FBXL20 Regulated Synaptic Plasticity To Promote The Pathogenesis Of Depression

BackgroundDepression is a mental disorder characterized by depressed mood,delayed thinking and decreased activity.According to the World Health Organization,the total number of patients with depression in the world was 322 million in 2015,and there were about 54 million patients in China,accounting for about 4.2%of the national population.According to the 2022 National Blue Book on Depression,about 95 million people in China suffer from depression.Due to its high heterogeneity and complexity,as well as high recurrence and suicide rates,depression seriously limits the social function and reduces the quality of life of patients.The existing pathogenesis of depression cannot fully explain the clinical manifestations of patients with depression,and there are still a considerable proportion of patients with poor treatment effect.Therefore,it is urgent to explore the pathogenesis of depression and find effective treatment strategies.Recent studies have found that synaptic plasticity may be involved in the occurrence and development of depression.So how does synaptic plasticity lead to depressive-like behavior?And what causes impaired synaptic plasticity under stress?None of this has yet been elucidated.Current studies suggest that plasticity changes in presynaptic terminals or postsynaptic membranes may play an important role in cognitive impairment and mood disorders.It has been reported that synaptic plasticity disorder is involved in the occurrence and development of a variety of neuropsychiatric diseases,but its role in major depressive disorder(MDD)and the underlying molecular mechanism are still unclear.In this study,we found significant synaptic damage in hippocampus of rats exposed to chronic unpredictable mild stress(CUMS).However,the specific mechanism is not clear,which is the focus of this study.This study is expected to further improve the pathogenesis of depression,and to provide potential new targets and scientific basis for the development of antidepressant drugs.Methods(1)Male Wistar rats(160-180 g)were randomly divided into Control group(blank control)and chronic unpredictable mild stress(CUMS)group.After six weeks of chronic unpredictable stress stimulation,the rats in CUMS group were tested for depression-like and anxiety-like behaviors by forced swimming test,sucrose preference test,open field test and elevated plus maze test.According to the behavioral results,the rats were divided into High-susceptible subpopulations(HS)and Low-susceptible subpopulations(LS).The stress hypersensitivity(HS)group was the depression-like behavior rats.Transmission electron microscopy was used to observe the number and morphology of synapses in the CA1 region of hippocampus,as well as the number and localization of vesicles in the anterior membrane.(2)Proteomic analysis of differentially expressed proteins in hippocampal CA1 region of normal and depression-like rats;The selected target proteins VGLUT1 and VAMP 1 were verified by Western blot and immunofluorescence experiments.(3)The stress model of primary neurons induced by Corticosterone(CORT)was constructed.The expression levels of VGLUT1 and VAMP1 proteins were knocked down by lentivirus transfection,and the glutamate release from presynaptic membrane was detected by glutamate transmitter fluorescent probe(LV-iGluSnFR-eGFP).(4)FBXL20,an E3 ubiquitin ligase that was highly expressed in the hippocampal CA1 region of depressed rats,was screened out based on transcriptomics and proteomics analysis.The effect of FBXL20 on ubiquitination of VGLUT1 and VAMP1 was verified by immunoprecipitation assay.(5)To knock down FBXL20 expression,adeno-associated virus(AAV)was injected into the hippocampal CA1 region of rats by stereotactic brain injection;Behavioral test was used to detect the effect of FBXL20 knockdown on the depression-like behavior of depressed rats.Transmission electron microscopy was used to observe the effect of FBXL20 knockdown on synaptic plasticity in depression-like rats.Results(1)CUMS-induced depression-like rats showed depression-like manifestations such as anhedonia and behavioral despair.The number of synapses in the hippocampal CA1 region of depression-like rats decreased,the length of the active area of the presynaptic membrane shortened,and the number of synaptic vesicles located in the active area of the presynaptic membrane decreased,showing synaptic plasticity damage.(2)Fluoxetine improved the anhedonia and behavioral despair of depression-like rats,and improved the synaptic plasticity damage caused by chronic stress,such as the reduction of the number of synapses in the hippocampal CA1 region,the shortening of the length of the anterior membrane active area,and the reduction of the number of synaptic vesicles in the presynaptic membrane active area.(3)Proteomics results showed that the protein expression levels of VGLUT1 and VAMP1 in the hippocampal CA1 region of depression-like rats were decreased,and fluoxetine treatment could reverse the decreased expression levels of VGLUT1 and VAMP1 in the hippocampal CA1 region of depression-like rats.(4)The expression of VGLUT1 and VAMP1 was decreased in primary neuronal stress model induced by CORT.Knockdown of VGLUT1 in cultured primary neurons resulted in decreased glutamate transmitter release.Knockdown of VAMP1 in cultured primary shinjigens resulted in reduced release of glutamate transmitters.(5)Co-immunoprecipitation showed increased ubiquitination of VGLUT1 and VAMP 1 proteins in the hippocampal CA1 region of depression-like rats.(6)Proteomics results showed that E3 ubiquitin ligase F-box and leucine-rich repeat protein 20(FBXL20,also known as SCRAPPER)protein expression levels in the hippocampal CA1 region of depressive-like rats were increased.(7)Knockdown of FBXL20 can up-regulate the protein expression of VGLUT1 and VAMP1 in the hippocampal CA1 region of depression-like rats and improve the depression-like behavior of the rats.(8)Knockdown of FBXL20 in the hippocampal CA1 region of depression-like rats can effectively improve the synaptic plasticity damage in the hippocampal CA1 region of depression-like rats,such as the reduction of the number of synapses,the shortening of the length of the anterior membrane active area,and the reduction of the number of synaptic vesicles located in the presynaptic membrane active area.ConclusionsChronic stress can promote the ubiquitination and degradation of synapse-associated proteins VGLUT1 and VAMP1 by up-regulating the expression of E3 ubiquitin ligase FBXL20 in the hippocampal CA1 region of rats.The decreased expression levels of VGLUT1 and VAMP 1 lead to decreased uptake and release of glutamate transmitters in neuronal synaptic vesicles,thereby affecting neuronal synaptic transmission,and finally promoting depression-like behavior in rats.In summary,the results of the present study revealed that stress stress may regulate the expression of VGLUT1/VAMP1 through FBXL20,leading to the damage of neuronal synaptic structure and function in the hippocampal CA1 region of rats,ultimately promoting the generation of depression-like behaviors.The results of this study improve the pathogenesis of depression and provide potential targets and scientific basis for the development of clinical antidepressant drugs.