Mechanism Of Abnormal Regulation Of UBE2O On Neuronal Death And Cognitive Function In Alzheimer’s Disease

Background:Alzheimer’s disease(AD)is essentially a neurodegenerative disease and is the most common type of dementia in the elderly.Ubiquitin proteasome system(UPS)is essential for protein homeostasis,its dysfunction is related to the pathogenesis of AD.Ubiquitin coupling enzyme E20(UBE2O)is a kind of E2-E3 hybrid enzyme,which is the main component of UPS.However,its role in the pathogenesis of AD has not been fully determined.Objective:To investigate the expression characteristics of UBE2O and its role in the pathogenesis of AD.The vector plasmids of different length truncated fragments of UBE2O were constructed to explore the specific functional domain and molecular mechanism of UBE2O in the pathological mechanism of AD.Ube2o knockout(Ube2o-KO)mice were constructed to study the effects of UBE2O on cognitive and memory function and anxiety.Methods:1.Expression characteristics of UBE2O and its role in the pathogenesis of AD(1)Western Blot was used to detect the expression of UBE2O in all tissues,organs and brain regions of adult wild type(WT)mice.(2)The cell distribution characteristics of UBE2O in the brain of WT mice were detected by immunofluorescence staining.The expression of UBE2O in neurons and glial cells of WT mice and the expression of UBE2O in different cell lines were detected by Western Blot.(3)The changes of UBE2O expression in cortex and hippocampus of WT mice at different ages were detected by Western Blot.(4)Changes of UBE2O expression in cortex,hippocampus and AD model cells of 6-month-old AD model mice(5×FAD).(5)Aβ1-42(1μM)or DMSO(1μM)was added to the neuronal cell culture medium,and the cell lysate was collected at 12h,24h and 36h,respectively,the expression of UBE2O was detected by Western blot.(6)The effect of UBE2O overexpression on the expression of amyloid β protein precursor(APP)of AD pathogenic mutation.(7)The effect of UBE2O expression on neuronal activity was detected by detecting the activity of lactate dehydrogenase(LDH)in cell culture medium.2.Construction of UBE2O plasmid vectorThe UBE2O expression vectors with different length truncated fragments were constructed through the steps of primer design,PCR amplification,restriction enzyme digestion of vector and target fragment,T4 DNA ligase connection and transformation.3.Construction and behavior analysis of Ube2o-KO miceThe F0 generation of Ube2o knockout mice(heterozygous)were caged together to obtain homozygous Ube2o knockout mice(Ube2o-KO).Ube2o-KO mice aged 3 months(3M)and 6 months(6M)and WT mice were selected for open field test,elevated cross maze test,new object recognition test and Y maze spontaneous alternation test to observe the effects of Ube2o gene knockout on anxiety,cognitive and memory function of mice.Results:1.The expression characteristics of UBE2O and its role in the pathogenesis of AD(1)UBE2O is highly expressed in the cortex and hippocampus of WT mice,and UBE2O is highly expressed in neurons compared with glial cells.(2)In the cortex and hippocampus of WT mice,the expression of UBE2O decreased with age,and the peak expression of UBE2O in cortex and hippocampus of WT mice was on the 17th day(P17)and 14th day(P14),respectively.(3)In addition,the expression of UBE2O in the cortex and hippocampus of AD mice was significantly lower than that of WT mice.(4)Aβ1-42 induced the decrease of UBE2O expression in neuron cells.(5)Overexpression of APP decreased the expression of UBE2O and promoted neuronal death,while increased expression of UBE2O decreased the expression of APP and saved neuronal death induced by APPswe.2.Construction of UBE2O plasmid vectorThe results of PCR and sequencing showed that the plasmid vector UBE2O was successfully constructed.3.Behavioral analysis of Ube2o-KO mice(1)Genotype identification and Western Blot results showed that homozygous Ube2o-KO mice were successfully obtained;(2)Behavioral analysis showed that Ube2o-KO mice showed decreased ability of recognition memory and working memory and more obvious anxiety.Conclusion:Our study suggests that the decreased expression level of UBE2O in AD,and the age-related decrease of UBE2O may promote the neuronal death of AD.The increased expression of UBE2O reduces the production of APP in the pathological process of AD,and saves the neuronal death caused by APP.Ube2o-KO mice showed more obvious anxiety and decreased ability of recognition memory.The construction of plasmid vectors with different truncated fragments of UBE2O laid a foundation for further study of the specific mechanism and functional domain of UBE2O.