Department of Anatomy, Hebei Medical University, Shijiazhuang, 050017, Hebei, China; Neuroscience Research Center, Hebei Medical University, Shijiazhuang, 050017, Hebei, China.
Department of Anatomy, Hebei Medical University, Shijiazhuang, 050017, Hebei, China.
Mol Cell Endocrinol. 2022 Aug 20;554:111711. doi: 10.1016/j.mce.2022.111711. Epub 2022 Jul 5.
Androgens play an important role in the regulation of hippocampal synaptic plasticity. While the classical molecular mechanism of androgen's genomic activity is their binding to intracellular androgen receptors (iARs), they can also induce rapid non-genomic effects through specific membrane androgen receptors (mARs). In this study, we aimed to localize and characterize these mARs in primary rat hippocampal neurons. Specific punctate fluorescent signals on the cell surface, observed by testosterone-fetal bovine serum albumin conjugated fluorescein isothiocyanate (T-BSA-FITC), indicated the presence of mARs in hippocampal neurons. T-BSA-FITC binding to the cell membrane was incompletely blocked by the iAR-antagonist flutamide, and mAR binding site was competitively bound by free testosterone (T). Most neurons expressing androgen membrane binding sites are glutamatergic (excitatory), although several are γ-aminobutyric acid (GABA)ergic (inhibitory). Confocal microscopy and live-cell imaging techniques were used to observe the real-time rapid effects of androgens on hippocampal dendritic spine morphology. Immunofluorescence cell staining was used to observe their effects on the postsynaptic density protein 95 (PSD95) and synapsin (SYN) synaptic markers. While androgens did not cause a short-term increase in dendritic spine density of rat primary hippocampal neurons, they promoted the transformation of dendritic spines from thin to mushroom, promoted dendritic spine maturation, increased dendritic spine surface area, and rapidly increased PSD95 and SYN expression in the primary hippocampal neurons. Hippocampal synaptosomes were prepared using the Optiprep and Percoll density gradient two-step centrifuge methods, and the gene expression profiles of the synaptosomes and hippocampus were compared using a gene chip; PSD95 mRNA expression was detected by reverse transcription-polymerase chain reaction. Several mRNAs were detected at the synaptic site, including PSD95. Finally, the Venus-PSD95 plasmid was constructed and transfected into HT22 cells, which is a mouse hippocampal neuronal cell line. The real-time effect of androgen on synaptic protein PSD95 was observed by fluorescence recovery after photobleaching experiments, which involved the translation process of PSD95 mRNA. In conclusion, our findings increased our understanding of how androgens exert the neuroprotective mechanisms on synaptic plasticity.
雄激素在调节海马突触可塑性方面发挥着重要作用。虽然雄激素的经典分子机制是与细胞内雄激素受体（iAR）结合，但它们也可以通过特定的膜雄激素受体（mAR）诱导快速的非基因组效应。在这项研究中，我们旨在定位和表征原代大鼠海马神经元中的这些 mAR。通过睾酮-胎牛血清白蛋白缀合的荧光素异硫氰酸酯（T-BSA-FITC）观察到细胞表面的特异性点状荧光信号，表明海马神经元中存在 mAR。T-BSA-FITC 与细胞膜的结合不能被 iAR 拮抗剂氟他胺完全阻断，mAR 结合位点可被游离睾酮（T）竞争性结合。表达雄激素膜结合位点的大多数神经元是谷氨酸能（兴奋性）的，尽管有几个是γ-氨基丁酸（GABA）能（抑制性）的。共聚焦显微镜和活细胞成像技术用于观察雄激素对海马树突棘形态的实时快速影响。免疫荧光细胞染色用于观察它们对突触后密度蛋白 95（PSD95）和突触小体（SYN）突触标记物的影响。虽然雄激素不会导致大鼠原代海马神经元树突棘密度的短期增加，但它们促进了树突棘从细到蘑菇的形态转化，促进了树突棘成熟，增加了树突棘表面积，并迅速增加了原代海马神经元中 PSD95 和 SYN 的表达。使用 Optiprep 和 Percoll 密度梯度两步离心法制备海马突触体，并使用基因芯片比较突触体和海马的基因表达谱；通过逆转录-聚合酶链反应检测 PSD95 mRNA 的表达。在突触部位检测到几种 mRNAs，包括 PSD95。最后，构建 Venus-PSD95 质粒并转染 HT22 细胞，这是一种小鼠海马神经元细胞系。通过荧光恢复后光漂白实验观察雄激素对突触蛋白 PSD95 的实时影响，该实验涉及 PSD95 mRNA 的翻译过程。总之，我们的研究结果增加了我们对雄激素如何发挥神经保护机制对突触可塑性的影响的理解。
