The Role And Mechanism Of NHE1 In Regulating Hippocampal Synaptic Plasticity In Depression

Background:With the rapid development of society and the frequent occurrence of various high-pressure events,depression has gradually become the most common mental disorder in the world,which characterized by long-term depression,lack of pleasure and behavioral despair.The clinical first-line antidepressant drugs are not effective and patients are prone to relapse after drug withdrawal,which reveal the multi-etiological mechanism of depression.Therefore,it is of great importance to make a more comprehensive understanding of the pathological mechanism of depression in order to find new targets for treating depression and to develop better diagnosis and treatment strategies.Recent research results have showed that neuronal synaptic plasticity disorder might play an important role in the occurrence and development of depression.Chronic and continuous stress often leads to abnormal adaptability of the brain,and the damage of synaptic plasticity including the structure and function,thus affecting the normal neural discharge,neuroendocrine and other activities,which may ultimately be an important incentive to promote depression,anxiety and other emotional disorders,but the mechanism is unclear.Intracellular acid-base balance is one of the important characteristics of maintaining a homeostatic cell environment,but so far,the role and mechanism of intracellular acid-base homeostasis imbalance in the pathogenesis of depression remain unclear.Na+/H+exchanger(NHE)belongs to a highly conservative evolutionary transporter family,which can exchange intracellular H+and extracellular Na+in an electrically neutral way and regulate the dynamic balance of intracellular pH(pHi).NHE1 is the most abundantly expressed subtype in the central nervous system,especially in the cortex,hippocampus and cerebellum,and is the main determinant of the intracellular pH homeostasis of mammalian neurons.However,its role in the regulation of synaptic plasticity and whether its abnormal expression is involved in the pathogenesis of depression remains unclear.Under this research background,we proposed to establish chronic unpredictable mild stress(CUMS)model as a classic depression animal model,and use a variety of experimental techniques and methods including proteomic sequencing analysis,electrophysiology,animal behavior tests molecular biological experiment,virology and transgenic mice combination to explore whether NHE1 is involved in the pathogenesis of depression by regulating synaptic plasticity under the effect of stress and the specific molecular mechanism of its role.Methods:(1)CUMS modeling in vivo:Male Wistar rats(120-140 g)were randomly divided into Control(no treatment,Ctrl)group and CUMS group.The rats in the CUMS group were given chronic unpredictable mild stress for 5 weeks.Then the two groups of rats were subjected to undergo behavioral tests to assess depression-like and anxiety-like behaviors,including the sucrose preference test,the forced swimming test,the open field test,and the elevated plus maze test.H+fluorescent probe was used to detect intracellular pH of neurons.Immunofluorescence,Golgi staining and transmission electron microscopy were used to detect neuronal structural plasticity such as dendritic spines and synaptic morphological number;Brain slice patch clamp recording technique for brain slice was used to record functional discharge activity.Proteomic sequencing was performed on hippocampal tissue from rats in the Control group and CUMS group which showed depressive-like behavior in behavioral tests.Bioinformatics analysis found there were a lot of differentially expressed proteins including NHE1 which regulates intracellular pH changes in the hippocampus CA1 region of depression-like rats.(2)Intracerebral injection modeling of adeno-associated virus knocking down NHE1:Adeno-associated virus knocking down NHE1 was constructed and injected into the bilateral hippocampal CA1(Cornu Ammonis 1)region of rats.Behavioral tests such as SPT(Sucrose Preference Test)and FST(Forced Swimming Test)were performed 2-3 weeks after virus infection.If there was no significant depression-like behavior,the rats were given chronic unpredictable mild stress stimulation for 2 weeks;The expression level of NHE1 protein,intracellular pH,neuronal structure and function changes after virus injection were separately observed by western blot,Gorgi staining,whole-cell patch clamp technology and electron microscopy.(3)In vitro experimental modeling:The mRNA expression level of NHE1 was verified by whole transcriptome sequencing and qPCR.The differential expression proteins related to the regulation of NHE1 expression were found through enrichment analysis.Bioinformatics analysis was used to find differentially expressed proteins related to the regulation of NHE1 protein expression.Primary hippocampal neurons were cultured to establish the cell stress model induced by Corticosterone(CORT)and to verify the degradation pathway of NHE1 protein under stress stimulation:The lysosomal inhibitor chloroquine was used to block the lysosomal degradation pathways.The proteasome inhibitor MG132 was used to block the proteasome degradation pathways.The colocalization of NHE1 and CUL4A(Cullin4A)was observed in endoplasmic reticulum(ER)by confocal technology and the ubiquitination level of NHE1 under stress stimulation was verified by immunoprecipitation technology.(4)Adeno-associated virus that overexpressed CUL4A was constructed and injected into the bilateral hippocampal CA1 region of Thyl-Cre mice.Behavioral tests such as sucrose preference test and forced swimming test were performed 2-3 weeks after virus infection;Intracellular pH indicator and confocal microscope were used to detect the change of neuronal pHi in the hippocampal CA1 region;Western blot was used to detect protein expression levels of CUL4A and NHE1;Whole-cell patch clamp technique recorded spontaneous firing and induced action potential changes of neurons.(5)The chemogenetic virus AAV-DIO-hM4Di-mCheery was constructed and injected into the hippocampal CA1 region of Thyl-Cre mice.The chemogenetic virus AAV-CaMKII-hM4Di-mCheery was constructed and injected into the hippocampal CA1 region of normal rats by stereotactic injection.2-3 weeks after the virus infection,the animals were intraperitoneally injected with saline or CNO(clozapine-N-oxide).Behavioral changes were detected by sucrose preference test and forced swimming test;The brain patch clamp technique was used to detect excitability of neurons before and after CNO administration.Results:(1)Compared with the Control group,the sucrose water consumed by CUMS group rats in the sucrose preference test was significantly reduced;In the forced swimming test,the immobility time of CUMS group increased significantly;In the open field test,there was no significant difference in the total distance of movement,but the time staying in the central grid of CUMS rats decreased,compared with the Control group;In the elevated plus maze test,compared with the Control group,the number of entering the open arm and the total time of staying in the open arm in CUMS group were both significantly reduced.Compared with Control group,the neuronal intracellular pH of hippocampal CA1 region in CUMS group was significantly lower;The complexity of neurons,the density of dendritic spines and the number of synapses decreased;Mini excitatory postsynaptic current and spontaneous excitatory postsynaptic current decreased in pyramidal neurons of CUMS group.These results suggest that chronic unpredictable mild stress for 5 weeks could induce depression-like behavior in rats,reduce intracellular pH of neurons and lead to abnormal plasticity of hippocampal neurons in structure and function.(2)The proteomic results showed that there were a lot of proteins differentially expressed in the CA1 region,among which the expression of NHE1 in the CA1 region of depression rats was significantly reduced.Further analysis found that function of NHE1 was closely related to the regulation of intracellular pH,suggesting that NHE1 might be a functional molecule involved in the occurrence and development of depression.(3)The results of behavioral tests showed that,compared with the AAV-eGFP group,after the knockdown of NHE1 in the CA1 region of normal rats,the sucrose preference in the sucrose preference test,the time of swimming and resting in the forced swimming test,the total distance of movement and the time of staying in the central area in the open field test,and the number of entering the open arm and the time of staying in the open arm in the elevated plus maze test had no significant effect.However,behavioral tests showed that CUMS stimulation for only 2 weeks was sufficient to induce significant depression-like behaviors in the rats knocking down NHE1,including a decrease in the percentage of sucrose consumption in the sucrose preference test and a significant increase in the immobility time in the forced swimming test.It is suggested that rats of knocking down NHE1 were more likely to exhibit depressive behavior after stress,compared with the AAV-eGFP group.(4)The results of qPCR showed that there was no statistically significant difference in the mRNA level of NHE1 between the Control group and the CUMS group,suggesting that the down-regulation of NHE1 protein level may be due to post-transcriptional or post-translational pathway regulation.COG(Cluster of homologous groups)enrichment analysis showed that the differentially expressed proteins were mainly concentrated in the post-translational modification pathway.In the differentially expressed proteins,CUL4A is an important member of the E3 ubiquitin ligase family that degrades proteins through ubiquitination,and its expression level in CUMS group is significantly increased,compared with the Control group.Western blot showed that treatment with proteasome inhibitor MG 132 significantly reverse the Cort-induced downregulation of NHE1 expression level,indicating that the down-regulation of NHE1 protein level was related to the proteasome degradation pathway.Immunofluorescence results showed that CUL4A and NHE1 were co-located in the endoplasmic reticulum.The results of immunoprecipitation showed that the NHE1 ubiquitination level increased with treatment time of CORT.These results suggest that CUL4A may target the ubiquitination modification of NHE1 and promote its degradation through proteasome pathway.(5)CUMS stimulation for 2 weeks induced significant depression-like behavior after CUL4A was overexpressed in excitatory pyramidal neurons in hippocampal CA1 of Thyl-Cre mice.With the overexpression of CUL4A,intracellular pH of CA1 neurons,the activity of glutamate synthetase,the spontaneous discharge and the evoked action potential of neurons decreased significantly accompanied by decreased expression levels of NHE1 protein,compared with the AAV-eGFP group.(6)Patch clamp whole-cell recording results showed that the evoked current threshold of action potential generated by hippocampal neurons of Thyl-Cre mice expressing hM4Di increased and neuronal excitability decreased after CNO was added into recording solution.After administration of CNO,the discharge frequency of excitatory neurons in rats expressing hM4Di decreased significantly under the same injection current induction condition.Behavioral test results found that Thyl-Cre mice with specific expression of hM4Di in pyramidal neurons in the CNO group,compared with the saline injection group,showed more immobile in both the forced swimming experiment and the tail suspension experiment.In rats with specific expression of hM4Di in excitatory neurons,intrabitoneal injection of CNO increased immobility time in forced swimming test,decreased consumption of sugar water in sucrose preference test,and exhibited depression-like behavior.Conclusion:(1)CUMS stimulation for 5 weeks could increase the expression of E3 ubiquitin ligase CUL4A in the hippocampal CA1 region of rats.CUL4A may degrade NHE1 protein by ubiquitination pathway,resulting in the reduction of H+ excretion in neurons,the decrease of pH value and the imbalance of acid-base homeostasis in cells,which in turn lead to the disorder of synaptic plasticity of pyramidal neurons in the CA1 region of hippocampus,the weakening of synaptic transmission efficiency,the reduction of neuronal excitability,and finally promote the depression-like behavior in animals.The results of our study improve the mechanism of neuroplasticity damage in the pathogenesis of depression caused by stress stress stimulation and provide potential targets and scientific basis for the discovery and screening of new antidepressants and new strategies for antidepressant treatment.