Molecular Mechanism of Axon Guidance by Second Messenger

描述（由申请人提供）：细胞内Ca 2+和环核苷酸依赖性信号传导均影响神经生长锥延伸的速率和方向，以响应netrin-1梯度，netrin-1是非洲爪蟾脊髓神经元中的一种可扩散导向分子。Netrin-1是一种在发育中的脊椎动物神经系统的中线高度表达的分泌蛋白，其在引导轴突至其靶细胞中充当引诱剂和排斥剂。netrin- 1的双功能作用是由DCC受体和DCC-UNC 5受体复合物分别激活吸引和排斥引起的。DCC介导的吸引需要高水平的细胞内Ca 2+和cAMP依赖性信号传导。相反，DCC-UNC 5介导的排斥需要低水平的细胞内Ca 2+以及cAMP和cGMP信号传导。吸引过程中细胞内高水平的Ca ~（2+）主要通过激活质膜上的L型Ca ~（2+）通道和内部储存的肌醇1，4，5-三磷酸受体（IP 3Rs）和ryanodme受体（RyRs）来调节。L型Ca通道或RyR的失活降低了细胞内Ca 2+的升高，导致排斥。另一方面，IP 3Rs或组合的L型Ca 2+通道和RyRs的失活导致netrin-1诱导的转向的损失。作为我们的主要目标，了解在神经系统发育过程中引导信号调节在建立功能性神经连接中的作用，我们建议确定cAMP/cGMP和Ca 2+依赖性信号的分子和细胞机制以及这两种信号在netrin-1诱导的生长锥反应中会聚的方式。本研究利用共聚焦成像系统高分辨率地检测细胞内钙离子在真实的时空动态变化，并通过电生理记录技术监测细胞生长锥内钙离子通道的特性，具体目的如下：1）研究netrin- 1信号对细胞内钙离子内流的调控作用; 2）确定netrin- 1信号转导过程中细胞内Ca ~（2+）库对Ca ~（2+）释放的调节作用：3）确定netrin受体激活诱导的质膜和内质网间Ca ~（2+）通道的功能偶联机制; 4）研究netrin-1信号转导通路诱导的cAMP/cGMP与Ca ~（2+）依赖性信号转导的相互作用。所提出的研究实施了一种独特的方法来阐明指导分子触发的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。

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.