The Effects Of Estrogen Receptor Beta Activity Modulation On Hippocampal Actin Polymerization And Learning And Memory Of Female Mice

Along with the increase of age,ageing related decline of estrogens（E₂）has been shown to play an important role in the impairment of learning and memory in dementias like Alzheimer’s disease（AD）.However,the mechanism involved in AD is still unclear and the effect of the existing treatment is undesirable.Therefore,to study the pathogenesis and treatment of AD have a profound influence on prevention and treatment of this disease as well as the development of medical care and family care.E₂plays an important role in women’s physiological development and improves the memory impariement in olds,though estrogen receptor（ER）regulates synaptic plasticity and learning and memory in the hippocampus.Estrogen receptor has been shown to participate in E₂-mediated regulating hippocampal synaptic plasticity,learning and memory.There are two types of ER: one is membrane receptor GPR30 located in intracellular membrane components including cytomembrane,mitochondrial membrane and endoplasmic reticulum membrane,which regulated physiological function via the second messenger mediated estrogen signaling.Another is the classical nuclear receptor,including ERα and ERβ.It is generally assumed that they can regulate the transcription of the target genes mainly through binding with DNA in the nucleus.Steroid receptor coactivator-1（SRC-1）is one of cofactors that are necessary for the transcriptional activation of steroid hormone nuclear receptors.SRC-1 is highly expressed in hippocampus and cerebral cortex,which is influenced by postnatal development and gonads resection.Some studies have shown that ovariectomy and nuclear receptor inhibitor significantly decreased the expression of synaptic protein and the density of dendritic spine in the hippocampus,while estrogen replacement therapy and the nuclear receptor agonists can increase the expression of synaptic protein and the density of dendritic spine,but themechanism of ERβ is unclear.Estrogen receptor beta（ERβ）was cloned in 1996,over the past 20 years,studies have suggested that ERβ is widely expressed in body and participate in the regulation of E₂ on a variety of brain functions and the expression and function in the different sections are not the same.For example,in the central nervous system,detected higher level ERβ in cerebral cortex,hippocampus and other area,it suggested that ERβ may play a mediating role of E₂ regulation on learning and memory.In the reproductive system,testis,ovaries and other tissues were detected the existence of ERβ,it may participate in the regulation of E₂ on the growth and differentiation of germ cells.In addition,ERβ also was detected in the cardiovascular,digestive and other systems and also has an important physiological significance.With the specific antagonist of ERβ or gene knockout technique,studies indicated that ERβ inhibition could significantly decreased hippocampal synaptic protein,the density of dendritic spines and impaired spatial learning and memory.With the agonist of ERβ improved synaptic plasticity and spatial learning and memory,suggesting that ERβ has an important role of regulation on learning and memory,however,the detail mechanism is still unclear.Actin is an important composition of cytoskeleton.The dynamic changes of the polymerization and depolymerization in actin is the basis of synaptic plasticity,learning and memory.G-actin,the monomeric form of actin,can be polymerized into fibriform F-actin induced by Profilin-1.Therefore,the change of F-actin/G-actin ratio is a common index of actin polymerization degree.Studies founded that mammalian target of rapamycincomplex 2（mTORC2）has asignificantinfluence on regulating actin cytoskeleton polymerization.Knock out Rictor,a core component of mTORC2,decreased actin polymerization and spine density,nevertheless,p-AKT（Ser473）activation,a downstream target molecules of Rictor,can block learning and memory impariment mentioned above.Those results suggested that mTORC2 plays a significant role in regulating actin cytoskeleton polymerization,learning and memory.Previous studies reported that ERβ can regulate the density of the dendritic spines and the behaviors of learning and memory,but whether ERβ is involved in this regulation onactin cytoskeleton polymerization remains unclear.Objective: To explore the function and the potential mechanism involving in ERβ regulating actin polymerization in hippocampal neurons and the learning and memory behaviors.Methods:1.In order to know the ERβ expression in hippocampus at different postnatal days,we use Western blot（WB）and Immunohistochemistry（IHC）experimental method in detection of male and female mice at different postnatal days（P0,P7,P14,P28,P56）to examine ERβ expression changes in the hippocampus.2.In order to investigate the effect of the activation ERβ of hippocampal actin cytoskeleton polymerization and learning and memory.Adult female mice were divided into 5 groups and treated for DMSO or ERβ agonist DPN（1.25,2.5,5.0mg/kg）subcutaneously one week after OVX,then to exanmine the change ofprotein molecule SRC-1,Rictor,p-AKT and actin cytoskeleton polymerization protein（p-cofilin/cofilin and Profilin-1）expression after OVX and know the DPN treatment best dose.Then under the condition of the best dose（5.0 mg/kg DPN）treatment examined the DPN in OVX mice hippocampus expression of these above-mentioned molecules,the dynamic changes of actin polymerization（F-actin/G-actin ratio）and hippocampal density of dendritic spines and the influence on learning and memory.3.Adult diestrus female mice,intraperitoneal administration of the same dose of ERβ antagonist PHTPP（100ug/kg）,divided into 5 groups: Control（vehicle control）and PHTPP injection of 1,3,5,7 days,to know clearly the best time of the PHTPP treatment regulation on the expression of SRC-1,Rictor,p-AKT,p-cofilin/cofilin and Profilin-1 in the hippocampus.Thenunder the condition of the best time（7d）,using Morris water maze investigated the change of learning memory behavior in the mice,then with WB,IHC and Golgi-cox-Impregnation to detect the change of SRC-1,Rictor,p-AKT,actin cytoskeleton protein（p-cofilin/cofilin and Profilin-1）expression and actin polymerization state（F-actin/G-actin ratio）and dendritic spine density in hippocampal region after inhibiting ERβ activity,in order to explore the relationship between the change of ERβ activity and learning and spatial memory.Results:1.The expression of ERβ was found at P0,P7,P14,P28,P56 in the hippocampus of male and female mice.High level was tested at P0,lower level at P7 and P14,then it increased to higher level at P28 and P56.In male mice the level at P28 was equal to the level at P0,while in female mice the level at P28 has exceeded P0.2.OVX can decline the expression of SRC-1,Rictor,p-AKT,the protein of actin cytoskeleton（p-cofilin/cofilin,Profilin-1）in the hippocampus.Profilin-1 was increased at 1.25mg/kg DPN,but the other molecule has no obvious change.The expression of all the above molecular was reversed by 2.5-5.0mg/kg DPN when compared with OVX and chosen 5.0 mg/kg DPN as the best dose.OVX impaired the learning and memory by Morris water maze and decreased the density of dendritic spines in the hippocampus by Golgi-cox-Impregnation and induced actin depolymerization by immunohistochemistry（IHC）and Western blot.It shown that expression of SRC-1,Rictor,p-AKT and actin cytoskeleton proteins and actin polymerization state（F-actin/G-actin ratio）in the hippocampus by OVX and rescued by 5.0 mg/kg DPN treatment.3.The expression of hippocampal SRC-1,Rictor,p-AKT,proteins of actin cytoskeleton（p-cofilin/cofilin,Profilin-1）with PHTPP treatment began to decline at 5d,except for Profilin-1 the other molecule expression was significantly decreased at 7d and chosen 7d as the best time.Under this condition,PHTPP impaired the learning and memory of the female mice by Morris water maze.It also decreased the density of dendritic spines in the hippocampus by Golgi-cox-Impregnation and downregulated the expression of SRC-1,Rictor,p-AKT and cytoskeleton proteins and actin depolymerization（downregulated F-actin/G-actin ratio）in the hippocampus by IHC and Western blot.Conclusions:1.The expression of ERβ was found in the postnatal hippocampus（P0-P56）of male and female mice,and then it reached higher level at P0 and in adults,showing a U-type trend.2.OVX significantly declined the expression of hippocampal SRC-1,Rictor,p-AKT and cytoskeleton proteins and the density of dendritic spines,promoted actin depolymerization and impaired the learning and memory and reversed by activating ERβ,the results indicated that activating ERβ may mediate to promote the actin cytoskeleton depolymerization to affect learning and memory eventually.3.ERβ activity inhibition significantly decreased the expression of hippocampal SRC-1,Rictor,p-AKT and cytoskeleton proteins and the density of dendritic spines.It also promoted the actin cytoskeleton depolymerization and impaired the learning and memory behavior of the animals.The results showed that inhibiting ERβ may regulate the dynamic changes of actin cytoskeletal polymerization depolymerization and finally induce the learning and memory impairment.In summary,in this study we used ovariectomy,Morris water maze,IHC,Western blot and Golgi-cox-Impregnation to examine the change of ERβ activity on spatial learning and memory in mice and the expression of SRC-1,mTORC2,the dynamic changes of actin cytoskeletal polymerization and depolymerization,combined with previous studies indicated that the involvement of ERβ in the regulation of SRC-1,mTORC2,actin cytoskeletal polymerization and depolymerization effected to synaptic plasticity,ultimately affect the behavior of learning and memory,the results also provided a new experimental basis to look for a new target for the prevention and treatment of AD.