Signalling mechanisms underlying Trk function in neonatal sympathetic neurons

NGF acting through the TrkA tyrosine kinase receptor promotes survival and axon growth of developing sympathetic neurons in vivo and in vitro. This thesis examines the signalling pathways and proteins used by Trk receptors to mediate neuronal survival, local axon growth, and growth cone regulation in neonatal rat sympathetic neurons of the superior cervical ganglion (SCG).First, I examined the involvement of key signalling motifs on the Trk receptor in mediating neurotrophin-dependent survival and local axon growth. Using recombinant adenoviruses, I expressed the BDNF receptor TrkB, which is not endogenously expressed in SCG neurons, either in wild-type form or mutated at defined effector binding sites. Ectopically expressed wild-type TrkB activated signalling pathways similarly to endogenous TrkA, and supported both neuronal survival and local axon growth in compartmented Campenot chambers. However, TrkB mutated at the Shc-binding site was impaired in its ability to activate PI3-kinase/Akt and MEK/ERK, and was a poor mediator of both neuronal survival and local axon growth. Furthermore, by using pharmacological inhibitors, I found that TrkB-mediated survival and local axon growth required both PI3-kinase and MEK/ERK signalling.Next, I investigated the contribution of Trk signalling to growth cone maintenance in sympathetic neurons. Acute inhibition of NGF/TrkA signalling rapidly collapsed growth cones, as did inhibition of either PI3-kinase or MEK/ERK. I then asked whether the chemorepellent Sema3F collapsed sympathetic growth cones by inhibiting TrkA-mediated growth signals. Sema3F did not disrupt NGF-induced activation of TrkA, Shc, or PLC-gamma but significantly reduced PI3-kinase and MEK/ERK activation. Furthermore, sustained hyper-activation of PI3-kinase and MEK/ERK partially blocked Sema3F-induced collapse, suggesting that Sema3F acts, at least in part, by inhibiting Trk-dependent pathways.Together, these data describe the role of Trk signalling in survival, local axon growth, and growth cone maintenance in cultured sympathetic neurons. The Shc-binding site was found to be critical for TrkB-mediated survival and local axon growth. The PI3-kinase and MEK/ERK pathways were found to be important for survival, local axon growth and growth cone maintenance. Furthermore, the chemorepellent Sema3F collapses sympathetic growth cones, at least in part, by interfering with key neurotrophin-induced signalling pathways