Iptakalim Alleviates Synaptic Damages Via Targeting Mitochondrial ATP Sensitive Potassium Channel In Depression

Depression is a common mood disorder,with major clinical manifestations including low mood,anhedonia,sleep and cognitive dysfunction,and even obvious suicidal tendencies in severe cases.According to statistics,the total number of people suffering from depression in the world has exceeded 300 million,and it will become the second most prevalent disease in the world.At present,the clinical treatment is mainly drug therapy,but the commonly used antidepressant drugs generally have the problems of slow onset,short duration,poor efficacy and so on.Therefore,it is urgent to further clarify the pathological mechanism of depression and develop rapid antidepressant drugs based on new targets.A large number of studies have shown that the volume of the hippocampal region in patients with depression and mice with depression model is significantly atrophied,and the number of synapses is also reduced to varying degrees.The structure and function of synapses are damaged,leading to synaptic plasticity disorder.Mitochondria are the main sites of ATP production and play a key role in maintaining presynaptic calcium homeostasis and neurotransmitter release.Studies have shown that depression leads to the deficiency of antioxidant enzymes and the increase of mitochondrial ROS in the body,resulting in mitochondrial dysfunction and impairing the transmission of synaptic neurotransmitters and synaptic plasticity.Mitochondrial fission and fusion plays a crucial role in maintaining mitochondrial functional homeostasis when the cell is under metabolic or environmental stress.Abundant studies have shown that the abnormal balance of mitochondrial fission and fusion is related to the occurrence and development of neurodegenerative diseases.However,it has not been reported whether mitochondrial fission and fusion is related to mitochondrial dysfunction and synaptic plasticity damage in depression.Therefore,studying and elucidating the influence of mitochondrial dynamic changes on synaptic plasticity and its mechanism in depression may provide new ideas for the development of "non-monoamine" antidepressant drugs and antidepressant therapy.Iptakalim(Ipt),as a novel ATP-sensitive potassium channel opener,is a small molecule of aromatic secondary amines designed and synthesized by Chinese scholars.Iptakalim can easily cross the blood-brain barrier.Current studies have shown that it has significant neuroprotective effects on neurons and astrocytes against necrosis and apoptosis in animal models such as stroke and Parkinson’s disease as well as in cellular models without the dose of dilating blood vessels and lowering blood pressure.Our previous studies have shown that Iptakalim exerts antidepressant protective effects by reducing inflammation and oxidative stress in the hypothalamus of mice,but the exact mechanism is not clear.Recent studies have shown that ATP-sensitive potassium channels in mitochondria are located in the inner membrane of mitochondria,composed of inward rectifying potassium channel subunits(MITOK)and ATP-binding domain(MITOSUR),which regulate the volume of mitochondria and maintain mitochondrial functional homeostasis.Therefore,in this study,in vivo(an animal model of depression established by chronic mild stress method)and in vitro(a cell model of damage induced by corticosterone stimulation),we explored the new mechanism of the antidepressant action of Iptakalim,and provided a reliable theoretical basis for the development of Iptakalim as a "non-monoamine" antidepressant.Objective:To study and reveal the effect of Iptakalim on synaptic plasticity of depression model mice,and to clarify the mechanism of Iptakalim improving depression-like behavior in mice.Methods:1.Preparation of depression model mice: male C57BL/6 mice aged 6-7 weeks were randomly divided into normal control group,model group,Iptakalim treatment group and fluoxetine treatment group.The in vivo mice model of depression was established by chronic mild stress(CMS)method.After modeling for 4 weeks,the mice were treated with Iptakalim or fluoxetine for 4 weeks.The sucrose preference test(SPT),forced swimming test(FST),tail suspension test(TST)and open field test(OFT)were used to evaluate the depression-like behavior of mice and the antidepressant effect of Iptakalim.2.The changes of neuronal synaptic structure and mitochondrial morphology and structure in the hippocampal region of mice were observed by Transmission electron microscopy(TEM).3.Immunofluorescence(IF)technique was used to detect the structural changes of neurite processes labeled by microtubule associated protein 2(MAP2)in the hippocampus of mice in each group.Cytochrome c oxidase Ⅳ(COX Ⅳ)as a marker of mitochondria was used to label changes of mitochondrial morphology and structure.And postsynaptic density 95(PSD95)and synaptophysin(SYN)expression changes were detected by IF.4.The primary neurons of mice were isolated and cultured,and the cell model of depression in vitro was established by corticosterone induction.Immunocytochemistry(ICC)staining was used to detect the process structure of MAP2-labeled neurons and the morphology and number of mitochondria of Mitotracker (?) Red CMXROS labeled neurons.5.N2 a cell lines were transfected with si RNA technology to observe the effect of down-regulation of Mitok subunit on the protective effects of Iptakalim.6.The functional changes of neuronal mitochondria in an in vitro model of depression were evaluated using ATP detection kit and mitochondrial membrane potential detection kit(JC-1).7.Methyl thiazolyl tetrazolium(MTT)and Lactic dehydrogenase(LDH)were used to detect cell activity.8.Western blotting was used to detect the expressions of synapse-related proteins PSD95 and SYN,as well as mitochondrial fission / fusion related proteins p-Drp1/Drp1 and OPA1 in mouse hippocampal tissues and cell samples.Results:1.Iptakalim improves depression-like behavior in depression model mice.Compared with the control group,the sucrose preference rate and body weight of depression model mice were significantly decreased,while the treatment with Iptakalim could significantly improve the sucrose preference rate and body weight loss of depression model mice.Compared with the depression model group,the immobility time in FST and TST was significantly shortened in the Iptakalim-treated mice,while the total distance in the OFT and the time of spontaneous activity in the central region were significantly increased.These results suggest that Iptakalim could improve the depression-like behaviors of depressed model mice.2.Iptakalim alleviates the damage of synaptic plasticity in the hippocampus of depression model mice.Iptakalim improved the synaptic structure damages,such as the decrease in the thickness of the postsynaptic dense and the decrease in the curvature of the synaptic interface,and the increase in the width of the synaptic cleft in CMS group.The results of tissue immunofluorescence showed that Iptakalim alleviated the atrophy,reduced length and density of MAP2-positive hippocampal neurons in CMS group.Further studies showed that Iptakalim partially reversed the down-regulated expression of synapse-related proteins PSD95 and SYN in the hippocampus induced by CMS.These results suggest that Iptakalim can improve the damage of synaptic plasticity in the hippocampal region of CMS induced depression mice.3.Iptakalim improves mitochondrial structure destruction and abnormal mitochondrial fission in hippocampal neurons induced by CMS in mice.The results of immunofluorescence showed that Iptakalim could improve the severe fragmentation of neuronal mitochondria in the hippocampal region of depression model mice.By TEM observation,it was found that Iptakalim partially reversed the damage and fragmentation of mitochondrial structure in the hippocampus of mice induced by CMS,and alleviated the damage of mitochondrial dynamics.Western blot results showed that Iptakalim could inhibit the abnormal increase of mitochondrial fission related protein p-Drp1/Drp1 and the significant decrease of mitochondrial fusion protein OPA1 in the hippocampus of CMS model mice,and inhibit the Drp1 transport from cytoplasm to mitochondria induced by CMS,thus improving the abnormal mitochondrial fission.These results indicate that Iptakalim can improve mitochondrial dysfunction by alleviating structural damage and excessive mitochondrial fission in the hippocampus of CMS model mice.4.Iptakalim reverses corticosterone-induced destruction of dendrites and synaptic structures in mice primary neurons.Immunofluorescence staining was used to observe the changes of dendritic structure of MAP2-positive neurons.It was found that Iptakalim could reverse the decrease of the branch length and number of neurite processes induced by corticosterone in a concentration-dependent manner.Western blot results showed that Iptakalim partially reversed the down-regulation of synapse-related protein PSD95 and SYN expression in corticosterone-induced neurons.These results suggest that Iptakalim can improve corticosterone-induced the destruction of neuronal dendrites and synaptic structures in a dose-dependent manner.5.Iptakalim improved the abnormal mitochondrial fission and mitochondrial dysfunction in mice primary neurons induced by corticosterone.Mitochondria labeled with Mitotracker Red probe were observed by laser confocal microscopy.The results showed that Iptakalim improved the degree of mitochondrial fragmentation and the destruction of reticular structure in mice neurons induced by corticosterone.Western blot results showed that Iptakalim partially reversed corticosterone-induced increase of mitochondrial division protein p-Drp1/Drp1 and down-regulation of mitochondrial fusion protein OPA1 expression.At the same time,through the detection of ATP content and mitochondrial membrane potential in neurons,it was found that Iptakalim increased the mitochondrial ATP content and mitochondrial membrane potential in a concentration-dependent manner.6.The effects of Iptakalim on improving mitochondrial structure and function depend on mitochondrial ATP-sensitive potassium channels.Changes in cell viability detected by MTT assay showed that corticosterone(100μM)induced decreased N2 a cell viability,while treatment with Iptakalim(100 μM)significantly alleviated the decreased corticosterone-induced cell viability.Si RNA interference was used to down-regulate the expression of Mitok,the ATP-sensitive potassium channel subunit in mitochondria.The results of laser confocal microscopy showed that down-regulation of Mitok subunit could partially reverse the effect of Iptakalim on corticosterone-induced mitochondrial structural damage.Western blot results also showed that down-regulation of Mitok subunit expression could partially reverse the effect of Iptakalim on the increase of mitochondrial division protein pDrp1/Drp1 and the decrease of mitochondrial fusion protein OPA1 expression.Similarly,the protective effects of Iptakalim on the promotion of cell ATP production and the enhancement of mitochondrial membrane potential were partially cancelled by downregulating the expression of Mito K.Studies have shown that the action of Iptakalim on mitochondrial ATP-sensitive potassium channels ameliorates corticosterone-induced abnormalities in neuronal mitochondrial structure and function.7.The effects of Iptakalim on mitochondrial ATP-sensitive potassium channels improves the synaptic plasticity abnormalities induced by corticosterone.It was found that Iptakalim inhibited corticosterone-induced down-regulation of synapse-related proteins PSD95 and SYN,and this effect was partially cancelled after down-regulation of Mitok subunit expression.After down-regulation of the Mitok subunit,the inhibition of corticosterone-induced LDH release by Iptakalim was partially cancelled.The results suggest that Iptakalim protects corticosterone-induced synaptic plasticity by acting on mitochondrial ATP-sensitive potassium channels.Conclusions:Iptakalim can improve abnormal mitochondrial fission,mitochondrial structural damage,decreased membrane potential and ATP production,thus improving synaptic function and playing an antidepressant role by acting on mitochondrial ATP-sensitive potassium channels.Innovations of the present study:1.To elucidate the new mechanism by which Iptakalim improves depression-like behavior in depressed model mice.Iptakalim can improve the structure and function of mitochondria by regulating mitochondrial fission and fusion,thus improving synaptic function and exerting antidepressant effects.2.To reveal the important role of mitochondrial ATP-sensitive potassium channels in the regulation of mitochondrial structure and functional homeostasis as well as synaptic plasticity by Iptakalim,providing a new target for the development of "nonmonoamine" antidepressant drugs.