TRIM9 Controls Growth Cone Responses to Netrin Through DCC and UNC5C

The guidance cue netrin-1 promotes both growth cone attraction and growth cone repulsion. How netrin-1 elicits diverse axonal responses, beyond engaging the netrin receptor DCC and UNC5 family members, remains elusive. Here, we demonstrate that murine netrin-1 induces biphasic axonal responses in cortical neurons: Attraction at lower concentrations and repulsion at higher concentrations using both a microfluidic-based netrin-1 gradient and bath application of netrin-1. We find that repulsive turning in a netrin gradient is blocked by knockdown of UNC5C, whereas attractive turning is impaired by knockdown of DCC. TRIM9 is a brain-enriched E3 ubiquitin ligase previously shown to bind and cluster the attractive receptor DCC at the plasma membrane and regulate netrin-dependent attractive responses. However, whether TRIM9 also regulated repulsive responses to netrin-1 remained to be seen. In this study, we show that TRIM9 localizes and interacts with both the attractive netrin receptor DCC and the repulsive netrin receptor, UNC5C. We find that deletion of murine Trim9 alters both attractive and repulsive axon turning and changes in growth cones size in response to murine netrin-1. TRIM9 was required for netrin-1-dependent changes in the surface levels of DCC and UNC5C in the growth cone during morphogenesis. We demonstrate that DCC at the membrane regulates the growth cone area and show that TRIM9 negatively regulates FAK activity in the absence of both repulsive and attractive concentrations of netrin-1. Together, our work demonstrates that TRIM9 interacts with and regulates both DCC and UNC5C during attractive and repulsive axonal responses to netrin-1.