Testosterone Improves Spatial Cognitive Dysfunction Via Androgen Non-genomic Effect In Tfm Mice

Studies have found that reduced male aging-related androgen levels are closely related to cognitive decline in older men and cognitive dysfunction in neurodegenerative diseases,and that supplementation with androgen can significantly improve older men and Alzheimer's disease(AD)patients' cognitive function,indicating that androgens play an important role in cognitive function.Most of the current research on the neuroprotective effects of androgens on improving cognitive function has focused on the classical genomic effects of androgens,and little is known about the role of androgen nongenomic effects in this process.In this study,we used androgen receptors(AR)deficient testicular female Mice(Tfm)mice as animal research models.Compared with normal male C57BL/6J male mice,Tfm male mice had significantly lower androgen levels,accompanied by spatial cognitive dysfunction.This study will use 3-month-old male C57BL/6J mice and Tfm mice as animal research models to observe whether androgen can improve spatial cognitive dysfunction of Tfm mice via other non-androgen receptors pathways in the absence of the classical androgen receptors pathway.Objective:To explore the effect of testosterone on learning and memory impairment in Tfm mice and its mechanism.Methods:Tfm male mice were screened by sex,PCR and gene sequencing.Single-injection of 3mg · kg-1 testosterone to littermate male mice,and serum serum testosterone levels were measured by ELISA.Tfm mice were injected with T-BSA-FITC,observation of androgen membrane binding site by laser confocal microscope.36 Tfm mice aged 3 months were randomly divided into vehicle group(Tfm group),testosterone group(T group),aromatase inhibitor + testosterone group(A+T group),and 12 male C57BL/6J were selected as homologous normal control group(Con group),Con group and Tfm group were injected intraperitoneally with sterile sesame oil,T group was intraperitoneally injected with T(3mg · kg-1 · 3day-1),and A+T group was intraperitoneally injected with testosterone combined with aromatase inhibitors Anastrozole(10 mg · kg-1 · day-1)continuously for 1 month.Morris water maze and shuttle box experiment were performed to test the spatial cognitive function after the administration.After the behavioral test,the brain was decapitated to prepare paraffin sections,and the hippocampal neurons and dendritic spine density in the hippocampus of mice were observed by hematoxylin-eosin staining,Nissl staining,and Golgi-cox staining.Western blot experiments were used to detect the levels of phosphorylated and total proteins of Mammalian target of rapamycin(mTOR),and the level of Postsynaptic density 95(PSD95)in hippocampus of mice.Results: PCR and gene sequencing results showed that there was a single base deletion at the 170-bp site in Tfm mice.ELISA results showed that testosterone(3mg · kg-1)was injected intraperitoneally and the testosterone metabolism was basically metabolized in male littermate male mice around 72 h.Therefore,3mg · kg-1 · 72h-1 was selected as the concentration and time of testosterone administration.The results of immunofluorescence experiments showed that androgen membrane binding sites existed in the hippocampal dentate gyrus of Tfm mice.The results of the Morris water maze experiment showed that compared with the Con group,the average escape latency and the distance to the platform increased significantly,while the number of crossing platforms decreased significantly in the Tfm group.Compared with the Tfm group,the average escape latency and the distance to the platform were significantly reduced,while the number of crossing the platform were significantly increased in the T and A+T groups.There was no statistical difference between mice in Con group,T group and A+T group.There was no significant difference in the results of the shuttle box experiment.Hematoxylin-eosin staining,Nissl staining,and Golgi-cox staining showed that compared with the Con group,neuron damage and dendritic spine density in the hippocampus,which was reduced in the Tfm group.Compared with the Tfm group,hippocampal neuron damage was reversed and dendritic spine density was increased in the T and A+T groups,while there was no statistical difference among the Con group,T and A+T groups.Western blot results showed that compared with the Con group,mTOR phosphorylated protein and PSD95 protein expression in the hippocampal region,which was decreased in the Tfm group.Compared with the Tfm group,mTOR phosphorylated protein and PSD95 protein expression in the hippocampal region of the T group and A+T group increased.There was no statistical difference among the Con group,T and A+T group.The above experimental results show that androgens can activate non-genomic effects mediated by mTOR phosphorylation,promote the expression of PSD95 protein,reduce hippocampal neuron damage and increase dendritic spine density,and improve spatial cognitive dysfunction in Tfm mice.Conclusion:1.Androgen membrane binding site in hippocampal neurons of Tfm male mice.2.Compared with C57 BL / 6J male mice,Tfm male mice have reduced androgen receptor defects,lower serum testosterone levels,accompanied by spatial cognitive dysfunction,hippocampal neuron damage and reduced dendritic spine density;hippocampal mTOR phosphorylated protein and PSD95 Protein levels were also significantly reduced.3.T supplementation treatment can improve spatial cognitive dysfunction in Tfm mice,reverse hippocampal neuron damage,increase dendritic spine density in hippocampal neurons,and significantly increase levels of PSD95 protein and mTOR phosphorylated protein in hippocampus.