| Alzheimer’s disease(AD)is a progressive neurodegenerative disease the most common form of dementia.Its clinical symptoms include memory disorders,aphasia,visual space agnosia,skill damage,executive dysfunction and the personality and behavior changes.Due to the slow onset and concealed progress,so far there is no effective treatment.According to statistics,the global number of patients with dementia has reached46,800,000 in 2015,among which 50%-75% were AD sufferings.At present,the number of patients with AD in China has been ranked the first in the world.The number of global dementia doubled every 20 years and will reach to 131 million people by 2050.At present,senile dementia has become one of the main lethal and disabling diseases in aging society,which brought heavy burden to the family and society.Many studies have reported that the number of female AD patients is about 3 folds than that of males,with higher occurrence in postmenopausal women.Therefore,the decline of ovarian estrogen in aging women has long been considered one of the main causes of AD.There are two sources of estrogen in brain,one is circulating estrogen from the ovary and other endocrine organs,the other one is neuronal estrogens(local estrogen)derived from androgens,which are catalyzed by aromatase.A large number of studies have confirmed that estrogen has significant neuroprotective effects such as rescue learning and memory and cognition disorder caused by ageing or other nerve injuries.Estrogen replacement therapy(ERT)has been used in clinical trials,but it is not always effective,thus how estrogen regulates synaptic plasticity,the basis of learning and memory,is urgently needed to investigate.Hippocampus is one of the most important structures responsible for learning and memory.Accumulated studies have shown that ovariectomy(OVX)can significantly decrease synaptic density and dendritic spine density in the hippocampus which can bereversed by estrogen replacement,indicating the fundamental role of estrogen on hippocampus.It has been reported that after OVX or during estrus cycle,CA1 synapse density and dendritic spine density fluctuated significantly.After blocking local estrogen synthesis,the synaptic density in CA1 also decreased significantly,indicating hippocampal estrogen is also involved in the regulation of the cytoskeleton remodeling.However,the specific mechanism of estrogen regulating the neuronal cytoskeleton has not been reported.It is well known that actin is one of the most important cytoskeletal proteins,its dynamic change from globular actin(G-actin)to filamentous actin(F-actin)is the basis of the morphological plasticity,which is one of the main aspect of synaptic plasticity.Additionally,Profilin and Jasplakinolide(JPK)can promote the G-actin converting to F-actin(actin polymerization),Cofilin and cytochalasin D(Cyto D)can promote the F-actin converting to G-actin(actin depolymerization).It has been reported that Rictor Rictor(rapamycin-insensitive companion of mTOR;the core component of mammalian target of rapamycin complex 2,mTORC2)can regulate the dynamic of actin cytoskeleton polymerization and this regulation is the key to convert the short term memory to long time memory.Studies have found that estrogen can regulate cytoskeletal polymerization in peripheral tissues and hippocampus,but in the hippocampus whether estrogen regulation on cytoskeleton involves Rictor and other mentioned actin cytoskeleton remodeling proteins is unclear.Estrogen exerts its function through two classes of receptors: classic nuclear receptor(ERα and ERβ),mediating genomic estrogen effects;or the novel membrane estrogen receptor GPR30(G-protein coupled membranous estrogen receptor;also named GPER1,G-protein coupled estrogen receptor 1),mediating rapid non-genomic estrogen effect.Recent studies have revealed the existence of both nuclear-and membrane ER in the hippocampus of animal species,and GPR30 has been shown to be involved in the regulation of hippocampal synaptic plasticity as well as cognition.However,whether GPR30 is involved in the regulation of estrogen on actin cytoskeleton in hippocampus the underlying mechanisms remain to be elucidated.In order to explore the specific mechanism of GPR30 regulation on the actin cytoskeleton of hippocampal neurons,in this study we conducted the following experiments:1.We examined the expression of GPR30 and the actin polymerization protein Profilin-1 in postnatal hippocampus of male and female mice(P0 to P56).We also examined the expression of GPR30 and Profilin-1 after OVX or aromatase inhibitors letrozole(LET)treatment.2.We treated adult female mice with OVX,OVX plus GPR30 agonist G1,or GPR30 antagonist G15,then examined the changes of learning and memory behavior,the CA1 dendritic spine density and F-actin/G-actin ratio.Additionally,levels of nuclear ERs and their coactivator SRC-1,Rictor and its downstream effector p-AKT,actin cytoskeleton remodeling protein Profilin-1 and Cofilin,postsynaptic protein PSD95 and Glu R1 were also examined under these treatments.3.The in vivo data were further verified with hippocampal cell line mHippo E-14.Moreover,levels of actin cytoskeleton regulatory proteins and synaptic proteins were also examined after treatment of mHippoE-14 cells G1,G15,SRC-1 inhibitor bufalin(Bu),PI3 K pathway inhibitor wortmannin(WM),actin polymerization promoter JPK or disruptor Cyto D,alone or combined.Main experimental results:1.The expression of GPR30 and Profilin-1 in the(P0-P56)hippocampus showed a V-type profile,high levels were detected at P0,and then it decreased at P7,and increased again and reached the highest level at P56.Moreover,expression of GPR30 and Profilin-1was significantly decreased by OVX and LET treatment.2.OVX significantly impaired learning and memory and decreased the density of dendritic spines of hippocampus.OVX also downregulated the expression of nuclear estrogen receptor and SRC-1,synaptic protein PSD-95 and GluR1,Rictor and p–AKT as well as Profilin-1 and the ratio of F-actin/G-actin.Cofilin,the actin cytoskeleton depolymerization protein,was significantly upregulated by OVX.GPR30 agonist G1 reversed but GPR30 antagonist G15 mimicked the effects of OVX.3.In vitro experiments showed that the in vivo effects of G1 or G15 could be fully demonstrated.Importantly,the activating effects of G1 could be blocked by inhibition of SRC-1 with Bu,PI3 K pathway with WM or disrupting actin polymerization with Cyto D;the activating effect of JPK could be blocked by G15.Main conclusions:1.Higher levels of GPR30 and Profilin-1 are detected in the newborn and adult hippocampus of male and female mice.Both ovary and local estrogen can regulate actin cytoskeleton polymerization.2.Estrogen regulates actin cytoskeleton polymerization dynamic by action on nuclear ERs and synaptic proteins,actin cytoskeleton remodeling proteins,dendrtic spine density and finally,learning and memory.The above effects are,at least in part,mediated by membrane estrogen receptor GPR30.3.GPR30 exerts its function may through nuclear estrogen receptors/ SRC-1 and/or PI3 K to regulate Rictor(mTORC2),then actin cytoskeleton remodeling proteins and synaptic proteins and ultimately regulates learning and memory. |
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