lncRNAs and Anesthetic-Induced Developmental Neurotoxicity

项目概述：2016年，美国食品和药物管理局警告说，重复或长期使用 三岁以下儿童全身麻醉药的过量使用可能会影响他们的大脑发育。此警告 引起了人们对儿科麻醉安全性的严重关切。有两个主要的障碍在 麻醉剂所致发育神经毒性(AIDN)的研究领域：1)到目前为止，大多数证据表明 AIDN是通过动物实验获得的。人体研究的结果仍然没有定论。2) 机制在很大程度上是未知的。这项拟议研究的目标是解决这两个障碍。第一, 我们建立了一种新的三维(3D)人迷你脑的诱导多能性体外培养系统 用于模拟人脑发育的干细胞。人类迷你脑更接近于发育中的人类 大脑，无论在结构上还是功能上，都超过了广泛使用的2D神经元。因此，人类迷你的应用 AIDN研究领域的大脑有助于弥合动物和人类研究之间的差距。我们的预赛 数据提供了第一个证据，表明临床上相关剂量的异丙酚或七氟醚，两种 儿科常用麻醉药，可诱导人迷你脑细胞死亡。其次，我们最近使用了一个 无偏筛选24,881个长非编码RNA(LncRNA)和35,923个表达 新生小鼠海马区的信使RNA。我们发现，lncRNA的表达水平 AK156531基因及其邻近的蛋白编码基因Neuronal Per Arnt Sim DomainProtein 4(NPAS4)分别是 异丙酚暴露后显著降低。已知的lncRNAs功能之一是调节其 附近的基因表达。我们发现，AK156531基因敲除降低了两个人迷你体内的NPAS4水平 脑和小鼠脑，强烈提示AK156531可能调节NPAS4的表达。NPAS4是 参与兴奋性/抑制性(E/I)平衡、学习和记忆以及神经保护。我们还发现， 新生儿异丙酚暴露引起小鼠多种不良反应(E/I失衡、神经元死亡和 记忆功能受损)。这些令人兴奋的发现，结合已报道的NPAS4的功能，表明 AK156531基因的异常表达可能直接参与了AIDN的发病。因此，我们建议利用 AK156531在AIDN中的作用和机制 并利用人类迷你大脑将这些发现转化为人类。我们假设 AK156531的下调可通过以下途径导致E/I失衡、神经元死亡和认知功能障碍 NPAS4信号转导。在该领域首次将人类迷你大脑与AK156531击倒相结合 和过度表达小鼠模型，以研究lncRNA参与的新机制。这项建议 有望为儿童精神疾病的神经发育后果提供新的机械学见解 麻醉剂暴露。这将进一步有助于开发更合理的相关神经保护策略 儿科麻醉剂的使用，并朝着更好地保证儿科麻醉使用的安全性迈进。

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.