At the developing neuromuscular junctions (NMJs), motoneurons send axons to innervate target muscle fibers and the growth cone-target contact induces development of synaptic specializations. However, little is known about the mechanisms involved in the formation of the nerve terminal. In this thesis work, I elucidated the retrograde signaling pathways underlying the development of presynaptic nerve terminal. Firstly, when cultured Xenopus spinal neurons were treated with muscle-derived neurotrophins, the neuronal survival was promoted dramatically. Surprisingly, the nerve-induced acetylcholine receptor (AChR) clustering on muscle was inhibited as a result of a reduction of agrin deposition along the treated neurites. These suggest neurotrophins, while up-regulate neuronal survival/outg...[ Read more ]

At the developing neuromuscular junctions (NMJs), motoneurons send axons to innervate target muscle fibers and the growth cone-target contact induces development of synaptic specializations. However, little is known about the mechanisms involved in the formation of the nerve terminal. In this thesis work, I elucidated the retrograde signaling pathways underlying the development of presynaptic nerve terminal. Firstly, when cultured Xenopus spinal neurons were treated with muscle-derived neurotrophins, the neuronal survival was promoted dramatically. Surprisingly, the nerve-induced acetylcholine receptor (AChR) clustering on muscle was inhibited as a result of a reduction of agrin deposition along the treated neurites. These suggest neurotrophins, while up-regulate neuronal survival/outgrowth, down-regulate the synaptogenic machinery. Secondly, mitochondria were found to be co-clustered with synaptic vesicles (SVs) at presynaptic sites in nerve-muscle cocultures or upon focal stimulation with growth factor-coated beads. The actin polymerization blocker partially inhibited bead-induced mitochondrial and SV clustering while the microtubule disrupting agent was ineffective. In contrast, serine/threonine phosphatase inhibitor suppressed SV but not mitochondrial clustering. These indicate that synaptogenic stimuli induce the clustering of mitochondria and SVs by a common cytoskeleton-mediated mechanism but involving different signaling molecules. Thirdly, using a mitochondrial membrane potential-sensitive probe, I found that mitochondria with higher potential tended to be localized at NMJ and within bead-induced presynaptic specializations. Transient suppression of mitochondrial activity by pharmacological agent inhibited the bead-induced SV clustering in neurites and the nerve-induced AChR clustering on muscle cells. These suggest that mitochondrial activity is an essential factor in the development of synaptic specializations. Lastly, I examined the signal transduction mechanism underlying presynaptic development. Specifically, the involvement of protein tyrosine kinases and phosphatases in regulating the formation of SV and mitochondrial clusters was studied. Taken together, these findings highlight the fundamental mechanisms involved in the development of the nerve terminal at the vertebrate NMJ.