min with either vehicle, 100 nM Ki16425 (Ki), 100 nM NBFI or the combination of Ki16425 and NBFI and then exposed for 10 min to either vehicle or 15

µM amitriptyline (Amitript) (A) or 5 µM mianserin (Mians).(B). Cell extracts were then analyzed for phospho-ERK1/2 and total ERK1/2. Values are the mean ± SEM of four experiments. *** P < 0.001 vs control (vehicle + vehicle); # P < 0.05, ### P < 0.001 by

ANOVA followed by Newman-Keuls post hoc test.