Molecular Mechanism of Axon Guidance by Second Messenger

描述(由申请人提供)：细胞内钙和环核苷酸依赖的信号都影响神经生长锥延伸的速度和方向，以响应Netrin-1梯度，Netrin-1梯度是非洲爪哇脊髓神经元中的一种可扩散的指导分子。Netrin-1是一种分泌蛋白，在脊椎动物发育中的神经系统中线高度表达，在引导轴突到达靶细胞时既是吸引剂，也是排斥剂。Netrin-1的双功能作用是通过分别激活DCC受体和DCC-uc5受体复合体来实现的。DCC介导的吸引力需要高水平的细胞内钙和cAMP依赖的信号转导。相反，DCC-unc5介导的排斥需要低水平的细胞内钙以及cAMP和cGMP信号。在吸引过程中，细胞内高水平的钙主要由质膜上L类钙通道和内库中的肌醇1，4，5-三磷酸受体(IP3Rs)和兰诺受体(RyRs)调节。L型钙通道或RyRs的失活可减少细胞内钙升高，从而导致斥力。另一方面，IP3Rs或L钙通道和RyRs结合的失活导致Netrin-1诱导的转折丧失。为了了解引导信号调节在神经系统发育过程中建立功能神经联系中的作用，我们提出了确定cAMP/cGMP和钙依赖信号的分子和细胞机制以及这两种信号在netrin-1诱导的生长锥反应中汇聚的方法。采用在单细胞水平上对生长锥行为进行定量分析的组合方法，使用圆盘扫描仪共聚焦成像系统以高分辨率实时和空间检测钙动态，并通过电生理记录监测生长锥中钙通道的特性，本研究的具体目的如下：1)研究Netrin-1信号对钙离子内流的调节作用；2)研究Netrin-1信号转导过程中细胞内钙离子释放的调控；3)研究Netrin受体激活引起的质膜和内质网钙通道的功能偶联机制；4)研究Netrin-1信号诱导的cAMP/cGMP与钙依赖信号之间的相互作用。建议的研究实现了一个独特的方法来阐明潜在的引导分子触发第二信使信号的细胞和分子转导事件。这些结果不仅有助于更好地了解神经发育的分子基础，还将为促进损伤后神经再生的潜在治疗应用提供见解。

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.