The characterization of signaling molecules involved in axonal guidance

During development, an axon encounters both attractive and repulsive guidance cues in the extracellular environment that act as "signposts" to direct the axon to its proper target. Precise axon pathfinding requires highly coordinated regulation of the cytoskeleton within the growth cone. This dynamic regulation manifests itself in axonal branching, turning, and pathfinding, presynaptic differentiation, and growth cone collapse and extension. Semaphorin 3A (Sema3A), a secreted guidance cue that primarily functions to repel axons from inappropriate targets, induces cytoskeletal rearrangements that result in growth cone collapse. These effects require intra-axonal messenger RNA translation. In chapter three, we show that transcripts for RhoA, a small guanosine triphosphatase (GTPase) that regulates the actin cytoskeleton, are localized to developing axons and growth cones, and this localization is mediated by an axonal targeting element located in the RhoA 3' untranslated region (UTR). Sema3A induces intra-axonal translation of RhoA mRNA and this local translation of RhoA is necessary and sufficient for Sema3A-mediated growth cone collapse. These studies indicate that local RhoA translation regulates the neuronal cytoskeleton and identify a new mechanism for the regulation of RhoA signaling.; The opposite side of Sema3A-mediated axon repulsion is netrin-1-mediated axon outgrowth. Axon outgrowth is essential for neuronal development and morphogenesis as well as for regeneration following axonal injury. The effects of netrin-1, a secreted molecule that regulates growth cone morphology and axonal outgrowth, depend upon the intracellular levels of cyclic AMP (cAMP). The specific pathways that regulate cAMP levels in developing axons and in response to netrin-1 signaling are unclear. In chapter four, we show that 'soluble' adenylyl cyclase (sAC), a noncanonical, calcium-regulated cAMP generating enzyme previously implicated in sperm maturation, is expressed in neurons and in developing axons and is the source of cAMP generated in response to netrin-1. Overexpression of sAC results in axonal outgrowth and increased growth cone elaboration, as measured by filopodial formation and growth cone size; while inhibition of sAC blocks netrin-1-induced axon outgrowth and growth cone elaboration. Taken together, these results indicate that netrin-1 signals through sAC generated cAMP, and identify a fundamental role for sAC in axonal development.