| Alzheimer’s disease(AD)is a neurodegenerative disease and the most common type of senile dementia.The main symptoms of AD are memory impairment,cognitive impairment,personality and behavioral changes and other neurological symptoms.AD seriously affect the social activity,work and life equality of patients.With the arrival of the aging society,AD has become a major health problem today.World Health Organization(WHO)survey shows that there are about 35 millions 600 thousands AD patients in the world and about 8 million AD patients in China,which brings huge economic and spiritual burden to the society.AD patients are mainly seen in old women,suggesting that estrogen deficiency caused by aging may be one of the risk factors for AD.There are two sources of estrogen in the central nervous system,one is circulating estrogen secreted predominantly by ovary(Circulating estrogen),and the other one is neuronal estrogen(Local estrogen)derived from androgens which is catalyzed by aromatase.The hippocampus is an important brain region involved in learning and memory.A large number of studies show that estrogens can regulate the hippocampus-dependent learning and memory.Our previous study showed the level of aromatase reduced in the hippocampus of AD mice,and letrozole,an inhi bitor of aromatase,could affect the learning and memory behavior of mice.These studies suggested that estrogen could regulate the hippocampus-dependent learning and memory,but the underlying mechanism is still unclear.Synaptic plasticity mainly includes functional plasticity and morphological plasticity,which is an important mechanism of learning and memory.The dynamic changes of actin cytoskeleton are the basis of morphological plasticity,which is mainly related to the changes of synaptic morphology,synaptic density and dendritic spine density.It was found that ovariectomy reduced the density of dendritic spines in the hippocampus of rats,which could be reversed by estrogen replacement therapy,but how estrogens regulate dendritic spine remains unclear.The cytoskeleton is mainly composed of actin;there are two forms of actin in neurons: globular actin(G-actin)monomers and fibrous actin(F-actin)polymers.The transformation of these two forms of actin is the basis of the dynamic changes of cytos keleton,which is closely related to synaptic plasticity.The dynamic changes of the cytoskeleton is regulated by several factors,Profilin-1 promotes the G-actin polymerization into F-actin,while Cofilin induces F-actin depolymerization into G-actin,and the function of Cofilin could be deactivated after its serine residues phosphorylation.Therefore,increasing the ratio of p-Cofilin(Ser3)/Cofilin ratio and the expression of Profilin-1 could reduce the depolymerization and increase the polymerization of cytoskeleton.It has been reported that the mammalian target of rapamycin complex 2(m TORC2)could regulate the phosphorylation level of Cofilin through the AKT signaling pathway and thus regulate the dynamic changes of actin cytoskeleton.Rictor is the core component of m TORC2,previous study has revealed that when Rictor was knockout,spatial memory,hippocampal dendritic spines density and the ratio of F-actin/G-actin were significantly decreased.Sparse studies showed that estrogen can regulate the actin cytoskeleton,but its mechanism is not clear.There are two classes of estrogen receptors: classic nuclear receptor(ERα and ERβ)and the novel membrane estrogen receptor(GPR30).Estrogen exerts its effect on the regulation of target gene transcription though nuclear receptors which need coactivator such as steroid receptor activator-1(SRC-1)for their transactivation;while it induces rapids cell response through non-genomic pathway that is mediated by GPR30.As the first discovered estrogen receptor,ERα gene was cloned successfully in 1986.Accumulated studies have shown that ERα is highly expressed in the hippocampus of many species.Many studies have shown that ERα has been involved in the estrogenic regulation on neuroprotection,synaptic proteins expression and learning and memory enhancement,but whether estrogen can affect learning and memory by regulating the dynamic changes of the hippocampal actin cytoskeleton and how ERα is involved in these processes are still unclear.In order to investigate the mechanism of estrogen and its receptor ERα regulate the cytoskeleton of hippocampal neurons,we conducted the following experiments in this paper:1.We examined the expression of estrogen signal,m TORC2 signal and cytoskeleton remodeling proteins in postnatal hippocampus of male and female mice(from newborn to old age).2.We treated C57 female mice with aromatase inhibitor LET then examined the level of SRC-1,m TORC2 activity and its downstream cytoskeleton remodeling proteins(Profilin-1,Cofilin and its phosphorylated form p-Cofilin(ser3))and the ratio of F-actin/G-actin.Then we used LET,E2,RNAi lentivirus against SRC-1 to treat hippocampal cell line m HippoE-14 and examined the level of SRC-1,mTORC2 activity and its downstream cytoskeleton remodeling proteins.3.We treated adult female mice with ERα antagonist MPP,OVX,OVX plus ERα agonist PPT,then examined the changes of spatial learning and memory,the CA1 dendritic spine density and the radio of F-actin/G-actin.Additionally,we also examined the levels of SRC-1,mTORC2 activity and cytoskeleton remodeling proteins.4.We used E2,specific antagonist and agonist of ERα(MPP,PPT),RNAi lentivirus against SRC-1(si SRC-1)or Rictor(si Rictor)to treat hippocampal cell line m Hippo E-14,and then we examined the level of SRC-1,m TORC2 activity and cytoskeleton remodeling proteins.5.We treated adult female mice with MPP and PHTPP then activated mTORC2 signal pathway by A-443654,then we examined the changes of learning and memory and the mTORC2 activity and the ratio F-actin/G-actin.Main experimental results:1.The results shown that high levels of aromatase were detected at P0 and P7,then it decreased at P14 and P28,and increased again at P56 and significantly decreased in old age.The expression of ERα and the ratio of p-AKT(Ser473)/AKT in the hippocampus of postnatal mice decreased gradually,high levels were detected at P0 and P7,and then it decreased gradually and further decreased in the old age compared to adult.The overall trend of the expression of SRC-1,Profilin-1and the ratio of p-Cofilin(ser3)/Cofilin was consistent,which showed a gradually increasing trend from newborn to adult and significantly decreased in the old age compared to adult mice.2.Intraperitoneal LET treatment significantly reduced the expression of ERα,SRC-1,mTORC2 activity and cytoskeleton remodeling proteins in the hippocampus of adult female mice in a dose-dependent manner.LET treatment significantly reduced the expression SRC-1,m TORC2 activity and cytoskeleton remodeling proteins in hippocampal cell line(mHippo E-14)and E2 treatment could reverse this phenomenon.We also found that the mTORC2 activity and cytoskeleton remodeling proteins significantly reduced by RNA interference lentivirus of SRC-1.Estrogen induced expression of m TORC2 signal and actin cytoskeleton regulatory proteins could be blocked by RNA interference lentivirus of SRC-1.3.Intraperitoneal MPP treatment significantly impaired spatial learning and memory and decreased the CA1 dendritic spines density of hippocampus.The levels of SRC-1,mTORC2 activity,cytoskeleton remodeling proteins and the ratio of G-actin/F-actin also were downregulated by MPP treatment.These changes were also seen in OVX animals,,and PPT could reverse the effect of OVX.4.E2 could significantly upregulate the expression of SRC-1,mTORC2 activity and cytoskeleton remodeling proteins,while MPP could block the effect of E2.The activating effect of PPT on mTORC2 activity and cytoskeleton remodeling proteins could b e blocked by RNA interference lentivirus of SRC-1 or Rictor.5.Enhancing the activity of mTORC2 signaling pathway by A-443654 could reverse the spatial learning and memory impairment and cytoskeleton depolymerization induced by MPP and PHTPP in adult female mice.Main conclusions:1.There are different developmental patterns of aromatase,ERα,SRC-1,Rictor,p-AKT(473),AKT,Prfilin-1,p-Cofilin(Ser3)and Cofilin in the hippocampus of mice from afterbirth to adulthood,but there were not obvious sex differences of the developmental patterns of those molecules,indicating that they may have different effects on the development and maturation of the hippocampus in the early postnatal period,but the effect may not have significant sex differences.However,in the hippocampus of aged mice,the expression of these molecules significantly decreased than that of adults,suggesting that the aging may be closely related to the dynamic changes of actin cytoskeleton and the decline of learning and memory.2.Inhibiting the synthesis of estrogen with LET could lead to the depolymerization of actin cytoskeleton,which is at least partially mediated by SRC-1.3.Estrogen could regulate the level of SRC-1,mTORC2 signal,actin cytoskeleton polymerization,hippocampal spines density of CA1 and spatial learning and memory,which is at least partially mediated by ERα.4.SRC-1/mTORC2 pathway may mediate the effect of ERα on the dynamic changes of the actin cytoskeleton and further affect hippocampus-based learning and memory behavior. |
PDF Full Text Request