Haloperidol induces neurotoxicity by the NMDA receptor downstream signaling pathway, alternative from glutamate excitotoxicity

Neurochemistry International 50 (2007) 976–982 www.elsevier.com/locate/neuint

Haloperidol induces neurotoxicity by the NMDA receptor downstream signaling pathway, alternative from glutamate excitotoxicity E. Zhuravliova, T. Barbakadze, N. Natsvlishvili, D.G. Mikeladze * Laboratory of Neurochemistry, Institute of Physiology 14 Gotua Street, Tbilisi 0160, Georgia Received 22 July 2006; received in revised form 19 September 2006; accepted 22 September 2006 Available online 7 November 2006

Abstract The NMDA receptor is believed to be important in a wide range of nervous system functions including neuronal migration, synapse formation, learning and memory. In addition, it is involved in excitotoxic neuronal cell death that occurs in a variety of acute and chronic neurological disorders. Besides of agonist/coagonist sites, other modulator sites, including butyrophenone site may regulate the N-methyl-D-aspartate receptor. It has been shown that haloperidol, an antipsychotic neuroleptic drug, interacts with the NR2B subunit of NMDA receptor and inhibits NMDA response in neuronal cells. We found that NMDA receptor was co-immunoprecipitated by anti-Ras antibody and this complex, beside NR2 subunit of NMDA receptor contained haloperidol-binding proteins, nNOS and Ras-GRF. Furthermore, we have shown that haloperidol induces neurotoxicity of neuronal cells via NMDA receptor complex, accompanied by dissociation of Ras-GRF from membranes and activation of c-Jun-kinase. Inclusion of insulin prevented relocalization of Ras-GRF and subsequent neuronal death. Haloperidol-induced dissociation of RasGRF leads to inhibition of membrane-bound form of Ras protein and changes downstream regulators activity that results in the initiation of the apoptotic processes via the mitochondrial way. Our results suggest that haloperidol induces neuronal cell death by the interaction with NMDA receptor, but through the alternative from glutamate excitotoxicity signaling pathway. # 2006 Elsevier Ltd. All rights reserved. Keywords: Haloperidol; Ras-GRF; NMDA receptor; Neurotoxicity

1. Introduction The NMDA receptor (NMDAR) is believed to be important in a wide range of nervous system functions including neuronal migration, synapse formation, learning and memory. The opening of NMDA receptors leads to an influx of cations including Ca2+, which initiates signal transduction cascades that in turn modulate synaptic strength. In addition, NMDAR is involved in excitotoxic neuronal cell death that occurs in a variety of acute and chronic neurological disorders (Waxman and Lynch, 2005). Among the many regulatory proteins activated by calcium entering cells through NMDARs are RasGTPases (H-, N- and K-Ras) (Sheng and Kim, 2002), which can be activated by a wide array of upstream signals, including RasGRF proteins. Ras-GRF is the representative of guanine nucleotide exchange factors (GEFs) specific for the Ras family

* Corresponding author. Tel.: +7 995 32 998571/374724; fax: +7 995 32 998571. E-mail address: [email protected] (D.G. Mikeladze). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.09.015

of small GTP-binding proteins, which initiate the nucleotide replacement cycle by catalyzing the exchange of GDP for GTP. Highly similar p140 Ras-GRF1 and p130 Ras-GRF2 proteins contain calmodulin-binding domains that promote activation of the Ras-GRFs in response to elevated calcium levels in cells (Farnsworth et al., 1995; Fam et al., 1997). There is much evidence implicating a critical role of Ras-GRF-MAPKdependent signaling pathway in synaptic plasticity, cognitive function of brain, long-term memory and emotional learning (Orban et al., 1999; Impey et al., 1999). The primary structure of Ras-GRF reveals the presence of a number of regulatory motifs presumably involved in diverse signaling control mechanisms and protein–protein interactions. It has been shown that NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts with Ras-GRF1 (Krapivinski et al., 2003), suggesting that besides Ca2+ Ras-GRF can be directly activated by NMDAR. It has been found that haloperidol, a therapeutically useful antipsychotic drug, inhibits neuronal NMDA responses and has neuroprotective effects against NMDA-induced neurotoxicity (Ilyin et al., 1996; Nishikawa et al., 2000). Results from

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Whittemore et al. (1997) suggest that a non-competitive allosteric modulator site is expressed by isoforms of the receptor containing the NR1/NR2B subunit mediates haloperidol’s action on NMDA receptor. The ligand binding experiments (Coughenour and Cordon, 1997), as well as point mutation studies (Brimecombe et al., 1998) showed that haloperidol interacts with polyamine/ifenprodil sensitive modulatory sites of the NR2B subunits. Inhibitory effects of haloperidol correlate with developmental changes in NMDA receptor composition, such that NMDA receptor activity in immature neurons, which that express mostly NR1/NR2B-containing receptors, is completely inhibited by these antagonists (Sinor et al., 2000; Lynch and Guttmann, 2002). In neurons with preferentially expressed NR1/ NR2B receptors haloperidol effectively block glutamate toxicity (Sinor et al., 2000). However, there are some evidence that haloperidol induces oxidative toxicity of neuronal cells by activation of NF-kB (Post et al., 1998) and apoptosis through p38 mitogen-activated protein kinase/c-Jun-NH2-terminal protein kinase pathway (Noh et al., 2000). Oxidative toxicity of haloperidol has emerged as pathogenic events of extrapyramidal side effects including tardive diskinesia, which imposes the major limitation on the use of this class of drugs (Marsden and Jenner, 1980; Diederich and Goctz, 1998). Nevertheless, the molecular pathways leading to haloperidol-induced cell death and mechanisms implying in attenuation of NMDAR-mediated neurotoxicity have not been differentiated yet and need further elucidation. It is clear that both neuroprotective and neurotoxic effects of haloperidol depend on cellular localization and the abundance of haloperidol sensitive NR2B subunits that transmit the extracellular signals through various downstream effectors. In this regard, most important targets of haloperidol may be proteins that directly interact with NR2B subunits (Krapivinski et al., 2003) and transmit extracellular signals by allosterically regulated protein–protein interactions. In this study, we have demonstrated that haloperidol induces neuronal cells death via NMDA receptor and dissociation of membrane-bound Ras-GRF. 2. Materials and methods 2.1. Cerebrocortical cultures Primary cortical cultures of mixed neurons and glia were derived from newborn rats (Wistar). Briefly, following dissociation in 0.027% trypsin, cerebral cortical cells were plated either 96-well multiwell plates or 35-mm dishes that had been coated previously overnight with 15 mg/ml poly-L-lysine and then with DMEM (Sigma, USA) culture medium supplemented with 10% fetal bovine serum. After removal of final coating solution, cells were seeded (106 ml1) in a serum free medium composed of a mixture of DMEM, contained 40 mg/ml gentamycin, and 60 mg/ml penicillin. Cells were cultured at 37 8C in a humidified atmosphere of 95% air and 5% CO2 and were used after 8–10 days.

2.2. MTT reduction cell viability assay Experiments were performed using glia/neuron cultured in 96-well plates. The effects of 1 mM glutamate, 10 mM MK-801, 10 mM haloperidol, 10 mM DTG and 100 ng/ml insulin on cell viability was assessed by a colorimetric assay based on the cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into a blue-colored formazan product by mitochondrial

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succinate dehydrogenase (Abe and Matsuki, 2000). Additions were made directly to the glia/neuron culture medium for 24 h. Cells then were washed twice with HEPES-buffered incubation medium (HBM) (140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.1 mM MgCl2, 1.2 CaCl2, 5.5 mM glucose and 20 mM HEPES, pH 7.4.) and incubated for 45 min at 37 8C in HBM containing MTT (0.5 mg/ml). After this period, the HBM was carefully removed, and the blue formazan product was solubilized in 300 ml of 100% dimethyl sulfoxide. The absorbance of each well was read at 570 nm.

2.3. LDH cell viability assay Cell death was estimated by the measurement of lactate dehydrogenase (LDH) (Miyamoto et al., 1989) released into the medium by dead or damaged cells after 24-h treatment with glutamate, haloperidol or other drugs. Briefly, LDH activity was quantified by the NADH oxidation rate, which was followed spectrophotometrically at 340 nm. Total LDH was estimated after the lysis of cells in the buffer containing Triton X-100 (1% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM dithiothreitol [DTT], 1 mM NaH2PO4, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 50 mM HEPES buffer, pH 7.5). The amount of LDH after each treatment, expressed as a percentage of the total LDH, reflects the percentage of dead or damaged cells.

2.4. Solubilization and purification of NMDAR complexes Membrane preparations from rat cortex or hippocampus was obtained after tissue homogenization in 20 vol ice-cold 0.32 M sucrose, containing 0.1 M PMSF, 3 mM EDTA, 5 U/ml aprotinin and 5 mg/ml pepstatin A. The homogenate was centrifuged at 1000  g for 10 min, the pellet was removed, and the supernatant was centrifuged at 20,000  g for 20 min. The pellet was resuspended in 20 mM Tris–HCl, pH 7.4 buffer, containing 0.1 mM PMSF, 2 mM EDTA, 5 mg/ml pepstatin and 5 U/ml aprotinin (buffer A) to yield a suspension of 5 mg protein/ml and frozen at 408 until use. Solubilization was performed by 1% sodium deoxycholate at detergent/protein ratio = 4/1 (mg/mg) in buffer A during 1 h at 4 8C and then centrifugation at 100,000  g for 1 h was followed. Final supernatant was dialyzed against 20 mM Tris–HCl (pH 7.4) containing 2 mM EDTA and 0.1 mM PMSF (buffer B) and was applied on dextrorphan-Sepharose column (1 cm  10 cm), which was pre-equilibrated by buffer A. The column was washed with 40 ml buffer A and matrix-binding proteins were eluted by 100 mM dextrorphan in buffer A. The eluates were dialyzed against buffer B and their binding activity was determined. Binding of dextrorphan to Sepharose 4B was carried out by Egly and Porath (1979).

2.5. Binding experiments [3H]Gpp(NH)p binding to solubilized or affinity-purified preparations were determined in buffer A, containing 200–300 mg/ml protein and 5 nM [3H]Gpp(NH)p. Non-specific binding was calculated after addition 0.5 mM of Gpp(NH)p into the medium. The incubation was carried out during 1 h at 4 8C and the mixture was filtered through Whatman GF/B filters pretreated with 0.05 polyethylenimine. Radioactivity retained on the filter was determined by liquid scintillation spectrophotometer.

2.6. Immunoprecipitation and immunoblotting After the treatment regiment, the medium was firs removed, the attached cells were treated by buffer containing 150 mM NaCl, 5 mM MgCl2, 1 mM PMSF, 1 mM DTT, 1 mM sodium phosphate, 50 mM HEPES and 0.05% SDSNa, pH 7.5 (buffer A) and incubated for 30 min at 4 8C. Lysed cells were centrifuged at 3000  g for 30 min and sediment proteins extracted overnight at 4 8C by 1% sodium deoxycholate in buffer A. The deoxycholate extract was centrifuged (32,000  g, 60 min, and 4 8C) and supernatant was incubated with 1–2 mg correspondence antibodies for 60 min at 4 8C. Protein A/G-agarose (25 ml) was added and the incubation continued for 2 h. Samples were centrifuged at 2500  g and the pellets were washed three times with 50 mM HEPES buffer, pH 7.5. Bound proteins were eluted by adding SDSPAGE sample buffer (20 ml) and boiled at 100 8C. Samples were then centrifuged and supernatants were used for immunoblotting.

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Polyclonal antibodies against the NMDA receptor e2 and e1 subunits, PSD95, NOS1 and Ras-GRF1 were obtained from Santa Cruz Biotechnology (Santa Cruz, USA). The samples were separated by SDS-polyacrylamide gel electrophoresis 15% gels and transferred to nitrocellulose sheets. After blocking with 5% bovine serum albumin and 0.05% Tween 20 in Tris–HCl-buffered saline, the sheets were incubated with primary antibodies in the blocking solution. Labeled bands were visualized using enhanced chemiluminescence (Amersham). The bands were analyzed by densitometric scanning. The amounts of the proteins were quantified from the intensity of the bands, which has linearity to the amounts of the samples applied to the gel.

2.7. Measurements of farnesylated Ras The amount of farnesylated Ras was determined according to the method of Goalstone et al. (1998) with slight modification. Briefly, glutamate or drugtreated neuronal/glial cells were lysed in 0.5 ml buffer containing 150 mM NaCl, 5 mM MgCl2, 1 mM PMSF, 1 mM DTT, 1 mM sodium vanadate, 1 mM sodium phosphate, 1% Triton X-100, 0.05% SDS, 10 mg/ml leupeptin, and 50 mM HEPES, pH 7.5. Crude lysates were sonicated and centrifuged at 10,000 rpm. Total protein from the supernatant was determined by the bicinchoninic acid assay (Pierce Biotechnology) and diluted to 1 mg/ml. Equal volumes of lysate and 4% Triton X-114 were combined, vortexed, and incubated at 37 8C for 3 min. Solutions were kept at room temperature until phases had separated. The lower lipid phase containing membrane-bound proteins was incubated with anti-Ras antibodies (H-Ras; Santa Cruz Biotechnology) and immunoprecipitated using Protein A/G-agarose. Samples were centrifuged at 2,500 rpm and pellets were washed three times with 50 mM HEPES buffer, pH 7.5. Bound proteins were eluted by adding SDS-PAGE sample buffer (20 ml) and boiled at 100 8C. Samples were then centrifuged and supernatants were used for immunoblotting.

2.8. Assay for NO synthesis Synthesis of NO was determined by assay of culture supernatants for nitrite (Kolker et al., 2001). Briefly, 300 ml of culture supernatant was allowed to react with 100 ml of 10% Griess reagent (Green et al., 1982) and 2,6 ml deionized water, and incubated at room temperature for 30 min. The optical density of the assays samples was measured spectrophotometrically at 548 nm. Fresh culture medium served as the blank in all experiments. Protein concentration was determined using a dye-binding method (BioRad). The data were treated by one-way ANOVA analysis. The data from each experiment were analyzed separately. Where a significant effect was observed in the ANOVA analysis, comparisons of those samples were made by the t-test.

Table 1 Cell viability after treatment of cells by glutamate, MK-801, haloperidol, DTG and insulin Reagent

Control 1 mM glutamate 10 mM haloperidol 10 mM haloperidol + 1 mM glutamate 10 mM haloperidol + 10 mM MK-801 10 mM haloperidol + 10 mM DTG 10 mM haloperidol + 100 ng/ml insulin

Viability (%) MTT-test

LDH-test

100 71.5  4.7 72.6  5.3 52.4  3.2 69.7  4.1 66.4  3.4 98.4  5.8

100 69.9  6.7 62.7  4.9 43.6  4.8 73.5  5.5 67.1  5.9 97.3  4.7

Cell viability after exposure to haloperidol, glutamate, sigma ligands or insulin for 24 h as assessed by the MTT and LDH tests. All data were normalized to control value (untreated or only preincubated cells). The data are presented as the means  S.E.M. for triplicate determination.

reduces cell survival and preincubation with 1 mM glutamate increases the toxic effects. The action of haloperidol was not eliminated neither by MK-801, nor sigma2 antagonist DTG, suggesting that pro-apoptogenic effect of haloperidol does not mediated by sigma-sites, or by channel proteins of NMDAR. In the next series of experiments, cytochrome c release from mitochondria, as the first indicator of mitochondrial ways of apoptosis was determined after haloperidol and glutamate treatments (Fig. 1). We found that both glutamate and haloperidol induce translocation of cytochrome c from mitochondria into cytosol, implying that pore-forming voltage-dependent anion channel (VDAC) open in both cases. Since NMDA receptor activation and elevation of [Ca2+], lead to increased generation of nitric oxide, we examined the effect of haloperidol on NO production. As shown in Table 2 treatment of cell by glutamate increases the production of NO. However, surprisingly addition of haloperidol to the cells reduces the glutamate-induced production of nitric oxide.

3. Results 3.1. Effects of haloperidol on the viability and production of nitric oxide in primary cortical culture cells Haloperidol is cytotoxic to neurons in a concentration dependent manner and causes cell death by oxidative stress and apoptosis (Post et al., 1998; Ukai et al., 2004). The precise mechanisms of the neuronal toxicity induced by haloperidol, as well as a target molecule for cellular action of haloperidol are poorly understood. Protein phosphatases (Gong et al., 1996), sigma2 receptors (Wei et al., 2006), mitochondrial electron transfer systems (Modica-Napolitano et al., 2003) may be candidates for effects of typical neuroleptics, including haloperidol. Thus, at first the effects of haloperidol on cell apoptosis were examined. To quantify the cell viability MTT assays and lactic dehydrogenase (LDH) test were used. As shown in Table 1, addition of 10 mM haloperidol to the cells

Fig. 1. Effect of glutamate, haloperidol and insulin on the levels of cytoplasmic cytochrome c, farnesylated Ras, membrane-bound Ras-GRF and phospho-JNK in primary neuronal/glial cells. Cells were incubated with 1mM glutamate or 10 mM haloperidol, or coincubated with 10 mM haloperidol and 100 ng/ml insulin for 18 h. Cell extracts were analyzed by Western blotting using the monoclonal anti-cytochrome c, anti-Ras, anti-Ras-GRF and anti-phospho-JNK antibodies. Farnesylated Ras from cell extract were isolated from cell extracts as described in Section 2. Blot is representative of two similar experiments.

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Table 2 NO production after treatment of cells by glutamate, haloperidol, MK-801 and DTG Reagent

Nitrite (nmol/ [mg 24 h])

Control 1 mM glutamate 10 mM haloperidol 10 mM haloperidol + 1 mM glutamate 10 mM haloperidol + 10 mM MK-801 10 mM haloperidol + 10 mM DTG

4.3  0.6 12.8  1.4 1.7  0.3 8.5  1.7 3.8  1.4 3.5  1.3

Synthesis of NO was determined after exposure of cells to haloperidol, glutamate or sigma ligands for 24 h as described in Section 2. All data were normalized to control value (untreated or only preincubated cells). The data are presented as the means  S.E.M. for triplicate determination.

Moreover, in the presence of haloperidol endogenous production of NO was reduced also. Thus, the cytotoxic effect of haloperidol is not mediated by activation of NO-synthase and correspondingly by increase of [Ca2+], suggesting that this process differs from glutamate-induced cell death. 3.2. Purification and co-immunoprecipitation of NR2A/ NR2B subunits of NMDA receptor by anti-Ras In recent years it has become clear that many neuronal modulator mechanisms may be co-coordinated by a group of binding proteins that both cluster NMDA receptors and link them to signaling pathways within the cell. The possible proteins participating in the formation of the macromolecular signaling complexes with NMDA receptors may be small Gproteins, which transmit signals to downstream effectors and play crucial roles in gene regulation. It has been shown that NR2B subunit of NMDA receptors directly interacts with RasGRF1 that promotes the release of GDP bound to Ras, allowing activation of downstream target proteins (Krapivinski et al., 2003). Correspondingly, Ras may be one of the most important targets allosterically regulated by various NMDA agonist/ antagonists including haloperidol. Haloperidol acts as selective allosteric modulator of NR2B subunit of NMDA receptors and block voltage-dependent channel (Lynch and Guttmann, 2002; Waxman and Lynch, 2005). Since protein–protein interactions appear to be an important determinant of specificity in signaling by neurotransmitter receptors, we investigated the cooperation between haloperidol- and GTP-binding sites in the partially purified NMDA receptors preparations. For purification of supramolecular complex of NMDA receptors the solubilization and affinity chromatography on dextrorphan-Sepharose was performed (see Section 2). Using [3]H-50 -guanylylimidodiphosphate (Gpp(NH)p), the pharmacological specificities of affinity-purified preparation were studied by competition binding experiments. We found that out of the series of NMDA antagonists tested, haloperidol is significantly more potent at decreasing [3H]Gpp(NH)p binding (IC50 = 12.5 nM). Phencyclidine (PCP), MK-801((+)-5methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10imine) and dextrorphan were less potent than haloperidol (Fig. 2). These data confirm the suggestion that the action of

Fig. 2. Inhibition of 5 nM [3H]GppNHp binding by MK-801 (*), pentazocine (!), SKF-10 047 (~) and haloperidol (&) in affinity-purified preparation of NMDA receptor. In the absence of haloperidol, 100% binding corresponds to the specific binding of the control. Binding was carried out as described in Section 2. The curve is a representative of three independent experiments. Specific binding in the absence of haloperidol for [3H]GppNHp was 2773  248 (dpm  S.E.M.).

haloperidol on the binding of guanine nucleotide is specific and not mediated by channel sites of NMDA receptor. For identification of proteins in the supramolecular complex of NMDA receptor, affinity-purified preparation was immunoprecipitated by anti-Ras and analyzed by Western blot. It was found that only the e2 (NR2A/NR2B) subunits of the NMDA receptors were present in immunoprecipitated preparations. In addition, it was revealed that this preparation contained RasGRF and nNOS and did not contain the e1 subunit of the NMDA receptor and PSD-95 (Fig. 3). Thus, it is possible to conclude that the supramolecular complex obtained after affinity chromatography contains the NR2A/NR2B subunits of the NMDA receptor, Ras and Ras-GRF (nNOS) and this

Fig. 3. Co-immunoprecipitation of NR2A/NR2B subunits of NMDA receptor and nNOS by anti-Ras. Affinity-purified preparation of NMDAR is precipitated using the Ras antibody, separated on SDS-PAGE, and analyzed using immunoblots probed with nNOS, NR2A/NR2B subunits and PSD95 antibodies as described in Section 2. Lines 1 and 2—nNOS in the preparation; lines 3 and 4— NR2A/NR2B in the preparation; line 5—PSD-95. The blot is a representative of three independent experiments.

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macromolecular complex is sensitive to haloperidol. These data indicate that binding of haloperidol to the butyrophenone sites of NMDA receptor may induce reduction of GTP-binding activity through Ras-GRF, presumably via direct allosteric protein–protein interaction. 3.3. Effect of haloperidol on the content of Ras-GRF and farnesylated Ras in the cells of rat primary cortical structure Ras-p140 GRF1 and p130 Ras-GRF2 are the predominant mediators of NMDAR activation of the Ras signaling pathway and inhibition of NMDAR by haloperidol should change the properties of transmitted signal. There is increasing evidence that the activity and subcellular localization of GEFs are highly regulated. Several GEFs are stimulated by protein–protein interactions or by phosphorylation and in many cases, GEF activation seems to be linked with relocalization (Schmidt and Hall, 2002). Moreover, it has been found that the specific lipid moiety attached to Ras can contribute to signaling specificity of GEFs (Gotoh et al., 2001). Thus, in the next series of experiments, the content of membrane-bound Ras-GRF and farnesylation states of Ras were examined in mixed neuronal/ glial cells after haloperidol treatment. Western blot analysis of cell lysates revealed that haloperidol decreased the content of membrane-bound Ras-GRF, whereas the levels of farnesylated Ras did not change (Fig. 1). These findings indicate that haloperidol promote the dissociation of Ras-GRF from target proteins, localized apparently in the supramolecular clusters of NMDA receptor. c-Jun-activated kinase (JNK) is implicated in neuronal apoptosis. This protein kinase was activated following exposure of cortical neurons to haloperidol and inclusion of insulin prevented neuronal death (Noh et al., 2000). To identify downstream effectors of Ras-GRF and to confirm that the JNK signaling pathway could be participated in this process we examined the effects of haloperidol and insulin on the activity of JNK and distribution of Ras-GRF. We found that the levels of phosphorylated JNK significantly increased in the presence of haloperidol (Fig. 1). However, in insulin-treated cells haloperidol does not change the viability of cells, or the content of phosphorylated JNK (Fig. 1). Accordingly, in presence of insulin the content of membrane-bound Ras-GRF was not altered by haloperidol treatment. These findings suggest a significant role for Ras-GRF signaling pathway in haloperidol-treated cells and its relevance to the regulation of JNK activity. 4. Discussion The putative mechanism of action of antipsychotic drugs (APDs) used in the treatment of schizophrenia has been extensively investigated. Haloperidol is typical APDs that improve positive symptoms of schizophrenia, e.g., hallucinations, but have a high propensity to cause various extrapyramidal side effects. There is a large body of evidence that haloperidol induces apoptosis by reducing cellular survival

signaling, which possibly contributes to the differential clinical therapeutic efficacy and expression of side effects in schizophrenia (Marsden and Jenner, 1980; Diederich and Goctz, 1998). Protein phosphatases (Gong et al., 1996), sigma2 receptors (Wei et al., 2006), mitochondrial electron transfer systems (Modica-Napolitano et al., 2003) may be candidates for the effect of typical APDs, however, the precise mechanisms of the neuronal toxicity induced by haloperidol, as well as a target molecule for cellular action of haloperidol are poorly understood. On the other hand, haloperidol acts as allosteric modulator of NMDAR-channel, reducing excitotoxic injury of neurons. In neurons with preferentially expressed NR1/NR2B receptors haloperidol effectively blocked glutamate toxicity (Sinor et al., 2000) and inhibitory effects of haloperidol correlate with developmental changes in NMDA receptor composition (Sinor et al., 2000; Lynch and Guttmann, 2002). Thus, the molecular pathways that lead to the cell death induced by haloperidol and mechanisms that imply in attenuation of NMDA-mediated neurotoxicity are not differentiated and need further elucidation. We found that in our experimental conditions with immature primary neuronal/glial cells haloperidol as well as glutamate induces apoptosis. However, in contrast to glutamate haloperidol mediates cell death that does not depend on the calcium influx and subsequent NO synthesis. Moreover, addition only haloperidol to the cells reduces the endogenous production of NO. Nevertheless, both compounds induce translocation of cytochrome c from mitochondria suggesting that pore-forming voltage-dependent anion channel open in both cases. These data suggest that glutamate-mediated apoptosis differ from haloperidol-induced cell death and distinct ways may involve in these processes. NMDA receptors are heteromeric receptors, composed of NR1 subunits in combination with NR2 subunits. Both NR1 and NR2 subunits are usually required to create a functional receptor, which most likely contains two NR1 subunits and two NR2 subunits (Furukawa et al., 2005). Both subunits have binding sites on their C-termini for a number of proteins, such as SAP-102, PSD-95, SynGAP, Src, PKA, calmodulin, aactinin and a variety of other proteins, including Ras-GRF (Sheng and Kim, 2002; Krapivinski et al., 2003). Ras-GRF1 and Ras-GRF2 are the predominant mediators of NMDAR activation of the Ras/Erk signaling pathway and maintain CREB activity in neurons from adult, but not neonatal animals, where Sos proteins function in this role instead (Tian et al., 2004). These findings imply that Ras-GRF1 and Ras-GRF2 play redundant roles in coupling NMDARs to Ras/Erk and CREB signaling, which is consistent with the fact that most CNS neurons contain both proteins. It is interesting to note that both Ras-GRF family members normally play a protective role in stroke-induced neuronal damage (Tian et al., 2004). We purified NMDA receptor from bovine brain by dextrorphan-Sepharose and found that besides haloperidolbinding NR2B subunits receptor complex contain GTP-binding proteins, apparently Ras. Using immunoprecipitation technique, we found that in addition to Ras this complex contain the Ras-GRF1 suggesting on direct interaction of Ras-GRF with

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NR2B. This observation agrees with the findings of Krapivinski et al. (2003), showing that Ras-GRF1 specifically binds only the NR2B subunit of NMDAR. Besides, our results indicate that occupation of modulatory sites of NMDAR by haloperidol reduces Ras guanine nucleotides exchange activity. Such negative cooperation may be result of dissociation of Ras-GRF from supramolecular complexes of NMDAR. Taking into account that Ras provides protection from apoptosis by attenuation of Jun N-terminal protein kinase (JNK) activity (Wolfman et al., 2002), these data suggest that Ras-GRF provides a major contribution against haloperidol-induced apoptosis. The JNK signaling pathway is implicated in neuronal apoptosis, which is strongly linked to the mitochondrial dysfunctions. The JNK-mediated mitochondrial pathology was reported following growth factor withdrawal in cerebellar granule cells (Whitfield et al., 2001; Harris and Johnson, 2001; Donovan et al., 2002), in response to various drugs including haloperidol. (Noh et al., 2000; Eminel et al., 2004). Apoptosis induced by withdrawal of trophic support and glutamate are mechanistically different. Caspase-independent apoptosis induced by glutamate is accompanied by strong activation of p38, whereas withdrawal of trophic support induces caspasedependent death accompanied by JNK-dependent phosphorylation of c-Jun (Cao et al., 2004). Our results agree with these observations, since in contrast to glutamate haloperidol activates phosphorylation of JNK and induce JNK-dependent translocation of cytochrome c from mitochondria. All together suggest that the Ras-GRF signaling pathway inhibited after treatment by haloperidol and activating JNK is likely related to the molecular machinery preceding apoptotic cell death. The antiapoptotic action of insulin has been demonstrated in several studies (Estevez et al., 1995; McDonald et al., 1996). It has been suggested that insulin and insulin-like factor have a pro-survival as well as a neuromodulatory function by acting on the PI3K/Akt pathway, linking survival directly to NMDAR and synaptic plasticity (van der Heide et al., 2005). Our data show that in presence of insulin, the viability of cells was increased and haloperidol does not affect the membrane localization of the Ras-GRF. The precise mechanism of action of insulin on the translocation/relocalization of the membraneassociated Ras-GRF remains unclear. Additional studies will be needed to determine insulin’s action, but is likely that Ras-GRF is recruited to the membranes in response to cellular activation by insulin receptors. There are some evidence, that Ras-GRF requires its DH domain to translocate to the membrane, to stimulate exchange on Ras, and to activate mitogen-activated protein kinase (MAPK) and these processes are regulated by the Rho family GTPases (Arozarena et al., 2000). Because insulin receptor substrate 53 (IRS53) links activated Rac1/Cdc42 to the regulation of the synaptic morphogenesis (Choi et al., 2005), it may be conclude that activation of insulin receptors leads to relocalization of Ras-GRF through IRS53 protein. Several GEFs are stimulated by protein–protein interactions or by phosphorylation. The structural domains of RasGRF1 include two pleckstrin homology (PH) domains, a coiled-coil motif (CC), an IQ motif, a Dbl homology domain (DH), a Ras

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exchange motif/PEST motif and a C-terminal CDC25 homology domain that is required for guanine exchange toward Ras (Schmidt and Hall, 2002). Pleckstrin homology domains interact with phosphoinositides in the lipid membrane (Kavran et al., 1998), can act as phosphotyrosine-binding domains (Zhou et al., 1995), and have been shown to interact with other proteins, such as the G subunit (Inglese et al., 1995). PH domain has been suggested to mediate the translocation of GEFs to membranes and to cytoskeletal structures, where its GTPase substrates are located. Interestingly, Ras-GRF1 possesses a second (N-terminal) PH domain, and it is this, not the one adjacent to the DH domain, that is required for membrane localization (Michiels et al., 1997; Stam et al., 1997). All together suggests that the subcellular localization of GEFs is a key aspect of their activity, and, in many cases, GEF activation seems to be intimately linked with relocalization. This relocalization of GEFs may be important for NMDAR function, presumable because of impaired Ras-derived signaling pathways. Haloperidol-induced dissociation of Ras-GRF leads to inhibition of membrane-bound form of Ras and decreases downstream regulators activities that results in the initiation of the apoptotic processes via JNK activation and release of apoptogenic factors from mitochondria. Further studies are necessary to determine which pathway mediate RasGRF-derived pro-apoptotic signal, as well as the nature and significance of interaction of NMDAR-subunits with the RasGRF, remains to be clarified. References Abe, K., Matsuki, A., 2000. Measurement of cellular 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and LDH release using MTT. J. Neurosci. Res. 38, 325–329. Arozarena, I., Aaronson, D.S., Matallanas, D., Sanz, V., Ajenjo, N., Tenbaum, S.P., Teramoto, H., Ighishi, T., Zabala, J.C.J., Gutkind, J.S., Crespo, P., 2000. The Rho family GTPase Cdc42 regulates the activation of Ras/MAP kinase by the exchange factor Ras-GRF. J. Biol. Chem. 275 (34), 26441– 26448. Brimecombe, J.C., Gallagher, M.J., Lynch, D.R., Aizenman, E., 1998. An NR2B point mutation affecting haloperidol and CP101,606 sensitivity of single recombinant N-methyl-D-aspartate receptors. J. Pharmacol. Exp. Therap. 286, 627–634. Cao, J., Semenova, M.M., Solovyan, V.T., Han, J., Coffey, E.T., Courtney, M.J., 2004. Distinct requirements for p38 and c-Jun N-terminal kinase stressactivated protein kinases in different forms of apoptotic neuronal death. J. Biol. Chem. 279, 35903–35913. Choi, J., Ko, J., Racz, B., Burette, A., Lee, J.-R., Kim, S., Na, M., Lee, H.W., Kim, K., Weinberg, J., Kim, E., 2005. Regulation of dendritic spine morphogenesis by insulin receptor substrate 53, a downstream effector of Rac1 and Cdc42 small GTPases. J. Neurosci. 25 (4), 869–879. Coughenour, L., Cordon, J., 1997. Characterization of haloperidol and trifluperidol as subtype-selective N-methyl-D-aspartate receptor antagonists using [3H]TCP and [3H]Ifenprodil binding in rat brain membranes. J. Pharmacol. Exp. Therap. 280, 584–592. Diederich, N., Goctz, C., 1998. Drug-induced movement disorders. Neurol. Clin. 16, 125–139. Donovan, N., Becker, E.B., Konishi, Y., Bonni, A., 2002. JNK phosphorylation and activation of BAD couples the stress-activated signaling pathway to the cell death machinery. J. Biol. Chem. 277, 40944–40949. Egly, J.-M., Porath, J., 1979. Change-transfer and water mediated chromatography. Part II. Adsorbtion of nucleotides and related compounds in acriflavin–Sephadex. J. Chromatogr. A 168, 35–47.

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