Adaptation Mechanisms in Long-range Growth Cone Guidance

描述（由申请人提供）：在发育过程中，复杂的神经网络的形成是通过对生长轴突的尖端（生长锥）的正确寻路来实现的，以响应指导线索。环境中存在的扩散分子和底物结合分子可以形成梯度，并作为远程化学引诱剂或化学驱避剂，引导生长锥到达其目标。在过去的二十年中，通过遗传和生物化学方法已经确定了几个引导分子家族及其受体。然而，定向生长锥运动的信号转导和调节机制才刚刚开始被阐明。我们的长期目标是了解决定轴突发育和再生的运动和方向性的分子和细胞机制，以响应指导线索和抑制分子，并制定治疗策略，以促进人类中枢神经系统损伤或疾病后特定轴突轨道的再生。生长锥引导说明了单个细胞如何与其环境相互作用的复杂性。神经元生长锥可以通过检测环境中极浅的分子梯度（低至1%），对化学引诱物的来源做出高度定向的极性和运动反应。然而，当一个平均浓度可以跨越几个数量级的引导线索梯度迁移时，生长锥也可以保持其灵敏度。因此，生长锥必须获得一种时间机制，可以在迁移期间重置生长锥的敏感性，这涉及适应。我们已经开发了一种定量分析方法来分析生长锥在一个确定的梯度引导线索中的转向决策。利用这种方法培养非洲爪蟾脊髓神经元，我们发现这些神经元的生长锥在向netrin-1梯度迁移时表现出连续的脱敏（反应丧失）和重敏（适应）阶段，netrin-1是许多生物体中神经回路形成所必需的进化保守的发育指导分子。在这篇论文中，我们的目的是研究生长锥对netrin-1的适应性反应的分子机制。具体而言，我们将重点研究与大肠癌（DCC）相关的即时信号机制，这是一种具有良好特征的netrin-1膜受体，结合生物化学，免疫细胞化学，Ca2+成像和生长锥转向试验

DESCRIPTION (provided by applicant): The intricate neural network formation during development is achieved by correct pathfinding of the tip of a growing axon, the growth cone, in response to guidance cues. Diffusible and substrate bound molecules presented in the environment can form gradients and function as long-range chemoattractants or chemorepellents to guide the growth cones to their targets. Over the last two decades, several families of guidance molecules and their receptors have been identified by genetic and biochemical approaches. The signal transduction and modulation mechanisms underlying the directed growth cone motility, however, have just begun to be elucidated. Our long-term goal is to understand the molecular and cellular mechanisms that determine the motility and directionality of developing and regenerating axons in response to guidance cues as well as inhibitory molecules and to develop therapeutic strategies to promote regeneration of specific axonal tracks after injury or diseases of human CNS. Growth cone guidance exemplifies the complexity of how a single cell interacts with its environment. The neuronal growth cones can respond with highly oriented polarity and motility toward the source of chemoattractant based on detecting a very shallow molecular gradient presented in the environment, which can be as low as 1%. However, a growth cone can also maintain its sensitivity when migrating up a gradient of guidance cue, the average concentration of which can span several orders of magnitude. A temporal mechanism must therefore be acquired by the growth cone that can reset the sensitivity of the growth cone during migration, which involves adaptation. We have developed a quantitative assay to analyze the steering decision of growth cones in a defined gradient of guidance cues. Using this approach with Xenopus spinal neurons in cultures, we have shown that the growth cones of these neurons exhibit consecutive phases of desensitization (loss of response) and resensitization (adaptation) when migrating up a gradient of netrin-1, an evolutionally conserved developmental guidance molecule essential for neural circuit formation in many organisms. In this proposal, we aim to investigate the molecular mechanisms underlying the adaptive growth cone responses to netrin-1. Specifically, we will focus on examining immediate signaling mechanisms associated with Deleted in Colorectal Cancer (DCC), a well characterized membrane receptors for netrin-1, with a combination of biochemistry, immunocytochemistry, Ca2+ imaging and growth cone turning assays