Molecular Mechanism of Axon Guidance by Second Messenger

描述（由申请人提供）：细胞内Ca2+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，这是非洲爪蟾脊髓神经元中的一种可扩散的引导分子。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的过程中起引诱剂和驱避剂的作用。netrin- 1的双重功能作用源于分别激活DCC受体和DCC- unc5受体复合物的吸引和排斥作用。dcc介导的吸引需要高水平的细胞内Ca2+和camp依赖性信号。相反，dcc - unc5介导的斥力需要低水平的细胞内Ca2+和cAMP和cGMP信号。吸引过程中细胞内的高水平Ca2+主要通过激活质膜上的l型Ca2+通道和内部储存中的肌醇1,4,5-三磷酸受体（IP3Rs）和色胺受体（RyRs）来调节。无论是l型钙通道还是ryr的失活都会降低细胞内Ca2+的升高，从而导致排斥。另一方面，IP3Rs或组合l型Ca2+通道和RyRs失活导致netrin-1诱导的转向丧失。我们的主要目标是了解引导信号调控在神经系统发育过程中建立功能性神经连接中的作用，我们建议确定cAMP/cGMP和Ca2+依赖信号的分子和细胞机制，以及这两个信号在netrin-1诱导的生长锥反应中收敛的方式。采用单细胞水平的生长锥行为定量分析，利用磁盘扫描仪共聚焦成像系统实时和高分辨率地检测Ca2+动态，并通过生长锥内的电生理记录监测Ca2+通道的特性，我们提出的研究的具体目的如下：1)确定响应netrin- 1信号的Ca2+进入调节；2)在netrin- 1信号传导过程中，通过内部Ca2+储存确定Ca2+释放的调控；3)确定网蛋白受体激活诱导质膜与内质网Ca2+通道的功能偶联机制；4)确定cAMP/cGMP与netrin-1信号诱导的Ca2+依赖性信号的相互作用。本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。

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.