Molecular Mechanism of Axon Guidance by Second Messenger

第二信使轴突引导的分子机制

• 批准号: 7493137
• 负责人: KYONSOO HONG
• 金额: $ 8.45万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-15 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7493137
• 关键词: Axon; Behavior; Cell membrane; Cells; Complex; Coupling; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Elevation; Endoplasmic Reticulum; Event; Goals; Growth Cones; Hand; Image; Injury; Inositol 1,4,5-Trisphosphate; Mediating; Molecular; Monitor; Nerve; Nerve Regeneration; Nervous system structure; Neurons; Property; Proteins; Rate; Receptor Activation; Regulation; Research; Resolution; Role; Second Messenger Systems; Signal Transduction; Spinal; System; Therapeutic; Time; Xenopus; axon guidance; base; human NTN1 protein; insight; nervous system development; netrin receptor; netrin-1; neurodevelopment; receptor; relating to nervous system; response; second messenger

DESCRIPTION (provided by applicant): Both intracellular Ca2+ and cyclic nucleotide-dependent signaling influence the rate and direction of a nerve growth cone extension in response to a netrin-1 gradient, a diffusible guidance molecule in Xenopus Iaevis spinal neurons. Netrin-1 is a secreted protein expressed highly in the midline of the developing vertebrate nervous system that acts as both an attractant and a repellent in guiding axons to their target cells. The bifunctional role of netrin- 1 results from activation of DCC receptor and DCC-UNC5 receptor complex for attraction and repulsion, respectively. The DCC-mediated attraction requires a high level of intracellular Ca2+ and cAMP-dependent signaling. Conversely, DCC-UNC5-mediated repulsion requires a low level of intracellular Ca2+ and both cAMP and cGMP signaling. The high level of intracellular Ca2+ during attraction is mainly regulated by activation of L-type Ca2+ channels in the plasma membrane and inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodme receptors (RyRs) in internal stores. Inactivation of either L-type Ca channels or RyRs reduces the intracellular Ca2+ elevation resulting in repulsion. On the other hand, inactivation of IP3Rs or combined L-type Ca2+ channels and RyRs results in a loss of netrin-1-induced turning. As our major goal to understand the role of guidance signal regulation in establishing functional neural connections during nervous system development, we propose to determine the molecular and cellular mechanisms of cAMP/cGMP and Ca2+-dependent signals and the means by which these two signals converge during netrin-1-induced growth cone response.Using combined approaches of quantitative analysis of growth cone behavior at a single cell level, detecting Ca2+ dynamics in real time and space with high resolution using a disk scanner confocal imaging system, and monitoring the properties of Ca2+ channels by electrophysiologic recordings in growth cones, our specific aims of the proposed research are as follows: 1) To determine the regulation of Ca2+ entry in response to netrin- 1 signaling; 2) To determine the regulation of Ca2+ release via internal Ca2+ stores during netrin- 1 signaling; 3) To determine the functional coupling mechanisms of Ca2+ channels between the plasma membrane and endoplasmic reticulum induced by netrin receptor activation; 4) To determine the interaction between cAMP/cGMP and Ca2+-dependent signaling induced by netrin-1 signaling. The proposed studies implement a unique approach to elucidate the cellular and molecular transduction events underlying guidance molecule triggered second messenger signaling. The results will contribute not only to a better understanding of the molecular basis of neural development, but also provide insights into potential therapeutic applications in promoting post-injury nerve regeneration.

描述（由申请人提供）：细胞内 Ca2+ 和环核苷酸依赖性信号传导都会影响响应 netrin-1 梯度（非洲爪蟾脊髓神经元中的可扩散引导分子）的神经生长锥延伸的速率和方向。 Netrin-1 是一种在发育中的脊椎动物神经系统中线高度表达的分泌蛋白，在引导轴突到达目标细胞方面既充当引诱剂又充当驱避剂。 netrin-1 的双功能作用是由于 DCC 受体和 DCC-UNC5 受体复合物的激活而分别产生吸引和排斥作用。 DCC 介导的吸引力需要高水平的细胞内 Ca2+ 和 cAMP 依赖性信号传导。相反，DCC-UNC5 介导的排斥需要低水平的细胞内 Ca2+ 以及 cAMP 和 cGMP 信号传导。吸引过程中细胞内高水平的 Ca2+ 主要通过质膜中 L 型 Ca2+ 通道以及内部储存中的肌醇 1,4,5-三磷酸受体 (IP3Rs) 和 ryanodme 受体 (RyRs) 的激活来调节。 L 型 Ca 通道或 RyR 的失活会降低细胞内 Ca2+ 的升高，从而导致排斥。另一方面，IP3Rs 或组合的 L 型 Ca2+ 通道和 RyRs 的失活会导致 netrin-1 诱导的转向丧失。作为我们了解引导信号调节在神经系统发育过程中建立功能性神经连接中的作用的主要目标，我们建议确定 cAMP/cGMP 和 Ca2+ 依赖性信号的分子和细胞机制，以及这两种信号在 netrin-1 诱导的生长锥反应期间汇聚的方式。采用单细胞水平生长锥行为定量分析的组合方法，实时和空间检测 Ca2+ 动态 使用圆盘扫描仪共焦成像系统进行高分辨率，并通过生长锥中的电生理记录监测 Ca2+ 通道的特性，我们拟议研究的具体目标如下： 1) 确定 Ca2+ 进入对 netrin-1 信号传导的调节； 2) 确定 netrin-1 信号传导期间通过内部 Ca2+ 储存对 Ca2+ 释放的调节； 3）确定netrin受体激活诱导的质膜与内质网之间Ca2+通道的功能耦合机制； 4) 确定cAMP/cGMP 与netrin-1 信号传导诱导的Ca2+ 依赖性信号传导之间的相互作用。拟议的研究采用了一种独特的方法来阐明引导分子触发第二信使信号传导的细胞和分子转导事件。这些结果不仅有助于更好地理解神经发育的分子基础，而且还为促进损伤后神经再生的潜在治疗应用提供了见解。