Signaling mechanisms that regulate cytoskeletal organization Downstream of Netrin-1 mediated axonal chemoattraction

The development of the nervous system is highly dependent on the differentiation of various neuronal and glial cell populations, efficient targeting of axons, establishment of neuronal connections and continuous branching/pruning and establishment of new connections. These processes are possible through migration, adhesion and cytoskeletal rearrangement processes that are common in most organogenesis. The extracellular matrix and guidance molecules interact with cell surface receptors that transduce signaling mechanisms intracellularly allowing for these cellular responses to take place.Netrins are bifunctional chemotropic cues that attract or repel different classes of axons by signaling through the netrin receptors Deleted in Colorectal Cancer (DCC) and the UNC5 homologues (UNC5a, b, c and d) during the development of the nervous system. This family of chemotropic molecules is a member of the laminin superfamily. Similar to laminin-integrin signaling, netrin-1's interaction with its receptor DCC results in the activation of the Rho GTPases Rac and Cdc42 and the promotion of a signaling cascade that leads to growth cone cytoskeletal and membrane remodeling. This thesis focused on further identifying the signaling molecules and complexes downstream of DCC that are required for netrin-1 mediated commissural neuron axon chemoattraction.In the first part of this thesis we have used an unbiased mass spectrometry approach to identify signaling molecules complexed to DCC in commissural neurons isolated from E12/13 spinal cord. From the mass spectrometry data obtained we chose to characterize Arp2/3 and 14-3-3. Arp2/3 co-immunoprecipitated with DCC in commissural neuron in a netrin-1-dependent manner. We further established that netrin-1 mediated commissural growth cone expansion and filopodia remodeling is blocked by wiskostatin, a potent chemical inhibitor of N-WASP activity towards the Arp2/3 complex. The treatment of the neurons with wiskostatin also inhibited netrin-dependent externalization of DCC. These results suggest that actin polymerization resulting from N-WASP/Arp2/3 complexes may be required to maintain DCC cell surface recycling during netrin-1 mediated axon guidance. The 14-3-3 epsilon adaptor protein was also identified as a DCC associated protein. However, its association with DCC decreased with the addition of netrin-1. The inhibition of 14-3-3 function using a function-blocking peptide resulted in the collapse of commissural growth cones which could not be recovered by the addition of netrin-1. In the second part of this thesis we identified β-Pix as a potential GEF downstream of DCC involved in the activation and propagation of Rac-1 and Cdc42 signaling. We found that a β-Pix/Git complex associates with DCC in commissural neurons. β-Pix is highly expressed in the developing spinal cord in various neuronal subpopulations. Additionally functional assays using the overexpression of β-Pix mutants showed that β-Pix complexes and association with Pak are important steps in the maintenance of netrin-1 induced changes in GC morphology, in commissural axon extension to the midline, and for cortical neuron branching. Using modified GTPase assays we further demonstrated that β-Pix is required for Cdc42 and Rac-1 activation downstream of netrin-1 and is associated with the GTP-bound GTPases. β-Pix may associate with the GTPases by binding to Pak, or alternatively, may bind directly, as has been demonstrated for the related protein α-Pix, resulting in allosteric activation of the GTPase. These results support a novel model, in which β-Pix/Git complexes play an integral part in the activation of Rac-1 and Cdc42 downstream of DCC.These findings provide novel insight into the signaling events activated by netrin-1 downstream of DCC during commissural axon guidance. They have also identified new questions related to how signal transduction mechanisms activated by netrin-1 regulate the cytoskeleton of the axonal growth cone.

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