Adaptation Mechanisms in Long-range Growth Cone Guidance

描述（由申请人提供）：发育期间复杂的神经网络形成是通过响应于引导线索对生长轴突（生长锥）的尖端进行正确寻路来实现的。在环境中存在的可扩散的和底物结合的分子可以形成梯度，并作为长程化学引诱剂或化学排斥剂发挥作用，以引导生长锥到达它们的靶点。在过去的二十年中，已经通过遗传和生物化学方法鉴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阐明。我们的长期目标是了解决定轴突发育和再生的运动性和方向性的分子和细胞机制，以响应引导线索以及抑制分子，并开发治疗策略，以促进人类CNS损伤或疾病后特定轴突轨迹的再生。生长锥的引导阐明了单个细胞如何与其环境相互作用的复杂性。神经元生长锥可以基于检测到环境中存在的非常浅的分子梯度（其可以低至1%）以高度定向的极性和朝向化学引诱物的来源的运动性来响应。然而，生长锥也可以保持其灵敏度时，迁移梯度的指导线索，其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，这种机制可以在迁移过程中重置生长锥的敏感性，这涉及到适应。我们已经开发了一种定量分析，以分析在一个定义的梯度的指导线索的生长锥的转向决策。使用这种方法与非洲爪蟾脊髓神经元在文化中，我们已经表明，这些神经元的生长锥表现出连续的阶段脱敏（响应损失）和重新敏化（适应）时，迁移的netrin-1，进化保守的发展指导分子在许多生物体中的神经回路形成的梯度。在这个建议中，我们的目标是调查的分子机制的适应性生长锥netrin-1的反应。具体而言，我们将集中研究与结直肠癌（DCC）相关的直接信号传导机制，DCC是netrin-1的一种良好表征的膜受体，结合生物化学，免疫细胞化学，Ca 2+成像和生长锥旋转测定

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