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
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