lncRNAs and Anesthetic-Induced Developmental Neurotoxicity

2016年，美国食品和药物管理局警告说，重复或长期使用 三岁以下的儿童使用全身麻醉药可能会影响他们的大脑发育。此警告 引起了对小儿麻醉安全性的严重关注。有两个主要障碍， 麻醉诱导的发育神经毒性（AIDN）的研究领域：1）迄今为止，大多数证据表明， 从动物研究中获得AIDN。人类研究的结果仍然没有定论。2)的 机制在很大程度上是未知的。这项研究的目的是解决这两个障碍。第一、 我们建立了一个新的体外三维（3D）人脑系统，使用诱导多能 用于模拟人类大脑发育的干细胞。人类的迷你大脑更类似于发育中的人类 大脑，无论是结构上还是功能上，都比广泛使用的2D神经元要好。因此，应用人体迷你 大脑在艾滋病研究领域的研究有助于弥合动物和人类研究之间的差距。我们的初步 数据提供了第一个证据，表明临床相关剂量的丙泊酚或七氟烷，两个 常用的小儿麻醉剂，在人类迷你大脑中诱导细胞死亡。其次，我们最近使用了一个 无偏方法筛选24，881个长非编码RNA（lncRNA）和35，923个长非编码RNA（lncRNA）的表达。 新生小鼠海马中的信使RNA。我们发现lncRNA的表达水平 AK 156531基因及其邻近的蛋白质编码基因Neuronal Per Arnt Sim domain protein 4（NPAS 4）， 在暴露于丙泊酚后显著降低。lncRNA的已知功能之一是调节它们的表达。 附近的基因表达我们发现，AK 156531的敲低降低了两种人类微环境中NPAS 4的水平， 脑和小鼠脑，强烈表明AK 156531可能调节NPAS 4表达。NPAS 4是 参与兴奋/抑制（E/I）平衡、学习和记忆以及神经保护。我们还发现 新生儿丙泊酚暴露在小鼠中引起多种不良反应（E/I失衡，神经元死亡， 记忆功能受损）。这些令人兴奋的发现，结合报道的NPAS 4功能，表明 AK 156531的异常表达可能直接导致AIDN的发生。因此，我们建议利用 AK 156531获得和丧失功能的方法，以检查AK 156531在AIDN中的作用和机制。 小鼠，并通过使用人类的迷你大脑来促进这些发现对人类的翻译。我们假设 AK 156531的下调导致E/I失衡、神经元死亡和认知功能障碍， NPAS 4信号传导。在该领域，人类迷你大脑将首次与AK 156531敲除相结合 和过表达小鼠模型来研究lncRNA参与的新机制。这项建议 有望为儿童神经发育的后果提供新的机制见解。 麻醉剂暴露这将进一步帮助开发更合理的神经保护策略， 儿科麻醉剂的使用，并朝着更好地保证儿科麻醉剂使用的安全性方向发展。

PROJECT SUMMARY: In 2016, the U.S. Food and Drug Administration warned that repeated or lengthy use of general anesthetics in children below the age of three might affect their brain development. This warning raises serious concerns regarding the safety of pediatric anesthesia. There are two main barriers in the research field of anesthetic-induced developmental neurotoxicity (AIDN): 1) So far, most of the evidence for AIDN was obtained from animal studies. The results from human studies remain inconclusive. 2) The mechanisms are largely unknown. The goal of this proposed study is to address both of these barriers. First, we established a new in vitro system of three-dimensional (3D) human mini brains using induced pluripotent stem cells for modeling human brain development. Human mini brains are more similar to developing human brains, both structurally and functionally, than the widely used 2D neurons. Thus, application of human mini brains in AIDN research field helps bridge the gap between the animal and human studies. Our preliminary data provided the first evidence showing that clinically relevant doses of either propofol or sevoflurane, two commonly used pediatric anesthetics, induced cell death in human mini brains. Second, we recently used an unbiased approach to screen the expression of 24,881 long non-coding RNAs (lncRNAs) and 35,923 messenger RNAs in neonatal mouse hippocampi. We discovered that the expression levels of the lncRNA AK156531 gene, and its nearby protein-coding gene Neuronal Per Arnt Sim domain protein 4 (NPAS4), were dramatically decreased following propofol exposure. One of the known functions of lncRNAs is to regulate their nearby gene expression. We found that knockdown of AK156531 decreased NPAS4 levels in both human mini brains and mouse brains, strongly suggesting that AK156531 might regulate NPAS4 expression. NPAS4 is involved in excitatory/inhibitory (E/I) balance, learning and memory, and neuroprotection. We also found that neonatal propofol exposure caused multiple adverse effects in mice (E/I imbalance, neuronal death, and impaired memory function). These exciting findings, combined with the reported function of NPAS4, suggest that the abnormally expressed AK156531 might directly contribute to AIDN. Thus, we propose to utilize AK156531 gain- and loss-of-function approaches to examine the role and mechanism of AK156531 in AIDN in mice, and to facilitate the translation of these findings to humans by using human mini brains. We hypothesize that downregulation of AK156531 contributes to E/I imbalance, neuronal death and cognitive dysfunction via NPAS4 signaling. For the first time in this field, human mini brains will be combined with AK156531 knockdown and overexpression mouse models to investigate the novel mechanisms of lncRNA involvement. This proposal is expected to provide new mechanistic insights into the neurodevelopmental consequences of pediatric anesthetic exposure. This will further aid in the development of more rational neuroprotective strategies related to pediatric anesthetic use, and movement towards a better assurance of safety for pediatric anesthesia use.