microRNAs and Anesthetic-Induced Developmental Neurotoxicity

描述(申请人提供)：越来越多的证据表明，在动物模型中，长期接触全身麻醉药会导致广泛的神经细胞死亡，继而导致长期记忆和学习障碍，这引发了人们对产科和儿科麻醉安全性的严重担忧。虽然麻醉剂诱导的神经毒性增加的潜在机制是复杂的，并且刚刚开始被理解，但我们令人兴奋的初步数据指出了microRNAs在神经毒性中的作用。MicroRNAs是一种内源性、小的、非编码的RNAs，通过抑制靶基因的表达，在正常发育和生理以及疾病中发挥强大的调节作用。具体地说，miR-21已被证明可以减少不同类型细胞的凋亡。根据我们的初步数据和其他人之前的报告，我们假设下调的miR-21导致的线粒体分裂增加与麻醉剂(异丙酚)的神经毒性有关。异丙酚最广泛地用于儿科和产科的镇静和麻醉。我们建议利用功能获得和功能丧失的方法来研究miR-21在异丙酚神经毒性中的作用，利用干细胞来源的神经元将研究结果翻译到人类，并探讨miR-21作用的以下分子机制：miR-21靶向并抑制程序性细胞死亡4(PDCD4)，它可以：1)激活蛋白激酶B(Akt)，2)减少线粒体分裂，延迟线粒体通透性转换孔(MPTP)的开放，以及3)减少细胞死亡。MiR-21表达下调，PDCD4表达上调，Akt活性减弱，线粒体分裂增加，这些都可能是异丙酚产生神经毒性的原因之一。我们最初令人兴奋的数据表明，异丙酚导致干细胞来源的人类神经元miR-21的下调。此外，miR-21基因敲除增加了小鼠发育神经元和人类神经元对异丙酚暴露的脆弱性。MiR-21过表达可减轻异丙酚诱导的神经细胞凋亡。此外，miR-21的降低伴随着Akt活性的降低，线粒体分裂的增加，以及人神经元MPTP开放的增加，这有力地支持了我们的假设。为了验证我们的假设，我们在未来五年提出了以下具体目标：1)研究miR-21在小鼠异丙酚神经毒性中的作用；2)确定miR-21在异丙酚神经毒性中的作用；以及3)确定miR-21/PDCD4/线粒体分裂途径在异丙酚对人神经元和小鼠脑中的神经毒性中的作用。这是一项具有高度临床相关性的研究，具有创新性，走在该领域的前沿。基于这项拟议研究的发现，我们可以制定更合理的神经保护策略，从而在确保儿科人群麻醉安全方面取得重大进展。

DESCRIPTION (provided by applicant): Growing evidence demonstrates that prolonged exposure to general anesthetics induces widespread neuronal cell death followed by long-term memory and learning disability in animal models, raising serious concerns about the safety of obstetric and pediatric anesthesia. Although the underlying mechanisms of increased anesthetic-induced neurotoxicity are complex and just beginning to be understood, our exciting preliminary data point to the role of microRNAs in neurotoxicity. MicroRNAs are endogenous, small, non- coding RNAs that are powerful regulators in normal development and physiology, and diseases through inhibition of target gene expression. Specifically, miR-21 has been shown to decrease apoptosis in varying cell types. Based on our preliminary data and previous reports by others, we hypothesized that the increased mitochondrial fission conferred by downregulated miR-21 contributes to the anesthetic (propofol) neurotoxicity. Propofol is most widely used for sedation and anesthesia in pediatric and obstetric medicine. We propose to utilize gain- and loss-of-function approaches to examine the role of miR-21 in propofol neurotoxicity in mice, translate the findings to humans using stem cell-derived neurons, and investigate the following molecular mechanisms underlying the roles of miR-21 effect: miR-21 targets and suppresses programmed cell death 4 (PDCD4), which can: 1) activate protein kinase B (Akt), 2) decrease mitochondrial fission, delay opening of mitochondrial permeability transition pore (mPTP), and 3) reduce cell death. Downregulated miR-21, upregulated PDCD4, attenuated activation of Akt, and increased mitochondrial fission are likely to contribute to the neurotoxicity conferred by propofol. Our initial exciting data indicate that propofol causes downregulation of miR-21 in stem cell-derived human neurons. In addition, miR-21 knockout increases the vulnerability of mouse developmental neurons and human neurons to propofol exposure. Overexpression of miR-21 attenuated propofol-induced apoptosis in cultured human neurons. Moreover, the reduction of miR-21 is accompanied with a decrease of Akt activation, an increase of mitochondrial fission, and an increase in mPTP opening in human neurons, strongly supporting our hypothesis. We propose the following Specific Aims for the next five years to test our hypotheses: 1) to examine the role of miR-21 in propofol neurotoxicity in mouse brains; 2) to determine the role of miR-21 in propofol neurotoxicity in human neurons; and 3) to determine the role of miR-21/PDCD4/mitochondrial fission pathway in propofol neurotoxicity in human neurons and mouse brains. This is a highly clinically relevant study that is innovative and at the forefront in this field. Based on the findings from this proposed study, we can develop more rational neuroprotection strategies, leading to major advances toward assuring the safety of anesthesia in pediatric populations.