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轴突寻径的机制。特别令人感兴趣的是将化学防腐剂对生长锥体的作用转化为坍塌的信号。我们的数据表明，12/15-脂氧合酶产生二十烷类化合物对于信号素3A诱导的崩溃是必要的和充分的(至少部分)。其他结果表明，这些二十烷类化合物的合成是一个级联反应的一部分，上游涉及胞浆磷脂酶A2(CPLA/2)，下游涉及蛋白激酶C(PKC)和黏附位点蛋白，它们的磷酸化可能引发黏附位点的解体和脱落。在一系列细胞生物学研究中，我们将验证这一途径调节崩溃过程中生长锥体外周脱离的假设。实验将集中在信号素3A的作用上。在反应性神经元的培养中，我们将研究生长、结构和黏附。在分离的富含信号素3A受体神经亲和素-1的生长锥样品中，我们将分析cPLA2的激活、二十烷类化合物的合成和PKC的刺激，以及粘附点蛋白的磷酸化。这些研究有望(I)为驱避剂激活的鲜为人知的信号机制提供新的见解，以及(Ii)建立一种新的信号通路，据信可引发生长锥粘附点的分解。对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，深入了解化学反应物对生长锥的作用可能为促进成年中枢神经系统的神经再生提供新的选择。

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.