EICOSANOID IN GROWTH CONE REPELLENT SIGNALING

该计划的主要目标是研究轴突在发育和再生过程中的寻路机制。 特别感兴趣的是将化学排斥剂对生长锥的作用转化为崩溃的信号传导。 我们的数据表明，12/15-脂氧合酶产生的类花生酸是必要的和足够的（至少部分）脑信号蛋白3A诱导的崩溃。 其他结果表明，这些类花生酸的合成是级联反应的一部分，涉及上游的胞质磷脂酶A2（cPLA/2）和下游的蛋白激酶C（PKC）和粘附位点蛋白，其磷酸化可能会触发粘附位点的解体和分离。在一系列的细胞生物学研究中，我们将测试这一假设，即该途径调节脱落的生长锥周边在崩溃。实验将集中在semaphorin 3A的影响。 在反应神经元的培养中，我们将研究生长结构和粘附。 在分离的生长锥制剂中富含脑信号蛋白3A受体，neurophilin-1，我们将分析cPLA 2激活，类花生酸合成和PKC刺激，以及粘附位点蛋白的磷酸化。这些研究预计（i）提供新的见解，很少了解信号机制激活的驱避剂和（ii）建立一个新的信号通路，被认为是触发拆卸的生长锥粘附网站。 生长锥寻路和排斥机制的分析是我们理解神经系统发育的基础，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.