Spatial regulation of neuronal survival, axon growth, and axon degeneration in compartmented cultures of sympathetic neurons.

摘要

In developing sympathetic neurons, nerve growth factor (NGF) interacts with its receptor tyrosine kinase, TrkA, on axon terminals to generate local signals that act within the axon terminals and retrograde signals that ultimately regulate gene expression in the nucleus. Foremost, this thesis investigates predictions of the signaling endosome hypothesis by examining the requirement of internalization and retrograde transport of NGF, and of retrograde arrival of phosphorylated TrkA in the cell bodies, in mediating two of the major biological functions of NGF in sympathetic neurons: neuronal survival and axonal growth. Data presented in this thesis demonstrate that the survival of sympathetic neurons can be maintained by binding of NGF to receptors on the axon terminals, without retrograde transport of the growth factor. Axon growth was attenuated when phosphorylation of Erkl/2 was prevented by inhibiting MEK kinase activity locally in distal axons, whereas inhibition of MEK in cell bodies/proximal axons had no such effect, suggesting that internalization of NGF/pTrkA complexes is required to activate MEK/Erk signaling locally in axons where it is required to promote axon growth. These data refute the hypothesis that retrograde transport of NGF/pTrkA-containing signaling endosomes is the exclusive mechanism by which retrograde signals are communicated. Instead they suggest that retrograde signals can reach the cell bodies in the absence of the NGF or the pTrkA that initiated them. They also suggest that endocytosis of NGF/pTrkA complexes may be important for aspects of NGF-induced signaling are distinct from retrograde signaling, including promoting local axon growth.; In other experiments, mechanisms that control local axon degeneration were investigated. Wallerian degeneration of transected distal axons and axonal degeneration due to NGF withdrawal were prevented by pharmacological inhibition of proteasome function, preserving their morphology and mitochondrial metabolic activity for at least 24h. Transected axons treated with the proteasome inhibitor MG 132 displayed persistent phosphorylation of Erk1/2, and co-application of a MEK inhibitor; U0126, restored the rapid Wallerian degeneration observed in untreated axons. (Abstract shortened by UMI.)