| The dramatic increase in the number of patients with Alzheimer’s Disease(AD)caused by the ageing is one of the most important factors threatening the global social security system.The main clinical manifestations of AD are spatial learning and memory dysfunction,cognitive dysfunction,loss of misconception,language impairment,and personality mental and behavioral abnormalities.There are currently about 50 million patients worldwide,and this number is expected to increase to 152 million by 2050.A la rge number of studies have confirmed that more women(mostly see after postmenopause)than men suffer from in AD at a ration about 3:1,thus the decline in estrogen levels(mainly 17β-estradiol,E2)caused by aging is considered to be one of the main pathogenic factors of AD.Clinical trials have confirmed that estrogen replacement therapy(ERT)can reverse the cognitive dysfunction caused by estrogen deficiency to a certain extent and thus contribute to the prevention and treatment of AD.However,the efficacy of ERT treatment is still controversial;and long-term ERT also has the risk of causing estrogen-responsive tumors such as breast cancer.Therefore,it is important to find new targets for the prevention and treatment of AD patients under conditions of estrogen deficiency.In the brain,the source of E2 could be circulating E2(mainly ovarian estrogen)that penetrating blood-brain-barrier,or derived from local de novo synthesis from cholesterol throught the action of estrogen synthase,aromatase(AROM).High levels of AROM immunireactivities have been detected in the hippocampus,the core for spatial lerning and memory;and E2 produced witin the hippocampus through AROM is uaually called hippocampal E2.Ovariectomy(OVX)and aromatase inhibitors such as letrozole(LET)are commonly used to prepare E2 deficiency models,and because LET can penetrate the blood-brain barrier,it is also commonly used to prepare brain especially hippocampal E2 deficient model.Our previous studies have found that OVX and LET can inhibit the hippocampal CA1 synaptic density,dendritic spine density and regulate the dynamic changes of actin cytoskeleton,indicating that both circulating E2 and hippocampal E2 play important role in regulating hippocampal synaptic plasticity and finally affect hippocampus-based spatial learning and memory,but the detail mechanisms under OVX and LET are still unclear.Serum and glucocorticoid-regulated protein kinase 1(SGK1)is a serine/threonine protein kinase that was discovered in 1993 since it was rapidly transcribed in rat breast cancer cells when stimulated by serum and/or glucocorticoids.SGK1 is not only regulated by post-translation phosphorylation and dephosphorylation but also by rapid regulation of transcriptional level.It is widely expressed in human body and has been shown to be invovled in regulating multiple functions such as ion channels,cell survival and migration,membrane transporters,cyclic adenylate binding element(CREB)and other transcriptional factors;it is also involved in cell proliferation and apoptosis,neural excitability,inflammation,hormone secretion and other processes.Sparse literatures have reported that SGK1 can improve the pathogenesis of AD.When activated by phosphorylation,SGK1 can induce the phosphorylation of downstream molecular signals and transcriptional activation factors,improve the expression of anti-apoptotic protein Mcl-1 thereby antagonize apoptosis induced by Aβ and promote cell survival.SGK1 can also reduce the formation and accumulation of Aβ by regulating some proteins such as FE65 through phosphorylation,which is beneficial to alleviate the disease of AD.However,the role of SGK1 in the estrogenic regulation of learning and memory has not been reported.The hippocampus is the most important brain area related to learning and memory.Accumulated studies have demonstrated that hippocampal synaptic plasticity(including molecular and morphological changes of synapse,the density of synapse,the strength of synaptic transmission,etc.)has important influence on the ability of learning and memory.The changes of synaptic protein levels are the basis of synaptic transmission and plasticity;the dynamic change of actin polymerization and depolymerization is the key event for spine density ans synapse density.Previously we have found that E2 deficiency not only reduced the expression level of synaptic proteins but also affected the dynamic changes of actin cytoskeleton polymerization.The main pathological changes in AD are plaque formation(senile plaque),neurofibrillary tangles,neuroinflammation and neuronal loss.The formation of plaques is the result of insoluble beta-amyloid protein(Aβ)deposition.Amyloid precursor protein(APP)can be cleaved by α,β and γ secretase,in which the cleavaged product of α secretase is soluble Aβ,while the cleavaged product of the β and γ secretase is insoluble Aβ.For insoluble Aβ,it has been found that it can be degradated by insulin degradation enzyme(IDE)and Neprilysin(NEP).E2 deficiency has been shown to aggravate Aβ deposition and alter the expression these Aβ metabolism-related proteins.Although it has been reported that SGK1 plays a role in reducing the generation and accumulation of Aβ,the role of SGK1 in the pathologic changes of AD under E2 regulation remains largely unknown.It has been well established that the regulatory role of estrogens involves estrogen receptors,including estrogen receptors(ERα and ERβ)and G Protein-coupled Receptor 30(GPR30).Previous studies in the research group have found that GPR30 is closely related to hippocampal synaptic plasticity that involves mTORC2 and its core component Rictor,but whether the effects of GPR30 and Rictor are related to the expression of SGK1 is unclear.In order to further explore the changes in the expression of E2-regulated hippocampal genes and the effect of SGK1 on learning and memory and finally provide some possible targets for the prevention or treatment of AD,the following studies were conducted in this project:1.To explore the effect of estrogen on the transcription of hippocampal genes,we prepared two estrogen deficiency models,OVX and LET injection,and used transcriptome microarray technology to screen the differential genes in OVX and LET mice.Informatics analysis was used to predict the molecules most relevant to estrogen and the results were validated with Western blot(WB)and qPCR.Transcriptome analysis showed that SGK1 expression was significantly decreased in both estrogen deficiency models.2.In order to explore the role of SGK1 in estrogenic regulation of learning and memory in mice,we constructed adeno-associated viruses with SGK1 overexpression,and then used OVX model and hippocampal stereotopic injection of virus to explore whether SGK1 can reverse the impairment of learning and memory behavior caused by estrogen deficiency.WB and qPCR were used to verify the effectiveness of the virus;then and water maze was used to test the learning and memory ability of mice.3.To explore the underlying mechanisms of SGK1 on the alterations of estrogen-related learning and memory behavior,by using the above models,WB was conducted to examine the changes of hippocampal synaptic related proteins(PSD95,GluR1 Spinophilin and Synaptophysin)and actin cytoskeleton remodeling related proteins(Profilin and Cofilin),Golgi staining was used to examine the changes of CA1 spine density and transmission electron microscopy was used toexamine the changes of hippocampal CA1 synapse density.4.In order to explore the effect of SGK1 on the pathological changes of AD,WB was used to detect the effect of hippocampal specific overexpression of SGK1 on the changes of Aβ protein and its metabolism-related proteins;Congo Red was used to examine the formation and alteration of senile plaques in the hippocampus.5.To investigate the role of SGK1 in the regulation of GPR30/mTORC2 pathway on hippocampal synaptic plasticity and learning and memory.Through in vivo and in vitro experiments,we used behavioral,Western blot and immunofluorescence techniques to detect the regulation of GPR30/mTORC2 pathway on learning and memory behavior in mice and its relationship with SGK1 expression.Main results:1.Transcriptome microarray results indicated when p<0.05 and fold change(fc)> 1.5,a total of 143 differentially expressed genes were screened with OVX/Sham;and there were 136 differential genes with LET/DMSO under the same screening conditions.Both OVX and LET caused upregulation of Gm10717 and Gm11116 as well as downregulation of SGK1 and Nfkbia.Genemania function prediction showed that SGK1 was the only one that associated with AROM and estrogen receptors,strongly suggesting SGK1 is the mediator for the estrogenic regulation on spatial learning and memory behavior of mice.2.Compared with the sham animals,OVX caused longer escape latency in finding the platform during the 5d learning phase;while on the memory test day(day 6),the number of times passed the platform and the time stayed in the target quadrant were significantly reduced by OVX.However,when the OVX mice were injected with SGK1 overexpression AAV in the hippocampus,the escape latency was shortened during the learning phase;and the number of times passing through the platform and the target quadrant residence time were significantly increased at the memory test(day 6)to levels comparable to that detected in the sham animals.3.Hippocampal specific overexpression of SGK1 induced improment on OVX-induced changes in synaptic and actin cytoskeletal proteisn as well as decreased in synaptic density and dendritic spine density.4.Overexpression of SGK1 in hippocampus reversed the changes of Aβ protein and its metabolization-related proteins caused by OVX,reduced the protein level of BACE1 and increase the level of IDE,but it did not affect Aβ deposition and the formation of senile plaques.5.Inactivation of GPR30 could inhibit E2-induced changes in mTORC2,SGK1,synaptic proteins and actin polymerization;activation of mTORC2 could reverse GPR30 induced spatial memory impairment and actin depolymerization;inhibition of Rictor could reduce the expression level of SGK1.Main conclusions:1.Both OVX and LET can induce significant changes in the expression of multiple genes in the hippocampus,and SGK1 is one of the most significantly downregulated genes.Bioinformatics indicates that it is a gene related to estrogen and its receptor,indicating that GK1 may play an important role in the regulation of the structure and function of the hippocampus by estrogen.2.Overexpression of SGK1 can improve hippocampal synaptic plasticity of OVX mice,inhibit the generation of Aβ and promote its metabolism,thus playing a key role in estrogenic regulation of spatial learning and memory.3.SGK1 may be involved in the GPR30/mTORC2 regulation of synaptic plasticity and learning and memory in hippocampus of mice.4.These results provide novel experimental evidences for elucidating the mechanism under estrogenic regulation on learning and memory as well as cognition,indicating thet SGK1 may be a potent preventive and therapeutic target against estrogen-related neurodegenerative diseases such as AD. |
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