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

干细胞分化和成体神经发生中昼夜节律与氧化还原信号之间的耦合

• 批准号: 10524773
• 负责人: Daniel Maxim Iascone
• 金额: $ 7.41万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10524773
• 关键词: ARNTL gene; Acute; Adult; Alzheimer' s Disease; Behavioral; Biological Models; Biology; Bromodeoxyuridine; CRISPR/Cas technology; Cell Differentiation process; Cell division; Cells; Circadian Dysregulation; Circadian Rhythms; Circadian gene expression; Coupling; Cre driver; Development; Disease; Down-Regulation; Embryonic Development; Exhibits; Fibroblasts; Gene Expression; Genes; Genetic; Genetic Recombination; Genetic Transcription; Glutamates; Glycolysis; Goals; Health; Hippocampus; Histology; Homeostasis; Hour; Human; Hydrogen Peroxide; Image; Knock-out; Knockout Mice; Label; Link; Mammals; Maps; Measures; Mediating; Metabolic; Metabolism; Modeling; Molecular; Mus; NADP; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; Oxidation-Reduction; Oxidative Phosphorylation; Oxidative Stress; Parkinson Disease; Pathway interactions; Pentosephosphate Pathway; Phase; Physiological Processes; Pluripotent Stem Cells; Post-Translational Protein Processing; Process; Production; Proliferating; Reactive Oxygen Species; Regulation; Reporter; Research; Research Training; Role; Signal Transduction; Stains; Stem cell pluripotency; Synapses; Tamoxifen; Testing; Thymidine; Time; Training; Transgenes; Transgenic Organisms; Venus; Visualization; adult neurogenesis; adult stem cell; age related; analog; biological adaptation to stress; cell type; circadian; circadian pacemaker; cofactor; directed differentiation; embryonic stem cell; excitatory neuron; genome editing; granule cell; human embryonic stem cell; in vitro Model; in vivo; in vivo Model; induced pluripotent stem cell; nerve stem cell; neurogenesis; neuronal circuitry; novel; pluripotency; self-renewal; sensor; stem cell differentiation; stem cell division; stem cell homeostasis; stem cell model; stem cells; tool; transcription factor

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

项目摘要 昼夜节律对于协调关键行为和生理过程的时间是必要的 哺乳动物[1-3]。但是，虽然我们对胚胎中昼夜节律基因功能的理解 发展正在迅速发展[4-6]，这是这些节奏出现的分子机制 在干细胞分化过程中仍然难以捉摸[7]。最近，由活性氧信号传导（氧化还原 信号传导已成为成人细胞代谢与昼夜节律之间的重要联系 功能[8，9]。通过生产氧化还原辅因子NADPH的信号传导 转录振荡的重要调节因子，影响核心昼夜节律基因的表达 通过氧化还原敏感的转录因子NRF2 [10]。 NRF2是胚胎干细胞的关键调节剂 多能性和自我更新，但是氧化还原信号是否有助于昼夜节律的发展 在分化的干细胞中，仍未探索[11]。 使用过氧化氢和PER2表达的荧光记者，我们建议同时 首次可视化单细胞中的活性氧和昼夜节律。通过结合 具有CRISPR/CAS9介导的基因组编辑方法的新型模型系统，我们将因果测试 氧化还原信号传导在人类诱导的多能干细胞中的昼夜节律发展中 指示分化为谷氨酸能神经元。使用成年海马神经发生作为体内模型 神经元分化系统，我们将进一步探索氧化还原耦合的功能 协调成人颗粒细胞的顺序发展和回路整合。 该提案的长期目标是创建一个新颖的模型系统，以探索通过 氧化还原与昼夜节律转录因子网络之间的哪些相互作用指导适当 开发的顺序。当前的提案试图调查氧化还原-circadian 耦合驱动多能和成年干细胞的分化，我们旨在描述一个一般范式 健康和疾病中代谢，细胞分裂和干细胞稳态的协调。 假设：我们假设氧化还原信号传导驱动干细胞中昼夜节律的发展 在多能丧失以及氧化还原信号和昼夜节律之间的相互调节之后 驱动细胞成熟。我们预测会破坏成年神经干细胞中的氧化还原耦合 通过急性NRF2敲除将引起细胞分裂和分化，但阻碍了 成人颗粒细胞的昼夜节律和正常成熟。我们将在 以下目的： AIM 1：在人类诱导的多能干细胞中，因果关系将氧化还原信号与昼夜节律的发展联系起来 AIM 2：确定NRF2 KO介导的氧化还原偶联破坏对成人神经发生的影响