Adaptation Mechanisms in Long-range Growth Cone Guidance

描述(申请人提供)：在发育过程中复杂的神经网络形成是通过正确寻找生长轴突尖端的路径来实现的，生长锥体响应指导线索。存在于环境中的可扩散和底物结合的分子可以形成梯度，并作为远程化学吸引剂或化学抵抗剂来引导生长锥体到达它们的目标。在过去的二十年里，已经通过遗传和生化方法鉴定了几个家族的导向分子和它们的受体。然而，定向生长锥运动背后的信号转导和调控机制才刚刚开始阐明。我们的长期目标是了解决定发育和再生轴突的运动性和方向性的分子和细胞机制，以响应指导信号和抑制分子，并开发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迹的再生。生长锥体指导说明了单个细胞如何与其环境相互作用的复杂性。神经元生长锥体可以通过检测到环境中存在的非常浅的分子梯度，以高度定向的极性和运动性对化学吸引剂的来源做出反应，分子梯度可以低至1%。然而，生长锥在向上移动引导线索的梯度时也可以保持其灵敏度，引导线索的平均浓度可以跨越几个数量级。因此，生长锥必须获得一种时间机制，以便在迁徙过程中重置生长锥的敏感性，这涉及到适应。我们开发了一种定量分析方法来分析生长锥体在确定的引导线索梯度中的导向决策。对培养的非洲爪哇脊髓神经元使用这种方法，我们已经证明，当向上迁移Netrin-1的梯度时，这些神经元的生长锥体呈现出脱敏(反应丧失)和再敏化(适应)的连续阶段。Netrin-1是一种进化上保守的发育指导分子，对许多生物体的神经回路形成至关重要。在这项建议中，我们的目标是研究对netrin-1的适应性生长锥反应的分子机制。具体地说，我们将结合生物化学、免疫细胞化学、钙离子成像和生长锥体翻转试验，重点研究与结肠直肠癌(DCC)中缺失相关的直接信号机制，DCC是一种具有良好特性的Netrin-1膜受体。

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