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Inhibition of glial inflammatory activation and neurotoxicity by tricyclic antidepressants
Neuropharmacology 55 (2008) 826–834
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Inhibition of glial inflammatory activation and neurotoxicity by tricyclic antidepressants Jaegyu Hwang a, Long Tai Zheng a, Jiyeon Ock a, Maan Gee Lee a, Sang-Hyun Kim a, Ho-Won Lee b, Won-Ha Lee c, Hae-Chul Park d, Kyoungho Suk a, * a
Department of Pharmacology, School of Medicine, Brain Science and Engineering Institute, CMRI, Kyungpook National University, 101 Dong-In, Joong-gu, Daegu 700-422, Republic of Korea Department of Neurology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea c Department of Genetic Engineering, School of Life Sciences and Biotechnology, Kyungpook National University, Daegu, Republic of Korea d Department of Medical Science, Korea University Ansan Hospital, Ansan, Gyeonggi, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2008 Received in revised form 26 May 2008 Accepted 23 June 2008
Glial activation and neuroinflammatory processes play an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and HIV dementia. Activated glial cells can secrete various proinflammatory cytokines and neurotoxic mediators, which may contribute to neuronal cell death. Inhibition of glial activation may alleviate neurodegeneration under these conditions. In the present study, the antiinflammatory and neuroprotective effects of tricyclic antidepressants were investigated using cultured brain cells as a model. The results showed that clomipramine and imipramine significantly decreased the production of nitric oxide or tumor necrosis factor-alpha (TNF-a) in microglia and astrocyte cultures. Clomipramine and imipramine also attenuated the expression of inducible nitric oxide synthase and proinflammatory cytokines such as interleukin-1b and TNF-a at mRNA levels. In addition, clomipramine and imipramine inhibited IkB degradation, nuclear translocation of the p65 subunit of NF-kB, and phosphorylation of p38 mitogen-activated protein kinase in the lipopolysaccharide-stimulated microglia cells. Moreover, clomipramine and imipramine were neuroprotective as the drugs reduced microglia-mediated neuroblastoma cell death in a microglia/ neuron co-culture. Therefore, these results imply that clomipramine and imipramine have antiinflammatory and neuroprotective effects in the central nervous system by modulating glial activation. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Antidepressant Microglia Astrocyte Inflammation Neuroprotection
1. Introduction Neuroinflammation is actively involved in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and HIV-associated dementia (Block et al., 2007). Neuroglia such as microglia and astrocytes are non-neuronal cells that provide physical and nutritional support for neurons in the central nervous system (CNS). Microglia, which constitute about 10% of all glial cells, are the primary immune cells in the CNS, and they are also considered to be the major cell type responsible for inflammation-mediated neurotoxicity (Liu and Hong, 2003). Under the neurodegenerative condition, microglia cells can be activated by various neurotoxic factors produced by injured neuronal cells (Gao et al., 2003; McGeer and McGeer, 2003). Activation of microglia is also observed in response to brain injury
* Corresponding author. Tel.: þ82 53 420 4835; fax: þ82 53 256 1566. E-mail address: [email protected] (K. Suk). 0028-3908/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2008.06.045
and after the exposure to lipopolysaccharide (LPS), interferon (IFN)-g, or b-amyloid. Activated microglia have the capability of producing proinflammatory cytokines and neurotoxic mediators such as tumor necrosis factor (TNF)-a, prostaglandin (PG) E2, interleukin (IL)-1b, IL-6, and free radicals such as nitric oxide (NO) and superoxide anion (Giulian et al., 1994; Zielasek and Hartung, 1996). These proinflammatory cytokines and neurotoxic mediators are thought to contribute to neuronal injuries and progression of the neuroinflammatory diseases (Gonzalez-Scarano and Baltuch, 1999; Minghetti and Levi, 1998). Over-activation of astrocytes also facilitates ongoing neurodegeneration by producing various neurotoxic factors (Suk, 2005). Astrocytes are the most numerous glial cell types in the brain and play an important role in the homeostatic control of the neuronal extracellular environment (Chen and Swanson, 2003). Upon inflammatory stimulation, astrocytes proliferate and produce diverse intercellular mediators such as NO and TNF-a (Suk, 2005). There is growing evidence that inflammatory mediators produced by activated astrocytes may also be involved in the pathogenesis of various neurodegenerative
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diseases (Aschner, 1998). Taken together, as inflammatory activation of microglia and astrocytes is often observed in neuronal injuries and actively involved in the initiation and progression of several neurodegenerative diseases (Block et al., 2007; McGeer and McGeer, 2003, 2004; McGeer et al., 1988), inhibition of glial activation and subsequent neuroinflammation may be an effective therapeutic approach against neurodegenerative diseases. Antidepressants are widely used for treating major and minor depression. There are four general types of antidepressants including tricyclics, monoamine oxidase inhibitors, selective serotonin reuptake inhibitors (SSRI), and serotonin and noradrenaline reuptake inhibitors (Stahl, 1998). Clomipramine and imipramine belong to tricyclic antidepressants and have an antidepressant effect through inhibiting the reuptake of the neurotransmitters norepinephrine and serotonin by neurons (Wille et al., 2008). Recently, several studies have demonstrated that antidepressant drugs have an effect that is related to modulation of cell-mediated immune responses. Clomipramine and imipramine reduced TNF-a, IL-1b, and IL-6 production by LPS-stimulated human monocytes (Xia et al., 1996). These antidepressants also induced apoptosis of human lymphocytes (Xia et al., 1996), and decreased production of IL-2 and IFN-g in human T cells or monocytes (Xia et al., 1997). It has been also reported that chronic treatment of imipramine reduced lymphocyte proliferation and IL-1/IL-2 production by splenocytes from a chronic mild stress-induced depression model of rats (Kubera et al., 1996). Selective serotonin reuptake inhibitors such as escitalopram and citalopram also showed immunomodulating effects. Citalopram inhibited differentiation of human monocytes into macrophage-like cells in vitro (Ying et al., 2002). Recent studies have demonstrated that the antidepressant drugs amitriptyline, imipramine, clomipramine, trazodone, and fluoxetine have antiinflammatory effects in carageenan-induced paw edema model (Abdel-Salam et al., 2003). It was also reported that amitriptyline and its metabolite nortriptyline reduced IL-1b and TNF-a secretion in LPS-activated mixed glia and microglia cultures (Obuchowicz et al., 2006). Imipramine, fluvoxamine, and reboxetine inhibited IL-6 and NO production by IFN-g-stimulated microglia cells (Hashioka et al., 2007). Although previous studies have well demonstrated antiinflammatory effects of antidepressants in the peripheral system, only a few reports have addressed these effects in brain glia cells. Moreover, the detailed molecular mechanisms underlying the antiinflammatory effects of tricyclic antidepressants in the CNS are not completely understood. The effects of tricyclic antidepressant on glial neurotoxicity have not been evaluated either. Here, the effect of tricyclic antidepressants on the inflammatory activation and neurotoxicity of microglia, as well as astrocytes, has been determined. This study showed that clomipramine and imipramine inhibited LPS-induced production of NO, TNF-a, and their gene expression in microglia. Clomipramine and imipramine also suppressed NF-kB and p38 mitogen-activated protein kinase (MAPK) activation in LPS-stimulated microglia cells. The antiinflammatory effects of these antidepressants were also found in astrocytes and macrophages. In addition, these antidepressants showed neuroprotective effects by attenuating microglial neurotoxicity in a microglia/neuron co-culture system.
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37 C, 5% CO2. B35 rat neuroblastoma (ATCC, CRL-2754) (Schubert et al., 1974), HAPI rat microglia (Cheepsunthorn et al., 2001), and RAW 264.7 macrophage cell lines (ATCC, TBI-71) were grown and maintained in DMEM supplemented with 10% heatinactivated FBS, penicillin (10 U/ml) and streptomycin (10 mg/ml) at 37 C, 5% CO2. Mouse primary microglia and astrocytes cultures were prepared by mild trypsinization as described previously with minor modifications (Saura et al., 2003). In brief, the forebrains of newborn Institute of Cancer Research (ICR) mice were chopped and dissociated by mechanical disruption using a nylon mesh. The cells were seeded in poly-D-lysine-coated flasks. After in vitro culture for 10–14 days, microglia cells were isolated from mixed glia cultures by mild trypsinization. Mixed glia cultures were incubated with a trypsin solution [0.25% trypsin, 1 mM EDTA in Hank’s balanced salt solution (HBSS)] diluted 1:4 in PBS containing 1 mM CaCl2 for 30–60 min. This resulted in the detachment of an upper layer of astrocytes in one piece, whereas microglia remained attached to the bottom of the culture flask. The detached layer of astrocytes was aspirated, and the remaining microglia were used for experiments. The prepared primary microglia cultures were more than 95% pure, as determined by isolectin B4 staining (data not shown). Astrocytes were isolated by shaking mixed glia cultures at 250 rpm overnight and then the culture medium was discarded. The purity of astrocyte cultures was greater than 95% as determined by glial fibrillary acidic protein (GFAP) immunocytochemical staining (data not shown). Animals used in the current research were acquired and cared for in accordance with the guidelines published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study was approved by the Institutional Review Board of Kyungpook National University School of Medicine. 2.2. Cell viability test Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Microglia, astrocytes or RAW 264.7 macrophages were seeded in triplicate at the density of 8 104 cells/well on 96-well plate. The cells were treated with antidepressants and LPS for 24 h. MTT was added to each well, and incubated for 4 h at 37 C. After culture media were discarded, dimethyl sulfoxide (DMSO) was added to dissolve the formazan dye. The optical density was measured at 540 nm. The data are representative of results obtained from three independent experiments, which was performed in triplicate (mean S.D., n ¼ 3). 2.3. Nitrite quantification NO secreted in glial culture supernatants was measured by Griess reagent as described (Lee and Suk, 2007). After microglia, astrocytes, or RAW 264.7 macrophages were treated with stimulating agents in 96-well plates, NO 2 concentration in culture supernatants was measured to assess NO production. Fifty microliters of sample aliquots were mixed with 50 ml of Griess reagent (1% sulfanilamide/0.1% naphthylethylene diamine dihydrochloride/2% phosphoric acid) in a 96-well plate and incubated at 25 C for 10 min. The absorbance at 550 nm was measured on a microplate reader. NaNO2 was used as the standard to calculate NO 2 concentrations. The data are representative of results obtained from three independent experiments, which was performed in triplicate (mean S.D., n ¼ 3). Interassay coefficient of variation (CV) was 9.9% for clomipramine or 8.6% for imipramine, respectively. 2.4. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated using TRIzol reagent (Molecular Research Center Inc., Cincinnati, OH) according to the manufacturer’s instruction. Reverse transcription was carried out using a Moloney murine leukemia virus (M-MLV) and oligo(dT) primer. PCR amplification using primer sets specific for inducible nitric oxide synthase (iNOS), TNF-a, IL-1b or b-actin was carried out at 94 C for 30 s, 55 C for 30 s, and 72 C for 1 min, and repeated 23 cycles followed by incubation at 72 C for 7 min. Nucleotide sequences of the primers were based on published cDNA sequences of mouse iNOS, TNF-a, IL-1b, or b-actin: iNOS forward, CCC TTC CGA AGT TTC TGG CAG CAG C; iNOS reverse, GGC TGT CAG AGC CTC GTG GCT TTG G; TNFa forward, CAT CTT CTC AAA ATT CGA GTG ACA A; TNF-a reverse, ACT TGG GCA GAT TGA CCT CAG; IL-1b forward, GCA ACT GTT CCT GAA CTC; IL-1b reverse, CTC GGA GCC TGT AGT GCA; b-actin forward, ATC CTG AAA GAC CTC TAT GC; b-actin reverse, AAC GCA GCT CAG TAA CAG TC. The b-actin was used as an internal control to evaluate relative expression of iNOS, TNF-a and IL-1b. The results are representative of three independent experiments.
2. Materials and methods
2.5. Immunofluorescence assay
2.1. Reagents and cell cultures
The effect of antidepressants on the nuclear translocation of a p65 subunit of NF-kB was examined by an immunofluorescence assay as described (Sung et al., 2007). For the detection of the intracellular location of p65 subunit of NF-kB, BV-2 microglia cells (1 105 cells/well in 24-well plates) were cultured on sterile cover slips in 24-well plates and treated with drugs and LPS. At 1 h after the LPS stimulation, the cells were fixed with methanol for 20 min at 20 C and washed with PBS for 5 min. The fixed cells were then permeabilized with 0.5% Triton X-100 in PBS for 1 h at room temperature, washed with 0.05% Tween-20 in PBS for 10 min and 0.05% Tween-20/1% BSA in PBS for 5 min. The permeabilized cells were
Bacterial lipopolysaccharide (LPS) (Escherichia coli serotype 055:B5), clomipramine, and imipramine were purchased from Sigma–Aldrich (St. Louis, MO). The drugs were dissolved in phosphate-buffered saline (PBS; 150 mM NaCl, 5 mM phosphate, pH 7.4). Recombinant mouse IFN-g was purchased from R&D Systems (Minneapolis, MN). A BV-2 murine microglia cell line (Bocchini et al., 1992) was grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), gentamicin (50 mg/ml) at
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then treated with 1 mg/ml of mouse monoclonal anti-human NF-kB p65 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 h at room temperature, washed with 0.05% Tween-20/1% BSA in PBS for 5 min. Cells were then incubated in a 1:2000 dilution of Alexa Fluor 488-labeled goat anti-mouse IgG antibody (Molecular Probes Inc., Eugene, OR) for 1 h at room temperature, and washed with 0.05% Tween-20 in PBS for 5 min and PBS for 5 min. Cells were then stained with 0.5 mg/ ml of Hoechst staining solution (Molecular Probes) for 20 min at 37 C and then washed. Finally, the cover slips with cells were dried in a 37 C oven for 45 min and mounted in a 1:1 mixture of xylene and malinol. The number of cells with p65 nuclear translocation was determined under a fluorescence microscope. More than 100 cells with p65 translocation were counted. The results are representative of three independent experiments. 2.6. Western blot analysis Cells were lysed in triple-detergent lysis buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride]. Protein concentration in cell lysates was determined using a protein assay kit (Bio-Rad, Hercules, CA). An equal amount of protein from each sample was separated by SDSpolyacrylamide gel electrophoresis (12% gel) and transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies [rabbit polyclonal anti-human IkB-a (Santa Cruz Biotechnology), rabbit polyclonal anti-human phospho-p38 MAPK (Cell Signaling Technology Inc., Beverly, MA), monoclonal anti-a-tubulin clone B-5-1-2 mouse ascites fluid (Sigma)] and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and anti-mouse; Amersham Biosciences) followed by enhanced chemiluminescence detection (Amersham Biosciences). The results are representative of three independent experiments. 2.7. Enzyme-linked immunosorbent assay (ELISA) TNF-a secreted in microglial culture supernatants was measured as described (Park et al., 2002) by specific ELISA using rat monoclonal anti-mouse TNF-a antibody as capture antibody and goat biotinylated polyclonal anti-mouse TNF-a antibody as detection antibody (ELISA development reagents; R&D Systems). The biotinylated anti-TNF-a antibody was detected by sequential incubation with streptavidin– horseradish peroxidase conjugate and chromogenic substrates. The data are representative of results obtained from three independent experiments, which was performed in triplicate (mean S.D., n ¼ 3). Interassay CV was 4.9% for clomipramine or 2.4% for imipramine, respectively.
Fig. 1. Effects of the antidepressants on NO production in LPS-activated BV-2 microglia. BV-2 microglia cells (8 104 cells/well in a 96-well plate) were incubated with 100 ng/ml of LPS in the presence or absence of antidepressants (10, 30 mM) for 24 h. The amounts of nitrite in the supernatants were measured using Griess reagent (A). Cell viability was examined by MTT reduction assays and the results were expressed as the percentage of surviving cells over control cells (B). The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
2.8. Microglia/neuron co-culture For the co-culture experiment, BV-2 microglia cells or HAPI rat microglia cells were seeded in triplicate at the density of 1.5 104 cells/well in 96-well plate. BV-2 microglia cells or HAPI rat microglia cells were pretreated with drugs for 30 min. Then, culture supernatants were discarded and 100 ng/ml of LPS was added together with B35 rat neuroblastoma cells stably expressing EGFP (3.75 104 cells/well), which was followed by the co-culture for 24 h. The numerical ratio of microglia to neuron was 1:2.5. Afterwards, the EGFP-positive cells were counted under a fluorescence microscope (Olympus IX 70, Tokyo, Japan). Images of three random fields per well were captured and analyzed by the MetaMorph imaging system (Universal Imaging Corp, West Chester, PA). LPS alone did not affect B35 neuroblastoma cell viability (data not shown). The results are representative of three independent experiments. 2.9. Statistical analysis Results were expressed as mean S.D. The data were analyzed by one-way ANOVA following the Student Newman Keul’s post hoc analysis using SPSS program (version 12.0). A value of P < 0.05 was considered statistically significant.
for their inhibitory activity (Fig. 2A). Clomipramine of 10–20 mM and imipramine of 5–15 mM inhibited microglial NO production in a dose-dependent manner (Fig. 2A) without a significant cytotoxicity (Fig. 2B). The LPS-induced NO production was similarly decreased by the tricyclic antidepressants in mouse primary microglia cultures (Fig. 2C) and HAPI rat microglia cells (Fig. 2D), indicating that the NO inhibitory effects of the antidepressants are not limited to BV-2 cells. The drugs at the concentration tested did not affect the viability of either primary microglia cultures or HAPI rat microglia cells (data not shown). The antiinflammatory activity of the tricyclic antidepressants clomipramine (15 mM) and imipramine (10 mM) was further studied. Because NO and TNF-a are the major neurotoxic factors under neuroinflammatory condition, the possibility of whether clomipramine and imipramine reduce the production of TNF-a has also been tested. Clomipramine and imipramine also decreased TNF-a production in LPS-stimulated BV-2 microglia cells (Fig. 3).
3. Results 3.1. Inhibitory effects of antidepressants on NO and TNF-a production in microglia First, the effects of clomipramine and imipramine (10, 30 mM) on NO production in LPS-stimulated BV-2 mouse microglia cell line were evaluated (Fig. 1A). In order to rule out the possibility that the reduction of NO production may be due to the cytotoxicity of the drugs, cell viability was evaluated (Fig. 1B). Because 30 mM of clomipramine and imipramine was apparently toxic to BV-2 microglia cells, the drugs at lower concentrations were reassessed
3.2. Antidepressants inhibited the expression of iNOS, IL-1b, and TNF-a genes In order to investigate the effects of clomipramine and imipramine on the gene expression of inflammatory mediators at the transcriptional level, the levels of iNOS, IL-1b, and TNF-a mRNA in the LPS-stimulated BV-2 microglia cells were determined by RT-PCR analysis. The antidepressants inhibited the LPS-induced expression of iNOS, IL-1b, and TNF-a, whereas the drugs alone did not induce any significant change of the gene expression (Fig. 4).
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Fig. 2. Effects of the antidepressants on NO production in the LPS-stimulated BV-2 and HAPI microglia cell lines or primary microglia cultures. BV-2 mouse microglia cells (A), mouse primary microglia cultures (C) or HAPI rat microglia cells (D) (8 104 cells/well in a 96-well plate) were incubated with 100 ng/ml of LPS in the presence or absence of clomipramine (10–20 mM) or imipramine (5–15 mM) for 24 h. The amounts of nitrite in the supernatants were measured using Griess reagent. Cell viability was examined by MTT reduction assays and the results were expressed as the percentage of surviving cells over control cells (B). The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
3.3. Antidepressants attenuated the LPS-induced NF-kB and p38 MAPK activation NF-kB and p38 MAPK are key upstream regulators that induce proinflammatory cytokines and iNOS gene expression in glia cells (Da Silva et al., 1997; Jongeneel, 1995). Therefore, it was determined whether the antiinflammatory effects of the antidepressants occurred through the blockade of NF-kB and p38 MAPK activation in BV-2 microglia cells. The processes of NF-kB activation include IkB degradation and a subsequent nuclear translocation of p65 subunit of NF-kB. LPS induced the translocation of p65 into the nucleus within 60 min after the stimulation, which was inhibited by the antidepressants as determined by an immunofluorescence assay (Fig. 5A) and subsequent enumeration of the cells with p65 translocation (Fig. 5B). Western blot analysis further showed that degradation of IkB-a after 30 min stimulation with LPS was markedly inhibited by the antidepressants (Fig. 5C). After stimulation of BV-2 microglia cells with LPS for 30 min, activation of p38 MAPK was also observed as determined by Western blot analysis using antibody specific for phospho-p38 MAPK. Clomipramine and imipramine significantly inhibited the phosphorylation of p38 MAPK (Fig. 5C). These results indicated that the antidepressants suppressed LPS-induced NF-kB and p38
MAPK activation in microglia, and that the inhibition of iNOS, IL-1b, and TNF-a gene expression by the antidepressants was likely due to the blockade of NF-kB and p38 MAPK pathways. 3.4. Antidepressants inhibited microglial neurotoxicity in a microglia/neuron co-culture model To investigate potential neuroprotective effects of clomipramine and imipramine in vitro, a microglia/neuron co-culture model was used. As various proinflammatory mediators produced by activated microglia can induce neuronal cell death and amplify progression of neuronal degeneration (Stoll and Jander, 1999; Streit et al., 1999), inhibition of microglial activation may be neuroprotective. Thus, microglial neurotoxicity and neuroprotective effects of a compound can be tested in a microglia and neuron co-culture, where activated microglia may induce neuronal cell death. A compound can be considered neuroprotective, if it protects neurons against the cytotoxic effect of activated microglia in the co-culture of microglia and neurons. This possibility of the antidepressants was tested using a co-culture of B35 neuroblastoma cells with LPS-activated BV-2 microglia cells. B35 neuroblastoma cells that have been stably transfected with EGFP expression construct were employed for the simple identification of neuroblastoma cells in the co-culture. BV-2 microglia cells were activated with LPS with or without
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pretreatment with the antidepressants, and then co-cultured with B35 neuroblastoma cells (see Fig. 6A for the co-culture scheme). The viability of B35 cells was measured by counting the EGFPexpressing cells after the co-culture under a fluorescence microscope, because B35 cells stably expressed EGFP. This co-culture system has been successfully used for the determination of microglial neurotoxicity (Kim et al., 2007). Clomipramine (10 mM) and imipramine (15 mM) almost completely inhibited B35 cell death in the co-culture (Fig. 6B, C). The neuroprotective effect of antidepressants was also examined in the co-culture of HAPI rat microglia cells and B35 rat neuroblastoma cells. A similar result of neuroprotection was obtained (data not shown). The results indicate that clomipramine and imipramine may be neuroprotective by suppressing microglial neurotoxicity. 3.5. Effects of the antidepressants on the inflammatory activation of RAW 264.7 macrophage cells and primary astrocyte cultures
Fig. 3. Effects of the antidepressants on TNF-a production in LPS-stimulated BV-2 microglia cells. BV-2 microglia cells (8 104 cells/well in a 96-well plate) were incubated with 100 ng/ml of LPS in the presence or absence of clomipramine (15 mM) and imipramine (10 mM) for 24 h. The amounts of TNF-a in the supernatants were measured by ELISA. The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
Effects of the antidepressants on the NO production in RAW 264.7 macrophage cells and primary astrocyte cultures were tested. Clomipramine and imipramine significantly decreased NO production in LPS-stimulated RAW 264.7 macrophage cells (Fig. 7A) and primary astrocyte cultures (Fig. 7B). When astrocytes were stimulated with LPS plus IFN-g, greater amounts of NO production were achieved, which were similarly inhibited by the antidepressants (Fig. 7C). These results indicated that the antidepressants also have antiinflammatory effects in peripheral macrophages and astrocytes.
Fig. 4. Effects of the antidepressants on proinflammatory cytokine genes expression in LPS-simulated BV-2 microglia cells. BV-2 cells were treated with LPS (100 ng/ml) in the absence or presence of antidepressants (15 mM of clomipramine or 10 mM of imipramine), and total RNA was isolated at 6 h after the treatment. The levels iNOS, IL-1b, and TNFa mRNA were determined by RT-PCR (upper) and then subjected to densitometric quantification (lower). Levels of iNOS, IL-1b and TNF-a mRNA were normalized to b-actin levels and expressed as a relative change in comparison to the LPS treatment, which was set to 100% (lane 2). The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
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Fig. 5. Blockade of NF-kB activation and p38 MAPK phosphorylation by the antidepressants. The BV-2 microglia cells were seeded in triplicate at the density of 1 105 cells/well in a 24-well plate. The cells were stimulated with 100 ng/ml of LPS in the absence or presence of clomipramine (15 mM) and imipramine (10 mM) that had been added 30 min before the stimulation. At 1 h after the LPS addition, subcellular location of NF-kB p65 subunit was determined by an immunofluorescence assay (A). The p65 protein was detected using anti-p65 antibody conjugated with fluorescein isothiocyanate (FITC). Representative images of cells are shown (left). Magnification, 200; scale bar, 50 mm. Boxed rectangular regions were enlarged (right). The number of cells with p65 nuclear translocation was determined and the percentage of cells with p65 translocation was calculated: more than 100 cells were counted (B). Total cell lysates obtained 10, 30 and 60 min after the LPS stimulation were subjected to Western blotting to assess the levels of IkB-a or phospho-p38 MAPK proteins (C) (upper). Quantification of IkB-a or phospho-p38 protein levels was performed by densitometric analysis (lower). Detection of a-tubulin was done to confirm the equal loading of the samples. The values were expressed as a percentage of maximal band intensity in the microglia cell culture treated with LPS alone, which was set to 100% (lane 3). The data are the mean S.D. (n ¼ 3), and are representative of three or more independent experiments. *P < 0.01, **P < 0.001; significantly different from the value in cells treated with LPS only.
4. Discussion In the present study, it has been demonstrated that tricyclic antidepressants such as clomipramine and imipramine inhibit
inflammatory activation of microglia and astrocytes. Clomipramine and imipramine significantly reduced NO or TNF-a production in LPS-stimulated BV-2 microglia cells, primary microglia or astrocyte cultures. The RT-PCR analysis showed that these antidepressants
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Fig. 6. Effects of the antidepressants on B35 rat neuroblastoma cell viability in a microglia/neuron co-culture. The BV-2 microglia cells were seeded in triplicate at the density of 1.5 104 cells/well in a 96-well plate. Microglia cells were pretreated with clomipramine (15 mM) or imipramine (10 mM) for 30 min. Culture supernatants were discarded and stimulated with 100 ng/ml of LPS. At the same time, rat B35 neuroblastoma cells (3.75 104 cells/well) stably expressing EGFP were added onto BV-2 cells, and then co-cultured for 24 h (A). After the co-culture for 24 h, the EGFP-positive cells were counted under a fluorescence microscope to evaluate B35 neuroblastoma cell death (B). Representative images of cells are shown (magnification, 100); scale bar, 100 mm. The number of fluorescent cells in several randomly chosen microscopic fields per well was determined, and the data were expressed as the mean S.D. (n ¼ 3) (C). The results are representative of three independent experiments. *P < 0.001; significantly different from the value in cells treated with LPS in the absence of antidepressants.
markedly suppressed the iNOS, TNF-a, and IL-1b gene expression at the transcriptional level. NF-kB and p38 MAPK pathways were at least partly involved in the antiinflammatory mechanisms of the antidepressants. In addition, these antidepressants showed neuroprotective effects by attenuating microglial neurotoxicity in a microglia/neuron co-culture system. In the resting state, microglia typically exist with ramified morphology and monitor the brain environment (Nimmerjahn et al., 2005). However, under the neurodegenerative conditions, they are activated and release various inflammatory mediators including proinflammatory cytokines and free radicals such as NO and superoxide anion (Aloisi, 2001). Because NO is one of the main proinflammatory mediators and plays an important role in neuroinflammatory diseases, the effects of the antidepressants on the NO production in LPS-stimulated microglia cells were examined. Based on the strong inhibitory effect of clomipramine and imipramine on the NO production, further studies were focused on these drugs. Clomipramine and imipramine as tricyclic antidepressants
exhibited a strong antiinflammatory property (such as inhibition of proinflammatory cytokine expression and production, NF-kB, and p38 MAPK) in brain microglia cells as well as astrocytes in the current study. These results are consistent with previous reports where clomipramine and imipramine decreased IL-1b and TNFa production in human monocytes (Xia et al., 1996), and imipramine inhibited IL-6 and NO production in IFN-g-activated 6-3 mouse microglia cell line (Hashioka et al., 2007). Clomipramine and imipramine in therapeutic doses reach the levels of 0.28–0.80 and 0.16–0.54 mM, respectively, in the plasma (Wille et al., 2008, 2001). Clomipramine and imipramine are reported to accumulate in brain tissue 12.5-fold and 32-fold higher than in plasma or serum levels, respectively (DeVane et al., 1984; Weigmann et al., 2000), which is comparable to the concentrations of the drugs used in the present study. NF-kB is a key transcription factor activated by several cellular signal transduction pathways that play an important role in the expression of proinflammatory cytokines and enzymes such as
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Fig. 7. Effects of the antidepressants on the LPS-activated NO production in RAW 264.7 macrophage cells or primary astrocyte cultures. Either RAW 264.7 macrophage cells or primary astrocyte cultures (8 104 cells/well in a 96-well plate) were incubated with 100 ng/ml of LPS or LPS/IFN-g (50 unit/ml) in the presence or absence of clomipramine (15 mM) or imipramine (10 mM) for 24 h. The amounts of nitrite in the supernatants were measured using Griess reagent (A, RAW 264.7 cells; B and C, primary astrocyte cultures). The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
iNOS, IL-1b, and TNF-a (Baeuerle and Henkel, 1994; Baldwin, 2001). The molecular mechanisms of NF-kB activation have been well studied, and they involve a cascade activation of cytoplasmic proteins and the ultimate nuclear translocation of p65 subunit of NF-kB (Delhase et al., 2000; Karin and Ben-Neriah, 2000). LPS causes the nuclear translocation of p65 subunit of NF-kB thorough IkB degradation. In the present study, it was found that clomipramine and imipramine inhibited IkB degradation and the subsequent nuclear translocation of p65 in BV-2 microglia cells (Fig. 5). Previously, tricyclic antidepressants including clomipramine and imipramine inhibited NO production in monocytes or microglia by the cAMP-dependent protein kinase (PKA) pathway (Hashioka et al., 2007; Xia et al., 1996). Because activation of the cAMP-dependent PKA pathway is believed to negatively regulate the NF-kB activation in a number of cell types (Delfino and Walker, 1999; Pahan et al., 1997), it is speculated that the cAMP–PKA pathway may be also involved in the antiinflammatory effects of the antidepressants in our model system. Additionally, it was found that clomipramine and imipramine inhibited LPS-induced activation of p38 MAPK (Fig. 5C), which has been previously implicated in the signal transduction pathways responsible for the induction of iNOS and TNF-a gene expression in glial cells or macrophages (Kim et al., 2004; Park et al., 2007; Pawate and Bhat, 2006). Taken together, the current results indicated that clomipramine and imipramine inhibited the gene expression of iNOS, TNF-a, and IL-1b in microglia, the mechanism of which at least in part might involve the inhibition of NF-kB and p38 MAPK activation. It should be, however, noted that the gene expression was measured by RT-PCR, which is only a semi-quantitative methodology. Activation of glial cells (microglia and astrocytes) and inflammatory products derived from them have been implicated in disease progression and pathology in several neuroinflammatory diseases such as Alzheimer’s disease, Parkinson’s disease, and HIV dementia (Block et al., 2007). Glial activation has both destructive
and protective effects on neuronal injury in neurodegenerative diseases. Over-activation of glia, however, may contribute to neurodegenerative processes through the production of various neurotoxic factors including free radicals and proinflammatory cytokines (Klegeris et al., 2007). In fact, a number of antiinflammatory agents, which inhibited glial activation or production of proinflammatory mediators under the CNS disease conditions, attenuated neuronal degeneration (Esposito et al., 2007; Lee et al., 2003; McGeer and McGeer, 2007; Tikka et al., 2001a,b). Thus, it is suggested that a search for the efficient antiinflammatory compounds that attenuate glial activation may lead to an effective therapeutic approach against many neurodegenerative conditions. A recent study has demonstrated that imipramine treatment protects LPS-induced apoptosis in hippocampus-derived neural stem cells through activation of the BDNF and MAPK/ERK pathway (Peng et al., 2008). The current study showed that clomipramine and imipramine protected neuroblastoma cells against microglial neurotoxicity in a microglia/neuron co-culture (Fig. 6). The neuroprotective effect of the antidepressants is likely due to the inhibition of microglia activation, but not protective action on the neuroblastoma cells, because the antidepressants-pretreated microglia cultures were washed before the addition of the neuroblastoma cells for the co-culture. The B35 neuroblastoma cells were not exposed to the antidepressants. Therefore, the neuroprotective activity of imipramine that has been previously observed (Peng et al., 2008) may be partly due to its inhibitory effects on the neurotoxic microglia activation. Although the co-culture of the LPSstimulated microglia with neuroblastoma cell line may not be the same as the in vivo conditions, it partially reflects the pathological condition where activated microglia influence the death and survival of neuronal cells in neurodegenerative diseases. Further studies are, however, required to evaluate a neuroprotective property of the antidepressants in the animal models of neurodegenerative diseases and to understand the precise molecular
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mechanisms of antiinflammatory actions of the antidepressants in vitro as well as in vivo. Nevertheless, the present study suggests the protective effects of the antidepressants against inflammationmediated neurodegeneration. Future works along this line will lead to a novel therapeutic use of the antidepressants for the treatment of neurodegenerative diseases and other inflammatory disorders. Acknowledgments This work was supported by grant No. R01-2006-000-103140 from the Basic Research Program of the Korea Science & Engineering Foundation and by the Innate Immunity Research Program (2008-04090) from the Korea Ministry of Education, Science and Technology. KS is a recipient of the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-005-J04202). JH, LTZ and JO were supported by the Brain Korea 21 Project in 2008. References Abdel-Salam, O.M., Nofal, S.M., El-Shenawy, S.M., 2003. Evaluation of the antiinflammatory and anti-nociceptive effects of different antidepressants in the rat. Pharmacol. Res. 48, 157–165. Aloisi, F., 2001. Immune function of microglia. Glia 36, 165–179. Aschner, M., 1998. Astrocytes as mediators of immune and inflammatory responses in the CNS. Neurotoxicology 19, 269–281. Baeuerle, P.A., Henkel, T., 1994. Function and activation of NF-kappa B in the immune system. Annu. Rev. Immunol. 12, 141–179. Baldwin, A.S., 2001. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J. Clin. Invest. 107, 241–246. Block, M.L., Zecca, L., Hong, J.S., 2007. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat. Rev. Neurosci. 8, 57–69. Bocchini, V., Mazzolla, R., Barluzzi, R., Blasi, E., Sick, P., Kettenmann, H., 1992. An immortalized cell line expresses properties of activated microglial cells. J. Neurosci. Res. 31, 616–621. Cheepsunthorn, P., Radov, L., Menzies, S., Reid, J., Connor, J.R., 2001. Characterization of a novel brain-derived microglial cell line isolated from neonatal rat brain. Glia 35, 53–62. Chen, Y., Swanson, R.A., 2003. Astrocytes and brain injury. J. Cereb. Blood Flow Metab. 23, 137–149. Da Silva, J., Pierrat, B., Mary, J.L., Lesslauer, W., 1997. Blockade of p38 mitogenactivated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes. J. Biol. Chem. 272, 28373–28380. Delfino, F., Walker, W.H., 1999. Hormonal regulation of the NF-kappaB signaling pathway. Mol. Cell. Endocrinol. 157, 1–9. Delhase, M., Li, N., Karin, M., 2000. Kinase regulation in inflammatory response. Nature 406, 367–368. DeVane, C.L., Simpkins, J.W., Stout, S.A., 1984. Cerebral and blood pharmacokinetics of imipramine and its active metabolites in the pregnant rat. Psychopharmacology (Berl.) 84, 225–230. Esposito, E., Di Matteo, V., Benigno, A., Pierucci, M., Crescimanno, G., Di Giovanni, G., 2007. Non-steroidal anti-inflammatory drugs in Parkinson’s disease. Exp. Neurol. 205, 295–312. Gao, H.M., Liu, B., Zhang, W., Hong, J.S., 2003. Critical role of microglial NADPH oxidase-derived free radicals in the in vitro MPTP model of Parkinson’s disease. FASEB J. 17, 1954–1956. Giulian, D., Li, J., Li, X., George, J., Rutecki, P.A., 1994. The impact of microglia-derived cytokines upon gliosis in the CNS. Dev. Neurosci. 16, 128–136. Gonzalez-Scarano, F., Baltuch, G., 1999. Microglia as mediators of inflammatory and degenerative diseases. Annu. Rev. Neurosci. 22, 219–240. Hashioka, S., Klegeris, A., Monji, A., Kato, T., Sawada, M., McGeer, P.L., Kanba, S., 2007. Antidepressants inhibit interferon-gamma-induced microglial production of IL-6 and nitric oxide. Exp. Neurol. 206, 33–42. Jongeneel, C.V., 1995. Transcriptional regulation of the tumor necrosis factor alpha gene. Immunobiology 193, 210–216. Karin, M., Ben-Neriah, Y., 2000. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu. Rev. Immunol. 18, 621–663. Kim, S., Ock, J., Kim, A.K., Lee, H.W., Cho, J.Y., Kim, D.R., Park, J.Y., Suk, K., 2007. Neurotoxicity of microglial cathepsin D revealed by secretome analysis. J. Neurochem. 103, 2640–2650. Kim, W.K., Jang, P.G., Woo, M.S., Han, I.O., Piao, H.Z., Lee, K., Lee, H., Joh, T.H., Kim, H. S., 2004. A new anti-inflammatory agent KL-1037 represses proinflammatory cytokine and inducible nitric oxide synthase (iNOS) gene expression in activated microglia. Neuropharmacology 47, 243–252. Klegeris, A., McGeer, E.G., McGeer, P.L., 2007. Therapeutic approaches to inflammation in neurodegenerative disease. Curr. Opin. Neurol. 20, 351–357. Kubera, M., Symbirtsev, A., Basta-Kaim, A., Borycz, J., Roman, A., Papp, M., Claesson, M., 1996. Effect of chronic treatment with imipramine on interleukin 1 and interleukin 2 production by splenocytes obtained from rats subjected to a chronic mild stress model of depression. Pol. J. Pharmacol. 48, 503–506.
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Inhibition of glial inflammatory activation and neurotoxicity by tricyclic antidepressants Jaegyu Hwang a, Long Tai Zheng a, Jiyeon Ock a, Maan Gee Lee a, Sang-Hyun Kim a, Ho-Won Lee b, Won-Ha Lee c, Hae-Chul Park d, Kyoungho Suk a, * a
Department of Pharmacology, School of Medicine, Brain Science and Engineering Institute, CMRI, Kyungpook National University, 101 Dong-In, Joong-gu, Daegu 700-422, Republic of Korea Department of Neurology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea c Department of Genetic Engineering, School of Life Sciences and Biotechnology, Kyungpook National University, Daegu, Republic of Korea d Department of Medical Science, Korea University Ansan Hospital, Ansan, Gyeonggi, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2008 Received in revised form 26 May 2008 Accepted 23 June 2008
Glial activation and neuroinflammatory processes play an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and HIV dementia. Activated glial cells can secrete various proinflammatory cytokines and neurotoxic mediators, which may contribute to neuronal cell death. Inhibition of glial activation may alleviate neurodegeneration under these conditions. In the present study, the antiinflammatory and neuroprotective effects of tricyclic antidepressants were investigated using cultured brain cells as a model. The results showed that clomipramine and imipramine significantly decreased the production of nitric oxide or tumor necrosis factor-alpha (TNF-a) in microglia and astrocyte cultures. Clomipramine and imipramine also attenuated the expression of inducible nitric oxide synthase and proinflammatory cytokines such as interleukin-1b and TNF-a at mRNA levels. In addition, clomipramine and imipramine inhibited IkB degradation, nuclear translocation of the p65 subunit of NF-kB, and phosphorylation of p38 mitogen-activated protein kinase in the lipopolysaccharide-stimulated microglia cells. Moreover, clomipramine and imipramine were neuroprotective as the drugs reduced microglia-mediated neuroblastoma cell death in a microglia/ neuron co-culture. Therefore, these results imply that clomipramine and imipramine have antiinflammatory and neuroprotective effects in the central nervous system by modulating glial activation. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Antidepressant Microglia Astrocyte Inflammation Neuroprotection
1. Introduction Neuroinflammation is actively involved in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and HIV-associated dementia (Block et al., 2007). Neuroglia such as microglia and astrocytes are non-neuronal cells that provide physical and nutritional support for neurons in the central nervous system (CNS). Microglia, which constitute about 10% of all glial cells, are the primary immune cells in the CNS, and they are also considered to be the major cell type responsible for inflammation-mediated neurotoxicity (Liu and Hong, 2003). Under the neurodegenerative condition, microglia cells can be activated by various neurotoxic factors produced by injured neuronal cells (Gao et al., 2003; McGeer and McGeer, 2003). Activation of microglia is also observed in response to brain injury
* Corresponding author. Tel.: þ82 53 420 4835; fax: þ82 53 256 1566. E-mail address: [email protected] (K. Suk). 0028-3908/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2008.06.045
and after the exposure to lipopolysaccharide (LPS), interferon (IFN)-g, or b-amyloid. Activated microglia have the capability of producing proinflammatory cytokines and neurotoxic mediators such as tumor necrosis factor (TNF)-a, prostaglandin (PG) E2, interleukin (IL)-1b, IL-6, and free radicals such as nitric oxide (NO) and superoxide anion (Giulian et al., 1994; Zielasek and Hartung, 1996). These proinflammatory cytokines and neurotoxic mediators are thought to contribute to neuronal injuries and progression of the neuroinflammatory diseases (Gonzalez-Scarano and Baltuch, 1999; Minghetti and Levi, 1998). Over-activation of astrocytes also facilitates ongoing neurodegeneration by producing various neurotoxic factors (Suk, 2005). Astrocytes are the most numerous glial cell types in the brain and play an important role in the homeostatic control of the neuronal extracellular environment (Chen and Swanson, 2003). Upon inflammatory stimulation, astrocytes proliferate and produce diverse intercellular mediators such as NO and TNF-a (Suk, 2005). There is growing evidence that inflammatory mediators produced by activated astrocytes may also be involved in the pathogenesis of various neurodegenerative
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diseases (Aschner, 1998). Taken together, as inflammatory activation of microglia and astrocytes is often observed in neuronal injuries and actively involved in the initiation and progression of several neurodegenerative diseases (Block et al., 2007; McGeer and McGeer, 2003, 2004; McGeer et al., 1988), inhibition of glial activation and subsequent neuroinflammation may be an effective therapeutic approach against neurodegenerative diseases. Antidepressants are widely used for treating major and minor depression. There are four general types of antidepressants including tricyclics, monoamine oxidase inhibitors, selective serotonin reuptake inhibitors (SSRI), and serotonin and noradrenaline reuptake inhibitors (Stahl, 1998). Clomipramine and imipramine belong to tricyclic antidepressants and have an antidepressant effect through inhibiting the reuptake of the neurotransmitters norepinephrine and serotonin by neurons (Wille et al., 2008). Recently, several studies have demonstrated that antidepressant drugs have an effect that is related to modulation of cell-mediated immune responses. Clomipramine and imipramine reduced TNF-a, IL-1b, and IL-6 production by LPS-stimulated human monocytes (Xia et al., 1996). These antidepressants also induced apoptosis of human lymphocytes (Xia et al., 1996), and decreased production of IL-2 and IFN-g in human T cells or monocytes (Xia et al., 1997). It has been also reported that chronic treatment of imipramine reduced lymphocyte proliferation and IL-1/IL-2 production by splenocytes from a chronic mild stress-induced depression model of rats (Kubera et al., 1996). Selective serotonin reuptake inhibitors such as escitalopram and citalopram also showed immunomodulating effects. Citalopram inhibited differentiation of human monocytes into macrophage-like cells in vitro (Ying et al., 2002). Recent studies have demonstrated that the antidepressant drugs amitriptyline, imipramine, clomipramine, trazodone, and fluoxetine have antiinflammatory effects in carageenan-induced paw edema model (Abdel-Salam et al., 2003). It was also reported that amitriptyline and its metabolite nortriptyline reduced IL-1b and TNF-a secretion in LPS-activated mixed glia and microglia cultures (Obuchowicz et al., 2006). Imipramine, fluvoxamine, and reboxetine inhibited IL-6 and NO production by IFN-g-stimulated microglia cells (Hashioka et al., 2007). Although previous studies have well demonstrated antiinflammatory effects of antidepressants in the peripheral system, only a few reports have addressed these effects in brain glia cells. Moreover, the detailed molecular mechanisms underlying the antiinflammatory effects of tricyclic antidepressants in the CNS are not completely understood. The effects of tricyclic antidepressant on glial neurotoxicity have not been evaluated either. Here, the effect of tricyclic antidepressants on the inflammatory activation and neurotoxicity of microglia, as well as astrocytes, has been determined. This study showed that clomipramine and imipramine inhibited LPS-induced production of NO, TNF-a, and their gene expression in microglia. Clomipramine and imipramine also suppressed NF-kB and p38 mitogen-activated protein kinase (MAPK) activation in LPS-stimulated microglia cells. The antiinflammatory effects of these antidepressants were also found in astrocytes and macrophages. In addition, these antidepressants showed neuroprotective effects by attenuating microglial neurotoxicity in a microglia/neuron co-culture system.
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37 C, 5% CO2. B35 rat neuroblastoma (ATCC, CRL-2754) (Schubert et al., 1974), HAPI rat microglia (Cheepsunthorn et al., 2001), and RAW 264.7 macrophage cell lines (ATCC, TBI-71) were grown and maintained in DMEM supplemented with 10% heatinactivated FBS, penicillin (10 U/ml) and streptomycin (10 mg/ml) at 37 C, 5% CO2. Mouse primary microglia and astrocytes cultures were prepared by mild trypsinization as described previously with minor modifications (Saura et al., 2003). In brief, the forebrains of newborn Institute of Cancer Research (ICR) mice were chopped and dissociated by mechanical disruption using a nylon mesh. The cells were seeded in poly-D-lysine-coated flasks. After in vitro culture for 10–14 days, microglia cells were isolated from mixed glia cultures by mild trypsinization. Mixed glia cultures were incubated with a trypsin solution [0.25% trypsin, 1 mM EDTA in Hank’s balanced salt solution (HBSS)] diluted 1:4 in PBS containing 1 mM CaCl2 for 30–60 min. This resulted in the detachment of an upper layer of astrocytes in one piece, whereas microglia remained attached to the bottom of the culture flask. The detached layer of astrocytes was aspirated, and the remaining microglia were used for experiments. The prepared primary microglia cultures were more than 95% pure, as determined by isolectin B4 staining (data not shown). Astrocytes were isolated by shaking mixed glia cultures at 250 rpm overnight and then the culture medium was discarded. The purity of astrocyte cultures was greater than 95% as determined by glial fibrillary acidic protein (GFAP) immunocytochemical staining (data not shown). Animals used in the current research were acquired and cared for in accordance with the guidelines published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study was approved by the Institutional Review Board of Kyungpook National University School of Medicine. 2.2. Cell viability test Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Microglia, astrocytes or RAW 264.7 macrophages were seeded in triplicate at the density of 8 104 cells/well on 96-well plate. The cells were treated with antidepressants and LPS for 24 h. MTT was added to each well, and incubated for 4 h at 37 C. After culture media were discarded, dimethyl sulfoxide (DMSO) was added to dissolve the formazan dye. The optical density was measured at 540 nm. The data are representative of results obtained from three independent experiments, which was performed in triplicate (mean S.D., n ¼ 3). 2.3. Nitrite quantification NO secreted in glial culture supernatants was measured by Griess reagent as described (Lee and Suk, 2007). After microglia, astrocytes, or RAW 264.7 macrophages were treated with stimulating agents in 96-well plates, NO 2 concentration in culture supernatants was measured to assess NO production. Fifty microliters of sample aliquots were mixed with 50 ml of Griess reagent (1% sulfanilamide/0.1% naphthylethylene diamine dihydrochloride/2% phosphoric acid) in a 96-well plate and incubated at 25 C for 10 min. The absorbance at 550 nm was measured on a microplate reader. NaNO2 was used as the standard to calculate NO 2 concentrations. The data are representative of results obtained from three independent experiments, which was performed in triplicate (mean S.D., n ¼ 3). Interassay coefficient of variation (CV) was 9.9% for clomipramine or 8.6% for imipramine, respectively. 2.4. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated using TRIzol reagent (Molecular Research Center Inc., Cincinnati, OH) according to the manufacturer’s instruction. Reverse transcription was carried out using a Moloney murine leukemia virus (M-MLV) and oligo(dT) primer. PCR amplification using primer sets specific for inducible nitric oxide synthase (iNOS), TNF-a, IL-1b or b-actin was carried out at 94 C for 30 s, 55 C for 30 s, and 72 C for 1 min, and repeated 23 cycles followed by incubation at 72 C for 7 min. Nucleotide sequences of the primers were based on published cDNA sequences of mouse iNOS, TNF-a, IL-1b, or b-actin: iNOS forward, CCC TTC CGA AGT TTC TGG CAG CAG C; iNOS reverse, GGC TGT CAG AGC CTC GTG GCT TTG G; TNFa forward, CAT CTT CTC AAA ATT CGA GTG ACA A; TNF-a reverse, ACT TGG GCA GAT TGA CCT CAG; IL-1b forward, GCA ACT GTT CCT GAA CTC; IL-1b reverse, CTC GGA GCC TGT AGT GCA; b-actin forward, ATC CTG AAA GAC CTC TAT GC; b-actin reverse, AAC GCA GCT CAG TAA CAG TC. The b-actin was used as an internal control to evaluate relative expression of iNOS, TNF-a and IL-1b. The results are representative of three independent experiments.
2. Materials and methods
2.5. Immunofluorescence assay
2.1. Reagents and cell cultures
The effect of antidepressants on the nuclear translocation of a p65 subunit of NF-kB was examined by an immunofluorescence assay as described (Sung et al., 2007). For the detection of the intracellular location of p65 subunit of NF-kB, BV-2 microglia cells (1 105 cells/well in 24-well plates) were cultured on sterile cover slips in 24-well plates and treated with drugs and LPS. At 1 h after the LPS stimulation, the cells were fixed with methanol for 20 min at 20 C and washed with PBS for 5 min. The fixed cells were then permeabilized with 0.5% Triton X-100 in PBS for 1 h at room temperature, washed with 0.05% Tween-20 in PBS for 10 min and 0.05% Tween-20/1% BSA in PBS for 5 min. The permeabilized cells were
Bacterial lipopolysaccharide (LPS) (Escherichia coli serotype 055:B5), clomipramine, and imipramine were purchased from Sigma–Aldrich (St. Louis, MO). The drugs were dissolved in phosphate-buffered saline (PBS; 150 mM NaCl, 5 mM phosphate, pH 7.4). Recombinant mouse IFN-g was purchased from R&D Systems (Minneapolis, MN). A BV-2 murine microglia cell line (Bocchini et al., 1992) was grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), gentamicin (50 mg/ml) at
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then treated with 1 mg/ml of mouse monoclonal anti-human NF-kB p65 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 h at room temperature, washed with 0.05% Tween-20/1% BSA in PBS for 5 min. Cells were then incubated in a 1:2000 dilution of Alexa Fluor 488-labeled goat anti-mouse IgG antibody (Molecular Probes Inc., Eugene, OR) for 1 h at room temperature, and washed with 0.05% Tween-20 in PBS for 5 min and PBS for 5 min. Cells were then stained with 0.5 mg/ ml of Hoechst staining solution (Molecular Probes) for 20 min at 37 C and then washed. Finally, the cover slips with cells were dried in a 37 C oven for 45 min and mounted in a 1:1 mixture of xylene and malinol. The number of cells with p65 nuclear translocation was determined under a fluorescence microscope. More than 100 cells with p65 translocation were counted. The results are representative of three independent experiments. 2.6. Western blot analysis Cells were lysed in triple-detergent lysis buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride]. Protein concentration in cell lysates was determined using a protein assay kit (Bio-Rad, Hercules, CA). An equal amount of protein from each sample was separated by SDSpolyacrylamide gel electrophoresis (12% gel) and transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies [rabbit polyclonal anti-human IkB-a (Santa Cruz Biotechnology), rabbit polyclonal anti-human phospho-p38 MAPK (Cell Signaling Technology Inc., Beverly, MA), monoclonal anti-a-tubulin clone B-5-1-2 mouse ascites fluid (Sigma)] and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and anti-mouse; Amersham Biosciences) followed by enhanced chemiluminescence detection (Amersham Biosciences). The results are representative of three independent experiments. 2.7. Enzyme-linked immunosorbent assay (ELISA) TNF-a secreted in microglial culture supernatants was measured as described (Park et al., 2002) by specific ELISA using rat monoclonal anti-mouse TNF-a antibody as capture antibody and goat biotinylated polyclonal anti-mouse TNF-a antibody as detection antibody (ELISA development reagents; R&D Systems). The biotinylated anti-TNF-a antibody was detected by sequential incubation with streptavidin– horseradish peroxidase conjugate and chromogenic substrates. The data are representative of results obtained from three independent experiments, which was performed in triplicate (mean S.D., n ¼ 3). Interassay CV was 4.9% for clomipramine or 2.4% for imipramine, respectively.
Fig. 1. Effects of the antidepressants on NO production in LPS-activated BV-2 microglia. BV-2 microglia cells (8 104 cells/well in a 96-well plate) were incubated with 100 ng/ml of LPS in the presence or absence of antidepressants (10, 30 mM) for 24 h. The amounts of nitrite in the supernatants were measured using Griess reagent (A). Cell viability was examined by MTT reduction assays and the results were expressed as the percentage of surviving cells over control cells (B). The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
2.8. Microglia/neuron co-culture For the co-culture experiment, BV-2 microglia cells or HAPI rat microglia cells were seeded in triplicate at the density of 1.5 104 cells/well in 96-well plate. BV-2 microglia cells or HAPI rat microglia cells were pretreated with drugs for 30 min. Then, culture supernatants were discarded and 100 ng/ml of LPS was added together with B35 rat neuroblastoma cells stably expressing EGFP (3.75 104 cells/well), which was followed by the co-culture for 24 h. The numerical ratio of microglia to neuron was 1:2.5. Afterwards, the EGFP-positive cells were counted under a fluorescence microscope (Olympus IX 70, Tokyo, Japan). Images of three random fields per well were captured and analyzed by the MetaMorph imaging system (Universal Imaging Corp, West Chester, PA). LPS alone did not affect B35 neuroblastoma cell viability (data not shown). The results are representative of three independent experiments. 2.9. Statistical analysis Results were expressed as mean S.D. The data were analyzed by one-way ANOVA following the Student Newman Keul’s post hoc analysis using SPSS program (version 12.0). A value of P < 0.05 was considered statistically significant.
for their inhibitory activity (Fig. 2A). Clomipramine of 10–20 mM and imipramine of 5–15 mM inhibited microglial NO production in a dose-dependent manner (Fig. 2A) without a significant cytotoxicity (Fig. 2B). The LPS-induced NO production was similarly decreased by the tricyclic antidepressants in mouse primary microglia cultures (Fig. 2C) and HAPI rat microglia cells (Fig. 2D), indicating that the NO inhibitory effects of the antidepressants are not limited to BV-2 cells. The drugs at the concentration tested did not affect the viability of either primary microglia cultures or HAPI rat microglia cells (data not shown). The antiinflammatory activity of the tricyclic antidepressants clomipramine (15 mM) and imipramine (10 mM) was further studied. Because NO and TNF-a are the major neurotoxic factors under neuroinflammatory condition, the possibility of whether clomipramine and imipramine reduce the production of TNF-a has also been tested. Clomipramine and imipramine also decreased TNF-a production in LPS-stimulated BV-2 microglia cells (Fig. 3).
3. Results 3.1. Inhibitory effects of antidepressants on NO and TNF-a production in microglia First, the effects of clomipramine and imipramine (10, 30 mM) on NO production in LPS-stimulated BV-2 mouse microglia cell line were evaluated (Fig. 1A). In order to rule out the possibility that the reduction of NO production may be due to the cytotoxicity of the drugs, cell viability was evaluated (Fig. 1B). Because 30 mM of clomipramine and imipramine was apparently toxic to BV-2 microglia cells, the drugs at lower concentrations were reassessed
3.2. Antidepressants inhibited the expression of iNOS, IL-1b, and TNF-a genes In order to investigate the effects of clomipramine and imipramine on the gene expression of inflammatory mediators at the transcriptional level, the levels of iNOS, IL-1b, and TNF-a mRNA in the LPS-stimulated BV-2 microglia cells were determined by RT-PCR analysis. The antidepressants inhibited the LPS-induced expression of iNOS, IL-1b, and TNF-a, whereas the drugs alone did not induce any significant change of the gene expression (Fig. 4).
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Fig. 2. Effects of the antidepressants on NO production in the LPS-stimulated BV-2 and HAPI microglia cell lines or primary microglia cultures. BV-2 mouse microglia cells (A), mouse primary microglia cultures (C) or HAPI rat microglia cells (D) (8 104 cells/well in a 96-well plate) were incubated with 100 ng/ml of LPS in the presence or absence of clomipramine (10–20 mM) or imipramine (5–15 mM) for 24 h. The amounts of nitrite in the supernatants were measured using Griess reagent. Cell viability was examined by MTT reduction assays and the results were expressed as the percentage of surviving cells over control cells (B). The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
3.3. Antidepressants attenuated the LPS-induced NF-kB and p38 MAPK activation NF-kB and p38 MAPK are key upstream regulators that induce proinflammatory cytokines and iNOS gene expression in glia cells (Da Silva et al., 1997; Jongeneel, 1995). Therefore, it was determined whether the antiinflammatory effects of the antidepressants occurred through the blockade of NF-kB and p38 MAPK activation in BV-2 microglia cells. The processes of NF-kB activation include IkB degradation and a subsequent nuclear translocation of p65 subunit of NF-kB. LPS induced the translocation of p65 into the nucleus within 60 min after the stimulation, which was inhibited by the antidepressants as determined by an immunofluorescence assay (Fig. 5A) and subsequent enumeration of the cells with p65 translocation (Fig. 5B). Western blot analysis further showed that degradation of IkB-a after 30 min stimulation with LPS was markedly inhibited by the antidepressants (Fig. 5C). After stimulation of BV-2 microglia cells with LPS for 30 min, activation of p38 MAPK was also observed as determined by Western blot analysis using antibody specific for phospho-p38 MAPK. Clomipramine and imipramine significantly inhibited the phosphorylation of p38 MAPK (Fig. 5C). These results indicated that the antidepressants suppressed LPS-induced NF-kB and p38
MAPK activation in microglia, and that the inhibition of iNOS, IL-1b, and TNF-a gene expression by the antidepressants was likely due to the blockade of NF-kB and p38 MAPK pathways. 3.4. Antidepressants inhibited microglial neurotoxicity in a microglia/neuron co-culture model To investigate potential neuroprotective effects of clomipramine and imipramine in vitro, a microglia/neuron co-culture model was used. As various proinflammatory mediators produced by activated microglia can induce neuronal cell death and amplify progression of neuronal degeneration (Stoll and Jander, 1999; Streit et al., 1999), inhibition of microglial activation may be neuroprotective. Thus, microglial neurotoxicity and neuroprotective effects of a compound can be tested in a microglia and neuron co-culture, where activated microglia may induce neuronal cell death. A compound can be considered neuroprotective, if it protects neurons against the cytotoxic effect of activated microglia in the co-culture of microglia and neurons. This possibility of the antidepressants was tested using a co-culture of B35 neuroblastoma cells with LPS-activated BV-2 microglia cells. B35 neuroblastoma cells that have been stably transfected with EGFP expression construct were employed for the simple identification of neuroblastoma cells in the co-culture. BV-2 microglia cells were activated with LPS with or without
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pretreatment with the antidepressants, and then co-cultured with B35 neuroblastoma cells (see Fig. 6A for the co-culture scheme). The viability of B35 cells was measured by counting the EGFPexpressing cells after the co-culture under a fluorescence microscope, because B35 cells stably expressed EGFP. This co-culture system has been successfully used for the determination of microglial neurotoxicity (Kim et al., 2007). Clomipramine (10 mM) and imipramine (15 mM) almost completely inhibited B35 cell death in the co-culture (Fig. 6B, C). The neuroprotective effect of antidepressants was also examined in the co-culture of HAPI rat microglia cells and B35 rat neuroblastoma cells. A similar result of neuroprotection was obtained (data not shown). The results indicate that clomipramine and imipramine may be neuroprotective by suppressing microglial neurotoxicity. 3.5. Effects of the antidepressants on the inflammatory activation of RAW 264.7 macrophage cells and primary astrocyte cultures
Fig. 3. Effects of the antidepressants on TNF-a production in LPS-stimulated BV-2 microglia cells. BV-2 microglia cells (8 104 cells/well in a 96-well plate) were incubated with 100 ng/ml of LPS in the presence or absence of clomipramine (15 mM) and imipramine (10 mM) for 24 h. The amounts of TNF-a in the supernatants were measured by ELISA. The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
Effects of the antidepressants on the NO production in RAW 264.7 macrophage cells and primary astrocyte cultures were tested. Clomipramine and imipramine significantly decreased NO production in LPS-stimulated RAW 264.7 macrophage cells (Fig. 7A) and primary astrocyte cultures (Fig. 7B). When astrocytes were stimulated with LPS plus IFN-g, greater amounts of NO production were achieved, which were similarly inhibited by the antidepressants (Fig. 7C). These results indicated that the antidepressants also have antiinflammatory effects in peripheral macrophages and astrocytes.
Fig. 4. Effects of the antidepressants on proinflammatory cytokine genes expression in LPS-simulated BV-2 microglia cells. BV-2 cells were treated with LPS (100 ng/ml) in the absence or presence of antidepressants (15 mM of clomipramine or 10 mM of imipramine), and total RNA was isolated at 6 h after the treatment. The levels iNOS, IL-1b, and TNFa mRNA were determined by RT-PCR (upper) and then subjected to densitometric quantification (lower). Levels of iNOS, IL-1b and TNF-a mRNA were normalized to b-actin levels and expressed as a relative change in comparison to the LPS treatment, which was set to 100% (lane 2). The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
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Fig. 5. Blockade of NF-kB activation and p38 MAPK phosphorylation by the antidepressants. The BV-2 microglia cells were seeded in triplicate at the density of 1 105 cells/well in a 24-well plate. The cells were stimulated with 100 ng/ml of LPS in the absence or presence of clomipramine (15 mM) and imipramine (10 mM) that had been added 30 min before the stimulation. At 1 h after the LPS addition, subcellular location of NF-kB p65 subunit was determined by an immunofluorescence assay (A). The p65 protein was detected using anti-p65 antibody conjugated with fluorescein isothiocyanate (FITC). Representative images of cells are shown (left). Magnification, 200; scale bar, 50 mm. Boxed rectangular regions were enlarged (right). The number of cells with p65 nuclear translocation was determined and the percentage of cells with p65 translocation was calculated: more than 100 cells were counted (B). Total cell lysates obtained 10, 30 and 60 min after the LPS stimulation were subjected to Western blotting to assess the levels of IkB-a or phospho-p38 MAPK proteins (C) (upper). Quantification of IkB-a or phospho-p38 protein levels was performed by densitometric analysis (lower). Detection of a-tubulin was done to confirm the equal loading of the samples. The values were expressed as a percentage of maximal band intensity in the microglia cell culture treated with LPS alone, which was set to 100% (lane 3). The data are the mean S.D. (n ¼ 3), and are representative of three or more independent experiments. *P < 0.01, **P < 0.001; significantly different from the value in cells treated with LPS only.
4. Discussion In the present study, it has been demonstrated that tricyclic antidepressants such as clomipramine and imipramine inhibit
inflammatory activation of microglia and astrocytes. Clomipramine and imipramine significantly reduced NO or TNF-a production in LPS-stimulated BV-2 microglia cells, primary microglia or astrocyte cultures. The RT-PCR analysis showed that these antidepressants
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Fig. 6. Effects of the antidepressants on B35 rat neuroblastoma cell viability in a microglia/neuron co-culture. The BV-2 microglia cells were seeded in triplicate at the density of 1.5 104 cells/well in a 96-well plate. Microglia cells were pretreated with clomipramine (15 mM) or imipramine (10 mM) for 30 min. Culture supernatants were discarded and stimulated with 100 ng/ml of LPS. At the same time, rat B35 neuroblastoma cells (3.75 104 cells/well) stably expressing EGFP were added onto BV-2 cells, and then co-cultured for 24 h (A). After the co-culture for 24 h, the EGFP-positive cells were counted under a fluorescence microscope to evaluate B35 neuroblastoma cell death (B). Representative images of cells are shown (magnification, 100); scale bar, 100 mm. The number of fluorescent cells in several randomly chosen microscopic fields per well was determined, and the data were expressed as the mean S.D. (n ¼ 3) (C). The results are representative of three independent experiments. *P < 0.001; significantly different from the value in cells treated with LPS in the absence of antidepressants.
markedly suppressed the iNOS, TNF-a, and IL-1b gene expression at the transcriptional level. NF-kB and p38 MAPK pathways were at least partly involved in the antiinflammatory mechanisms of the antidepressants. In addition, these antidepressants showed neuroprotective effects by attenuating microglial neurotoxicity in a microglia/neuron co-culture system. In the resting state, microglia typically exist with ramified morphology and monitor the brain environment (Nimmerjahn et al., 2005). However, under the neurodegenerative conditions, they are activated and release various inflammatory mediators including proinflammatory cytokines and free radicals such as NO and superoxide anion (Aloisi, 2001). Because NO is one of the main proinflammatory mediators and plays an important role in neuroinflammatory diseases, the effects of the antidepressants on the NO production in LPS-stimulated microglia cells were examined. Based on the strong inhibitory effect of clomipramine and imipramine on the NO production, further studies were focused on these drugs. Clomipramine and imipramine as tricyclic antidepressants
exhibited a strong antiinflammatory property (such as inhibition of proinflammatory cytokine expression and production, NF-kB, and p38 MAPK) in brain microglia cells as well as astrocytes in the current study. These results are consistent with previous reports where clomipramine and imipramine decreased IL-1b and TNFa production in human monocytes (Xia et al., 1996), and imipramine inhibited IL-6 and NO production in IFN-g-activated 6-3 mouse microglia cell line (Hashioka et al., 2007). Clomipramine and imipramine in therapeutic doses reach the levels of 0.28–0.80 and 0.16–0.54 mM, respectively, in the plasma (Wille et al., 2008, 2001). Clomipramine and imipramine are reported to accumulate in brain tissue 12.5-fold and 32-fold higher than in plasma or serum levels, respectively (DeVane et al., 1984; Weigmann et al., 2000), which is comparable to the concentrations of the drugs used in the present study. NF-kB is a key transcription factor activated by several cellular signal transduction pathways that play an important role in the expression of proinflammatory cytokines and enzymes such as
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Fig. 7. Effects of the antidepressants on the LPS-activated NO production in RAW 264.7 macrophage cells or primary astrocyte cultures. Either RAW 264.7 macrophage cells or primary astrocyte cultures (8 104 cells/well in a 96-well plate) were incubated with 100 ng/ml of LPS or LPS/IFN-g (50 unit/ml) in the presence or absence of clomipramine (15 mM) or imipramine (10 mM) for 24 h. The amounts of nitrite in the supernatants were measured using Griess reagent (A, RAW 264.7 cells; B and C, primary astrocyte cultures). The data were expressed as the mean S.D. (n ¼ 3), and are representative of results obtained from three independent experiments. *P < 0.05, **P < 0.01; significantly different from the value in cells treated with LPS only.
iNOS, IL-1b, and TNF-a (Baeuerle and Henkel, 1994; Baldwin, 2001). The molecular mechanisms of NF-kB activation have been well studied, and they involve a cascade activation of cytoplasmic proteins and the ultimate nuclear translocation of p65 subunit of NF-kB (Delhase et al., 2000; Karin and Ben-Neriah, 2000). LPS causes the nuclear translocation of p65 subunit of NF-kB thorough IkB degradation. In the present study, it was found that clomipramine and imipramine inhibited IkB degradation and the subsequent nuclear translocation of p65 in BV-2 microglia cells (Fig. 5). Previously, tricyclic antidepressants including clomipramine and imipramine inhibited NO production in monocytes or microglia by the cAMP-dependent protein kinase (PKA) pathway (Hashioka et al., 2007; Xia et al., 1996). Because activation of the cAMP-dependent PKA pathway is believed to negatively regulate the NF-kB activation in a number of cell types (Delfino and Walker, 1999; Pahan et al., 1997), it is speculated that the cAMP–PKA pathway may be also involved in the antiinflammatory effects of the antidepressants in our model system. Additionally, it was found that clomipramine and imipramine inhibited LPS-induced activation of p38 MAPK (Fig. 5C), which has been previously implicated in the signal transduction pathways responsible for the induction of iNOS and TNF-a gene expression in glial cells or macrophages (Kim et al., 2004; Park et al., 2007; Pawate and Bhat, 2006). Taken together, the current results indicated that clomipramine and imipramine inhibited the gene expression of iNOS, TNF-a, and IL-1b in microglia, the mechanism of which at least in part might involve the inhibition of NF-kB and p38 MAPK activation. It should be, however, noted that the gene expression was measured by RT-PCR, which is only a semi-quantitative methodology. Activation of glial cells (microglia and astrocytes) and inflammatory products derived from them have been implicated in disease progression and pathology in several neuroinflammatory diseases such as Alzheimer’s disease, Parkinson’s disease, and HIV dementia (Block et al., 2007). Glial activation has both destructive
and protective effects on neuronal injury in neurodegenerative diseases. Over-activation of glia, however, may contribute to neurodegenerative processes through the production of various neurotoxic factors including free radicals and proinflammatory cytokines (Klegeris et al., 2007). In fact, a number of antiinflammatory agents, which inhibited glial activation or production of proinflammatory mediators under the CNS disease conditions, attenuated neuronal degeneration (Esposito et al., 2007; Lee et al., 2003; McGeer and McGeer, 2007; Tikka et al., 2001a,b). Thus, it is suggested that a search for the efficient antiinflammatory compounds that attenuate glial activation may lead to an effective therapeutic approach against many neurodegenerative conditions. A recent study has demonstrated that imipramine treatment protects LPS-induced apoptosis in hippocampus-derived neural stem cells through activation of the BDNF and MAPK/ERK pathway (Peng et al., 2008). The current study showed that clomipramine and imipramine protected neuroblastoma cells against microglial neurotoxicity in a microglia/neuron co-culture (Fig. 6). The neuroprotective effect of the antidepressants is likely due to the inhibition of microglia activation, but not protective action on the neuroblastoma cells, because the antidepressants-pretreated microglia cultures were washed before the addition of the neuroblastoma cells for the co-culture. The B35 neuroblastoma cells were not exposed to the antidepressants. Therefore, the neuroprotective activity of imipramine that has been previously observed (Peng et al., 2008) may be partly due to its inhibitory effects on the neurotoxic microglia activation. Although the co-culture of the LPSstimulated microglia with neuroblastoma cell line may not be the same as the in vivo conditions, it partially reflects the pathological condition where activated microglia influence the death and survival of neuronal cells in neurodegenerative diseases. Further studies are, however, required to evaluate a neuroprotective property of the antidepressants in the animal models of neurodegenerative diseases and to understand the precise molecular
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