Netrin-1 signaling : cellular consequences and molecular mechanisms

Abstract

During the embryonic development of the nervous System, groups of multipotent cells proliferate, migrate, and differentiate to become neurons. The new born neurons need to make precise connections with their targets. These may be a great distance away from their soma. To make these connections, neurons send their axon through a biochemically complex environment and their growth cones may make many guidance decisions on the ways to their targets. The existence of axon guidance molecules was inferred by the early classical neuroanatomists. We now know of multiple examples of secreted or membrane-bound proteins that either attract or repel axonal growth cônes by causing the growth cône to collapse or extend. The molecular mechanisms that regulate growth cone collapse or extension are closely linked to reorganization of the cytoskeleton. The small Rho GTPases, Cdc42, Rac, and Rho play key roles in neuronal growth cônes by regulating the organization of the cytoskeleton. Netrins are a small family of secreted guidance eues that are implicated in the attraction and repulsion of axons during the development of the nervous System. The netrin-1 receptor deleted in colorectal cancer (DCC) is highly expressed by commissural neurons in the developing spinal cord. The findings described here show that DCC is present at the tips of filopodia and the edges of lamellipodia of HEK293T, NG108-15 neuroblastoma-glioma cells, and the growth cones of embryonic rat spinal commissural neurons. Furthermore, netrin-1 protein causes an increase in filopodia number and surface area of embryonic rat commissural neuron growth cônes and the cell fines when transfected to express DCC. Further experiments indicated that netrin-1 activates the small GTPases Cdc42 and Racl in the cell lines and the embryonic rat commissural spinal cord neurons. The activation of Cdc42 and Racl by netrin-1 requires DCC. Furthermore, netrin-1 causes increased phosphorylation of Pakl, an effector for both Cdc42 and Racl. Expression of a dominant negative form of N-WASP, an effector for Cdc42, blocks the netrin-1 induced morphological changes in the growth cones of commissural neurons. Evidence is presented that netrin-1 induces the formation of a complex of proteins interacting with the intra-cellular domain of DCC that includes Nckl, Cdc42, Racl, Pakl, and N-WASP. The findings described lead to a model whereby netrin-1 binding to DCC triggers the activation of Cdc42 and Racl, which leads to actin based membrane extension and changes in growth cone morphology