A Preliminary Study Of The Effects And Mechanism Of Hippocampal Aromatase On Synaptic Plasticity And Spatial Learning And Memory In Male APP/PS1 Transgenic Dementia Mice

The increasing incidence of Alzheimer’s disease(AD)with population aging is one of the most serious global challenges we have to face.AD is a neurodegenerative disease of aging and is the most common cause of dementia.Due to the fact that there are few specific treatment drugs and diagnosis for this disease at present as well as the long duration of more than ten years and the final symptoms of dementia,AD have brought serious mental and economic burdens to families and society.According to the Centers for Disease Control and Prevention(CDC),in 2014 the 4% of the total deaths in the United States is AD patients(https://www.cdc.gov/ features/Alzheimer-disease-deaths/index.html)and it is ranked one of six major causes of death in the United States.It is well known that the main clinical manifestations of AD patients are progressive decline of cognition,including memory loss,mental disorder,speech function disorders,personality and behavior change,directional and judgement and decline in other cognitive abilities,and finally the losing ability to self-care.At present,the pathogenesis and prevention strategies of AD are still controversial,and the drug research and development targeting at Aβ has largely failed.Therefore,it is urgent to find new targets for the prevention and treatment of AD.AD is mainly seen in postmenopausal women,so the lack of ovarian estrogen is considered as one of the major factors leading to AD.However,AD also has a certain incidence in elderly men,and its pathogenesis is not well understood.In some clinical investigations and studies,it was found that serum testosterone of AD patients was significantly lower than that of normal elderly men,which aroused more interest in the study of the relationship between androgen and AD.The androgen is catalyzed to estrogen by aromatase(AROM;thus called endogenous estrogen,mainly 17β-estradiol,E2),and binding to the estrogen receptors(including nuclear receptor ERα,ERβ and membrane receptor GPR30).The previous work have found that orchiectomy did not affect the learning and memory behavior of mice,but when treated with AROM inhibitor to decrease the production of E2,significant learning and memory impairment was noticed.These results strongly indicate a limited role of testicular androgens but the profound role of E2 in the regulation of learning and memory behavior.AROM is a complex of cytochrome P450 and NADPH-cytochrome P450 reductase.Studies have shown that estrogen catalyzed by aromatase have regulatory functions in neurodevelopment and synaptic plasticity.Moreover,in mammalian brain,hippocampus is the most important structure that related to learning and memory in the medial temporal lobe system.Accumulated studies have demonstrated that hippocampus is one of the earliest damaged areas in the AD brain,its synaptic plasticity changes,including the alteration of synaptic transmission,the changes of synaptic proteins,synapse density and spine density,are the basis of learning,memory and cognition.High levels of AROM mRNA and proteins have been detected in the hippocampus but its function is not well documented.Few studies have reported the regulation of AROM on the spatial learning and memory behavior of normal female mice,mostly by systemic injection of AROM inhibitors.However,the effects of specific regulation of hippocampal AROM on the dementia behavior of male APP/PS1 mice have not been reported.In an attempt to explore the effects and mechanism of hippocampal AROM on synaptic plasticity and spatial memory of APP/PS1 transgenic mice so as to provide novel preventive and therapeutic target for AD,in the current studies we conducted the following experiments:1.The changes of hippocampal AD pathology-related proteins(Aβ,p-tau,ADAM10,BACE1,NEP and IDE)in male APP/PS1 and C57 mice aged 2,4,6,8 and 10 months were detected by Western Blot and the time points of significant changes in the hippocampal expression of APP/PS1 mice were determined.2.The changes of hippocampal aromatase and synapse related proteins(Synaptophysin,PSD95,Spinophilin,GluR1)in male APP/PS1 and C57 mice aged 2,4,6,8 and 10 months were detected by Western Blot,and the time points of significant changes in the hippocampal expression of APP/PS1 mice were also determined.3.Combining the above experimental results,male APP/PS1 and C57 mice aged 4,6 and 8 months were selected to test their spatial learning and memory behavior through Morris Water Maze,and the time point when APP/PS1 mice showed obvious spatial memory impairment was determined.4.Combining with the time point between significant-decreased hippocampal AROM and impairment of learning and memory behavior,the hippocampus of 6 months APP/PS1 mice were injected with over-expression AROM AAV virus,then we examined the changes of spatial learning and memory using classic Morris Water Maze test at 9 months old.Main experimental results and conclusions:1.The expression of AD pathology-related proteins was significantly changed at 8 months in the hippocampus of C57 mice,while they were significantly changed in the hippocampus of APP/PS1 mice at 6 months.2.The expression of AROM in the hippocampus of C57 mice did not change until significant decrease at 10 months old.However,there was a significant decrease in the 6 months old APP/PS1 mice,which maintained a low level thereafter.There were no significant aging-related changes in the expression of the four synapse related proteins in the C57 mice at the time point detected,while in the hippocampus of APP/PS1 hippocampus they decreased at different levels.3.Morris Water Maze results showed that compared with the age-matched C57 mice,4 months and 6 months old APP/PS1 mice did not shown any difference in finding the platform during the 5d learning phase and the time stayed in the target quadrant and the times passed the platform on the memory test day(day 6).While 8 months old APP/PS1 mice showed longer escape latency in finding the platform during the 5d learning phase;and on the memory test day(day 6),the time stayed in the target quadrant and the times passed the platform were significantly reduced.The above results indicated that APP/PS1 mice were normal at 4 months and 6 months old,but showed obvious learning and memory impairment at 8 months old.4.By injecting the AROM overexpression virus into the hippocampus of 6-monthold APP/PS1 mice and conducting behavioral tests at the age of 9 months,it was found that control APP/PS1 mice had obvious learning and memory impairment,while hippocampus-specific AROM overexpression did not showed any learning and memory impairment.The above results demonstrated that endogenous estrogen catalyzed by AROM plays an important role in hippocampus-dependent spatial learning and memory behavior and an increase of the hippocampal AROM level could prevent the occurrence of dementia seen in normal aged APP/PS1 mice,indicating hippocampal AROM may be an important target for the prevention or treatment of AD.