Mechanisms of Neural Stem Cell Mechanoregulation

神经干细胞机械调节机制

• 批准号: 10160962
• 负责人: Sanjay Kumar
• 金额: $ 28.78万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10160962
• 关键词: 3-Dimensional; AMOT gene; Actins; Address; Adult; Affinity; Alkynes; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Animal Model; Astrocytes; Atomic Force Microscopy; Azides; Biochemical; Biocompatible Materials; Biomechanics; Biophysics; Brain; Cell Lineage; Cells; Chemistry; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cues; Cytoskeleton; Development; Disease; Dissection; Engineering; Event; Extracellular Matrix; Goals; Hippocampus (Brain); Hour; Hyaluronic Acid; Hybrids; Hydrogels; Kinetics; Learning; Life; Maps; Mechanics; Mediating; Memory; Myosin ATPase; Neurodegenerative Disorders; Neurons; Oligodendroglia; Oligonucleotides; Parkinson Disease; Pathway interactions; Pharmacology; Play; Population; Process; Property; Rattus; Regenerative Medicine; Relaxation; Role; Signal Transduction; Stress; Structure; System; Technology; Testing; Time; Tissue Engineering; Tissues; Viral; base; beta catenin; cell type; cellular targeting; crosslink; cycloaddition; design; experience; genome-wide; improved; in vivo; innovation; interest; loss of function; mechanical force; mechanical properties; nerve stem cell; neurodevelopment; neurogenesis; neuromechanism; neuroregulation; novel; polyacrylamide hydrogels; relating to nervous system; repaired; rho GTP-Binding Proteins; scaffold; screening; stem cell biology; stem cell fate; stem cell niche; stem cell self renewal; stem cells; tool; viscoelasticity

PROJECT SUMMARY/ABSTRACT Mechanical forces within the material microenvironment are increasingly recognized as important regulators of stem cell self-renewal and differentiation. Over the past decade we have been exploring these concepts in the context of adult hippocampal neural stem cells (NSCs), which generate neurons throughout adult life and play key roles in learning, memory, and disease processes. In our first period of R01 support, we have shown that extracellular matrix (ECM) stiffness cues can act through Rho GTPase- and myosin-dependent contractility to influence lineage commitment within a defined temporal window. Moreover, manipulation of these stiffness- sensing pathways in vivo can control hippocampal neurogenesis in a manner that is predictable from culture studies. More recently, we have created reversibly-stiffening oligonucleotide-crosslinked materials and applied this technology to narrow this window to 12-36 h and to begin elucidating key signals that are activated during this period to induce lineage commitment. In this renewal application, we now propose to build upon these advances by tackling two key questions of high general interest within the stem cell field: First, do the mechanoregulatory signaling relationships observed in simplified 2D systems hold in more complex 3D microenvironments, particularly ones with dynamic mechanical properties analogous to those encountered in vivo? Second, precisely how do the signals triggered by mechanical inputs (e.g. Rho GTPase-dependent myosin contraction) interface with the signals canonically understood to regulate NSC neurogenesis? In Aim 1, we will investigate mechanosensitive lineage commitment in 3D by applying new click-crosslinked hyaluronic acid hydrogels with tunable stiffness. We will also innovate upon these materials by incorporating reversible oligonucleotide-based crosslinks that allow variable degrees of stress relaxation, and then use these materials to ask if we can shift the time window of mechanosensitive lineage commitment. In Aim 2, we will investigate integration of mechanotransductive signaling and canonical pro-neurogenic signaling in the control of NSC neurogenesis. Specifically, we will test the hypothesis that mechanosensitive lineage commitment is controlled by a master signaling circuit involving YAP, angiomotin, and b-catenin. We will also apply genome-wide CRISPR gain/loss-of-function screens to identify additional candidates, which we will then characterize and incorporate into this regulatory framework. Successful completion of these studies will not only dramatically improve the field’s understanding of how mechanical signals influence NSC lineage commitment but offer a new intellectual roadmap and set of tools that will be broadly applicable to all stem cell types.

项目摘要/摘要 材料微环境中的机械力越来越被认为是重要的调节剂 干细胞自我更新和分化。在过去的十年中，我们一直在探索这些概念 成人海马神经元细胞（NSC）的背景，该细胞在整个成人生活中产生神经元并发挥作用 在学习，记忆和疾病过程中的关键作用。在我们的R01支持的第一阶段，我们已经表明 细胞外基质（ECM）刚度提示可以通过Rho GTPase-和肌球蛋白依赖性收缩性起作用 影响定义的临时窗口内的血统承诺。此外，操纵这些刚度 体内感测途径可以以培养的方式控制海马神经发生 研究。最近，我们创建了可逆的寡核苷酸交联材料并应用的 这项将此窗口范围缩小到12-36 h的技术，并开始阐明在 这一时期引起了血统的承诺。在此续订应用程序中，我们现在建议以此为基础 通过解决干细胞领域中高普遍兴趣的两个关键问题的进步：首先，执行 在简化的2D系统中观察到的机械调节信号传导关系在更复杂的3D中存放 微环境，尤其是具有类似于在 体内？其次，准确地说是如何由机械输入触发的信号（例如Rho GTPase依赖性肌球蛋白 收缩）与信号的接口在规范上理解以调节NSC神经发生？在AIM 1中，我们将 通过施加新的点击链接透明质酸，调查3D中的机械敏感谱系承诺 具有可调刚度的水凝胶。我们还将通过合并可逆的来对这些材料进行创新 基于寡核苷酸的交联，可以允许变化的应力放松，然后使用这些材料 询问我们是否可以改变机械敏感谱系承诺的时间窗口。在AIM 2中，我们将调查 在NSC控制中，机械转移信号传导和规范的促启动信号传导的整合 神经发生。特别是，我们将测试机械敏感谱系承诺的假设 由涉及YAP，血管敏和B-catenin的主信号传导电路。我们还将应用全基因组CRISPR 获得/功能丧失屏幕以识别其他候选者，然后我们将表征并合并 进入这个监管框架。这些研究的成功完成不仅将大大改善 菲尔德对机械信号如何影响NSC血统承诺的理解，但提供了新的知识分子 路线图和一组工具，这些工具将广泛适用于所有干细胞类型。