Molecular Mechanism of Axon Guidance by Second Messenger

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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.