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. 2017 Dec 14;8(70):115490-115502.
doi: 10.18632/oncotarget.23302. eCollection 2017 Dec 29.

Antidepressants, sertraline and paroxetine, increase calcium influx and induce mitochondrial damage-mediated apoptosis of astrocytes

Chee-Kin Then  1   2 , Kao-Hui Liu  3   4 , Ming-Hsuan Liao  1 , Kuo-Hsuan Chung  5   6 , Jia-Yi Wang  1   7 , Shing-Chuan Shen  1   8   9
Affiliations

Antidepressants, sertraline and paroxetine, increase calcium influx and induce mitochondrial damage-mediated apoptosis of astrocytes

Chee-Kin Then et al. Oncotarget. .

Abstract

The impacts of antidepressants on the pathogenesis of dementia remain unclear despite depression and dementia are closely related. Antidepressants have been reported may impair serotonin-regulated adaptive processes, increase neurological side-effects and cytotoxicity. An 'astroglio-centric' perspective of neurodegenerative diseases proposes astrocyte dysfunction is involved in the impairment of proper central nervous system functioning. Thus, defining whether antidepressants are harmful to astrocytes is an intriguing issue. We used an astrocyte cell line, primary cultured astrocytes and neuron cells, to identify the effects of 11 antidepressants which included selective serotonin reuptake inhibitors, a serotonin-norepinephrine reuptake inhibitor, tricyclic antidepressants, a tetracyclic antidepressant, a monoamine oxide inhibitor, and a serotonin antagonist and reuptake inhibitor. We found that treatment with 10 μM sertraline and 20 μM paroxetine significantly reduced cell viability. We further explored the underlying mechanisms and found induction of the [Ca2+]i level in astrocytes. We also revealed that sertraline and paroxetine induced mitochondrial damage, ROS generation, and astrocyte apoptosis with elevation of cleaved-caspase 3 and cleaved-PARP levels. Ultimately, we validated these mechanisms in primary cultured astrocytes and neuron cells and obtained consistent results. These results suggest that sertraline and paroxetine cause astrocyte dysfunction, and this impairment may be involved in the pathogenesis of neurodegenerative diseases.

Keywords: antidepressants; astrocyte apoptosis; calcium overload; mitochondrial damage.

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Conflict of interest statement

CONFLICTS OF INTEREST We have confirmed that there are no known conflicts of interest associated with this publication.

Figures

Figure 1
Figure 1. Sertraline and paroxetine reduce astrocyte viability
Astrocyte viability was determined after treatment with indicated concentrations of antidepressants for 48 h by an MTT assay. Data were collected from three independent experiments and statistically analyzed by Student’s t-test, and results are shown as the mean ± SD. *p<0.05, **p<0.01, and ***p<0.001 compared to the control group.
Figure 2
Figure 2. Sertraline and paroxetine induce dose-dependent intracellular calcium elevation in astrocytes
(A) Fluorescence imaging of [Ca2+]i using Fluo-4 was conducted after 1, 3, and 6 h of 10 μM sertraline, 20 μM paroxetine, and 20 μM citalopram treatment. (B, C) Quantitative data were collected by flow cytometry. Significant induction of fluorescence intensity was seen in cells exposed to sertraline and paroxetine compared to the control. (D, E) Sertraline and paroxetine triggered dose-dependent induction of intracellular calcium level. Data were collected from three independent experiments and statistically analyzed by Student’s t-test, and results are shown as the mean ± SD. *p<0.05, ***p<0.001 compared to the control group.
Figure 3
Figure 3. Sertraline and paroxetine induce mitochondrial membrane damage with depleted ATP production and trigger ROS generation in astrocytes
(A, B) Astrocyte cells were treated with sertraline, paroxetine, and citalopram for 1.5, 3, 6, 12, and 24 h, and DiOC6 staining was used to examine the damage of mitochondrial membranes. The distribution of cells according to their mitochondrial membrane potential is shown in each panel of the figure, and means of DiOC6 intensity of different groups are compared in histograms. (C, D) ATP production was measured to monitor the mitochondrial function. (E, F) ROS generation was observed at 24 h after sertraline and paroxetine treatment. The DCF fluorescence intensity was measured by flow cytometric analysis, and the ratio of cells located at the M1 phase was quantified. H2O2 treatment is the positive control for ROS production. Data were collected from three independent experiments and statistically analyzed by Student’s t-test, and results are shown as the mean ± SD. *p<0.05, ***p<0.001 compared to the control group.
Figure 4
Figure 4. Sertraline and paroxetine activate the intrinsic apoptotic pathway in astrocytes
Cell death was either (A) observed by microscopy or (B) quantified by a flow cytometric analysis. (C) Cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) proteins were determined by a Western blot analysis.
Figure 5
Figure 5. Sertraline and paroxetine reduced cell viability in primary astrocytes, neurons, and their primary mixed culture
(A) Cell viability of different cells with indicated experimental conditions was detected by an MTT assay at 12 h after treatment. Data were collected from three independent experiments, and results are shown as the mean ± SD. (B) Fluorescence imaging of [Ca2+]i of primary astrocytes using Fluo-4 was conducted after 1, 3, and 6 h of 10 μM sertraline, 20 μM paroxetine, and 20 μM citalopram treatment. (C, D) Quantitative data were collected by flow cytometry. (E) Mitochondrial damage of primary astrocytes was observed after 12 and 24 h of sertraline, paroxetine, and citalopram treatment. The DiOC6 fluorescence intensity was measured by a flow cytometric analysis. Data were collected from three independent experiments and statistically analyzed by Student’s t-test, and results are shown as the mean ± SD. *p<0.05, ***p<0.001 compared to the control group.
Figure 6
Figure 6. Sertraline- and paroxetine-induced cell death was not associated with glutamate receptors, P2 receptor or TRPA1 receptor
Astrocytes were pretreated with (A) NBQX, MK-801, NS-102, (B) PPADs, and HC-030031, and then exposed to sertraline and paroxetine. Cell viability was analyzed by an MTT assay. Data were collected from three independent experiments and statistically analyzed by Student’s t-test, and results are shown as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001 compared to the control group.
Figure 7
Figure 7. Working model related to sertraline- and paroxetine-induced astrocyte apoptosis
Treatments with sertraline and paroxetine led to induction of intracellular calcium, mitochondrial hyperpolarization followed by mitochondrial damage, and reactive oxygen species (ROS) generation. Ultimately, caspase-3 and poly(ADP-ribose) polymerase (PARP) proteins were activated, and apoptosis occurred.
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