Modulation of GABAA receptor subtypes by neurosteroids and anesthetics: Identification of molecular targets for neuroprotection

几种天然存在的神经类固醇具有神经保护、镇静、抗焦虑和麻醉的特性。这些化合物在神经元和星形胶质细胞中合成并释放，作为GABAA受体的强效变构调节剂，这是一个令人着迷的概念。与神经类固醇类似，苯二氮卓类药物（BZDs）和全身麻醉剂可增强GABAA受体功能，从而引起镇静、抗焦虑、健忘症、肌肉松弛和无意识。GABAA受体是一种五聚体离子通道，可以由19种蛋白质亚基组装而成，从而产生了大量具有不同生理功能的受体亚型。含有α 1亚基的受体参与镇静作用，α 2受体参与抗焦虑作用，α 5受体参与bzd的遗忘作用。脑内内源性神经类固醇的合成是由线粒体转运蛋白（TSPO）的配体调节的。我们最近发现，TSPO激动剂XBD173增强神经甾体生成，增加GABAA受体介导的抑制，并在没有镇静和耐受性的情况下引起大鼠和人的焦虑缓解。在另一项研究中，我们发现了神经类固醇异孕酮和异丙酚对GABAA受体介导的突触传递的协同作用。文献中有证据表明，不同的GABAA受体亚型对神经类固醇的敏感性并不相同，其中含有δ亚基的受体是非常敏感的受体之一。此外，我们观察到XBD173在没有镇静的情况下产生抗焦虑作用，这提示了α - 1- gabaa受体不是内源性合成神经类固醇的主要靶点的假设。本应用程序的主要目的是研究增强的神经甾体生成如何在突触，细胞和网络水平上调节最重要的GABAA受体亚型的功能。具体来说，XBD173将被测试其改变海马中抑制性突触传递和信号处理的潜力。此外，我们的目标是确定GABAA受体亚型有助于神经类固醇和麻醉药物之间潜在的协同相互作用。通过研究神经类固醇如何逆转长期增强（与学习和记忆相关的细胞）和细胞凋亡的有害缺氧效应的分子和细胞机制，我们打算确定参与介导神经类固醇诱导的神经保护的突触和突触外GABAA受体。为了解决这些问题，我们将对野生型、δ亚基敲除小鼠和仅表达一种bzd敏感GABAA受体亚型的点突变小鼠的神经元进行光遗传学、光学和电生理监测。总之，本提案旨在阐述神经类固醇的分子作用及其与麻醉药的相互作用，以评估未来在临床麻醉中的应用。

Several naturally occurring neurosteroids possess neuroprotective, sedative, anxiolytic and anaesthetic properties. It is a fascinating concept that these compounds, synthetized in and released by neurons and astrocytes, act as powerful allosteric modulators of GABAA receptors. Similar to neurosteroids, benzodiazepines (BZDs) and general anaesthetics enhance GABAA receptor function, thereby causing sedation, anxiolysis, amnesia, muscle relaxation and unconsciousness. GABAA receptors are pentameric ion channels that can assemble from nineteen protein subunits, giving rise to a large number of receptor subtypes with different physiological functions. Receptors containing alpha1-subunits contribute to the sedative, alpha2-receptors to the anxiolytic and alpha5-receptors to the amnestic effects of BZDs. Endogenous neurosteroid synthesis in the brain is regulated by ligands of the mitochondrial translocator protein (TSPO). We recently showed that the TSPO agonist XBD173 enhances neurosteroidogenesis, increases GABAA receptor-mediated inhibition and causes anxiolysis in rats and humans in the absence of sedation and tolerance development. In another study we identified synergistic interactions between the neurosteroid allopregnanolone and propofol on GABAA receptor-mediated synaptic transmission. There is evidence in the literature that dissimilar GABAA receptor subtypes are not equally sensitive to neurosteroids, with delta-subunit containing receptors being among the very responsive ones. In addition, our observation that XBD173 produces anxiolysis in the absence of sedation prompts the hypothesis that alpha1-GABAA receptors are not a major target for endogenously synthesized neurosteroids. The primary goal of this application is to investigate how enhanced neurosteroidogenesis modulates the function of the most important GABAA receptor subtypes on the synaptic, cellular and network level. Specifically, XBD173 will be tested for its potential to alter inhibitory synaptic transmission and signal processing in the hippocampus. Furthermore, we aim to identify GABAA receptor subtypes contributing to potentially synergistic interactions between neurosteroids and anaesthetic drugs. By investigating the molecular and cellular mechanisms how neurosteroids reverse detrimental hypoxic effects on long-term potentiation, a cellular correlate for learning and memory, and apoptosis we intend to identify synaptic and extrasynaptic GABAA receptors involved in mediating neurosteroid-induced neuroprotection. To address these issues, we will employ optogenetics, optical and electrophysiological monitoring of neuronal in wild type and delta-subunit knockout mice and point-mutated mice expressing only one BZD-sensitive GABAA receptor subtype. Altogether, the present proposal intends to elaborate the molecular actions of neurosteroids and their interactions with anaesthetic agents in order to evaluate future applications in clinical anaesthesia.