EICOSANOID IN GROWTH CONE REPELLENT SIGNALING

该计划的主要目标是研究在发育和再生过程中涉及轴突寻径的机制。特别感兴趣的是将化学驱虫剂在生长锥上的作用转化为崩溃的信号。我们的数据表明，由12/15-脂氧合酶产生的类二十烷化合物对于信号蛋白3a诱导的崩溃是必要和充分的（至少部分）。其他研究结果表明，这些类二十烷醇的合成是级联反应的一部分，上游是胞质磷脂酶A2 (cPLA/2)，下游是蛋白激酶C （PKC）和粘附位点蛋白，它们的磷酸化可能引发粘附位点的分解和脱离。在一系列的细胞生物学研究中，我们将测试这一途径在塌陷过程中调节生长锥外围的脱离的假设。实验将重点关注信号蛋白3A的作用。在反应神经元的培养中，我们将研究其生长结构和粘附性。在富集信号蛋白3A受体neurophilin-1的分离生长锥制剂中，我们将分析cPLA2的激活、类20蛋白合成和PKC的刺激，以及粘附位点蛋白的磷酸化。这些研究有望(i)为驱蚊剂激活的鲜为人知的信号机制提供新的见解，（ii）建立一种新的信号通路，被认为可以触发生长锥粘附位点的分解。对生长锥寻径和排斥机制的分析是我们理解神经系统发育的基础，而深入了解化学排斥剂对生长锥的作用可能会揭示促进成人中枢神经系统神经再生的新选择。

The broad goals of the proposed program are to study mechanisms involved in axonal pathfinding during development and regeneration. Of specific interest is the signaling that translates a chemorepellent's action on the growth cone into collapse. Our data indicate that generation of eicosanoids by 12/15-lipoxygenase is necessary and sufficient (at least partially) for semaphorin 3A-induced collapse. Other results suggest that synthesis of these eicosanoids is part of a cascade involving, upstream, cytosolic phospholipases A2 (cPLA/2) and, downstream, protein kinase C (PKC) and adhesion site proteins, whose phosphorylation may trigger adhesion site disassembly and detachment. In a series of cell biological studies we will test the hypothesis that this pathway regulates detachment of the growth cone periphery during collapse. Experiments will be focused on the effects of semaphorin 3A. In cultures of responsive neurons we will study growth structure and adhesion. In isolated growth cone preparations enriched in the semaphorin 3A receptor, neurophilin-1, we will analyze cPLA2 activation, eicosanoid synthesis and PKC stimulation, as well as phosphorylation of adhesion site proteins. These studies are expected (i) to provide new insights into the little understood signalling mechanisms activated by repellants and (ii) to establish a novel signalling pathway that is believed to trigger disassembly of growth cone adhesion sites. The analysis of the mechanisms of growth cone pathfinding and repulsion is fundamental to our understanding of nervous system development, and insight into the action of chemorepellants on growth cones is likely to reveal new options for promoting nerve regeneration in the adult CNS.