The Role And Mechanism Of KCC2-mediated Disturbance Of Chloride Homeostasis In Chronic Stress-induced Depressive-like Behavior

Objective:Numerous studies have shown that the gamma-aminobutyric acid（GABA）system is involved in the pathogenesis of major depressive disorder and is an important potential target for the treatment of depression.In mature neurons,the potassium-chloride cotransporter 2（K⁺-Cl^-cotransporter 2,KCC2）transports potassium and chloride ions from the intracellular to the extracellular,and is the main way for cells to pump intracellular chloride ions out,playing a key role in maintaining low intracellular chloride concentrations.GABA type A receptor（GABA_AR）-mediated synaptic inhibition is mainly dependent on chloride inward flow following channel opening.Defective expression of KCC2 reduces the driving force of chloride inward flow when GABA_ARs channels are opened,and weakens the inhibitory synaptic transmission mediated by GABA_ARs.This paper explores the role and mechanism of KCC2-mediated disturbance of chloride homeostasis in chronic stress-induced depressive-like behavior and seeks new targets and options for restoring chloride homeostasis.Methods:Chronic social defeat stress（CSDS）was applied to establish a mouse model of depression.Social behavior was detected according to the social interaction test（SIT）.Sucrose preference test（SPT）wasdetected to evaluate the reward-related behavior of CSDS mice.Novel-object recognition test was used to detect the effect of CSDS on learning and memory of mice.Open-field test was used to evaluate the effect of CSDS on movement ability and anxiety of mouse.Tail suspension test（TST）,forced swimming test（FST）were used to detect the despair behavior.Western blotting（WB）was used to detect the protein expression.Lentivirus（LV）was used for KCC2 silence or overexpression.Subthreshold social defeat stress（SSDS）was applied to detect the susceptibility of stress in mice.A fluorescent probe（N-（Ethoxycarbonylmethyl）-6-methoxyquinolinium bromide,MQAE）was used to detect the intracellular chloride concentration.Patch clamp technology was used to record neuronal GABA reverse potential(E_GABA)and miniature inhibitory postsynaptic currents（mIPSCs）to detect the inhibitory transmission.Stereotactic technology was used to inject virus or cannula implantation.MTT kit was used to detect the survive rate of RAW264.7 cell.Ultra-high-speed centrifugation was used to extract exosomes.Nanoparticle tracking analysis and immunoelectron microscopy were used to detect whether the particle size and morphology meet the exosomal standards.By the use of PKH67membrane dye kit,combining confocal imaging to detect whether the exosomes deliver Bume to the CNS.Determination of Bume content in Bume-Exo by high performance liquid chromatography.Results:（1）After CSDS procedure,the interaction ratio（IR）of susceptible mice was significantly lower than those in the control mice in SIT.Susceptible mice had reduced sucrose preference in SPT.（2）By WB it was found that the expression of KCC2 in d Hip（dorsal hippocampus,d Hip）and（ventral hippocampus,v Hip）of susceptible mice was significantly decreased.（3）Knockdown of KCC2 in d Hip by the use of LV（sh RNA-KCC2）promoted mice susceptibility to stress.（4）Overexpression（OE）of KCC2 in d Hip by the use of lentivirus reversed the behavioral deficit induced by CSDS and improve depressive-like behavior.（5）It was found that the intracellular chloride concentration of CSDS-susceptible mice was higher manifested with weaker probe fluorescence intensity.And the E_GABA in susceptible mice had a depolarized shift.（6）The amplitude of mIPSCs was significantly reduced in susceptible mice.（7）KCC2-OE could effectively reverse the shift of E_GABA induced by CSDS.（8）KCC2-OE reversed the decrease in amplitude of mIPSCs induced by CSDS.（9）Bume reversed the CSDS-induced E_GABA depolarization shift.（10）After incubation of the brain slices with Bume,and it was found that Bume reversed the decrease in mIPSCs amplitude induced by CSDS.（11）Direct injection of Bume into the dorsal hippocampus increased CSDS-induced depressive-like behaviors.（12）After incubation of RAW264.7 cells with Bume for 24 h,the exosomes were extracted and found to have high TSG101 protein expression but no Calnexin protein expression,and the particle size distribution and morphology of the extracts were consistent with the exosome criteria.（13）Bume-Exo was found to contain Bume in a high performance liquid chromatography experimental study.（14）PKH67-labelled exosomes was well-distributed in BLA,m PFC,NAc,d Hip in 6 hours after transnasal delivery.（15）Nasal administration of Bume-Exo reversed the decrease in interaction ratio induced by CSDS.Bume-Exo administered nasally reversed the decrease in socruse preference rate induced by CSDS.Bume-Exo reversed the CSDS-induced increase in immobility time in TST.（16）Bume-Exo reversed the CSDS-induced decrease in the amplitude of mIPSCs.Conclusion:The expression of KCC2 in d Hip of susceptible mice decreased significantly.CSDS increases the intracellular chloride concentration of neurons in d Hip of susceptible mice,accompanied by functional deficits in the GABA system.KCC2 knockdown in d Hip increases stress susceptibility of mice,and overexpression of KCC2 in d Hip reverses the CSDS-induced GABA system deficits and depressive-like behavior.The NKCC1 inhibitor Bume reversed CSDS-induced GABA system deficits and improved depressive-like behavior.Transnasal administration of Bume-Exo exerts promising antidepressant effects and provides a new strategy for the treatment of depression.