Mechanisms of Neural Stem Cell Mechanoregulation

项目摘要/摘要 在干细胞微环境的结构、力学和维度中编码的生物物理线索是 现在被认为是自我更新和差异化的重要调节者。在过去的15年多里，包括 在R01支持的两个时期，我们一直在探索该法规的机制和翻译方面 海马神经干细胞(NSCs)，它能在成年后产生新的神经元，并有助于 神经疾病和修复。我们为该领域对 干细胞机制生物学，包括NSC谱系决定最敏感的发现 细胞外基质(ECM)在受限的时间窗口内的机制，在此期间僵硬提示是 由包括RhoA GTP酶、肌动蛋白/肌球蛋白、血管球蛋白、YAP和β-连环蛋白的信号网络处理。 我们的发现提出了干细胞领域普遍感兴趣的两个重要问题，这将成为 为我们的续签申请提供基础。首先，是什么分子机制控制着NSC的机械敏感谱系 三维(3D)ECM中的承诺，以及这些机制与二维(2D)有何不同 ECM？我们将建立在我们令人兴奋的最新发现的基础上，即转录因子Egr1是一个关键的、3D特有的 神经干细胞机械敏感性谱系承诺的调节因子。第二，干细胞是如何动态整合的 在几分钟到几小时的时间尺度上的机械输入，以触发功能上重要的信号事件？这里 我们将利用我们的初步研究，其中我们已经探索了机械敏感信号事件的时间 使用不匹配的DNA交联粘弹性水凝胶和允许定时激活的光遗传试剂 RhoA和CDC42的激活。我们有两个具体目标：在目标1中，我们将通过 哪个Egr1使用2D和3D的组合来控制3D矩阵中的机械敏感性谱系承诺 工程生物材料、基于候选对象的分子研究和筛选以确定与Egr1相关的靶点 神经发生。在目标2中，我们将调查来自2D ECM的僵硬提示是如何在距离 小时时间尺度在几小时到几天的时间内进行整合，以控制世系承诺。这一目标的工作建立在 我们观察到，RhoA的定时光遗传刺激和粘性(损失)的掺入 弹性ECM均抑制神经干细胞的神经发生。我们将确定这两个项目的关键监管时间表 并确定它们是否通过共同的机制影响世系承诺。整合 目的，我们将调查3D ECM中机械敏感性谱系承诺的时间依赖性，并询问是否 RhoA刺激和应力松弛通过Egr1抑制神经发生。我们的工作将加速 菲尔德对干细胞如何感知和作用于机械信号以指导命运决定的理解，这是一个问题 具有很高的基本价值和翻译价值。我们还将结合几种创新的方法，包括 光遗传学、粘弹性ECM、基因组编辑和测序/筛选技术。我们希望我们的 研究将提供可应用于其他ECM和干细胞系统的智能路线图。

PROJECT SUMMARY/ABSTRACT Biophysical cues encoded in the structure, mechanics, and dimensionality of the stem cell microenvironment are now appreciated as important regulators of self-renewal and differentiation. For the past 15+ years, including two periods of R01 support, we have been exploring mechanistic and translational aspects of this regulation in hippocampal neural stem cells (NSCs), which generate new neurons into adulthood and contribute to neurological disease and repair. We have made several important contributions to the field’s understanding of stem cell mechanobiology, including the discovery that NSC lineage decisions are maximally sensitive to extracellular matrix (ECM) mechanics within a restricted temporal window, during which stiffness cues are processed by a signaling network that includes RhoA GTPase, actin/myosin, angiomotin, YAP, and β-catenin. Our discoveries raise two important questions of general interest to the stem cell field, which will serve as the foundation for our renewal application. First, what molecular mechanisms govern NSC mechanosensitive lineage commitment in three-dimensional (3D) ECMs, and how do these mechanisms differ from two-dimensional (2D) ECMs? We will build on our exciting recent discovery that the transcription factor Egr1 is a critical, 3D-specific regulator of NSC mechanosensitive lineage commitment. Second, how do stem cells dynamically integrate mechanical inputs on the time scale of minutes to hours to trigger functionally important signaling events? Here we will leverage our preliminary studies in which we have probed the timing of mechanosensitive signaling events with mismatched DNA-crosslinked viscoelastic hydrogels and optogenetic reagents that allow timed activation of RhoA and Cdc42 activation. We have two specific aims: In Aim 1, we will investigate mechanisms through which Egr1 controls mechanosensitive lineage commitment in 3D matrices using a combination of 2D and 3D engineered biomaterials, candidate-based molecular studies, and screens to identify Egr1 targets relevant to neurogenesis. In Aim 2, we will investigate how stiffness cues from 2D ECMs are triggered on the minutes-to- hours time scale are integrated over hours to days to control lineage commitment. Work in this aim builds on our observation that timed optogenetic stimulation of RhoA and incorporation of viscous (loss) properties into elastic ECMs both suppress NSC neurogenesis. We will identify critical regulatory time scales for both perturbations and determine if they influence lineage commitment through common mechanisms. To integrate aims, we will investigate the time-dependence of mechanosensitive lineage commitment in 3D ECMs and ask if RhoA stimulation and stress relaxation act through Egr1 to suppress neurogenesis. Our work will accelerate the field’s understanding of how stem cells sense and act upon mechanical signals to guide fate decisions, a problem of high fundamental and translational value. We will also marry several innovative approaches, including optogenetics, viscoelastic ECMs, genome editing, and sequencing/screening technologies. We expect that our studies will provide an intellectual roadmap that can be applied to other ECM and stem cell systems.