NADPH oxidase regulates growth cone guidance

活性氧（ROS）可以作为信号分子介导免疫中的生理功能， 细胞增殖、分化和迁移。ROS是否具有主要的信号传导功能， 信使在轴突生长和指导目前还不清楚。神经生长锥是一个高度能动的 在神经元过程的尖端的结构，引导它们在发育过程中适当的靶细胞， 神经系统的再生。生长锥整合了来自环境的分子信息 并通过多个信号级联将其转导以影响潜在的细胞骨架动力学。而大多数 主要的第二信使系统与调节定向生长锥运动有关， ROS的作用尚未确定。本研究有两个主要目标，重点是ROS产生 通过烟酰胺腺嘌呤二核苷酸磷酸-（NADPH）氧化酶（Nox）：（1）测定细胞和 ROS控制神经突生长的分子机制;以及（2）确定ROS是否作为第二个 信使下游的具体指导线索，以控制轴突的生长和指导。四个中央 假设（1）生理水平ROS是最佳的，且是粘附介导的神经突所需的 增长;（2）Src酪氨酸激酶是神经元生长锥中ROS信号传导的关键靶标;（3）神经元Nox 2衍生的ROS调节轴突寻路;以及（4）特异性轴突引导线索，如slit 2控制轴突寻路。 在体外和体内通过Nox 2衍生的ROS进行寻路。该项目将利用两个优秀的 模型系统来测试这些假设：大型的Aesthesia生长锥，用于生长的定量活细胞成像 视锥运动和细胞内活性氧在体外和发展斑马鱼胚胎成像和操纵 体内轴突发育。体外生长锥导向测定、新型荧光染料和生物传感器 分别特异于过氧化氢和Src活性，先进的成像技术，嵌合分析， Nox 2缺陷型斑马鱼品系以及视网膜神经节细胞特异性Nox 2突变型鱼类品系将用于 具体目的有两个：（1）确定细胞和分子机制 ROS控制神经突生长的机制。(2)第二个目的是确定神经元Nox 2在轴突中的作用。 视网膜神经节细胞的寻路。拟议的工作是高度创新的，因为它调查ROS作为一个 一组新的信号分子在轴突生长和指导，并开发了几个新的斑马鱼系 适用于研究Nox在神经系统中的功能。总之，这些研究有可能导致 在神经发育和再生领域的突破性发现。此外，由于基础 轴突生长和导向机制在物种间高度保守，这些研究将影响 开发用于神经变性疾病和中枢神经系统损伤的抗氧化剂治疗。

Reactive oxygen species (ROS) can act as signaling molecules mediating physiological functions in immunity, cell proliferation, differentiation, and migration. Whether ROS have a major signaling function as second messengers in axonal growth and guidance is currently unclear. The neuronal growth cone is a highly motile structure at the tip of neuronal processes, guiding them to appropriate target cells during development and regeneration of the nervous system. The growth cone integrates molecular information from the environment and transduces it via multiple signaling cascades to affect underlying cytoskeletal dynamics. Whereas most major second messenger systems have been implicated in regulating directional growth cone movement, such a role has not been established for ROS. The present study has two major objectives focusing on ROS produced by nicotinamide adenine dinucleotide phosphate-(NADPH) oxidase (Nox): (1) to determine the cellular and molecular mechanism by which ROS control neurite growth; and (2) to determine whether ROS act as second messengers downstream of specific guidance cues to control axonal growth and guidance. The four central hypotheses state that (1) a physiological level of ROS is optimal and required for adhesion-mediated neurite growth; (2) Src tyrosine kinase is a key target of ROS signaling in neuronal growth cones; (3) neuronal Nox2-derived ROS regulate axonal pathfinding; and (4) specific axon guidance cues such as slit2 control axonal pathfinding via Nox2-derived ROS both in vitro and in vivo. This project will take advantage of two excellent model systems to test these hypotheses: large Aplysia growth cones for quantitative live cell imaging of growth cone motility and intracellular ROS in vitro and developing zebrafish embryos for imaging and manipulating axonal development in vivo. In vitro growth cone guidance assays, novel fluorescent dyes and biosensors specific for hydrogen peroxide and Src activity, respectively, advanced imaging techniques, chimeric analysis of Nox2-deficient zebrafish lines as well as retinal ganglion cell-specific Nox2-mutant fish lines will be used to address the following two Specific Aims: (1) The first aim is to determine the cellular and molecular mechanism by which ROS in control neurite growth. (2) The second aim is to determine the role of neuronal Nox2 in axonal pathfinding of retinal ganglion cells. The proposed work is highly innovative because it investigates ROS as a novel group of signaling molecules in axonal growth and guidance and develops several new zebrafish lines suitable for studying Nox function in the nervous system. In summary, these studies have the potential of leading to breakthrough findings in the field of neuronal development and regeneration. Furthermore, since basic mechanisms of axonal growth and guidance are highly conserved across species, these studies will impact the development of antioxidant treatments for neurodegenerative diseases and central nervous system injuries.