Glucocorticoid Receptors (GR) in Mitochondria: The Role in Chronic Stress

已有研究表明，慢性应激在临床上与患者抑郁的形成有关，荷尔蒙被认为与某些情绪障碍的临床表现有关。慢性束缚应激引起啮齿类动物脑区的形态重组，同时伴随着行为的改变。尽管这些效应的确切机制尚不清楚，但越来越多的数据表明，神经保护和线粒体功能的改变可能在调节各种形式的突触和神经可塑性方面发挥重要作用；我们试图研究在慢性应激过程中激素对线粒体功能的调节。 建立皮质神经元培养，以确定糖皮质激素受体在线粒体中的定位和功能。低浓度(100 NM)和高浓度(1000 NM)皮质酮作用于培养的皮质神经元1.5h后，糖皮质激素受体移位到线粒体。与线粒体功能增强相一致，线粒体编码基因细胞色素氧化酶I(Coxi)(在其启动子区域有GRE)在处理24小时后在线粒体中的表达也增加。然而，治疗三天后，1uM皮质酮导致线粒体GR水平下降，100 nm皮质酮治疗没有。同样，与100 nm皮质酮相比，高(1uM)和低(100 Nm)皮质酮处理1d后线粒体膜电位均有不同程度的升高，只有高(1uM)皮质酮处理3d后线粒体膜电位显著降低。此外，与100 nm皮质酮和100 nm皮质酮相比，高(1µM)和低(100 Nm)皮质酮处理1d后线粒体膜电位显著降低，而高浓度(1uM)皮质酮处理3d后线粒体膜电位显著降低。为了确定慢性应激下的情况，我们发现慢性应激后前额叶皮质组织线粒体部分的糖皮质激素受体水平显著下降，提示在体外高浓度和长期皮质酮处理后也有类似的变化。这些研究可能为糖皮质激素调节线粒体功能和神经元信号的机制提供更多的见解。此外，这项研究还有可能有助于更全面地了解慢性应激和激素调节细胞可塑性和弹性的机制，并有助于改进治疗方法的未来发展。 糖皮质激素在调节神经可塑性方面起着重要的双向作用，低剂量可增强神经可塑性和空间记忆行为，而长期高剂量则产生抑制作用。我们进行了一系列实验来阐明这种双相作用的机制，发现糖皮质激素受体(GRs)在皮质酮处理后与抗凋亡蛋白Bcl2形成复合体，并在低或高剂量皮质酮急性处理后与Bcl2移位到原代皮质神经元的线粒体中。然而，在治疗三天后，皮质酮水平高，但不是低，导致线粒体中GR和Bcl-2水平下降。此外，线粒体功能的三个独立指标-线粒体钙保持能力、线粒体氧化和膜电位也受到长期皮质酮处理的倒U型调节。皮质酮对线粒体功能的这种调节与神经保护相关：即低剂量皮质酮治疗显示出神经保护作用，而高剂量皮质酮治疗则增强了海人藻酸(KA)诱导的皮质神经元的毒性。与体外研究一样，大剂量皮质酮长期治疗后，前额叶皮质线粒体中Bcl2水平显著降低，GR水平也显著降低。这些发现有可能有助于更全面地了解糖皮质激素和慢性应激调节细胞可塑性和弹性的机制，并为未来改进的治疗方法的发展提供信息。

Chronic stress has been shown to be associated clinically with formation of depression in patients and hormones are known to mediate certain clinical manifestations of mood disorders. Chronic restraint stress induces a morphological reorganization in the areas of rodent brain, effects which are also accompanied by behavioral changes. Although the precise mechanisms underlying these effects remain to be elucidated, increasing data suggests that an alteration in neuroprotection and mitochondrial functions may play an important role in regulating various forms of synaptic and neural plasticity; we have sought to investigate the mitochondrial functions regulated by hormones during chronic stress. Cortical neuronal cultures were established in order to determine the localization and function of glucocorticoid receptors in the mitochondria. Glucocorticoid receptors translocated into mitochondria after 1.5 hour treatment with low concentration (100 nM) and high concentration (1,000 nM) of corticosterone in cultured cortical neurons. Consistent with the enhancement of mitochondrial function, mitochondrially encoded gene cytochrome oxidase I (COXI) (has GRE in its promoter region) expression was also increased in mitochondria fraction after 24 hours treatment. However, after three days of treatment, 1uM corticosterone resulted in a decrease in GR levels in mitochondria and 100nM corticosterone treatment did not. Similarly, mitochondrial membrane potential were enhanced after one day treatment with high (1uM) and low (100nM) concentration of corticosterone in a similar extend, and only high concentration (1uM) significantly decreased in mitochondrial membrane potential after 3 day treatment in comparison to the 100nM corticosterone. In addition, mitochondrial oxidation were enhanced after one day treatment with high (1uM) and low (100nM) concentration of corticosterone in a similar extend, and only high concentration (1uM) significantly decreased in mitochondrial membrane potential after 3 day treatment in comparison to the 100nM corticosterone and untreated control. To determine the situation under chronic stress, we found that glucocorticoid receptor levels in mitochondria were significantly decreased in the mitochondrial fraction from prefrontal cortex tissue after chronic stress, suggesting a similar change after high concentration and long-term corticosterone treatment in vitro. These studies may provide additional insights into the mechanisms by which glucocorticoid regulate mitochondrial function and neuronal signaling. Furthermore, this research also has the potential to contribute to a more complete understanding of the mechanisms by which chronic stress and hormones regulate cellular plasticity and resilience and to the future development of improved therapeutics. Glucocorticoids play an important biphasic role in modulating neural plasticity, with low doses enhancing neural plasticity and spatial memory behavior, whereas chronic, higher doses producing inhibition. We undertook a series of experiments to elucidate the mechanisms underlying these biphasic effects, and found that glucocorticoid receptors (GRs) formed a complex with the anti-apoptotic protein Bcl-2 in response to corticosterone treatment, and translocated with Bcl-2 into mitochondria after acute treatment with low or high doses of corticosterone in primary cortical neurons. However, after three days of treatment, high, but not low, corticosterone resulted in a decrease in GR and Bcl-2 levels in mitochondria. In addition, three independent measures of mitochondrial functionmitochondrial calcium holding capacity, mitochondrial oxidation, and membrane potentialwere also regulated by long-term corticosterone treatment in an inverted U-shape. This regulation of mitochondrial function by corticosterone correlated with neuroprotection: that is, treatment with low doses of corticosterone demonstrated a neuroprotective effect, whereas treatment with high doses of corticosterone enhanced kainic acid (KA)-induced toxicity of cortical neurons. As with the in vitro studies, Bcl-2 levels in the mitochondria of the prefrontal cortex were significantly decreased, along with GR levels, after long-term treatment with high dose corticosterone. These findings have the potential to contribute to a more complete understanding of the mechanisms by which glucocorticoids and chronic stress regulate cellular plasticity and resilience, and to inform the future development of improved therapeutics.