Diffusible or substrate-bound guidance molecules are responsible for guiding the growth of axons towards their target cells in the developing nervous system. Several families of guidance molecules and their receptors have been identified, but the signaling mechanisms in the neuronal cytoplasm are largely unknown. In this project, we propose to carry out in vitro studies of signal transduction events underlying the turning response of the growth cone of cultured Xenopus spinal neurons induced by microscopic gradients of three guidance molecules: netrin-1, brain-derived neurotrophic factor (BDNF), and a secreted form of myelin-associated glycoprotein (MAG). In part I, we will carry out quantitative analysis of the turning responses of the growth cone in gradients of these guidance molecules and the modulatory actions of extracellular matrix components and electrical activity. We will also examine the hypothesis that similar cellular mechanisms are used by the growth cone to detect gradients of guidance cues in either diffusible or substrate-bound form. In part II, we will examine the role of calcium and cAMP gradients within the growth cone cytoplasm in mediating the growth cone turning responses, and determine the relationship between calcium and cAMP signals induced by these guidance molecules. In part III, we will examine various stages of cytoplasmic signaling from receptor activation to cytoskeleton rearrangement. We will study the mechanisms linking receptor activation to cytoplasmic signaling, the role of the Rho-family of small GTPases, filopodial activity and the dynamics of actin filaments and microtubules during the early phase of the turning response. In part IV, we will characterize the phenomenon of adaptation and resensitization of the growth cone response during its pathfinding towards a source of netrin-1 or BDNF and examine potential underlying mechanisms. These studies offer a unique opportunity in elucidating the cellular transduction events underlying the action of three defined guidance molecules. The results will contribute to our understanding of the normal development of the nervous system and provide insights into potential therapeutic approaches in promoting nerve regeneration after injury.
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