Neuronal life or death linked to depression treatment: the interplay between drugs and their stress-related outcomes relate to single or combined drug therapies

Abstract

Depression is a serious medical condition, typically treated by antidepressants. Conventional monotherapy can be effective only in 60-80% of patients, thus modern psychiatry deals with the challenge of new methods development. At the same moment, interactions between antidepressants and the occurrence of potential side effects raise serious concerns, which are even more exacerbated by the lack of relevant data on exact molecular mechanisms. Therefore, the aims of the study were to provide up-to-date information on the relative mechanisms of action of single antidepressants and their combinations. In this study, we evaluated the effect of single and combined antidepressants administration on mouse hippocampal neurons after 48 and 96 h in terms of cellular and biochemical features in vitro. We show for the first time that co-treatment with amitriptyline/imipramine + fluoxetine initiates in cells adaptation mechanisms which allow cells to adjust to stress and finally exerts less toxic events than in cells treated with single antidepressants. Antidepressants treatment induces in neuronal cells oxidative and nitrosative stress, which leads to micronuclei and double-strand DNA brakes formation. At this point, two different mechanistic events are initiated in cells treated with single and combined antidepressants. Single antidepressants (amitriptyline, imipramine or fluoxetine) activate cell cycle arrest resulting in proliferation inhibition. On the other hand, treatment with combined antidepressants (amitriptyline/imipramine + fluoxetine) initiates p16-dependent cell cycle arrest, overexpression of telomere maintenance proteins and finally restoration of proliferation. In conclusion, our findings may pave the way to better understanding of the stress-related effects on neurons associated with mono- and combined therapy with antidepressants.

Keywords: Amitriptyline; Antidepressants; Depression; Fluoxetine; Imipramine; Neurons.

Fig. 1

Antidepressants-mediated effects in mouse hippocampal neurons (HT-22 cells). Chemical structure of amitriptyline (a), imipramine (d), fluoxetine (g), amitriptyline + fluoxetine (j), imipramine + fluoxetine (m). Cells were treated for 48 and 96 h with wide range of antidepressants concentrations and MTT assay was performed (b, e, h, k, n) to chose one concentration for further studies and then the effects of single 10 µM and combined 5 + 5 µM antidepressants on morphological characteristics (c, f, i, l, o) were evaluated. Magnification of the objective lens × 10. Bars indicate SD, n = 3, ***^{/^^^}p < 0.001, **^{/^^}p < 0.01, *^{/^}p < 0.05, no indication—no statistical significance (one-way ANOVA and Dunnett’s a posteriori test)

Fig. 2

Antidepressants-mediated effects on oxidative/nitrosative stress and antioxidant defense. HT-22 cells were treated for 48 and 96 h and the effects of antidepressants on superoxide, nitric oxide and thiol production (a–e) were evaluated. Red fluorescence—dihydroethidium (ROS), green—4-amino-5-methylamino-2′,7′-difluoro-fluorescein diacetate (NO), blue fluorescence—Thiol Tracker (Thiol). Magnification of the objective lens × 10. Bars indicate SD, n = 3, ***^{/^^^}p < 0.001, **^{/^^}p < 0.01, *^{/^}p < 0.05, no indication—no statistical significance (one-way ANOVA and Dunnett’s a posteriori test) (Color figure online)

Fig. 3

Stress-related effects of antidepressants on cellular and biochemical features. Mouse hippocampal neurons were treated for 48 and 96 h and then the effects of antidepressants on ATP generation (a), cell cycle progression (b), micronuclei generation (c), γH2AX formation (d) were evaluated. Representative images of micronuclei formation (e) and γH2AX activity (f) are shown. Blue fluorescence—Hoechst 33342, red—Texas Red. Magnification of the objective lens × 10. Bars indicate SD, n = 3, ***^{/^^^}p < 0.001, **^{/^^}p < 0.01, *^{/^}p < 0.05, no indication—no statistical significance (one-way ANOVA and Dunnett’s a posteriori test) (Color figure online)

Fig. 4

Antidepressants-mediated effect on cellular protein content. HT-22 cells were treated with antidepressants for 48 and 96 h and densitometry analysis of NF-κB (b), p16 (c), p21 (d), p27 (e), p53 (f), TRF1 (g), TRF2 (h), calnexin (i), NuMa (j), cleaved caspase 3 (k), Bcl-2 (l) was evaluated. Representative Western Blots are presented (a). Bars indicate SD, n = 3, ***^{/^^^}p < 0.001, **^{/^^}p < 0.01, *^{/^}p < 0.05, no indication—no statistical significance (one-way ANOVA and Dunnett’s a posteriori test)

Fig. 5

Proposed molecular mechanisms underlying the complex and multi-stage processes of hippocampal cell response to antidepressants