Mechanisms of Axonal Pathfinding Controlled by Netrin-1

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During development, molecules in the immature brain steer axons to their target areas, following long and complex pathways. Proper pathfinding is required for the establishment of correct synaptic connections, and disturbances in the intricately regulated process of axonal pathfinding are thought to cause a variety of neurodevelopmental disorders. Netrin-1 is a secreted, attractive axon guidance cue that is crucial for the proper formation of the nervous system. The mechanisms by which netrin-1 mediates its axon outgrowth promoting effects are largely unknown, and the pathways identified thus far seem incongruent: cAMP/PKA signaling, intra-axonal protein synthesis and degradation, and regulation of the activity of Rho family GTPases, proteins with l<ey roles in the regulation of the axonal cytoskeleton. The goal of this application is to understand how these disparate signaling mechanisms are integrated to result in the characteristic axonal outgrowth and growth cone turning response that characterize netrin-1 signaling. Based on preliminary studies, I propose that netrin-1 regulates axon growth and pathfinding through a signaling mechanism that requires the formation of a signaling complex consisting of PARS, PAR6, cdc42, and aPKC^. In this complex, PAR6 recruits the ubiquitin E3 ligase Smurfl leading to degradation of RhoA. I further hypothesize that the formation of this complex is triggered by the local translation of PARS rather than recruitment of PARS from the cell body. The specific aims of this proposal are designed to validate this model and to determine the importance of local PARS translation in an in vivo model. As part of a long-term effort to understand how molecules such as netrin-1 regulate axonal outgrowth and pathfinding, the aims of this proposal are: (I) To demonstrate the role of Smurfl-dependent ubiquitination of RhoA in netrin-1 signaling, (II) to establish the role of local translation of PARS in netrin-1 signaling, and (III) to genetically dissect the role of axonally localized PARS mRNA in axon pathfinding in vivo. The results obtained from these experiments will create the foundation for a novel and unified framework for understanding netrin-1 signaling.