Coupling between circadian rhythms and redox signaling in stem cell differentiation and adult neurogenesis

项目摘要 昼夜节律是协调关键的行为和生理过程的时间所必需的 哺乳动物[1-3]。然而，虽然我们对生物钟基因在胚胎中的功能的理解 发展正在迅速推进[4-6]，这些节律出现的分子机制 在干细胞分化过程中仍然难以捉摸[7]。最近，由活性氧物种(氧化还原)发出的信号 信号)已经成为成人细胞新陈代谢和昼夜节律之间的重要联系 函数[8，9]。通过产生氧化还原辅因子NADPH的戊糖磷酸途径的信号转导 转录振荡的重要调节因子，影响核心生物钟基因的表达 通过氧化还原敏感的转录因子NRF2[10]。Nrf2是胚胎干细胞的重要调节因子。 多能性和自我更新，但氧化还原信号是否有助于昼夜节律的发展 干细胞的分化仍然完全没有被探索[11]。 利用过氧化氢和PER2表达的荧光记者，我们建议同时 首次在单个细胞中可视化活性氧物种和昼夜节律。通过将这一点结合起来 新的模型系统与CRISPR/Cas9介导的基因组编辑方法，我们将因果测试的作用 氧化还原信号在人诱导多能干细胞昼夜节律发展中的作用 定向分化为谷氨酸能神经元。成年海马神经发生作为活体模型的实验研究 神经分化系统，我们将进一步探讨氧化还原-昼夜节律偶联在 协调成体出生的颗粒细胞的顺序发育和回路整合。 这项提议的长期目标是创建一个新的模型系统，通过 氧化还原和昼夜节律转录因子网络之间的哪种相互作用引导适当的 发展的顺序时间。虽然目前的提案试图调查氧化还原昼夜节律 耦合驱动多能干细胞和成体干细胞的分化，我们的目标是描述一个通用的范例 在健康和疾病中新陈代谢、细胞分裂和干细胞动态平衡的协调。 假设：我们假设氧化还原信号驱动干细胞昼夜节律的发展 在失去多能性之后，氧化还原信号和昼夜节律之间的相互调节 推动细胞成熟。我们预测破坏成年神经干细胞的氧化还原-昼夜节律偶联 通过急性Nrf2基因敲除会诱导细胞分裂分化，但会阻碍肿瘤的发展。 成体出生颗粒细胞的昼夜节律和正常成熟。我们将在 以下目标： 目的1：人诱导的多能干细胞中氧化还原信号与昼夜节律发育的因果联系 目的2：确定NRF2KO介导的氧化还原-昼夜节律偶联的破坏对成人神经发生的影响

Project Summary Circadian rhythms are necessary to coordinate the timing of key behavioral and physiological processes in mammals [1-3]. However, while our understanding of the function of circadian clock genes in embryonic development is rapidly advancing [4-6], the molecular mechanisms through which these rhythms emerge during stem cell differentiation remain elusive [7]. Recently, signaling by reactive oxygen species (redox signaling) has emerged as an essential link between cellular metabolism and circadian rhythms in adult function [8, 9]. Signaling from the pentose phosphate pathway through production of redox cofactor NADPH is an important regulator of transcriptional oscillations, influencing the expression of core circadian clock genes through the redox-sensitive transcription factor NRF2 [10]. NRF2 is a crucial regulator of embryonic stem cell pluripotency and self-renewal, but whether redox signaling contributes to the development of circadian rhythms in differentiating stem cells remains completely unexplored [11]. Using fluorescent reporters of the hydrogen peroxide and Per2 expression, we propose to simultaneously visualize reactive oxygen species and circadian rhythms in single cells for the first time. By combining this novel model system with CRISPR/Cas9-mediated genome editing approaches, we will causally test the role of redox signaling in the development of circadian rhythms in human induced pluripotent stem cells undergoing directed differentiation to glutamatergic neurons. Using adult hippocampal neurogenesis as an in vivo model system for neuronal differentiation, we will further explore the function of redox-circadian coupling in coordinating the sequential development and circuit integration of adult-born granule cells. The long-term objective of this proposal is to create a novel model system to explore the mechanisms through which reciprocal interaction between redox and circadian transcription factor networks direct the proper sequential timing of development. While the current proposal seeks to investigate how redox-circadian coupling drives the differentiation of pluripotent and adult stem cells, we aim to describe a general paradigm for the coordination of metabolism, cell division, and stem cell homeostasis in health and disease. Hypothesis: We hypothesize that redox signaling drives the development of circadian rhythms in stem cells following the loss of pluripotency, and that reciprocal regulation between redox signaling and circadian rhythms drives the cellular maturation. We predict that disrupting redox-circadian coupling in adult neural stem cells through acute Nrf2 knockout will induce cell division and differentiation, but hinder the development of circadian rhythms and normal maturation of adult-born granule cells. We will test this hypothesis in the following aims: Aim 1: Causally link redox signaling to circadian rhythm development in human induced pluripotent stem cells Aim 2: Determine impact of Nrf2 KO-mediated disruption of redox-circadian coupling on adult neurogenesis