The Molecular Mechanism Of Prefrontal GABA Ergic Dysfunction In CUMS-induced Depression

Depression is a mental disorder characterized by a pervasive and persistent low mood in clinic,and often accompanied by other neurological diseases and chronic physiological diseases,such as anxiety,parkinson’s disease,multiple sclerosis,psoriasis,systemic lupus erythematosus,cardiovascular disease.Sociological factors,biological factors and psychological factors could contribute to the process,treatment and prognosis of depression.The risk factors of the depression are sex,age,race,marriage,socioeconomic status and educational level,genetic,life events and stress.In terms of its pathogenesis,chronic stress to the genetically susceptible individuals leads to the dysfunctions of monoamine,brain-derived neurotrophic factor and hypothalamus-pituitary-adrenal axis,which induce the neuron atrophy in brain reward circuits,such as the prefrontal cortex,nucleus accumbens and amygdala,in the depression patients and depression-like animals.In view of the complex pathogenesis,the current treatment of depression mainly includes: antidepressant drugs（tricyclic antidepressants,monoamine oxidase inhibitors,selective serotonin reuptake inhibitor,selective serotonin and norepinephrine reuptake inhibitors;psychotherapy;electrocoagulation therapy;fast antidepressant drugs.At present,the theory of interaction between stress and genetic factors is widely recognized.Neuroanatomical studies have shown that patients with depressive disorders have structural abnormalities in the specific brain and neural circuits associated with mood,reward,and executive functions.These highly interconnected "marginal" regions are presumably associated with depressive and antidepressant effects.Accumulating evidences from large number of autopsy and neuroimaging studies have showed that depressive patients have reduced gray matter volume and decreased glial density in the prefrontal cortex and hippocampus,which are thought to mediate the emotional aspects of depression,such as valuables and guilt.The prefrontal cortex has a wide range of connections and projections in the brain regions of learning and memory,and researchers have increasingly realized that the prefrontal cortex plays an important role in emotional processing.microRNA（miRNA）is a class of newly discovered endogenous non-coding small RNAs with 22 nucleotides.miRNAs regulate the expression of genes at post-transcriptional levels.As an important part of epigenetic regulation,recent studies have shown that miRNAs are specifically expressed or enriched in the brain or central nervous system.Depressive patients are often presented with imbalance neurological function,which is accompanied by changes in the expression of many miRNAs.miRNAs can participate in the pathophysiology of depression by regulating physiological processes such as neurogenesis,neurodevelopment,synaptic plasticity and neurotrophic factors.To reveal the molecular mechanism of the prefrontal cortex in the pathogenesis of stress-induced depression,we first used high-throughput sequencing to detect the changes of transcriptional and mi RNA expression profiles in the prefrontal cortex.The following results were obtained by bioinformatics analysis and subsequent experiments: 1）Mice were subjected to CUMS for consecutive three weeks.The ratio of the stay time of the M arm to the stay time in the total arm and the ratio of sugar water preference were significantly lower（P<0.05）than that in the control group,and the immobile time in the forced swimming group was significantly increased（P<0.01）.2)The following mRNA were significantly down-regulated in CUMS-induced depressive-like behavior mice,such as slc6a11,hap1,gad1,gad2,gng4,slc32a1,doc2 g,slc32a1,magel2,prkcd,ngfr,dusp1.On the other hand,the level of hba-a2,beta-s,hba-a1,hbb-b1,nr1d1,flot2 expression were significantly up-regulated.3)The analysis of signaling pathway enrichment indicated that GABAergic pathway,dopaminergic,synaptic vesicle cycling,amphetamines/ morphine addiction,neurotrophic factor signaling,MAPK signaling and chemokine-cytokine receptor signaling which were involved in chronic stress-induced depression pathophysiology process.4)Small RNA high-throughput sequencing analysis showed that 18 miRNAs’ expression level in the depression-like behavior group were significantly increased.The four significantly reduced miRNAs were mmu-miR-384-5p,mmu-miR-1264-3p,mmu-miR-217-5p,mmu-miR-490-5p in the depression-like behavior group.In addition,novel_mir₄6,novel_mir₂14,novel_mir₂13,novel_mir₁0,novel_mir₃9,novel_mir₁28,novel_mir₅4 were firstly detected in the prefrontal cortex.5)miRNA targeted genes were predicted by three miRNA prediction software（Targetscan,RNA22 and miRDB）,and then overlapped with the our DEG data to construct the prefrontal miRNA/mRNA regulatory network.6)To further validate the accuracy of RNA sequencing data,we selected five mRNAs,including two significantly elevated mRNAs,three down mRNAs and four novel miRNAs for qRT-PCR quantification.Combined high-throughput sequencing analysis results with the preliminary observation in our lab,we focus on the study of molecular and its epigenetic mechanism underlying GABAergic dsysfunction in the prefrontal cortex from CUMS-induced depression.To address the GABA terminal deterioration and its molecular mechanism,we combined multiple approaches to strengthen our results.For instance,the alternations in the molecular mechanism of GABA release/uptake were examined by high-throughput sequencing and qRT-PCR to quantify the expressions of both mRNAs and mi RNAs,western-blot to quantify their encoded proteins and double luciferase reporter was used to verify the interaction between miRNA and mRNA.1)The expression of GAD67,VGAT,GAT-3 protein involved in GABA synthesis,transport and reabsorption were significantly lower in the medial prefrontal cortices from CUMS-induced depression mice than in those from control mice.2)The expression of GAD67,VGAT,GAT-3 mRNA involved in GABA synthesis,transport and reabsorption were significantly decreased in the medial prefrontal cortices from CUMS-induced depression mice than that of control mice.3)Bioinformatics analysis showed that two regions of GAD67 3’UTR（769-775,902-908）,two regions of VGAT 3’UTR（622-628）and GAT-3 3’UTR（272-278,1393-1399）binds to miRNA-144-3p.miRNA-15b-5p was predicted to bind to the 1740-1747 region of the 3’UTR.A region of VGAT 3’UTR（608-614）was a binding site of miRNA-582-5p.miRNA-879-5p was predicted to interaction on 486-492 in GAT-3 3’UTR.4)miR-15b-5p,miRNA-879-5p,miRNA-144-3p and miRNA-582-5p were significantly increased expressed in the prefrontal cortex of the frontal cortex.5)There was the negative correlation between the network of mRNA/miRNA,which related to GABA synthesis,transport and reabsorption.6)Double luciferase report in vitro showed that our data suggest that GTA-3 was directly targeted by miR-15b-5p and miRNA-879-5p,VGAT was targeted by miRNA-144-3p and miRNA-582-5p,as well as GAD67 was targeted by miRNA-144-3p.The stress-induced incompatibility among the subcellular compartments of the GABAergic neurons as well as the incoordination between GABAergic and glutamatergic neurons lead to imbalanced neural networks in the medial prefrontal cortex,which may be one of the bases for depressive mood.In the future study,we need to investigate the causal relationship between the miRNA/mRNA regulatory network of the GABAergic neuron in the animal model.It is necessary to solve the following problems: whether the changes in miRNAs expression can regulate the phenotype of depression;the altered expression level of miRNAs whether play a role in antidepressant therapy by affecting the expression of targeted genes.