The effect of fluoxetine on astrocyte autophagy flux and injured mitochondria clearance in a mouse model of depression

Abstract

Although multiple hypotheses had been proposed to clarify the causes of depression, the accurate pathogenesis and effective treatment of depression still need to be solved. Pathological change of astrocytes has been recognized to play a pivotal role in depression. Fluoxetine is the first selective serotonin reuptake inhibitor, however, the underlying mechanisms of fluoxetine are incompletely excavated. Emerging evidence shows that fluoxetine promotes autophagic processes in tumor cells. However, whether astrocytic autophagy gets involved in the cytoprotection of fluoxetine on astrocytes in depression treatment remains unexplored. Here we prepared chronic mild stress (CMS)-induced mouse model and treated mice with fluoxetine (10 mg/kg) for 4 weeks to determine the correlation between proautophagic effect of fluoxetine and astrocyte protection in depression. Primary hippocampal astrocytes were cultured to investigate the potential mechanism of fluoxetine in regulating astrocyte autophagy. We found that fluoxetine (10 mg/kg) treatment promoted autophagosome formation and increased clearance of injured mitochondria, consequently protected astrocytes in CMS model mice. Fluoxetine (10 μM) could also promote the autophagic flux unblocked via enhancing fusion of autophagosomes with lysosomes in primary astrocytes. Moreover, fluoxetine promoted mitophagy by increased colocalization of autophagosomes and mitochondria, eliminating damaged mitochondria in corticosterone-treated astrocytes. Further in vitro study showed that p53 presence is required for fluoxetine activated autophagy flux and fluoxetine promotes astrocytic autophagy in a p53-dependent mechanism. Collectively, this work gives us insights into a novel approach to treat depression depending on astrocytes, and provides a promising molecular target for the development of antidepressant drugs besides regulating neurotransmitters.

Fig. 1. Fluoxetine improved behavioral symptoms and alleviated astrocyte damage in CMS model mice.

a The program of CMS model preparation and drug administration in the present study. b Percentage of sucrose preference in control and CMS groups in the presence or absence of fluoxetine treatment. n = 15 in each group. c The immobility time in forced swimming test and in d tail suspension test. n = 15. e Immunofluorescence of GFAP⁺ astrocytes in whole mouse brain slice, scale bar: 500 μm. f Immunofluorescence of GFAP⁺ cells in mouse hippocampus, scale bar: 100 μm and g stereology quantitative counting of GFAP⁺ cells in dentate gyrus of hippocampus. n = 6, data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. Con group, ^#p < 0.05 vs. CMS group

Fig. 2. Fluoxetine ameliorated mitochondrial impairment and promoted autophagosome formation in hippocampus of CMS mice.

a Transmission electron microscope showed that mitochondria were normal in control and fluoxetine-treated group (red arrows). Mitochondria were severely disrupted in CMS group and the disruption was significantly ameliorated in fluoxetine-treated CMS group (red arrows). Several autophagosomes were observed in fluoxetine alone and fluoxetine-treated CMS group (yellow arrows). b Immunofluorescence of LC3 protein in mouse brain, scale bar: 50 μm. c Representative western blotting of LC3 and p62 expressions, and d, e quantitative analysis of protein levels. n = 5, data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con, ^#p < 0.05 vs. CMS group

Fig. 3. Fluoxetine attenuated corticosterone-induced apoptosis and activated autophagy in primary cultured astrocytes.

a–c Primary astrocytes were subjected to fluoxetine in the concentration of 0.1, 1.0, and 10 μM and d–f treated with 10 μM fluoxetine for 2, 6, 12, and 24 h. Classic autophagic markers, LC3II/I and p62, were analyzed using western blotting. g CCK-8 assay detected the cytoprotection of fluoxetine on corticosterone-inhibited astrocyte survival. h Hoechst staining showed the antiapoptotic effect of fluoxetine on corticosterone-induced cell death, at 20× magnification. i Representative image and j, k quantitative analysis of LC3II/I and p62 expression in the treatment of FLX (10 μM) and CORT (1.2 mM). Data are representative of four independent experiments and are expressed as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. respective control groups

Fig. 4. Fluoxetine promotes autophagic flux in primary astrocytes effectively.

a Primary astrocytes were immunostained with GFAP (green), LC3 (red), and DAPI (blue) simultaneously. LC3 puncta significantly increase after 10 μM fluoxetine treatment. b, c Quantitative analysis of LC3 puncta immunofluorescence intensity by flow cytometry in each group. d Immunofluorescence staining of astrocytes transfected with mTag-Wasabi-LC3 plasmid. A part of GFP-LC3 puncta is degraded in lysosomal acidic environment, while RFP-LC3 is resistant to it. After fluoxetine treatment, puncta of RFP-LC3 are much more than those of GFP-LC3. e, f Quantitative analysis of autophagosomes and autolysosomes per cell in each group. Scale bar: 100 μm. Data are representative of three independent experiments and are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. Con group, ^#p < 0.05, ^##p < 0.01 vs. Con group, ^$$p < 0.01 vs. CORT group

Fig. 5. Mitophagy was induced by fluoxetine in primary astrocytes.

a Immunofluorescence for colocalization of LC3 and mitochondria in primary astrocytes. Cells were treated with 10 μM fluoxetine whose mitochondria were labeled by MitoTracker Deep Red. Each green spot represents one autophagosome. Scale bar: 20 μm. b Pearson’s correlation coefficient for colocalization of LC3 and mitochondria. c Immunofluorescence for colocalization of mitochondria (MitoTracker Green) and lysosomes (LysoTracker Red) in primary astrocytes. Scale bar: 60 μm. d Pearson’s correlation coefficient for colocalization of lysosomes and mitochondria. e–h Western blotting and quantitative analysis of Parkin and TOMM20 expressed in cytoplasm and mitochondria. Data are representative of four independent experiments and are expressed as mean ± SD. *p < 0.05 vs. Con group, ^#p < 0.05, ^##p < 0.01 vs. Con group^{, $}p < 0.05, ^$$p < 0.01 vs. CORT group

Fig. 6. p53 mediated proautophagic effect of Fluoxetine in astrocytes.

a Representative western blotting and quantitative analysis of p53 trafficking from cytoplasm into nucleus. b Immunofluorescence for nuclear translocation of p53 induced by fluoxetine in astrocytes. Scale bar: 50 μm. c Western blotting analysis for the effect of p53 inhibitor, PFT-α on the expressions of p-mTOR, Atg7, LC3II/I, p62, and beclin-1 in astrocytes. d Western blotting analysis for the effect of fluoxetine on LC3II/I, p62 protein levels in wild-type and p53^−/− MEF cells. Data are representative of four independent experiments and are expressed as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. respective Con groups

Fig. 7. Fluoxetine produced cytoprotection on astrocytes via promoting autophagy.

a Fluorescence images of intracellular ROS (green). Cells were stained with H₂DCF-DA (20 μM) following fluoxetine treatment and captured under the fluorescence microscope. Scale bar: 50 μm. b Flow cytometry analysis of mitochondrial ROS stained by MitoSOX. c, d Quantitative analysis for fluorescence intensity of intracellular ROS and mitochondrial ROS, respectively. e CCK-8 assay for the effect of 3-MA, CQ, and BafA1 on fluoxetine-protected cell viability. Data are representative of three independent experiments and are expressed as mean ± SD. **p < 0.01 vs. Con group, ^#p < 0.05, ^##p < 0.01 vs. CORT group, ^$p < 0.05 vs. CORT+FLX group

Fig. 8. Fluoxetine protected astrocytes in vivo and ameliorated depressive-like behaviors via promoting autophagy.

a, b Immunofluorescence images and cell counts of hippocampal GFAP⁺ astrocytes in fluoxetine and 3-MA treated CMS mice. Scale bar: 500 μm. c, d Immunofluorescence images of astrocytic morphology and quantitative analysis of total branch numbers in each group. Scale bar: 20 μm, n = 6. e, f Immobility time of fluoxetine and 3-MA treated CMS mice in FST and TST, respectively. n = 12, data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. Con group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. CMS group^{, $}p < 0.05, ^$$p < 0.01 vs. CMS+FLX group