Fluoxetine Protects Against Depression Via Promoting Autophagy In Astrocytes

Depression is a common psychiatric disease characterized by anhedonia, low motivation, pessimism and self-accusation. Patients sufferring from major depressive disorder would have the propensity to suicide, there are approximately 850,000 cases of tragedies taking place every year. World Health Organization predicts depression as a chronic disease will rank only second to hypertension. At present, more than 120,000, 000 people live under the shadow of depression and there are few rapid, robust and sustained antidepressants used clinically. Thus, searching for more effective antidepressants and exploring new therapy is an urgent task.Fluoxetine, the first selective serotonin reuptake inhibitor, is clinically used in the form of fluoxetine hydrochloride, which is approved for the treatment of depression, bulimia nervosa and obsessive-compulsive disorder. Fluoxetine improves patients’ emotion via upregulating the concentration of extracellular 5-HT in several depression-related brain regions such as prefrontal cortex, hippocampus and striatum. Previous studies indicated that fluoxetine exerts neuroprotective, anti-inflammatory and anti-tumor effects. Beyond its influence on the monoaminergic neurotransmission, fluoxetine exihibits effects on autophagic processes. It has been reported that fluoxetine induces pro-autophagic effect in human adipose-derived stem cells and it has become a novel pro-autophagic agent.Autophagy is a self-degradative process by which it balances the energy sources and plays role of eliminating misfold or aggregated proteins, dysfunctional organelles such as mitochondria, endoplasmic reticulum, peroxisomes and pathogens. Thus, autophagy is generally considered as a survival mechanism. Currently, most researches focus on the regulation of fluoxetine in neuronal autophagy while study about astrocytic autophagy in the treatment of depression is nearly blank.Of all the neural cells, astrocytes rank the first place in the number (90%) and distribution which exist between neurons and capillary, they contribute to brain homeostasis in a couple of ways, including consisting of blood-brain barrier (BBB), releasing growth factors, buffering extracellular K+, regulating neurotransmitter release and the brain immune response. Astrocytes release several neurotrophic factors (including CNF, BDNF and GDNF) and various neuron supporters (such as glycoproteins promoting axon growth, cell adhesion molecules and laminins), whose function is isolating and sustaining neurons. Post-mortem analysis of patients with depression indicates that there is a decrease in the density of cortical glia. At the meantime, it is confirmed that the expression of GFAP and GFAP positive cells significantly decline in hippocampus or PFC in animals of depression model. With the further study on depression, some non-monoamine-based mechanisms have been found, astrocyte dysfunction may be an important pathophysiological mechanism.C57BL/6J mice were used to establish chronic mild stress (CMS)-induced anhedonia model of depression and corticosterone was used to simulate depression model in vitro. We detected the effect of fluoxetine on autophagy and mitophagy in primary hippocampal astrocytes. Then, we observed the protection of astrocytes by fluoxetine in vivo and in vitro and performed behavioral tests to observe the depressive- like behaviors of mice when autophagy was inhibited.AIM:C57BL/6J mice were used to investigate the protection of astrocytes by fluoxetine in depression and elucidate the critical role of autophagy in fluoxetine action and depression.METHODS:1. Chronic mild stress (CMS) was used to establish depression model in vivo.2. Transmission electron microscopy was performed to observe the mitochondria and autophagosomes in astrocytes.3. The expression of LC3 and p62 was detected via Western blotting.4. Primary hippocampal astrocytes were transfected with mTag-Wasabi-LC3 plasmid to observe the effect of fluoxetine on the autophagy in astrocytes.5. H2DCF-DA and MitoSOX were applied to label intracellular and mitochondrial ROS detected by immunofluorescence and flow cytometry, CCK-8 kit was used to measure cell viability of astrocytes.6. Primary astrocytes were transfected with GFP-LC3 plasmid, MitoTracker deep red and LysoTracker were taken to label mitochondria and lysosomes respectively. The colocalization of mitochondria and autophagosomes or lysosomes were observed by fluorescence microscope.7. CMS mice were injected autophagy inhibitor 3-MA intraperitoneally and intracerebral ventricularly, the number of astrocytes and their protuberance were observed by fluorescence microscope.8. The depressive-like behaviors of mice were evaluated by forced swim test (FST) and tail suspension test (TST) post injection of 3-MA.RESULTS:1. Fluoxetine ameliorated depressive-like behaviors of CMS mice.C57BL/6J mice were subjected to chronic mild stress and treated with fluoxetine. Sucrose preference of fluoxetine-treated CMS group was increased by 11% compared with saline-treated CMS group. The immobility time of fluoxetine-treated CMS group was declined by 38% and 34% compared with saline-treated CMS group.2. Fluoxetine significantly ameliorated mitochondrial impairment and promoted autophagosome formation in hippocampus of CMS mice.C57BL/6J mice were subjected to chronic mild stress and treated with fluoxetine. Mitochondria were normal in control and fluoxetine-treated group. 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-treated group and fluoxetine-treated CMS group (yellow arrows).2. Fluoxetine induced autophagy in primary hippocampal astrocytesPrimary astrocytes were subjected to fluoxetine in the concentration of 0.1,1.0, 10μM and treated with 10μM fluoxetine for 2,6,12,24h. Classic autophagic markers LC3 II/I and p62 expressions were analyzed by Western blotting. Fluoxetine significantly upregulated ratio of LC3 Ⅱ/Ⅰ in the concentration of 10μM (P< 0.05) and downregulated p62 (P< 0.01). Fluoxetine increased ratio of LC3Ⅱ/Ⅰ by 76%(P < 0.01) and decreased p62 expression by 72%(P< 0.01) compared with control group.3. Fluoxetine promoted autophagic flux of primary astrocytes effectively.Primary astrocytes were immunostained with GFAP, LC3 and DAPI simultaneously. LC3 puncta were significantly increased after 10μM fluoxetine treatment (P< 0.01) and were decreased after corticosterone treatment (P< 0.05). While fluoxetine could increase puncta of LC3 by 10 times after corticosterone treatment (P< 0.01). Immunofluorescence staining of astrocytes transfected with mTag-Wasabi-LC3 plasmid. A part of GFP-LC3 puncta were degraded in lysosomal acidic environment while RFP-LC3 was resistant to it. After fluoxetine treatment, puncta of both RFP-LC3 and GFP-LC3 were elevated by 18 and 15 times respectively (P<0.05).4. Mitophagy induced by fluoxetine in primary astrocytesPrimary astrocytes were treated with 10μM fluoxetine whose mitochondria were labeled by MitoTracker Deep Red. Each green spot represents one autophagosome. Compared with control group, colocalization of mitochondria (MitoTracker Green) and lysosomes (LysoTracker Red) were significantly decreased in primary astrocytes (P< 0.05) and overlay of lysosomes and mitochondria were decreased (P< 0.01), expression of cytoplasmic Parkin was elevated (P< 0.05), expression of mitochondrial Parkin was declined (P< 0.05) and TOMM20 was increased (P< 0.01) after corticosterone treatment. Following fluoxetine treatment, colocalization of autophagosomes and mitochondria, lysosomes and mitochondria was increased by 63% and 49% respectively. Expression of mitochondrial Parkin was elevated by 37% compared with vehicle-treated corticosterone group.5. Fluoxetine induced cytoprotection via promoting autophagy in vitro.Cells were stained with H2DCF-DA and MitoSOX to label intracellular and mitochondrial ROS, corticosterone treatment elevated intracellular and mitochondrial ROS (P< 0.01) and cell viability was declined (P< 0.01). Fluoxetine treatment decreased ROS (P< 0.05) level by 25% and enhanced cell viability by 56% compared with vehicle-treated corticosterone group (P< 0.05). While autophagy was inhibited, ROS level was elevated by 22%(P< 0.05) and cell viability was declined by 30%(P < 0.05).6. Fluoxetine induced cytoprotection in vivo and ameliorated depressive-like behaviors via promoting autophagy.The total number of protuberance in CMS mice hippocampal astrocytes was significantly decreased (P< 0.05) and GFAP positive cells were declined (P< 0.01). Behavioral tests indicated that immobility times of CMS mice were increased (P< 0.05). After fluoxetine administration, the number of protuberance was doubled and GFAP positive cell number were increased by 38%(P< 0.05), immobility time of forced swim test was decreased by 38%(P< 0.01) and immobility time of tail suspension test was reduced by 34%(P< 0.001). When autophagy inhibitor 3-MA was given, compared with fluoxetine treatment group, the number of protuberance was reduced (P< 0.01), GFAP positive cells were decreased (P< 0.05) and the depressive-like behaviors were aggravated, immobility of forced swim test was elevated (P< 0.01) and of tail suspension test was increased (P< 0.05).CONCLUSIONS:1. Dysfunction of astrocytic autophagy was related to the pathogenesis of depression.2. Fluoxetine reduced death of astrocytes in depression via promoting mitophagy thereby protecting astrocytes.The major contributions of the present study:1. Our study reveals the critical role of autophagy in depression and deepens the recognition of the pathogenesis of depression.2. Our study elucidates that fluoxetine exerts protection of astrocytes in CMS-induced depression and provides a new thinking for the development of antidepressants.