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Sertraline promotes hippocampus-derived neural stem cells differentiating into neurons but not glia and attenuates LPS-induced cellular damage
Progress in Neuro-Psychopharmacology & Biological Psychiatry 36 (2012) 183–188
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Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
Sertraline promotes hippocampus-derived neural stem cells differentiating into neurons but not glia and attenuates LPS-induced cellular damage Zheng-Wu Peng a, 1, Yun-Yun Xue a, 1, Hua-Ning Wang a, Huai-Hai Wang a, Fen Xue a, Fang Kuang b, Bai-Ren Wang b, Yun-Chun Chen a, Li-Yi Zhang c, Qing-Rong Tan a,⁎ a b c
Department of Psychosomatic Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China Institute of Neuroscience, Fourth Military Medical University, Xi'an 710032, China 102 Hospital, Changzhou 213003, China
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Article history: Received 28 July 2011 Received in revised form 17 August 2011 Accepted 17 August 2011 Available online 25 August 2011 Keywords: Neural stem cells Neuronal differentiation Neuroprotective effect Sertraline
a b s t r a c t Sertraline is one of the most commonly used antidepressants in clinic. Although it is well accepted that sertraline exerts its action through inhibition of the reuptake of serotonin at presynaptic site in the brain, its effect on the neural stem cells (NSCs) has not been well elucidated. In this study, we utilized NSCs separated from the hippocampus of fetal rat to investigate the effect of sertraline on the proliferation and differentiation of NSCs. The study demonstrated that sertraline had no effect on NSCs proliferation but it significantly promoted NSCs to differentiate into serotoninergic neurons other than glia cells. Furthermore, we found that sertraline protected NSCs against the lipopolysaccharide-induced cellular damage. These data indicate that sertraline can promote neurogenesis and protect the viability of neural stem cells. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Depression is one of the most prevalent forms in psychopathology, and afflicts people in their whole life span owing to the recurrent feature (Burcusa and Iacono, 2007; Klerman and Weissman, 1988; Lecrubier, 2001). It is well accepted that complex dysregulation of the serotoninergic system plays a crucial role in the occurrence of the depressive disorders (Schloss and Williams, 1998). Some studies also suggest that inflammation and cell-mediated immune activation are important factors in the pathogenesis of depression (Maes et al., 1990, 1991; Mikova et al., 2001). Recent studies further implicate that the regulation of neurogenesis in adult brain is a possible target for the action of antidepressant drugs (Kodama et al., 2004; Manev et al., 2001). Selective serotonin reuptake inhibitor (SSRI) drugs produce relatively rapid blockade of serotonin (5-HT) transporters and elevate the serotonin concentration in synaptic cleft. However, their therapeutic effect lags for 3 to 4 weeks in clinic. In recent years, more
Abbreviations: SSRIs, selective serotonin reuptake inhibitors; NSCs, neuronal stem cells; BDNF, brain-derived neurotrophic factor; DMEM, Dulbecco's modified Eagle's medium; bFGF, fibroblast growth factor-basic; EGF, epidermal growth factor; EDTA, ethylene diamine tetraacetic acid; WST-1, 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; PB, phosphate buffer; BSA, bovine serum albumin; RT, room temperature; LDH, lactate dehydrogenase; LPS, lipopolysaccharide. ⁎ Corresponding author. Fax: +86 29 83293951. E-mail address: [email protected] (Q.-R. Tan). 1 Zheng-Wu Peng and Yun-Yun Xue contributed equally to this work. 0278-5846/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2011.08.014
and more data from pathological observation have shown that hippocampal atrophy always occurs in depressed patients (Chen et al., 2010; Sapolsky, 2000; Sheline et al., 1996), and antidepressants could promote neurogenesis demonstrated in vivo (Encinas et al., 2006; Hitoshi et al., 2007; Marlatt and Lucassen, 2010). Many researchers have focused on the role of neuronal stem cells (NSCs) in the functional mechanisms of antidepressants (Chiou et al., 2006; Huang et al., 2007). Sertraline is a widely used selective serotonin reuptake inhibitor (SSRI), whose antidepressive efficacy has been well established in clinical trials (Fava et al., 2000; Lepine et al., 2004; Schneider et al., 2003; Swenson et al., 2003). Studies have demonstrated that chronic but not acute administration of sertraline stimulates neurogenesis (Malberg and Blendy, 2005), and may have a beneficial effect on neurons (Peng et al., 2008). Increasing evidences have indicated that sertraline has a variety of protective effects on neurodegeneration caused by oxidative stress or disrupted energy metabolism (Kumar and Kumar, 2009), changing hippocampal structure (Bossini et al., 2007), modulating ATP and adenosine levels (Pedrazza et al., 2007), and up-regulating brain-derived neurotrophic factor (BDNF) (Matrisciano et al., 2009). It is shown that sertraline also slows disease progression and increase neurogenesis in the mouse model of Huntington's disease (Duan et al., 2008). However, there have only a few studies on the effect of sertraline on the NSCs, and no data are available whether sertraline acts on the proliferation or differentiation of NSCs. To further explore the pharmacology of sertraline, in this experiment, we used cell culture to investigate the effect of sertraline on NSCs from embryonic rat hippocampus.
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2. Materials and methods
2.5. Immunohistochemistry
2.1. Animals
To verify the identity of NSCs, neurospheres were collected and postfixed in 4% paraformaldehyde for 2 h at 4 °C before being moved into 20% sucrose in 0.1 M phosphate buffer (PB) overnight at 4 °C for cryoprotection. Frozen sections (10 μm) were cut with a cryostat and mounted onto gelatinized slides. Single cells were plated onto the poly-L-lysine coated coverslips. After 6 h of attachment, the cells on the slides were fixed in 4% paraformaldehyde in PBS, and then processed for immunocytochemical staining. For examination of NSCs differentiation, cells were seeded on poly-D-lysine-coated coverslips in triplicate at the density of 2 × 10 4 cells/well. After the cells were treated with sertraline, immunohistochemistry were carried out. The cells were fixed in a PBS solution containing 4% paraformaldehyde for 1 h, and washed 3 times with PBS. A PBS solution containing 1% bovine serum albumin (BSA) and 0.3% Triton was then added to the cells for 30 min at room temperature (RT). After removing this blocking reagent, cells were incubated in a humidity chamber at 4 °C overnight with the first antibodies composed of mouse anti-nestin (1:500, Abcam, U.K.), mouse anti-Tuj (1:500, Sigma, USA), mouse anti-CNPase (1:500, Abcam, U.K.), rabbit antiGFAP (1:2000, Millipore, USA) and rabbit anti-TPH1 (1:200, EPITOMICS, USA) diluted in blocking reagent. Then cells were washed 3 times with PBS and incubated for 2 h in the dark at room temperature in the presence of the fluorescent secondary antibodies (Alexa fluor 594 donkey anti-mouse IgG, 1:800, and Alexa fluor 488 donkey anti-rabbit IgG, 1:400, Invitrogen, USA). Finally, the cells were incubated with DAPI for 20 min at room temperature to stain the cellular nuclei. Finally, the coverslips were mounted onto slides in PBS/ glycerol (vol/vol). The preparations were analyzed under a fluorescent Olympus BX-51 microscope or a laser scanning confocal microscope (FV-1000, Olympus), and the positive cell was measured by using Image-Pro plus software (version 6.0, Media Cybernetics, USA).
The experimental protocol used in this study was approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and was conducted according to the Guidelines for Animal Experimentation of the university. Sprague–Dawley rats were purchased from Experimental Animal Center of the university. All efforts were made to minimize animals' suffering and to keep the numbers of animals used to a minimum. Mature female Sprague–Dawley rats (3 months old) at proestrus were placed overnight with mature males. And the females were examined for pregnancy by the presence of a vaginal plug or spermatozoa in the vagina.
2.2. Neural stem cell culture Embryonic brains between E14.5 and E16.5 were dissected under a stereomicroscope, and single cell suspensions of cortex and hippocampus were obtained by mechanical dissociation. Cells were primarily plated at a density of 1 × 10 5 cells/ml in culture bottle (4 ml/bottle), and grown in serum-free Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 20 ng/ml bFGF (human recombinant, Peprotech, 100-18B, USA), 20 ng/ml EGF (rat recombinant, Peprotech, 400-25, USA), B-27 and N-2 supplement (Gibic, USA), and penicillin and streptomycin (Tropepe et al., 1999). After 7 days' culture, the primary neurospheres were passaged. Briefly, the neurospheres were collected and dissociated with 0.05% trypsin and 200 μM EDTA for 10 min at 37 °C, then mechanically triturated with fire-polished glass pipettes. Then trypsin inhibitor (Sigma, USA) was added and laid for 5–8 min at room temperature to stop digestion. After filtering with 200 wells filter, filtered liquid was centrifuged for 5 min at 1 000 r/min. Then the single cells were resuspended. To assess the self-renewal ability, neurospheres were dissociated into a single cell suspension, and were re-plated at a density of 2 × 10 4 cells/ml in 24-well plates (0.4 ml/well). Cells were then cultured under the same condition as described above for a further 7 days and new neurospheres grew (Hitoshi et al., 2002). For the differentiation of neural stem cells, single cells were plated onto polylysine (50 mg/ml, Sigma, USA)-coated coverslips, in a 24-well plate, and the medium was changed to DMEM/F12 with 5% fetal bovine serum (Sigma, USA) to allow differentiation.
2.3. Drug treatment Sertraline (Pfizer) was dissolved by alcohol, and then diluted to different concentrations of stock solutions (10 folds of final concentration) by stem cell medium, with the final the concentration of alcohol at 0.5%. After replanting, NSCs were treated with sertraline by adding stock solutions to the medium.
2.4. Determination of cell viability Cell viability was determined by 2-(4-Iodophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1, Roche) assay. Neural stem cells were seeded at a density of 5 × 103 cells/well in 96-well plate. WST-1 was added to each well, and incubated for 4 h at 37 °C. The optical density was measured at 450 nm by a microplate reader. The data are representative as an average obtained from three independent experiments, which was performed at least 5 times (mean ± S.D., n = 5).
2.6. Western blotting analysis Cells were lysed with SDS-PAGE sample buffer composed of 62.5 mM Tris–HCl, 2% w/v SDS, 10% glycerol, 50 mM DTT, and 0.1% w/v bromphenol blue, and the insoluble materials were separated by centrifugation at 12,000 g for 10 min. The supernatant was heated at 100 °C for 10 min, and cooled on ice for 30 min afterwards. Electrophoresis was carried out by SDS-PAGE by using 10% polyacrylamide in accordance with routine protocols. Then the proteins in PAGE were transferred onto nitrocellulose membranes and blocked in blocking solution containing 5% defatted milk powder, 0.1% Tween20 in TBS for 1 h at RT with gentle shaking. After being washed in TBS for 3 times 8 min each, the following primary antibodies were used for incubation overnight at 4 °C: mouse anti-Tuj (1:2000, Sigma, USA), mouse anti-CNPase (1:1000, Abcam, U.K.), rabbit antiGFAP (1:5000, Millipore, USA) and mouse anti β-actin antibody (1:10,000, sigma, USA) as a loading control. Then the membranes were washed 3 times in TBS again, and incubated with peroxidaseconjugated goat anti-rabbit IgG or peroxidase-conjugated goat antimouse IgG in TBST for 1 h. After washing 3 times in TBS for 8 min each, the membrane was detected using a chemiluminescence detection kit (Supersignal west pico chemiluminescent substrate, Thermo, USA), and the immunoreactive proteins were then visualized on X-ray film and digitized. 2.7. Measurement of lactate dehydrogenase (LDH) activity In order to estimate the protective effect of sertraline on lipopolysaccharide (LPS) induced cell death, the level of lactate dehydrogenase (LDH) released from damaged cells into culture media was measured at 24 h and 48 h after the cells were treatment of cells with 200 ng/ml LPS. Cell culture media were collected and release of
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lactate dehydrogenase (LDH) from cells was evaluated using a Cytotoxicity Detection Kit (NJJC, China). Absorbance was measured at 492 nm with a microplate reader. The data are presented as the value averaged from three separate experiments (6 wells per one experiment). 2.8. Data analysis Statistical analysis was performed with SPSS software (SPSS Inc., Chicago, IL). All the data were shown as means ± SEM. Differences were subjected to statistical analysis by one-way ANOVA. Differences were considered significant when P b 0.05, and considered as highly significant when P b 0.01.
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As shown in Fig. 4, by using the immunofluorescent staining, the percentage of numbers of differentiated neurons (Tuj +/DAPI + cell) were significantly increased in 5 μM (df = 4, F = 121.94, P = 0.000) and 10 μM (P = 0.001) sertraline-treated group. There was no significant difference between other smaller doses of sertraline groups and control (Fig. 4C). The triple-staining immunofluorescent staining was used to further identify the serotoninergic feature of differentiated neurons. Neurons immunoreactive for TPH1 (marker for serotoninergic neurons, green), Tuj (marker for differentiated neurons, red), as well as stained by DAPI (nuclei staining; blue) were detected at 7 days after sertraline treatment (Fig. 4B). 3.3. The protective effect of sertraline on LPS-induced cell damage in NSCs
3. Results 3.1. Identification of the NSCs and effects of sertraline on their proliferation Immunofluorescent staining demonstrated that both the neurospheres and single NSCs expressed nestin, the marker for NSCs (Fig. 1). Sertraline did not affect the cell viability, as observed 1 week after treatment at the doses tested. Inversely, the cell viability was significantly decreased at 20 μM and 50 μM of sertraline (df = 6, F = 44.901, P = 0.000) administration. Therefore, the doses of 20 μM and 50 μM were not chosen for the subsequent experiments. Ability of proliferation of NSCs was analyzed by measuring the diameter of neurospheres. The diameter of NSCs neurospheres was significantly decreased in 20 μM (df = 5, F = 24.50, P = 0.000) and even no neurospheres were formed in 50 μM group. There were no significant differences detected in other small doses-given groups (Fig. 2A, B). The results indicate that the proliferation of NSCs is not facilitated after direct administration of sertraline, but is inhibited when the concentrations of sertraline are high. 3.2. Effects of sertraline on differentiation of NSCs As shown in Fig. 3, after treatment with sertraline, the expression of GFAP were significantly decreased in 1 μM (df = 4, F = 121.94, P = 0.017), 5 μM (P = 0.000) and 10 μM (P = 0.005) sertralinetreated groups as demonstrated by Western blot analysis. And the expression of CNPase was significantly decreased as well in 5 μM (df = 4, F = 2.507, P = 0.025) sertraline-treated group, in comparison with the control. On the other hand, the expression of Tuj (the marker for differentiated neurons) was evidently increased in 5 μM (df = 4, F = 42.748, P = 0.000) and 10 μM (P = 0.001) group. The result indicating that sertraline promotes NSCs differentiated predominantly into neurons but inhibits its differentiation to glia cells.
After treated with different does of sertraline and LPS (200 ng/ml) for 24 or 48 h, as shown in Fig. 5, 5 μM sertraline treatment significantly eased the decrease of cell viabilities induced by LPS at 24 h (df = 3, F = 5.1, P = 0.03) and 48 h (df = 3, F = 13.654, P = 0.000) and diminished the leakage of LDH at 24 h (df = 3, F = 5.783, P = 0.007) and 48 h (df = 3, F = 5.036, P = 0.003), compared to control. And 1 μM sertraline decreased LDH level at 48 h (P = 0.27), but no detectable effect on cell viability. These findings imply that sertraline attenuates the cellular damage of NSCs induced by LPS. 4. Discussion The hippocampus has been shown undergoing morphological changes in response to stress, including atrophy and loss of CA3 pyramidal neurons after exposure to physical or psychosocial stress (McEwen, 1999), and antidepressant treatment attenuates neuronal cell death and increases neurogenesis in the dentate gyrus of the hippocampus (Jin et al., 2009; Malberg et al., 2000; Santarelli et al., 2003). It seems that increased neurogenesis in hippocampus by the administration of antidepressant is necessary in altered behavior in the stress-induced depression animal models and patients. Although the mechanism is largely unknown, recent evidence suggests that antidepressants influence important signaling pathways that regulate neuroplasticity and cell survival (Vaidya and Duman, 2001). NSCs have been defined as cells with the capacity of self-renewal and multi-lineage differentiation (Tropepe et al., 1999). In adult central nervous system (CNS), thousands of NSCs exist in the hippocampal dentate gyrus (Cameron and McKay, 2001). These cells may contribute to tissue repair in some neurological diseases and screen the candidate agents for neurogenesis in neurodegenerative diseases (Hitoshi et al., 2002). By using the in vitro culture of neural stem cells from hippocampus of fetal rats, our study demonstrated that sertraline could not
Fig. 1. Nestin staining for neurospheres (A) and single neural stem cells (NSCs) (B) and effect of sertraline on the cell viability of NSCs (C, n = 6 for each). In A and B the right microphotographs are nestin staining, the middle is DAPI staining to show the nuclei, and the right is the merged images of former two. Note in C that lower doses of sertraline administration did not affect the cell viability of NSCs, and high doses of sertraline even had inhibitive action. **P b 0.01 vs. other groups. Bar: 100 μm.
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Fig. 2. Microphotographs at left (A) and histograms at right (B) showing the effect of different concentrations of sertraline on the diameter of neurospheres observed 1 week following administration (n = 8 each group). Note that lower doses (0.5–10 μM) of sertraline did not affect the diameter of neurospheres, while 20 μM group significantly decreased the diameter of neurospheres, compared with other groups. In 50 μM group, no neurospheres were formed (data not shown). **P b 0.01 vs. other group. Bar: 100 μm.
increase proliferation and viability of NSCs. More importantly, by using triple-staining immunofluorescent and Western bolt assay, we provided the evidence that sertraline not only promoted NSCs differentiating into serotoninergic neuronal, but also inhibited NSCs differentiating into glial cells (Figs. 3, 4). In our study, after 7 days, 5 μM of sertraline stimulated the differentiation of fetus rat NSCs, mostly into TPH1 + neurons and decreased the proteins level of maker of oligodendroglial cell (CNPase) and astrocyte (GFAP). This observation is in agreement with previous reports in other antidepressants, such as lithium (Wexler et al., 2008) and fluoxetine (Chen et al., 2007). The ratio of Tuj + cell was 63.4% in 5 μM sertraline, higher than 54.1% in control group. Recent studies have shown evidences that major depression is accompanied by the activation of the inflammatory-response system (O'Brien et al., 2004; Penninx et al., 2003). LPS is a highly
proinflammatory component of outer member of gram-negative bacteria and has been tested as a stress model for inducing apoptosis (Gayle et al., 2002). We showed that sertraline can protect LPS-induced cellular damage. Administration of 5 μM of sertraline to 200 μg/ml LPS-treated NSCs reduced the LDH release and increased cell viability significantly compared to control group. This observation was in agreement with previous reports about other kind of antidepressants (Chiou et al., 2006). The mechanism underlying the neuroprotection by sertraline treatment on NSCs remains undetermined. But prior reports suggested that sertraline might provide neuroprotection by decreasing the expression of proinflammatory cytokine. Sutcigil et al. (2007) demonstrated that sertraline therapy might have exerted immunomodulatory effects through a decrease in the proinflammatory cytokine IL-12 and an increase in the anti-inflammatory cytokines IL-4 and TGF-β1, which may influence neurogenesis greatly (Monje et al., 2003).
Fig. 3. Evaluation of protein levels of GFAP (expressed by astrocytes), CNPase (expressed by oligodendrocytes), and Tuj (marker for differentiated neurons) in each group determined by Western blot (n = 5 each group). A is the representative bands at each group. B, C, and D are densitometric analysis for GFAP, CNPase and Tuj, respectively. * P b 0.05 vs. control. **P b 0.01 vs. control.
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Fig. 4. Microphotographs (A, B) and histograms (C) showing the effect of sertraline on differentiation of NSCs. (A) Immunofluorescent staining for Tuj, GFAP and their merged images of differentiated NSCs. (B) Detection of the protein expression of Tuj, DAPI, TPH1, and merged images in the same differentiated NSCs. (C) Statistical analysis for the percentage of number of Tuj-positive cells in different doses of sertraline-treated groups. Note that the percentage of numbers of Tuj+/DAPI+ cells (mean ± SD of 5 five separate experiments) was increased in 5 and 10 μM group. *P b 0.05 vs. control, **P b 0.01 vs. control. Bar: 100 μm.
From the therapeutic aspect of antidepressant treatment, altered levels of serotonin, neurogenesis in hippocampus, neuronal protection are closely associated with the pharmacologic action of antidepressants (Lesch, 2001; Malberg, 2004; McKernan et al., 2009). In this study, our
results indicated that sertraline elevated the serotoninergic neuronal differentiation, and protected NSCs from LPS-induced apoptosis. These results enrich our understanding for the pharmacological mechanism of sertraline. It is needed to note that the cells used in this experiment
Fig. 5. Evaluation of the effect of sertraline on cell viability and LDH level in NSCs at 24 h and 48 h after LPS treatment (n = 5 for each). Note that sertraline eased the decrease cell viability and diminished the elevation of LDH level in LPS-treated NSCs. * P b 0.05 vs. control, **P b 0.01 vs. control.
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were from normal animal. Thus, study on the effect of sertraline on NSCs from stressed animals is needed to be further investigated. Acknowledgments We are indebted to Dr. Bai-Ren Wang for his valuable advice and support. This work was supported by the National Natural Science Foundation (30870886, 30700259), the National Science and Technology Supporting Plan of the eleventh five-year (43730018-8) and the National Key Technology Support Program of China (2009BAI77B07). References Bossini L, Tavanti M, Lombardelli A, Calossi S, Polizzotto NR, Galli R, et al. Changes in hippocampal volume in patients with post-traumatic stress disorder after sertraline treatment. J Clin Psychopharmacol 2007;27:233–5. Burcusa SL, Iacono WG. Risk for recurrence in depression. Clin Psychol Rev 2007;27: 959–85. Cameron HA, McKay RD. Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 2001;435:406–17. Chen SJ, Kao CL, Chang YL, Yen CJ, Shui JW, Chien CS, et al. Antidepressant administration modulates neural stem cell survival and serotoninergic differentiation through bcl-2. Curr Neurovasc Res 2007;4:19–29. Chen MC, Hamilton JP, Gotlib IH. Decreased hippocampal volume in healthy girls at risk of depression. Arch Gen Psychiatry 2010;67:270–6. Chiou SH, Chen SJ, Peng CH, Chang YL, Ku HH, Hsu WM, et al. Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell. Biochem Biophys Res Commun 2006;343:391–400. Duan W, Peng Q, Masuda N, Ford E, Tryggestad E, Ladenheim B, et al. Sertraline slows disease progression and increases neurogenesis in N171-82Q mouse model of Huntington's disease. Neurobiol Dis 2008;30:312–22. Encinas JM, Vaahtokari A, Enikolopov G. Fluoxetine targets early progenitor cells in the adult brain. Proc Natl Acad Sci USA 2006;103:8233–8. Fava M, Rosenbaum JF, Hoog SL, Tepner RG, Kopp JB, Nilsson ME. Fluoxetine versus sertraline and paroxetine in major depression: tolerability and efficacy in anxious depression. J Affect Disord 2000;59:119–26. Gayle DA, Ling Z, Tong C, Landers T, Lipton JW, Carvey PM. Lipopolysaccharide (LPS)induced dopamine cell loss in culture: roles of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide. Brain Res Dev Brain Res 2002;133:27–35. Hitoshi S, Alexson T, Tropepe V, Donoviel D, Elia AJ, Nye JS, et al. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 2002;16:846–58. Hitoshi S, Maruta N, Higashi M, Kumar A, Kato N, Ikenaka K. Antidepressant drugs reverse the loss of adult neural stem cells following chronic stress. J Neurosci Res 2007;85:3574–85. Huang YY, Peng CH, Yang YP, Wu CC, Hsu WM, Wang HJ, et al. Desipramine activated Bcl-2 expression and inhibited lipopolysaccharide-induced apoptosis in hippocampus-derived adult neural stem cells. J Pharmacol Sci 2007;104:61–72. Jin Y, Lim CM, Kim SW, Park JY, Seo JS, Han PL, et al. Fluoxetine attenuates kainic acidinduced neuronal cell death in the mouse hippocampus. Brain Res 2009;1281: 108–16. Klerman GL, Weissman MM. The changing epidemiology of depression. Clin Chem 1988;34:807–12. Kodama M, Fujioka T, Duman RS. Chronic olanzapine or fluoxetine administration increases cell proliferation in hippocampus and prefrontal cortex of adult rat. Biol Psychiatry 2004;56:570–80. Kumar P, Kumar A. Possible role of sertraline against 3-nitropropionic acid induced behavioral, oxidative stress and mitochondrial dysfunctions in rat brain. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:100–8. Lecrubier Y. The burden of depression and anxiety in general medicine. J Clin Psychiatry 2001;62(Suppl. 8):4–9. [discussion 10–1]. Lepine JP, Caillard V, Bisserbe JC, Troy S, Hotton JM, Boyer P. A randomized, placebocontrolled trial of sertraline for prophylactic treatment of highly recurrent major depressive disorder. Am J Psychiatry 2004;161:836–42. Lesch KP. Serotonergic gene expression and depression: implications for developing novel antidepressants. J Affect Disord 2001;62:57–76.
Maes M, Bosmans E, Suy E, Vandervorst C, De Jonckheere C, Raus J. Immune disturbances during major depression: upregulated expression of interleukin-2 receptors. Neuropsychobiology 1990;24:115–20. Maes M, Bosmans E, Suy E, Vandervorst C, DeJonckheere C, Raus J. Depression-related disturbances in mitogen-induced lymphocyte responses and interleukin-1 beta and soluble interleukin-2 receptor production. Acta Psychiatr Scand 1991;84: 379–86. Malberg JE. Implications of adult hippocampal neurogenesis in antidepressant action. J Psychiatry Neurosci 2004;29:196–205. Malberg JE, Blendy JA. Antidepressant action: to the nucleus and beyond. Trends Pharmacol Sci 2005;26:631–8. Malberg JE, Eisch AJ, Nestler EJ, Duman RS. Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci 2000;20:9104–10. Manev H, Uz T, Smalheiser NR, Manev R. Antidepressants alter cell proliferation in the adult brain in vivo and in neural cultures in vitro. Eur J Pharmacol 2001;411:67–70. Marlatt MW, Lucassen PJ, van Praag H. Comparison of neurogenic effects of fluoxetine, duloxetine and running in mice. Brain Res 2010;1341:93–9. Matrisciano F, Bonaccorso S, Ricciardi A, Scaccianoce S, Panaccione I, Wang L, et al. Changes in BDNF serum levels in patients with major depression disorder (MDD) after 6 months treatment with sertraline, escitalopram, or venlafaxine. J Psychiatr Res 2009;43:247–54. McEwen BS. Stress and hippocampal plasticity. Annu Rev Neurosci 1999;22:105–22. McKernan DP, Dinan TG, Cryan JF. “Killing the Blues”: a role for cellular suicide (apoptosis) in depression and the antidepressant response. Prog Neurobiol 2009;88: 246–63. Mikova O, Yakimova R, Bosmans E, Kenis G, Maes M. Increased serum tumor necrosis factor alpha concentrations in major depression and multiple sclerosis. Eur Neuropsychopharmacol 2001;11:203–8. Monje ML, Toda H, Palmer TD. Inflammatory blockade restores adult hippocampal neurogenesis. Science 2003;302:1760–5. O'Brien SM, Scott LV, Dinan TG. Cytokines: abnormalities in major depression and implications for pharmacological treatment. Hum Psychopharmacol 2004;19: 397–403. Pedrazza EL, Senger MR, Pedrazza L, Zimmermann FF, de Freitas Sarkis JJ, Bonan CD. Sertraline and clomipramine inhibit nucleotide catabolism in rat brain synaptosomes. Toxicol In Vitro 2007;21:671–6. Peng Q, Masuda N, Jiang M, Li Q, Zhao M, Ross CA, et al. The antidepressant sertraline improves the phenotype, promotes neurogenesis and increases BDNF levels in the R6/2 Huntington's disease mouse model. Exp Neurol 2008;210:154–63. Penninx BW, Kritchevsky SB, Yaffe K, Newman AB, Simonsick EM, Rubin S, et al. Inflammatory markers and depressed mood in older persons: results from the Health, Aging and Body Composition study. Biol Psychiatry 2003;54:566–72. Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, et al. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 2003;301:805–9. Sapolsky RM. The possibility of neurotoxicity in the hippocampus in major depression: a primer on neuron death. Biol Psychiatry 2000;48:755–65. Schloss P, Williams DC. The serotonin transporter: a primary target for antidepressant drugs. J Psychopharmacol 1998;12:115–21. Schneider LS, Nelson JC, Clary CM, Newhouse P, Krishnan KR, Shiovitz T, et al. An 8week multicenter, parallel-group, double-blind, placebo-controlled study of sertraline in elderly outpatients with major depression. Am J Psychiatry 2003;160: 1277–85. Sheline YI, Wang PW, Gado MH, Csernansky JG, Vannier MW. Hippocampal atrophy in recurrent major depression. Proc Natl Acad Sci USA 1996;93:3908–13. Sutcigil L, Oktenli C, Musabak U, Bozkurt A, Cansever A, Uzun O, et al. Pro- and antiinflammatory cytokine balance in major depression: effect of sertraline therapy. Clin Dev Immunol 2007;2007:76396. Swenson JR, O'Connor CM, Barton D, Van Zyl LT, Swedberg K, Forman LM, et al. Influence of depression and effect of treatment with sertraline on quality of life after hospitalization for acute coronary syndrome. Am J Cardiol 2003;92:1271–6. Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, der Kooy DV. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 1999;208:166–88. Vaidya VA, Duman RS. Depression—emerging insights from neurobiology. Br Med Bull 2001;57:61–79. Wexler EM, Geschwind DH, Palmer TD. Lithium regulates adult hippocampal progenitor development through canonical Wnt pathway activation. Mol Psychiatry 2008;13:285–92.
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Sertraline promotes hippocampus-derived neural stem cells differentiating into neurons but not glia and attenuates LPS-induced cellular damage Zheng-Wu Peng a, 1, Yun-Yun Xue a, 1, Hua-Ning Wang a, Huai-Hai Wang a, Fen Xue a, Fang Kuang b, Bai-Ren Wang b, Yun-Chun Chen a, Li-Yi Zhang c, Qing-Rong Tan a,⁎ a b c
Department of Psychosomatic Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China Institute of Neuroscience, Fourth Military Medical University, Xi'an 710032, China 102 Hospital, Changzhou 213003, China
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Article history: Received 28 July 2011 Received in revised form 17 August 2011 Accepted 17 August 2011 Available online 25 August 2011 Keywords: Neural stem cells Neuronal differentiation Neuroprotective effect Sertraline
a b s t r a c t Sertraline is one of the most commonly used antidepressants in clinic. Although it is well accepted that sertraline exerts its action through inhibition of the reuptake of serotonin at presynaptic site in the brain, its effect on the neural stem cells (NSCs) has not been well elucidated. In this study, we utilized NSCs separated from the hippocampus of fetal rat to investigate the effect of sertraline on the proliferation and differentiation of NSCs. The study demonstrated that sertraline had no effect on NSCs proliferation but it significantly promoted NSCs to differentiate into serotoninergic neurons other than glia cells. Furthermore, we found that sertraline protected NSCs against the lipopolysaccharide-induced cellular damage. These data indicate that sertraline can promote neurogenesis and protect the viability of neural stem cells. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Depression is one of the most prevalent forms in psychopathology, and afflicts people in their whole life span owing to the recurrent feature (Burcusa and Iacono, 2007; Klerman and Weissman, 1988; Lecrubier, 2001). It is well accepted that complex dysregulation of the serotoninergic system plays a crucial role in the occurrence of the depressive disorders (Schloss and Williams, 1998). Some studies also suggest that inflammation and cell-mediated immune activation are important factors in the pathogenesis of depression (Maes et al., 1990, 1991; Mikova et al., 2001). Recent studies further implicate that the regulation of neurogenesis in adult brain is a possible target for the action of antidepressant drugs (Kodama et al., 2004; Manev et al., 2001). Selective serotonin reuptake inhibitor (SSRI) drugs produce relatively rapid blockade of serotonin (5-HT) transporters and elevate the serotonin concentration in synaptic cleft. However, their therapeutic effect lags for 3 to 4 weeks in clinic. In recent years, more
Abbreviations: SSRIs, selective serotonin reuptake inhibitors; NSCs, neuronal stem cells; BDNF, brain-derived neurotrophic factor; DMEM, Dulbecco's modified Eagle's medium; bFGF, fibroblast growth factor-basic; EGF, epidermal growth factor; EDTA, ethylene diamine tetraacetic acid; WST-1, 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; PB, phosphate buffer; BSA, bovine serum albumin; RT, room temperature; LDH, lactate dehydrogenase; LPS, lipopolysaccharide. ⁎ Corresponding author. Fax: +86 29 83293951. E-mail address: [email protected] (Q.-R. Tan). 1 Zheng-Wu Peng and Yun-Yun Xue contributed equally to this work. 0278-5846/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2011.08.014
and more data from pathological observation have shown that hippocampal atrophy always occurs in depressed patients (Chen et al., 2010; Sapolsky, 2000; Sheline et al., 1996), and antidepressants could promote neurogenesis demonstrated in vivo (Encinas et al., 2006; Hitoshi et al., 2007; Marlatt and Lucassen, 2010). Many researchers have focused on the role of neuronal stem cells (NSCs) in the functional mechanisms of antidepressants (Chiou et al., 2006; Huang et al., 2007). Sertraline is a widely used selective serotonin reuptake inhibitor (SSRI), whose antidepressive efficacy has been well established in clinical trials (Fava et al., 2000; Lepine et al., 2004; Schneider et al., 2003; Swenson et al., 2003). Studies have demonstrated that chronic but not acute administration of sertraline stimulates neurogenesis (Malberg and Blendy, 2005), and may have a beneficial effect on neurons (Peng et al., 2008). Increasing evidences have indicated that sertraline has a variety of protective effects on neurodegeneration caused by oxidative stress or disrupted energy metabolism (Kumar and Kumar, 2009), changing hippocampal structure (Bossini et al., 2007), modulating ATP and adenosine levels (Pedrazza et al., 2007), and up-regulating brain-derived neurotrophic factor (BDNF) (Matrisciano et al., 2009). It is shown that sertraline also slows disease progression and increase neurogenesis in the mouse model of Huntington's disease (Duan et al., 2008). However, there have only a few studies on the effect of sertraline on the NSCs, and no data are available whether sertraline acts on the proliferation or differentiation of NSCs. To further explore the pharmacology of sertraline, in this experiment, we used cell culture to investigate the effect of sertraline on NSCs from embryonic rat hippocampus.
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2. Materials and methods
2.5. Immunohistochemistry
2.1. Animals
To verify the identity of NSCs, neurospheres were collected and postfixed in 4% paraformaldehyde for 2 h at 4 °C before being moved into 20% sucrose in 0.1 M phosphate buffer (PB) overnight at 4 °C for cryoprotection. Frozen sections (10 μm) were cut with a cryostat and mounted onto gelatinized slides. Single cells were plated onto the poly-L-lysine coated coverslips. After 6 h of attachment, the cells on the slides were fixed in 4% paraformaldehyde in PBS, and then processed for immunocytochemical staining. For examination of NSCs differentiation, cells were seeded on poly-D-lysine-coated coverslips in triplicate at the density of 2 × 10 4 cells/well. After the cells were treated with sertraline, immunohistochemistry were carried out. The cells were fixed in a PBS solution containing 4% paraformaldehyde for 1 h, and washed 3 times with PBS. A PBS solution containing 1% bovine serum albumin (BSA) and 0.3% Triton was then added to the cells for 30 min at room temperature (RT). After removing this blocking reagent, cells were incubated in a humidity chamber at 4 °C overnight with the first antibodies composed of mouse anti-nestin (1:500, Abcam, U.K.), mouse anti-Tuj (1:500, Sigma, USA), mouse anti-CNPase (1:500, Abcam, U.K.), rabbit antiGFAP (1:2000, Millipore, USA) and rabbit anti-TPH1 (1:200, EPITOMICS, USA) diluted in blocking reagent. Then cells were washed 3 times with PBS and incubated for 2 h in the dark at room temperature in the presence of the fluorescent secondary antibodies (Alexa fluor 594 donkey anti-mouse IgG, 1:800, and Alexa fluor 488 donkey anti-rabbit IgG, 1:400, Invitrogen, USA). Finally, the cells were incubated with DAPI for 20 min at room temperature to stain the cellular nuclei. Finally, the coverslips were mounted onto slides in PBS/ glycerol (vol/vol). The preparations were analyzed under a fluorescent Olympus BX-51 microscope or a laser scanning confocal microscope (FV-1000, Olympus), and the positive cell was measured by using Image-Pro plus software (version 6.0, Media Cybernetics, USA).
The experimental protocol used in this study was approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and was conducted according to the Guidelines for Animal Experimentation of the university. Sprague–Dawley rats were purchased from Experimental Animal Center of the university. All efforts were made to minimize animals' suffering and to keep the numbers of animals used to a minimum. Mature female Sprague–Dawley rats (3 months old) at proestrus were placed overnight with mature males. And the females were examined for pregnancy by the presence of a vaginal plug or spermatozoa in the vagina.
2.2. Neural stem cell culture Embryonic brains between E14.5 and E16.5 were dissected under a stereomicroscope, and single cell suspensions of cortex and hippocampus were obtained by mechanical dissociation. Cells were primarily plated at a density of 1 × 10 5 cells/ml in culture bottle (4 ml/bottle), and grown in serum-free Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 20 ng/ml bFGF (human recombinant, Peprotech, 100-18B, USA), 20 ng/ml EGF (rat recombinant, Peprotech, 400-25, USA), B-27 and N-2 supplement (Gibic, USA), and penicillin and streptomycin (Tropepe et al., 1999). After 7 days' culture, the primary neurospheres were passaged. Briefly, the neurospheres were collected and dissociated with 0.05% trypsin and 200 μM EDTA for 10 min at 37 °C, then mechanically triturated with fire-polished glass pipettes. Then trypsin inhibitor (Sigma, USA) was added and laid for 5–8 min at room temperature to stop digestion. After filtering with 200 wells filter, filtered liquid was centrifuged for 5 min at 1 000 r/min. Then the single cells were resuspended. To assess the self-renewal ability, neurospheres were dissociated into a single cell suspension, and were re-plated at a density of 2 × 10 4 cells/ml in 24-well plates (0.4 ml/well). Cells were then cultured under the same condition as described above for a further 7 days and new neurospheres grew (Hitoshi et al., 2002). For the differentiation of neural stem cells, single cells were plated onto polylysine (50 mg/ml, Sigma, USA)-coated coverslips, in a 24-well plate, and the medium was changed to DMEM/F12 with 5% fetal bovine serum (Sigma, USA) to allow differentiation.
2.3. Drug treatment Sertraline (Pfizer) was dissolved by alcohol, and then diluted to different concentrations of stock solutions (10 folds of final concentration) by stem cell medium, with the final the concentration of alcohol at 0.5%. After replanting, NSCs were treated with sertraline by adding stock solutions to the medium.
2.4. Determination of cell viability Cell viability was determined by 2-(4-Iodophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1, Roche) assay. Neural stem cells were seeded at a density of 5 × 103 cells/well in 96-well plate. WST-1 was added to each well, and incubated for 4 h at 37 °C. The optical density was measured at 450 nm by a microplate reader. The data are representative as an average obtained from three independent experiments, which was performed at least 5 times (mean ± S.D., n = 5).
2.6. Western blotting analysis Cells were lysed with SDS-PAGE sample buffer composed of 62.5 mM Tris–HCl, 2% w/v SDS, 10% glycerol, 50 mM DTT, and 0.1% w/v bromphenol blue, and the insoluble materials were separated by centrifugation at 12,000 g for 10 min. The supernatant was heated at 100 °C for 10 min, and cooled on ice for 30 min afterwards. Electrophoresis was carried out by SDS-PAGE by using 10% polyacrylamide in accordance with routine protocols. Then the proteins in PAGE were transferred onto nitrocellulose membranes and blocked in blocking solution containing 5% defatted milk powder, 0.1% Tween20 in TBS for 1 h at RT with gentle shaking. After being washed in TBS for 3 times 8 min each, the following primary antibodies were used for incubation overnight at 4 °C: mouse anti-Tuj (1:2000, Sigma, USA), mouse anti-CNPase (1:1000, Abcam, U.K.), rabbit antiGFAP (1:5000, Millipore, USA) and mouse anti β-actin antibody (1:10,000, sigma, USA) as a loading control. Then the membranes were washed 3 times in TBS again, and incubated with peroxidaseconjugated goat anti-rabbit IgG or peroxidase-conjugated goat antimouse IgG in TBST for 1 h. After washing 3 times in TBS for 8 min each, the membrane was detected using a chemiluminescence detection kit (Supersignal west pico chemiluminescent substrate, Thermo, USA), and the immunoreactive proteins were then visualized on X-ray film and digitized. 2.7. Measurement of lactate dehydrogenase (LDH) activity In order to estimate the protective effect of sertraline on lipopolysaccharide (LPS) induced cell death, the level of lactate dehydrogenase (LDH) released from damaged cells into culture media was measured at 24 h and 48 h after the cells were treatment of cells with 200 ng/ml LPS. Cell culture media were collected and release of
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lactate dehydrogenase (LDH) from cells was evaluated using a Cytotoxicity Detection Kit (NJJC, China). Absorbance was measured at 492 nm with a microplate reader. The data are presented as the value averaged from three separate experiments (6 wells per one experiment). 2.8. Data analysis Statistical analysis was performed with SPSS software (SPSS Inc., Chicago, IL). All the data were shown as means ± SEM. Differences were subjected to statistical analysis by one-way ANOVA. Differences were considered significant when P b 0.05, and considered as highly significant when P b 0.01.
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As shown in Fig. 4, by using the immunofluorescent staining, the percentage of numbers of differentiated neurons (Tuj +/DAPI + cell) were significantly increased in 5 μM (df = 4, F = 121.94, P = 0.000) and 10 μM (P = 0.001) sertraline-treated group. There was no significant difference between other smaller doses of sertraline groups and control (Fig. 4C). The triple-staining immunofluorescent staining was used to further identify the serotoninergic feature of differentiated neurons. Neurons immunoreactive for TPH1 (marker for serotoninergic neurons, green), Tuj (marker for differentiated neurons, red), as well as stained by DAPI (nuclei staining; blue) were detected at 7 days after sertraline treatment (Fig. 4B). 3.3. The protective effect of sertraline on LPS-induced cell damage in NSCs
3. Results 3.1. Identification of the NSCs and effects of sertraline on their proliferation Immunofluorescent staining demonstrated that both the neurospheres and single NSCs expressed nestin, the marker for NSCs (Fig. 1). Sertraline did not affect the cell viability, as observed 1 week after treatment at the doses tested. Inversely, the cell viability was significantly decreased at 20 μM and 50 μM of sertraline (df = 6, F = 44.901, P = 0.000) administration. Therefore, the doses of 20 μM and 50 μM were not chosen for the subsequent experiments. Ability of proliferation of NSCs was analyzed by measuring the diameter of neurospheres. The diameter of NSCs neurospheres was significantly decreased in 20 μM (df = 5, F = 24.50, P = 0.000) and even no neurospheres were formed in 50 μM group. There were no significant differences detected in other small doses-given groups (Fig. 2A, B). The results indicate that the proliferation of NSCs is not facilitated after direct administration of sertraline, but is inhibited when the concentrations of sertraline are high. 3.2. Effects of sertraline on differentiation of NSCs As shown in Fig. 3, after treatment with sertraline, the expression of GFAP were significantly decreased in 1 μM (df = 4, F = 121.94, P = 0.017), 5 μM (P = 0.000) and 10 μM (P = 0.005) sertralinetreated groups as demonstrated by Western blot analysis. And the expression of CNPase was significantly decreased as well in 5 μM (df = 4, F = 2.507, P = 0.025) sertraline-treated group, in comparison with the control. On the other hand, the expression of Tuj (the marker for differentiated neurons) was evidently increased in 5 μM (df = 4, F = 42.748, P = 0.000) and 10 μM (P = 0.001) group. The result indicating that sertraline promotes NSCs differentiated predominantly into neurons but inhibits its differentiation to glia cells.
After treated with different does of sertraline and LPS (200 ng/ml) for 24 or 48 h, as shown in Fig. 5, 5 μM sertraline treatment significantly eased the decrease of cell viabilities induced by LPS at 24 h (df = 3, F = 5.1, P = 0.03) and 48 h (df = 3, F = 13.654, P = 0.000) and diminished the leakage of LDH at 24 h (df = 3, F = 5.783, P = 0.007) and 48 h (df = 3, F = 5.036, P = 0.003), compared to control. And 1 μM sertraline decreased LDH level at 48 h (P = 0.27), but no detectable effect on cell viability. These findings imply that sertraline attenuates the cellular damage of NSCs induced by LPS. 4. Discussion The hippocampus has been shown undergoing morphological changes in response to stress, including atrophy and loss of CA3 pyramidal neurons after exposure to physical or psychosocial stress (McEwen, 1999), and antidepressant treatment attenuates neuronal cell death and increases neurogenesis in the dentate gyrus of the hippocampus (Jin et al., 2009; Malberg et al., 2000; Santarelli et al., 2003). It seems that increased neurogenesis in hippocampus by the administration of antidepressant is necessary in altered behavior in the stress-induced depression animal models and patients. Although the mechanism is largely unknown, recent evidence suggests that antidepressants influence important signaling pathways that regulate neuroplasticity and cell survival (Vaidya and Duman, 2001). NSCs have been defined as cells with the capacity of self-renewal and multi-lineage differentiation (Tropepe et al., 1999). In adult central nervous system (CNS), thousands of NSCs exist in the hippocampal dentate gyrus (Cameron and McKay, 2001). These cells may contribute to tissue repair in some neurological diseases and screen the candidate agents for neurogenesis in neurodegenerative diseases (Hitoshi et al., 2002). By using the in vitro culture of neural stem cells from hippocampus of fetal rats, our study demonstrated that sertraline could not
Fig. 1. Nestin staining for neurospheres (A) and single neural stem cells (NSCs) (B) and effect of sertraline on the cell viability of NSCs (C, n = 6 for each). In A and B the right microphotographs are nestin staining, the middle is DAPI staining to show the nuclei, and the right is the merged images of former two. Note in C that lower doses of sertraline administration did not affect the cell viability of NSCs, and high doses of sertraline even had inhibitive action. **P b 0.01 vs. other groups. Bar: 100 μm.
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Fig. 2. Microphotographs at left (A) and histograms at right (B) showing the effect of different concentrations of sertraline on the diameter of neurospheres observed 1 week following administration (n = 8 each group). Note that lower doses (0.5–10 μM) of sertraline did not affect the diameter of neurospheres, while 20 μM group significantly decreased the diameter of neurospheres, compared with other groups. In 50 μM group, no neurospheres were formed (data not shown). **P b 0.01 vs. other group. Bar: 100 μm.
increase proliferation and viability of NSCs. More importantly, by using triple-staining immunofluorescent and Western bolt assay, we provided the evidence that sertraline not only promoted NSCs differentiating into serotoninergic neuronal, but also inhibited NSCs differentiating into glial cells (Figs. 3, 4). In our study, after 7 days, 5 μM of sertraline stimulated the differentiation of fetus rat NSCs, mostly into TPH1 + neurons and decreased the proteins level of maker of oligodendroglial cell (CNPase) and astrocyte (GFAP). This observation is in agreement with previous reports in other antidepressants, such as lithium (Wexler et al., 2008) and fluoxetine (Chen et al., 2007). The ratio of Tuj + cell was 63.4% in 5 μM sertraline, higher than 54.1% in control group. Recent studies have shown evidences that major depression is accompanied by the activation of the inflammatory-response system (O'Brien et al., 2004; Penninx et al., 2003). LPS is a highly
proinflammatory component of outer member of gram-negative bacteria and has been tested as a stress model for inducing apoptosis (Gayle et al., 2002). We showed that sertraline can protect LPS-induced cellular damage. Administration of 5 μM of sertraline to 200 μg/ml LPS-treated NSCs reduced the LDH release and increased cell viability significantly compared to control group. This observation was in agreement with previous reports about other kind of antidepressants (Chiou et al., 2006). The mechanism underlying the neuroprotection by sertraline treatment on NSCs remains undetermined. But prior reports suggested that sertraline might provide neuroprotection by decreasing the expression of proinflammatory cytokine. Sutcigil et al. (2007) demonstrated that sertraline therapy might have exerted immunomodulatory effects through a decrease in the proinflammatory cytokine IL-12 and an increase in the anti-inflammatory cytokines IL-4 and TGF-β1, which may influence neurogenesis greatly (Monje et al., 2003).
Fig. 3. Evaluation of protein levels of GFAP (expressed by astrocytes), CNPase (expressed by oligodendrocytes), and Tuj (marker for differentiated neurons) in each group determined by Western blot (n = 5 each group). A is the representative bands at each group. B, C, and D are densitometric analysis for GFAP, CNPase and Tuj, respectively. * P b 0.05 vs. control. **P b 0.01 vs. control.
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Fig. 4. Microphotographs (A, B) and histograms (C) showing the effect of sertraline on differentiation of NSCs. (A) Immunofluorescent staining for Tuj, GFAP and their merged images of differentiated NSCs. (B) Detection of the protein expression of Tuj, DAPI, TPH1, and merged images in the same differentiated NSCs. (C) Statistical analysis for the percentage of number of Tuj-positive cells in different doses of sertraline-treated groups. Note that the percentage of numbers of Tuj+/DAPI+ cells (mean ± SD of 5 five separate experiments) was increased in 5 and 10 μM group. *P b 0.05 vs. control, **P b 0.01 vs. control. Bar: 100 μm.
From the therapeutic aspect of antidepressant treatment, altered levels of serotonin, neurogenesis in hippocampus, neuronal protection are closely associated with the pharmacologic action of antidepressants (Lesch, 2001; Malberg, 2004; McKernan et al., 2009). In this study, our
results indicated that sertraline elevated the serotoninergic neuronal differentiation, and protected NSCs from LPS-induced apoptosis. These results enrich our understanding for the pharmacological mechanism of sertraline. It is needed to note that the cells used in this experiment
Fig. 5. Evaluation of the effect of sertraline on cell viability and LDH level in NSCs at 24 h and 48 h after LPS treatment (n = 5 for each). Note that sertraline eased the decrease cell viability and diminished the elevation of LDH level in LPS-treated NSCs. * P b 0.05 vs. control, **P b 0.01 vs. control.
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were from normal animal. Thus, study on the effect of sertraline on NSCs from stressed animals is needed to be further investigated. Acknowledgments We are indebted to Dr. Bai-Ren Wang for his valuable advice and support. This work was supported by the National Natural Science Foundation (30870886, 30700259), the National Science and Technology Supporting Plan of the eleventh five-year (43730018-8) and the National Key Technology Support Program of China (2009BAI77B07). References Bossini L, Tavanti M, Lombardelli A, Calossi S, Polizzotto NR, Galli R, et al. Changes in hippocampal volume in patients with post-traumatic stress disorder after sertraline treatment. J Clin Psychopharmacol 2007;27:233–5. Burcusa SL, Iacono WG. Risk for recurrence in depression. Clin Psychol Rev 2007;27: 959–85. Cameron HA, McKay RD. Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 2001;435:406–17. Chen SJ, Kao CL, Chang YL, Yen CJ, Shui JW, Chien CS, et al. Antidepressant administration modulates neural stem cell survival and serotoninergic differentiation through bcl-2. Curr Neurovasc Res 2007;4:19–29. Chen MC, Hamilton JP, Gotlib IH. Decreased hippocampal volume in healthy girls at risk of depression. Arch Gen Psychiatry 2010;67:270–6. Chiou SH, Chen SJ, Peng CH, Chang YL, Ku HH, Hsu WM, et al. Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell. Biochem Biophys Res Commun 2006;343:391–400. Duan W, Peng Q, Masuda N, Ford E, Tryggestad E, Ladenheim B, et al. Sertraline slows disease progression and increases neurogenesis in N171-82Q mouse model of Huntington's disease. Neurobiol Dis 2008;30:312–22. Encinas JM, Vaahtokari A, Enikolopov G. Fluoxetine targets early progenitor cells in the adult brain. Proc Natl Acad Sci USA 2006;103:8233–8. Fava M, Rosenbaum JF, Hoog SL, Tepner RG, Kopp JB, Nilsson ME. Fluoxetine versus sertraline and paroxetine in major depression: tolerability and efficacy in anxious depression. J Affect Disord 2000;59:119–26. Gayle DA, Ling Z, Tong C, Landers T, Lipton JW, Carvey PM. Lipopolysaccharide (LPS)induced dopamine cell loss in culture: roles of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide. Brain Res Dev Brain Res 2002;133:27–35. Hitoshi S, Alexson T, Tropepe V, Donoviel D, Elia AJ, Nye JS, et al. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 2002;16:846–58. Hitoshi S, Maruta N, Higashi M, Kumar A, Kato N, Ikenaka K. Antidepressant drugs reverse the loss of adult neural stem cells following chronic stress. J Neurosci Res 2007;85:3574–85. Huang YY, Peng CH, Yang YP, Wu CC, Hsu WM, Wang HJ, et al. Desipramine activated Bcl-2 expression and inhibited lipopolysaccharide-induced apoptosis in hippocampus-derived adult neural stem cells. J Pharmacol Sci 2007;104:61–72. Jin Y, Lim CM, Kim SW, Park JY, Seo JS, Han PL, et al. Fluoxetine attenuates kainic acidinduced neuronal cell death in the mouse hippocampus. Brain Res 2009;1281: 108–16. Klerman GL, Weissman MM. The changing epidemiology of depression. Clin Chem 1988;34:807–12. Kodama M, Fujioka T, Duman RS. Chronic olanzapine or fluoxetine administration increases cell proliferation in hippocampus and prefrontal cortex of adult rat. Biol Psychiatry 2004;56:570–80. Kumar P, Kumar A. Possible role of sertraline against 3-nitropropionic acid induced behavioral, oxidative stress and mitochondrial dysfunctions in rat brain. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:100–8. Lecrubier Y. The burden of depression and anxiety in general medicine. J Clin Psychiatry 2001;62(Suppl. 8):4–9. [discussion 10–1]. Lepine JP, Caillard V, Bisserbe JC, Troy S, Hotton JM, Boyer P. A randomized, placebocontrolled trial of sertraline for prophylactic treatment of highly recurrent major depressive disorder. Am J Psychiatry 2004;161:836–42. Lesch KP. Serotonergic gene expression and depression: implications for developing novel antidepressants. J Affect Disord 2001;62:57–76.
Maes M, Bosmans E, Suy E, Vandervorst C, De Jonckheere C, Raus J. Immune disturbances during major depression: upregulated expression of interleukin-2 receptors. Neuropsychobiology 1990;24:115–20. Maes M, Bosmans E, Suy E, Vandervorst C, DeJonckheere C, Raus J. Depression-related disturbances in mitogen-induced lymphocyte responses and interleukin-1 beta and soluble interleukin-2 receptor production. Acta Psychiatr Scand 1991;84: 379–86. Malberg JE. Implications of adult hippocampal neurogenesis in antidepressant action. J Psychiatry Neurosci 2004;29:196–205. Malberg JE, Blendy JA. Antidepressant action: to the nucleus and beyond. Trends Pharmacol Sci 2005;26:631–8. Malberg JE, Eisch AJ, Nestler EJ, Duman RS. Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci 2000;20:9104–10. Manev H, Uz T, Smalheiser NR, Manev R. Antidepressants alter cell proliferation in the adult brain in vivo and in neural cultures in vitro. Eur J Pharmacol 2001;411:67–70. Marlatt MW, Lucassen PJ, van Praag H. Comparison of neurogenic effects of fluoxetine, duloxetine and running in mice. Brain Res 2010;1341:93–9. Matrisciano F, Bonaccorso S, Ricciardi A, Scaccianoce S, Panaccione I, Wang L, et al. Changes in BDNF serum levels in patients with major depression disorder (MDD) after 6 months treatment with sertraline, escitalopram, or venlafaxine. J Psychiatr Res 2009;43:247–54. McEwen BS. Stress and hippocampal plasticity. Annu Rev Neurosci 1999;22:105–22. McKernan DP, Dinan TG, Cryan JF. “Killing the Blues”: a role for cellular suicide (apoptosis) in depression and the antidepressant response. Prog Neurobiol 2009;88: 246–63. Mikova O, Yakimova R, Bosmans E, Kenis G, Maes M. Increased serum tumor necrosis factor alpha concentrations in major depression and multiple sclerosis. Eur Neuropsychopharmacol 2001;11:203–8. Monje ML, Toda H, Palmer TD. Inflammatory blockade restores adult hippocampal neurogenesis. Science 2003;302:1760–5. O'Brien SM, Scott LV, Dinan TG. Cytokines: abnormalities in major depression and implications for pharmacological treatment. Hum Psychopharmacol 2004;19: 397–403. Pedrazza EL, Senger MR, Pedrazza L, Zimmermann FF, de Freitas Sarkis JJ, Bonan CD. Sertraline and clomipramine inhibit nucleotide catabolism in rat brain synaptosomes. Toxicol In Vitro 2007;21:671–6. Peng Q, Masuda N, Jiang M, Li Q, Zhao M, Ross CA, et al. The antidepressant sertraline improves the phenotype, promotes neurogenesis and increases BDNF levels in the R6/2 Huntington's disease mouse model. Exp Neurol 2008;210:154–63. Penninx BW, Kritchevsky SB, Yaffe K, Newman AB, Simonsick EM, Rubin S, et al. Inflammatory markers and depressed mood in older persons: results from the Health, Aging and Body Composition study. Biol Psychiatry 2003;54:566–72. Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, et al. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 2003;301:805–9. Sapolsky RM. The possibility of neurotoxicity in the hippocampus in major depression: a primer on neuron death. Biol Psychiatry 2000;48:755–65. Schloss P, Williams DC. The serotonin transporter: a primary target for antidepressant drugs. J Psychopharmacol 1998;12:115–21. Schneider LS, Nelson JC, Clary CM, Newhouse P, Krishnan KR, Shiovitz T, et al. An 8week multicenter, parallel-group, double-blind, placebo-controlled study of sertraline in elderly outpatients with major depression. Am J Psychiatry 2003;160: 1277–85. Sheline YI, Wang PW, Gado MH, Csernansky JG, Vannier MW. Hippocampal atrophy in recurrent major depression. Proc Natl Acad Sci USA 1996;93:3908–13. Sutcigil L, Oktenli C, Musabak U, Bozkurt A, Cansever A, Uzun O, et al. Pro- and antiinflammatory cytokine balance in major depression: effect of sertraline therapy. Clin Dev Immunol 2007;2007:76396. Swenson JR, O'Connor CM, Barton D, Van Zyl LT, Swedberg K, Forman LM, et al. Influence of depression and effect of treatment with sertraline on quality of life after hospitalization for acute coronary syndrome. Am J Cardiol 2003;92:1271–6. Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, der Kooy DV. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 1999;208:166–88. Vaidya VA, Duman RS. Depression—emerging insights from neurobiology. Br Med Bull 2001;57:61–79. Wexler EM, Geschwind DH, Palmer TD. Lithium regulates adult hippocampal progenitor development through canonical Wnt pathway activation. Mol Psychiatry 2008;13:285–92.