Visualization of glucocorticoid receptor in the brain of green fluorescent protein–glucocorticoid receptor knockin mice
Visualization of glucocorticoid receptor in the brain of green fluorescent protein–glucocorticoid receptor knockin mice
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DOI：
10.1016/j.neuroscience.2005.06.071
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发表时间：
2005-12
期刊：
影响因子：
3.3
通讯作者：
T. Usuku;M. Nishi;M. Morimoto;J. Brewer;L. Muglia;T. Sugimoto;M. Kawata
中科院分区：
文献类型：
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作者：
T. Usuku;M. Nishi;M. Morimoto;J. Brewer;L. Muglia;T. Sugimoto;M. Kawata
糖皮质激素通过糖皮质激素受体(GR)在中枢神经系统发挥包括应激反应在内的多种神经内分泌效应。GRs在与配体结合后从细胞质转运到细胞核，然后发挥转录活性。尽管GR在体内的实际性质很重要，但在维持神经元回路的脑组织中，GR的亚细胞动力学仍然不清楚。为了解决这个问题，我们培育了绿色荧光蛋白(GFP)-GR敲门小鼠，其GR已被功能上与内源性GR无法区分的GFP-GR融合蛋白所取代。在GFP-GR敲击小鼠的固定脑切片上，绿色荧光的分布与GR免疫反应相似。通过激光共聚焦扫描显微镜荧光发射指纹图谱减去自体荧光，可以确定GFP-GR在海马CA3区的核定位，与其他区域相比，GR的亚细胞定位尚未解决。为了检测GFP-GR在体内的亚细胞转运，我们对GFP-GR敲击小鼠进行了肾上腺切除。肾上腺切除后2天，GFP-GR从胞核移位至胞浆和突起。此外，激光扫描细胞仪原位荧光强度定量显示整个GFP-GR的表达水平增加。然后，我们研究了在分离培养和组织切片中活的海马神经元中GFP-GR亚细胞定位的动态变化。在缺乏配体的情况下，GFP-GR不仅定位于核周，也定位于神经突起，并观察到配体处理后的核移位。这是第一个在更生理的条件下观察GR在小鼠脑内亚细胞运输的报告。本研究结果为体内和体外GR动力学研究提供了新的途径。
Glucocorticoids exert various neuroendocrinological effects, including stress response, in the central nervous system via glucocorticoid receptor (GR). GRs are transported from the cytoplasm to the nucleus upon ligand binding, and then exert the transcriptional activity. Although it is important for unraveling the actual property of the GR in vivo, subcellular dynamics of the GR are still unclear within the brain tissue in which the neuronal circuitry is maintained. To address this issue, we generated green fluorescent protein (GFP)–GR knockin mice, whose GR has been replaced by a GFP–GR fusion protein that is functionally indistinguishable from endogenous GR. In fixed brain sections of the GFP–GR knockin mice, the distribution of the green fluorescence was similar to that of GR immunoreactivity. By subtracting autofluorescence using fluorescent emission fingerprinting method with confocal laser scanning microscope, nuclear localization of GFP–GR was identifiable in the hippocampal CA3 subregion, where subcellular localization of the GR has been unsolved compared with other areas. To examine the subcellular trafficking of GFP–GR in vivo, we performed adrenalectomy on the GFP–GR knockin mice. GFP–GR was translocated from the nucleus to the cytoplasm and neurites two days after adrenalectomy. Furthermore, laser scanning cytometry by which fluorescence intensity in situ can be quantitatively measured revealed the entire GFP–GR expression level was increased. We then examined the dynamic changes in the subcellular localization of GFP–GR in living hippocampal neurons both in dissociated culture and in tissue slices. GFP–GR was localized in not only the perikarya but also neurites in the absence of ligand, and nuclear translocation following ligand treatment was observed. This is the first report visualizing subcellular trafficking of the GR in the mouse brain in more physiological condition. The present results propose new avenues for the research of the GR dynamics both in vitro and in vivo.