The protective effect of tianeptine on Gp120-induced apoptosis in astroglial cells: role of GS and NOS, and NF-κB suppression

Abstract

Background and purpose: Tianeptine is an antidepressant affecting the glutamatergic system. In spite of its proven clinical efficacy, molecular effects of tianeptine are not entirely clear. Tianeptine modulates cytokine expression in the CNS and protects the hippocampus from chronic stress effects. HIV infection is associated with inflammation and neuronal loss, causing HIV-associated dementia (HAD). The human immunodeficiency virus type-1 glycoprotein gp120 has been proposed as a likely aetiological agent of HAD. In this study, we determined whether tianeptine protects astroglial cells from the neurodegenerative effects of gp120.

Experimental approach: Human astroglial cells were treated with gp120 and tianeptine, and viability and apoptosis was monitored by TUNEL, annexin V, and activated caspase-3 staining and flow cytometry. Protein levels of glutamine synthase (GS), inducible and constitutive nitric oxide synthases (iNOS, cNOS) and nuclear factor κB (NF-κB) pathway were determined by Western blot analysis. The respective activities were assessed indirectly by measuring glutamine and nitrite concentrations or by luciferase reporter assays.

Key results: Tianeptine showed an anti-apoptotic effect and prevented caspase-3 activation by gp120. The mechanism of tianeptine's action involved GS and cNOS stabilization and iNOS suppression. Moreover, tianeptine increased IκB-α levels in the absence of gp120 and blocked its degradation in response to gp120. This correlated with the suppression of basal and gp120-induced NF-κB transcriptional activity.

Conclusions and implications: Tianeptine clearly exerts neuroprotective effects in vitro by suppressing the molecular pro-inflammatory effects of gp120. Studies in animal models should be performed to evaluate the potential of tianeptine as a treatment for HAD.

Figure 1

Tianeptine prevents gp120-induced apoptotic cell death in cultured astroglial cells. (A) Lipari astroglial cells were treated with gp120 for 72 h. Tianeptine was added 1 h before the addition of gp120. Cell mortality was assessed by % trypan blue positive cell counts after enzymatic cell detachment by trypsin. Data represent the mean of eight independent experiments ± SEM. The * indicates a statistically significant difference at P < 0.05 for gp120-treated astrocytes versus control; # indicates a statistically significant change at P < 0.05 for gp120 plus tianeptine versus gp120 only-treated astrocytes. (B) Incubation of Lipari astroglial cells with gp120 (10 nM) for 48 h induced mainly apoptotic cell death, as assessed by FITC-Annexin V/7-AAD double labelling followed by FACS bi-parametric analysis. Tianeptine (10, 50, 100 µM) prevented the effect of gp120. Cells (0.8 × 10⁴ cm⁻²) were seeded 24 h before the treatment. Annexin V columns show the percentage of total Annexin V positive cells, early (Annexin V positive 7-AAD negative) and -late (double stained) apoptotic cells; 7-AAD only columns show the percentage of Annexin V negative, 7-AAD positive cells which correspond mainly to necrotic cells. Data represent the mean ± SEM of three independent experiments. *, # − see (A). (C) Representative dot plots of FITC fluorescence versus 7-AAD fluorescence for the experiment shown in (B). Unstained AnnV – cells stained with 7-AAD, but not with FITC-Annexin V. (D) Incubation of U373 astroglial cells with gp120 for 48 h leads to caspase-3 activation as shown by the increased binding to FITC-conjugated z-VAD-fmk. Tianeptine antagonized the caspase-3 cleavage in response to gp120. U373 astroglial cells were treated and untreated with tianeptine at different concentrations and then stimulated or not with gp120 (10 nM). 48 h later the cells were incubated with FITC-z-VAD-fmk and processed for FACS analysis, followed by FlowJo analysis of FITC-positive gate. The experiment was performed four times in triplicate. *, # − statistical analysis was performed as in (A). (E) Representative dot plots of FITC fluorescence versus side-scatter for the experiment shown in (D).

Figure 2

Tianeptine enhances glutamine synthase (GS) levels and activity in astroglial cells. (A) The decrease in glutamine concentration in the supernatant of astroglial cells incubated with gp120 is reversed by tianeptine. Treatment of astroglial cells with gp120 (10 nM) for 24 h produced a reduction in glutamine formation compared with the untreated control. Tianeptine (10, 50, 100 µM) reversed this effect. Data represent the mean ± SEM of five independent experiments. *P < 0.05 when compared with control; #P < 0.05 as compared to gp120 alone-treated cells. (B) The effect of gp120 on GS expression levels in astroglial cells. Incubation of astroglial cells with gp 120 (10 nM) for 24 h dose dependently reduced the GS expression. Pretreatment for 2 h with tianeptine (10, 50, 100 µM) antagonized this effect. Blots are representative of four independent experiments. Normalized optical density of GS signal from four experiments is shown next. Data represent the mean ± SEM.

Figure 3

Tianeptine effect on nitric oxide and nitric oxide synthases. (A) Tianeptine prevents the transient decrease in nitrite levels in the supernatant of astroglial cells incubated with gp120. Treatment of astroglial cells with gp120 (10 nM) for 30 min produced an initial decrease of nitrite levels compared with untreated controls. Tianeptine (10, 50, 100 µM) reversed this effect dose dependently. Data represent the mean ± SEM of five independent experiments normalized to and expressed as percentage of nitrite concentration in untreated cells. *P < 0.05 when compared with control; #P < 0.05 tianeptine-treated versus gp 120 only treated cells. (B) The effect of gp120 on nNOS levels in human cultured astroglial cells. Incubation of cells with gp 120 (10 nM) for 24 h dose dependently reduced the nNOS expression as measured by Western blotting analysis. Tianeptine (10, 50, 100 µM), antagonized this effect dose dependently. Blots are representative of four independent experiments. Normalized optical density of nNOS signal from four experiments is shown next. Data represent the mean ± SEM. (C) Tianeptine prevents a later increase in nitrite levels in the supernatant of astroglial cells incubated with gp120 for 24 h. The experiment was performed as in (A) but the nitrate levels were assessed after 24 h. Data represent the mean ± SEM of three independent experiments. *,# − see (A). (D) Changes in protein levels of iNOS in a similar experiment to that in (B). Blots are representative of three independent experiments. Normalized optical density of nNOS signal from three experiments is shown below. Data represent the mean ± SEM.

Figure 4

Tianeptine interferes with NF-κB signalling by stabilizing IκB-α in astroglial cells. (A) Time-course analysis of IκB-α protein levels in response to gp120 stimulation. The astroglial cells (seeded 1.5 × 10⁴ cm⁻² cells 24 h before the treatments) were stimulated with 10 nM gp120 and lysed in RIPA buffer at indicated time points. Lysates were processed and 5 µg of total protein lysates were loaded on the 10% SDS-PAGE gel and subsequently analysed by Western blotting. The blot is representative of three independent experiments. The graph represents the normalized mean OD of IκB-α signal divided by mean OD of actin signal from three independent blots ± SEM. (B) Tianeptine stabilizes IκB-α in the absence of gp120 stimulation. The cells seeded as in (A) were treated for 1 h 40 min with different concentrations of tianeptine and processed as in (A) for Western blotting. The graph represents the normalized OD of IκB-α signal from analysed as in (A). (C) Tianeptine inhibits the degradation of IkBa induced by gp120. The astroglial cells were seeded as in (A) and stimulated with gp120 for 35 min. Tianeptine at different doses was added 1 h before the administration of gp120. The cells were processed for Western blotting as in (A). The graph shows the normalized OD of IκB-α signal analysed as in (A) from 3 independent experiments. (D) NF-κB transcriptional activity is suppressed by tianeptine. The astroglial cells were transfected with pNfkB-Luc and pRL reporter plasmids. Twenty hours post-transfection, the cells were stimulated with 10 nM gp120 and/or tianeptine (50 µM) for 5 h or left untreated. Each treatment was performed six times. Luciferase activity and internal control renilla activity are the mean of six experimental points. Data represent the mean ± SEM of a representative experiment, which was performed three times. *P < 0.05 when compared with control; #P < 0.05 tianeptine and gp120 versus gp120 alone-treated cells.

Figure 5

(A) Dose-response curve for effects of tianeptine on cell viability and enzyme activities counteracting pro-apoptotic effects of gp120. The response was calculated according to Figure 1 as the increase in cell viability (i.e. 100% – % trypan blue positive cells) compared with viability in the presence of 10 nM gp120. For glutamine synthase (GS) and nNOS activity (measured indirectly by nitrite concentration), the data from Figures 2A and 3A were recalculated, assuming that the gp120-induced suppressed activity corresponds to 0, while 100% activity corresponds to control untreated cells. Note that GS activity and nitrite levels are superimposable. (B) The schematic representation of molecular mechanisms induced by tianeptine. The primary effect of tianeptine may involve the block of gp120-induced cNOS down-regulation and IκB-α degradation. This may prevent the induction of iNOS and decrease in cNOS levels, thus stabilizing low physiological NO levels, which finally increase GS activity. High GS activity is fundamental for protection from apoptosis. Inhibition of NF-κB by tianeptine may attenuate other pro-inflammatory responses to gp120 comprising an increased production of pro-inflammatory cytokines. *, activated caspase-3.