Moclobemide upregulated Bcl-2 expression and induced neural stem cell differentiation into serotoninergic neuron via extracellular-regulated kinase pathway

Abstract

1. Moclobemide (MB) is an antidepressant drug that selectively and reversibly inhibits monoamine oxidase-A. Recent studies have revealed that antidepressant drugs possess the characters of potent growth-promoting factors for the development of neurogenesis and improve the survival rate of serotonin (5-hydroxytrytamine; 5-HT) neurons. However, whether MB comprises neuroprotection effects or modulates the proliferation of neural stem cells (NSCs) needs to be elucidated. 2. In this study, firstly, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to demonstrate that 50 microM MB can increase the cell viability of NSCs. The result of real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that the induction of MB can upregulate the gene expressions of Bcl-2 and Bcl-xL. By using caspases 8 and 3, ELISA and terminal dUTP nick-end labeling (TUNEL) assay, our data further confirmed that 50 microM MB-treated NSCs can prevent FasL-induced apoptosis. 3. The morphological findings also supported the evidence that MB can facilitate the dendritic development and increase the neurite expansion of NSCs. Moreover, we found that MB treatment increased the expression of Bcl-2 in NSCs through activating the extracellular-regulated kinase (ERK) phosphorylation. 4. By using the triple-staining immunofluorescent study, the percentages of serotonin- and MAP-2-positive cells in the day 7 culture of MB-treated NSCs were significantly increased (P<0.01). Furthermore, our data supported that MB treatment increased functional production of serotonin in NSCs via the modulation of ERK1/2. In sum, the study results support that MB can upregulate Bcl-2 expression and induce the differentiation of NSCs into serotoninergic neuron via ERK pathway.

Figure 1

Cultivation of neural stem cells (NSCs) and evaluation of the cell viability in MB-treated NSCs by MTT. (a) NSCs aggregated and formed neurosphere under serum-free medium culture. (b–d) The expressions of nestin (b and d: red fluorescent) and DAPI (c: blue fluorescent; d: merged image) were detected in neurosphere by immunofluorescent assay (bar: 50 μm). (e–f) The GFAP-positive (e: green fluorescent) and MAP-2 positive (green fluorescent) cells were detected in the differentiated NSCs (bar: 20 μm; a–f were performed by three separate experiments). (g) The cell viability of NSCs was analyzed using MTT assay. NSCs were treated with 0, 10, 25, 50, 75, 100, 200, and 400 μM MB for 24, 48, and 72 h. Data (mean±s.d. of six separate experiments) are expressed as percentages of the control value (no MB). ^*P<0.05, as compared to the control. (h) Growth curve of NSC cells: 10⁴ NSCs were plated on 6 cm culture dishes with and without MB. After treating with 50 and 200 μM MB for 24, 48, and 72 h, the number of NSCs was counted by hemocytometer, three counts for each point. The 0.1% DMSO was used as a vehicle control.

Figure 2

Detection of Bcl-2 expression in MB-treated NSCs by real-time RT–PCR and immunofluorescent assay. (a) The RNA levels of Bcl-2, Bcl-xL, MAP2, and nestin were detected in day-1, -3, -5 MB-treated NSCs by real-time RT–PCR. Data shown here are the mean±s.d. of three experiments. C: control; M: MB; ^*P<0.05, ^**P<0.01, as compared to the control (no MB). (b–g) By using immunofluorescent assay, the positive signals of Bcl-2 (b, d, e, and g; red fluorescent), DAPI (c, d, f, and g; blue fluorescent), and merged images were identified in MB-treated (b–d) and no MB-treated (e–g) NSCs (bar: 100 μm; b–g were performed by three separate experiments).

Figure 3

Evaluation of FasL-induced apoptotic activity in NSCs treated with and without moclobemide. (a) The annexin-5 activities were measured in NSCs treated with MB and without MB by using flow cytometry. (b, c) The apoptotic activities of caspase 8 and caspase 3 were detected by ELISA assay. (d) The severity of DNA fragmentation was analyzed by TUNEL assay. The control group (Ctrl: no adding FasL; first column in a–d), the group of NSCs without MB treatment (no MB; second column in a–d), and the group of MB-treated NSCs (third column in a–d). Data shown here are the mean±s.d. of three experiments; ^**P<0.01.

Figure 4

Effects of MB on the neurite development and neurite expansion. (a–d) MB-treated NSCs projected the neurite (arrows) and were differentiated into the dendritic formations with long process (arrowheads: primary branches; bar: 40 μm). The morphological developments of (e) the number of neurite, (f) neurite length, and (g) primary dendrities of the neuronal differentiation were observed and measured in day-1, day-3, day-5, and day-7 cultures of NSCs treated with MB and without MB. Data shown here are the mean±s.d. of three experiments; ^*P<0.05; ^**P<0.01, as compared to the control (without MB). The 0.1% DMSO was used as a vehicle control.

Figure 5

The treatment effects of caspase inhibitors and PD98059 on NSCs with and without MB. (a) The treatment effects of Z-VAD-FMK (a pan-caspase inhibitor), Z-DEVD-FMK (a selective inhibitor of caspase-3), and PD98059 (ERK1/2 inhibitor) on NSCs were examined. Data shown here are the mean±s.d. of three experiments. Control: no MB treatment. (^**P<0.01; MB-treated NSCs compared to control group) (^#P<0.01; PD98059-treated NSCs compared to control group). (b) The neurite development of differentiated NSCs was specifically inhibited by PD98059. (c) The neuronal formation and neurite expansion in MB-treated NSCs were observed (bar: 30 μm). The 0.1% DMSO was used as a vehicle control.

Figure 6

MB upregulated Bcl-2 expression through the phosphorylation of ERK1/2 in NSCs. (a) Detection of the protein amount of Bcl-2, phosphorylated ERK1/2, and total of ERK1/2 in day-1, day-3 and day-5 NSCs without MB treatment by Western blotting assay. (b) The expressions of Bcl-2 and phosphorylated ERK1/2 were upregulated in day-3 and day-5 MB-treated NSCs (no addition of PD98059). (c) The expression levels of Bcl-2 and phosphorylated ERK1/2 in MB-treated NSCs were specifically inhibited by PD98059. Data shown here are the mean±s.d. of three experiments. ^**P<0.01.

Figure 7

MB promotes NSCs differentiation into serotoninergic neurons. (a) Detection of the protein expression of 5-HT (green fluorescent), MAP2 (red fluorescent), and DAPI (blue fluorescent) in the same differentiated NSCs by using immnunofluorescent assay (bar: 20 μm). The percentages of (b) 5-HT-positive cells, (c) MAP2-positive cells, and (d) both of the 5-HT/MAP2-positive cells in the MB-treated and no MB-treated NSCs were measured by immunofluorescent signals. (b, c) The induction rates of 5-HT-positive cells and MAP2-positive cells in MB-treated NSCs. MB were significantly inhibited by PD98059. Data shown here are the mean±s.d. of three experiments. (^*P<0.05, ^**P<0.01; MB-treated group compared to non-MB-treated group) (^#P<0.05, ^##P<0.01; MB- and PD98059-treated group compared to only MB-treated group). The 0.1% DMSO was used as a vehicle control.

Figure 8

Detection of 5-HT levels in the culture medium of NSCs by HPLC-ECD. Typical chromatograms obtained from (a) a standard mixture containing 3,4-dihydroxyphenylacetic acid (DOPAC; 5.23 min), dopamine (DA; 6.87 min), hydroxyindoleacetic acid (5-HIAA; 7.78 min), homovanillic acid (HVA; 10.90 min), and 5-hydroxytryptamine (5-HT; 15.67 min); analysis was completed within 20 min. (b) A baseline collected from medium only. (c) A sample collected from MB-treated NSC medium. (d) The levels of 5-HT were measured by HPLC-ECD in day 1, day 3, day 5, and day 7 of the culture medium of NSCs treated with MB and no MB. Data shown here are the mean±s.d. of six experiments. (^*P<0.05, ^**P<0.01; MB-treated group via no MB-treated group) (^#P<0.05, ^##P<0.01; MB- and PD98059-treated group via only MB-treated group). The 0.1% DMSO was used as a vehicle control.