Mechanistic Study Of Agomelatine Improving The Cerebral Pathology And Memory Behavior Of APP/PS1 Transgenic Mice By Down-regulating Ube2c

Objective:Alzheimer’s disease（AD）is a progressive neurodegenerative disease characterized by memory loss,cognitive decline and advanced dementia.One of the pathological features of AD is the deposition of a large number of diffused plaques and neuritic plaques centered on amyloid-βprotein（Aβ）.The pathogenesis of AD is very complicated,which maybe results in the failure of many single-target drugs in the development of new drugs for AD.Therefore,it is of great significance to elucidate the pathogenesis of AD and to design and develop multi-target drugs for the prevention and treatment of AD.It has been reported that ubiquitin-conjugating enzyme 2C（Ube2c）might be involved in the development of AD.The expression level of Ube2c in the brain is closely related to cognitive function,but it is still unclear for the molecular mechanism of Ube2c in AD.The latest researches show that Ube2c is involved in the regulation of autophagy,and Ube2c overexpression can inhibit autophagy,while inhibition of Ube2c can promote autophagy.Autophagy is a major intracellular degradation pathway for the clearance of damaged organelles and misfolded peptides.Previous studies have indicated that autophagy is involved in the pathogenesis of neurodegenerative diseases including AD.Defective autophagy and highly expressed Ube2c have been found in AD patients and AD model mice.However,there is insufficient supportive evidence that whether high expression of Ube2c in AD could lead to autophagic dysfunction,by which promotes Aβpathology in the brain and impaires cognitive function.Meanwhile,it will also remain a challenge to search for clinically available Ube2c inhibitors if Ube2c inhibition could be effective to treat AD.Therefore,the present study further confirmed the high expression of Ube2c gene in the brains of AD patients and APP/PS1 transgenic mice by using bioinformatics analysis and AD animal model.Then,we used AAV-shRNA-mediated knockdown of Ube2c in the APP/PS1 mice and in vitro BV2 cells to investigate the effects of Ube2c knockdown on autophagy,AD-like pathological features and cognitive behavior in APP/PS1 transgenic mice.In addition,we systematically explored the effects of AGO on Ube2c expression,Aβneuritic plaques,cognitive behavior and hippocampal synaptic plasticity in APP/PS1transgenic mice by intraperitoneal injection of AGO.The purpose of this study is to explore a novel mechanism of AD pathogenesis and to provide new strategies and targets for the treatment of AD.Methods:1.Bioinformatics analysis and experimental confirmation of Ube2cThe differential expression of Ube2c between AD patients and normal people was firstly analyzed through AlzData database.On this basis,we further confirmed the changes of Ube2c expression in the hippocampus of 8-month APP/PS1 transgenic mice by immunoblotting and immunofluorescence.2.AAV-shRNA-mediated Ube2c knockdown in vitro and in vivo experimentsBy using AAV-shRNA-mediated Ube2c knockdown in BV2 cells and APP/PS1 mice,we investigated:（1）the effect of Ube2c knockdown on the expression of Ube2c protein,Aβclearance and autophagy-related proteins in BV2 cells;（2）the effect of Ube2c knockdown on Aβpathology in the hippocampus and learning and memory of APP/PS1transgenic mice.3.In vivo and in vitro experiments of AGO interventionFor in vitro experiment,Aβ-treated BV2 cells were used as an AD cell model.We observed the effects of AGO on Ube2c expression,Aβclearance and autophagy-related proteins in the BV2 cells by immunoblotting.For in vivo experiment,8-month-old APP/PS1 transgenic mice and wild-type littermates（WT）were used in this study,which were randomly divided into three groups according to different genotype and treatment.AGO（10 mg/kg）or equivalent vehicle（Veh）was intraperitoneally injected into mice once a day for 30 consecutive days.Thereafter,the m RNA-seq of hippocampi in the mice was measured and the differential genes were analyzed.By using immunofluorescence,we examined the number and distribution of Aβplaque and microglia in the hippocampal slices of mice.Furthermore,we also detected the levels of soluble Aβ₄₀and Aβ₄₂in the hippocampal tissues of mice by using meso scale discovery（MSD）method.Behavioral tests,including spontaneous alternative Y maze and Morris water maze,were performed to test the learning and memory function of mice.By recording field excitatory postsynaptic potentials（f EPSPs）in the hippocampal CA1 region of mice,high frequency stimulation-induced long-term potentiation（LTP）and paired pulse stimulation-induced paired-pulse facilitation（PPF）were observed respectively.Results:1.Abnormal high expression of Ube2c in the brain of patients and transgenic mice of ADBy analyzing AlzData database,we found that the levels of Ube2c in the hippocampus and cortex of AD patients were significantly higher than that of normal people,which was supported by our animal experimental results showing that Ube2c protein expression was also higher in the hippocampus and cortex of APP/PS1 mice.Notably,the immunofluoresence of hippocampal slices displayed that there was a significantly higher Ube2c expression in the Aβplaque-deposited area.Additionally,the immunofluoresence of Ube2c and Iba1-positive microglia had also a well co-localization,indicating that Ube2c might be mainly expressed in microglia.Furthermore,the results of in vitro BV2 cell culture experiments showed that there was a notable increase of Ube2c expression（P<0.001）in Aβ-treated cells.2.The effects of Ube2c knockdown on microglial autophagy,Aβpathology and learning and memory behavior in APP/PS1 miceThe experiments of Ube2c knockdown in BV2 cells showed that the Ube2c protein expression level in sh Ube2c group,compared to normal control group,had a significant decrease（P<0.05）,as well as a downward tendency of p62 expression and an obvious increment of LC3BII/LC3BI（P<0.01）.In addition,there was a notable decline of Ube2c expression（P<0.01）,p62 expression（P<0.001）and Aβlevel（P<0.05）in Aβ_1-42+sh Ube2c group compared with Aβ_1-42group,while the ratio of LC3BII/LC3BI had an increase（P<0.05）.These data suggested that down-regulation of Ube2c in BV2 cells indeed promoted microglial autophagy and Aβclearance.In the in vivo experiments of AAV transfection-mediated Ube2c knockdown in the hippocampus of APP/PS1 mice,we firstly verified a significant down-regulation of Ube2c in the hippocampus of sh Ube2c-treated APP/PS1 mice compared with shRNA ctrl-treated APP/PS1 mice（P<0.05）.And then,the hippocampal coronal sections stained with Thioflavin S（Thio S）displayed that Aβplaques were markedly increased in shRNA ctrl-treated APP/PS1 mice compared to shRNA ctrl-treated WT mice,while both the number（P<0.01）and area（P<0.05）of Aβplaques were obviously decreased in sh Ube2c-treated APP/PS1 mice.Meantime,the results of MSD detection showed that the levels of Aβ₄₀（P<0.05）and Aβ₄₂（P<0.01）were clearly declined in sh Ube2c-treated APP/PS1 mice compared to shRNA ctrl-treated APP/PS1 mice.Furthermore,Y maze test revealed that the percentage of correct spontaneous alternations displayed an improvement in APP/PS1-sh Ube2c group compared with APP/PS1-shRNA ctrl group（P=0.096）.Additionally,Morris water maze test showed that,compared to shRNA ctrl-treated APP/PS1 mice,the average escape latencies in sh Ube2c-treated APP/PS1 mice were remarkably shorter on the 6th day in place navigation test（P<0.05）,and the percentage of swimming time in the target quadrant（P<0.05）and the number of platform crossings（P=0.067）were both elevated.These results implied that Ube2c knockdown ameliorated working memory and long-term memory in APP/PS1 mice.3.The effects of AGO on Ube2c expression in APP/PS1 mice and microglial autophagyIn the m RNA sequencing after treatment with AGO,we found that compared with WT+Veh group,1822 genes showed differential expression in APP/PS1+Veh group,in which 959 differential genes（52.6%）were reversed in APP/PS1 mice treated with AGO.Cluster analysis of these reversed genes showed that the positive roles of AGO were mainly concentrated in cell homeostasis and Ca²⁺regulation in the hippocampus of APP/PS1 mice.Meanwhile and importantly,our m RNA sequencing analysis also showed that AGO clearly inhibited the high expression of Ube2c in the hippocampus of APP/PS1mice.In deed,the following immunoblotting experiment in BV2 cells showed that Ube2c expression in Aβ_1-42-treated BV2 cells was significantly higher than that in Aβ_1-42-untreated group（P<0.05）,while the Ube2c high expression was significantly descended in Aβ_1-42+AGO group（P<0.001）.Meanwhile,there was a marked increase in autophagy-related protein LC3BII/LC3BI（P<0.05）and an evident decrease in the autophagy substrate p62expression and intracellular Aβlevel in Aβ_1-42+AGO group（P<0.01）compared to Aβ_1-42group.Moreover,the Aβclearance effect of BV2 cells induced by AGO and Ube2c knockdown was inhibited by autophagy inhibitors 3-methyladenine（3-MA）and chloroquine（CQ）.These data suggested that AGO,by inhibiting Ube2c expression,enhanced the phagocytosis and autophagy in BV2 cells to eliminate intracellular Aβ.4.The effects of AGO on AD-like pathology in the hippocampus and learning and memory of APP/PS1 miceThe results of colocalization with Iba 1 antibody and Thio S staining in the brain slices exhibited that there was a remarkable increase of the number and area of Aβplaques in Veh-treated APP/PS1 mice compared with Veh-treated WT mice,which was obviously rescued by AGO treatment（APP/PS1+AGO）（P<0.05）.Meanwhile,the levels of Aβ₄₀and Aβ₄₂were evidently decreased in AGO-treated APP/PS1 mice compared to Veh-treated APP/PS1 mice by using MSD detection（P<0.05）.In addition,compared to Veh-treated WT mice,Veh-treated APP/PS1 mice displayed a obvious rise in the area（P<0.001）and number（P<0.01）of microglia.However,these rises were reversed by the treatment of AGO（APP/PS1+AGO）（P<0.01）.In Y maze test,the total number of arm entries did not show any significant difference among four groups（P>0.05）.Nevertheless,compared with WT+Veh group,Veh-treated APP/PS1 mice showed lower percentage of correct spontaneous alternations（P<0.01）,whereas AGO-treated APP/PS1 mice showed an increase（P<0.05）.In Morris water maze test,the results of place navigation test showed that APP/PS1+Veh group showed significantly longer escape latency than WT+Veh group from the 3rd day（3rd day:P<0.01;4th day:P<0.001;5th day:P<0.001）,while the escape latency of AGO-treated APP/PS1 mice（APP/PS1+AGO）was significantly shorter than that of Veh-treated APP/PS1 mice on 4th（P<0.05）and 5th day（P<0.001）.In the probe test,compared to WT+Veh group,the swimming time in the target quadrant displayed a less percentage in Veh-treated APP/PS1 mice（P<0.05）,whereas that was elevated in AGO-treated APP/PS1 mice（APP/PS1+AGO）（P<0.05）.In reversal place navigation test,compared with WT+Veh group,Veh-treated APP/PS1 mice exhibited significantly longer escape latency during four days（7th day:P<0.05;8th day:P<0.001;9th day:P<0.001;10th day:P<0.001）.Especially,AGO-treated APP/PS1 mice（APP/PS1+AGO）showed shorter escape latency than the Veh-treated APP/PS1 mice on the 9th（P<0.05）and 10th day（P<0.001）.In the reversal probe test,the percentage of swimming time in the target quadrant showed an apparent reduce in APP/PS1+Veh group than WT+Veh group（P<0.05）,while that was increased in AGO-treated APP/PS1 mice（APP/PS1+AGO）（P<0.05）.5.The effects of AGO on hippocampal synaptic plasticity of APP/PS1 miceBefore high-frequency stimulation,f EPSP amplitude in each group increased with the increase of stimulation current intensity,and there was no significant difference in f EPSP amplitude among three groups at the same stimulation current intensity（P>0.05）.However,Veh-treated APP/PS1 mice showed a significant reduction in the percentage of f EPSP slope at 30th min and 60th min post-HFS compared with Veh-treated WT mice（30min:P<0.05;60 min:P<0.01）,which was significantly reversed by AGO treatment（30min:P<0.05;60 min:P<0.01）.And no significant changes of the percentage of PPF slope were found among the three groups（P>0.05）.Conclusions:1.Highly expressed Ube2c gene in various brain regions of AD patients and Ube2c protein in hippocampus and cortex of APP/PS1 transgenic mice indicate that Ube2c might be involved in the pathogenesis of AD.2.The highly expressed Ube2c had a well co-localization with microglia and Aβin the hippocampus of APP/PS1 transgenic mice,implying that the Ube2c high expression might affect Aβelimination in microglia.3.Ube2c knockdown in BV2 cells enhanced expression levels of autophagy-related protein and a decreased Aβlevel,indicating that Ube2c high expression could inhibit microglial autophagy and Aβelimination.Ube2c knockdown in the hippocampus of APP/PS1 mice improved Aβpathology and memory behavior,confirming the pathogenicity of Ube2c high expression in AD.4.AGO reduced neuritic plaques,enhanced synaptic plasticity and improved cognitive behavior in APP/PS1 mice,suggesting that AGO might be beneficial for the treatment of AD.Furthermore,AGO significantly suppressed Ube2c expression in Aβ-treated BV2 cells(via 5-HT_2Creceptor)and AD mice,as well as enhanced microglia autophagy,implying that 5-HT_2Cmediated the neuroprotection of AGO,and Ube2c and autophagy-related molecules might become novel therapeutic targets of anti-AD drugs in the future.