The Coordination of Netrin Signal Transduction via TUBB3 and JNK1 in Axon Guidance

Microtubule dynamics plays an important role in axon guidance, but much still remains to be learned about the specifics of the interaction. TUBB3 is a neuronal specific microtubule subunit and TUBB3 mutations perturb microtubule dynamics and axon pathfinding, suggesting that TUBB3 may be required for axon guidance in the developing nervous system. Here, we report that DCC, a receptor of netrin-1, interacted directly to TUBB3 both in vitro and in vivo. Importantly, netrin-1 increased TUBB3/DCC interactions and the inhibition of Src family kinase or disruption of microtubule dynamics abolished these interactions. TUBB3 siRNA or shRNA reduced netrin-1-induced axon outgrowth in both E3 chicken dorsal spinal cord explants and E15 mouse cortical neurons and chick commissural axon turning in vitro and disrupted commissural axon projection in vivo. These results suggest that netrin-1 mediated signaling through DCC is linked directly to microtubule dynamics and that this link may play an essential role in netrin-mediated axon guidance. Microtubule dynamics is regulated by microtubule associated proteins (MAPs) and the JNK family of MAPKs, one of the major regulators of MAPs, plays a key role in neural survival, migration, and polarity as well as axon regeneration. However, no role for JNK activation in netrin-1 signaling had been identified previously. Here we show that JNK1is required for netrin signaling in the developing nervous system. Netrin-1 acting through either DCC or DSCAM increased JNK1 activity in both HEK cells and primary neurons. DCC and DSCAM co-expression further enhanced netrin-1-induced JNK1 activity in vitro. In contrast, netrin-1 had no effect on JNK2 or JNK3 activity. Phospho-JNK is strongly expressed in commissural axons before and as they crossed the floor plate, and netrin-1 stimulation dramatically increased the level of endogenous phospho-JNK in commissural axon growth cones. As predicted, inhibition of JNK signaling either by JNK1 RNA interference (RNAi) or the JNK inhibitor suppressed netrin-1-induced neurite outgrowth and axon attraction and JNK1 knockdown in ovo interfered with spinal cord commissural axon projection and pathfinding. Together, these results suggest that JNK1 may be involved in the coordination of DCC and DSCAM in netrin-1-mediated attraction.