Effects Of ZIP9-induced DHT On Learning And Memory And Hippocampal Synaptic Plasticity Of Mice

Alzheimer’s disease(AD)is an age-related neurodegenerative disease with progressive cognitive and behavioral disorders as the main clinical manifestations.It has been found that androgen has a neuroprotective effect on learning and memory in patients with AD,but the mechanism is not clear.The previous study of our team found that testicular feminization mutation(Tfm)mice with androgen receptor(AR)deficient had a lower serum androgen level and impaired learning and memory.After androgen supplementation,the learning and memory ability of Tfm mice was significantly improved,suggesting that Tfm mice have androgen binding sites other than classic ARs,which mediates the improvement of learning and memory by androgen.Recent research had revealed that Zrt-and Irt-like protein 9(ZIP9)was an G protein-coupled receptor of androgen,which could mediate the biological effects of androgens in various cells.However,it is not clear whether ZIP9 mediates the effect of androgen on learning and memory in Tfm mice.In this study,we proposed to explore whether androgen could affect learning and memory and hippocampal synaptic plasticity in Tfm mice through ZIP9,and to clarify the effect mechanism.Furthermore,we verified whether androgen can affect the learning and memory of AD animal model-APP/PS1 mice through this mechanism,so as to provide experimental evidence for androgen to improve AD.Part One The effects of ZIP9-induced DHT on learning and memory and hippocampal synaptic plasticity in Tfm miceObjective: Using androgen receptor-deficient Tfm mice and wild type(wildtype,WT)mice as the research object,we observed the expression of ZIP9 in hippocampal CA1 region,and ZIP9 knockdown efficiency after infection with adeno-associated virus AAV9-ZIP9-RNAi.Then we investigated the effects of dihydrotestosterone(DHT)supplementation after pre-knockdown of hippocampal ZIP9 on learning and memory,density of dendritic spines and postsynaptic density95(PSD95),Drebrin and synaptophysin(SYP)in Tfm mice hippocampal CA1 region.Methods:1.The expression of ZIP9 in the hippocampal CA1 region of WT and Tfm mice was detected by Western blot.2.After the Tfm mice were injected with AAV9-ZIP9-RNAi in the CA1 region of the hippocampus,the knockdown efficiency of ZIP9 in the CA1 region of the hippocampus was detected by Western blot.3.After hippocampal ZIP9 knockdown,Tfm mice were given DHT supplement.The learning and memory of Tfm mice were detected by Y maze,new object recognition and Morris water maze test.4.After hippocampal ZIP9 knockdown,Tfm mice were given DHT supplement.The density of dendritic spines of neurons in hippocampal CA1 region was observed by Golgi-Cox staining.5.After hippocampal ZIP9 knockdown,Tfm mice were given DHT supplement.The expression of synaptic proteins PSD95,Drebrin and SYP in hippocampal CA1 region was observed by immunohistochemical staining and Western blot.Results:1.The results of western blot showed that there was no statistical difference in the expression of ZIP9 protein in the CA1 region of the hippocampus between WT mice and Tfm mice.2.The results of western blot experiments showed that the expression of ZIP9 in hippocampal CA1 region of Tfm mice decreased significantly after the infection with AAV9-ZIP9-RNAi.3.The behavioral experiment results showed that Tfm mice exhibit obvious learning and memory defects compared to WT mice.After supplementation with DHT,the learning and memory ability of Tfm mice was significantly improved,while the effects of DHT on learning and memory were inhibited after hippocampal ZIP9 knockdown.4.The results of Golgi-Cox staining showed that the dendritic spine density in hippocampal CA1 region of Tfm mice were decreased significantly compared to WT mice.The density of dendritic spines in Tfm mice were increased significantly after supplementation of DHT,while the effect of DHT on increasing dendritic spine density was significantly inhibited after hippocampal ZIP9 knockdown of Tfm mice.5.The results of immunohistochemical staining and Western blot showed that the expression of synaptic proteins PSD95,Drebrin and SYP in hippocampal CA1 region of Tfm mice was significantly lower than that of WT mice.The expression of PSD95,Drebrin and SYP protein of Tfm mice was significantly increased after supplementation of DHT,while the effects of DHT on increasing the expression of PSD95,Drebrin and SYP were significantly inhibited after hippocampal ZIP9 knockdown of Tfm mice.Summary:1.ZIP9 was normally expressed in hippocampal CA1 region of Tfm mice.And ZIP9-induced DHT improved learning and memory of Tfm mice.2.ZIP9-induced DHT increased the density of dendritic spines and the expression of synaptic proteins PSD95,Drebrin and SYP in hippocampal CA1 region of Tfm mice.Part two The effects of ZIP9-induced DHT on ERK1/2-e IF4 E pathway and PSD95 protein expression in hippocampal neuronsObjective: Firstly,Tfm and WT mice were treated with DHT after hippocampal ZIP9 knockdown to observe whether ZIP9-induced DHT affected the phosphorylation of extracellular signal-regulated 1/2(ERK1/2)and eukaryotic initiation factor 4E(e IF4E)in hippocampal CA1 region.Subsequently,mouse hippocampal neuron HT22 cells were treated with DHT after knockdown or overexpression of ZIP9 to observe whether ZIP9 was involved in the effects of DHT on the expression of PSD95 protein and the phosphorylation of ERK1/2 and e IF4 E.HT22 cells overexpressing ZIP9 were pretreated with SCH772984,an ERK1/2 specific inhibitor,or e FT508,an e IF4 E specific inhibitor,to explore whether ZIP9 induced DHT to affect PSD95 protein through ERK1/2-e IF4 E pathway.Methods:1.After hippocampal ZIP9 knockdown,Tfm mice were given DHT supplement,the phosphorylation of ERK1/2 and e IF4 E in CA1 region were detected by Western blot.2.After stable knockdown of ZIP9,HT22 cells were treated with DHT.The expression of PSD95 protein and the phosphorylation of ERK1/2 and e IF4 E were observed by Western blot and immunofluorescence staining.3.After stable overexpression of ZIP9,HT22 cells were treated with DHT.The expression of PSD95 protein and the phosphorylation of ERK1/2 and e IF4 E were observed by Western blot and immunofluorescence staining.4.After stable overexpression of ZIP9,HT22 cells were pretreated with SCH772984,a specific inhibitor of ERK1/2,and the effects of DHT on the phosphorylation of ERK1/2 and e IF4 E and the expression of PSD95 protein were observed by Western blot and immunofluorescence staining.5.After stable overexpression of ZIP9,HT22 cells were pretreated with e FT508,a specific inhibitor of e IF4 E,and the effects of DHT on e IF4 E phosphorylation and the PSD95 protein expression were observed by Western blot and immunofluorescence staining.Results:1.The results of Western blot showed that ERK1/2 and e IF4 E phosphorylation in the hippocampal CA1 region of Tfm mice were significantly lower than those of WT mice.After DHT supplementation,the phosphorylation levels of ERK1/2 and e IF4 E in hippocampus CA1 region of Tfm mice were upregulated.And ZIP9 knockdown in hippocampal CA1 region of Tfm mice significantly inhibited the upregulation of phosphorylation levels of ERK1/2 and e IF4 E by the administration of DHT.2.The results of Western blot and immunocytofluorescent staining showed that DHT significantly up-regulated the PSD95 protein expression and ERK1/2,e IF4 E phosphorylation in HT22 cells.And ZIP9 knockdown could significantly prohibit the promotion of PSD95 protein expression and ERK1/2,e IF4 E phosphorylation.3.Western blot and immunocytofluorescent staining results showed that DHT significantly increased PSD95 protein expression and ERK1/2,e IF4 E phosphorylation in HT22 cells.And ZIP9 overexpression could further enhance the effects of DHT on increasing of PSD95 protein expression and ERK1/2,e IF4 E phosphorylation.4.Western blot and immunocytochemical fluorescence staining results showed that the phosphorylation of ERK1/2 and e IF4 E,as well as the expression of PSD95 protein in HT22 cells were increased after DHT administration.After ZIP9 overexpression,DHT promoted ERK1/2,e IF4 E phosphorylation,and PSD95 protein expression in HT22 cells more significantly.Pretreatment with 100 n M SCH772984 significantly prohibited the increase of ERK1/2 phosphorylation by DHT administration in ZIP9-overexpressed HT22 cells,and prohibited e IF4 E phosphorylation,andalso inhibited PSD95 protein expression either.5.The results of Western blot and immunofluorescence staining showed that DHT significantly increased e IF4 E phosphorylation and PSD95 protein expression in HT22 cells.After overexpression of ZIP9,the effect of DHT on promoting e IF4 E phosphorylation and PSD95 protein expression in HT22 cells was furtherly enhanced.Pretreatment with 25 n M e FT508 significantly inhibited the phosphorylation of e IF4 E and the expression of synaptic protein PSD95.Summary:1.ZIP9-induced DHT promoted the phosphorylation of ERK1/2 and e IF4 E in hippocampal CA1 region of Tfm mice2.ZIP9-induced DHT promoted the expression of synaptic protein PSD95 and the phosphorylation of ERK1/2,e IF4 E in HT22 cells.3.ZIP9-induced DHT promoted the expression of PSD95 protein in HT22 cells through ERK1/2-e IF4 E pathway.Part three Effects of ZIP9-induced DHT on learning and memory and hippocampal synaptic plasticity in APP/PS1 miceObjective: In this part,we took APP/PS1 mice,an animal model of AD,as the research object.By castrating and knocking down hippocampal ZIP9,we observed the effects of DHT supplementation on learning and memory,density of dendritic spines in hippocampal CA1 region,the expression of synaptic proteins PSD95,Drebrin,SYP and the activity of ERK1/2-e IF4 E signal pathway in APP/PS1 mice,so as to clarify the effects of ZIP9-induced DHT on learning and memory and hippocampal synaptic plasticity in APP/PS1 mice.Methods:1.After castration and knockout of hippocampal ZIP9 in APP/PS1 mice,the effects of DHT supplementation on learning and memory were detected by Y maze,new object recognition and Morris water maze test.2.After castration and knockout of hippocampal ZIP9 in APP/PS1 mice,After castration of APP/PS1 mice,the effects of DHT supplementation on synaptic proteins PSD95,Drebrin and SYP in hippocampal CA1 were observed by immunohistochemical staining and western blot.3.After castration and knockout of hippocampal ZIP9 in APP/PS1 mice,Golgi-Cox staining was used to observe the effects of DHT supplementation on the density of dendritic spines in hippocampal CA1 neurons.4.After castration and knockout of hippocampal ZIP9 in APP/PS1 mice,the effects of DHT supplementation on the phosphorylation of ERK1/2 and e IF4 E in hippocampal CA1 region of APP/PS1 mice after castration was detected by western blot.Results:1.The results of Y maze,new object recognition and Morris water maze test revealed that the learning and memory of APP/PS1 mice reduced significantly after castration and DHT supplementation could rescue the decline of learning and memory ability of castrated APP/PS1 mice.After ZIP9 knockdown,effects of DHT on rescuing learning and memory were significantly inhibited.2.The results of immunohistochemical staining and western blot showed that the expression of synaptic proteins PSD95,Drebrin,and SYP in the hippocampal CA1 region of APP/PS1 mice were significantly reduced after castration.DHT supplementation could rescue the decline of PSD95,Drebrin,and SYP protein expression in castrated APP/PS1 mice,while these effects induced by DHT were significantly inhibited after ZIP9 knockdown.3.Golgi-Cox staining experiment results revealed that the dendritic spine density of neurons in CA1 region of hippocampus of APP/PS1 mice was significantly reduced after castration.DHT supplementation could rescue the decline of dendritic spine density in the hippocampal CA1 region of castrated APP/PS1 mice,however,the effect induced by DHT was significantly inhibited after ZIP9 knockdown in the hippocampal CA1 region.4.Western blot results revealed that the activity of the ERK1/2-e IF4 E pathway in hippocampal CA1 region of APP/PS1 mice was significantly reduced after castration.DHT supplementation could save the decrease of ERK1/2-e IF4 E pathway activity in hippocampal CA1 region of castrated APP/PS1 mice.However,the effect of DHT on saving ERK1/2-e IF4 E pathway activity was significantly inhibited after ZIP9 knockdown.Summary:1.ZIP9-induced DHT improved learning and memory in APP/PS1 mice.2.ZIP9-induced DHT increased the density of dendritic spines and the expression of synaptic proteins PSD95,Drebrin and SYP in hippocampal CA1 region of APP/PS1 mice.3.ZIP9-induced DHT increased the activity of ERK1/2-e IF4 E signal pathway in hippocampal CA1 region of APP/PS1 mice.Conclusions:1.ZIP9-induced DHT increased dendritic spine density and synaptic proteins expression in Tfm mice hippocampi,thereby improving learning,memory.2.ZIP9-induced DHT promoted the expression of synaptic proteins in hippocampal neurons by activating the ERK1/2-e IF4 E signaling pathway.3.ZIP9-induced DHT increased the dendritic spine density and the expression of synaptic proteins in the hippocampus of APP/PS1 mice by activating the ERK1/2-e IF4 E signaling pathway,thereby affecting learning and memory.