During the development of vertebrate neuromuscular junctions (NMJs), path-finding axonal growth cones of motoneurons differentiate into nerve terminals capable of releasing the neurotransmitter acetyIchoIine (ACh) contained within synaptic versicles (SVs) clustered at the active zone. ACh activates its receptors (AChRs) on target muscle cells to generate action potentials. Delineating the molecular signaling events that bring about the presynaptic clustering of SVs is therefore crucial for understanding how the NMJ develops. The presynaptic differentiation was studied through the following three aspects - tyrosine kinase signaling, the involvement of the cytoskeleton and the formation of the varicosities with the use of the Xenopus spinal motoneurons as the model system. Previously we h...[ Read more ]

During the development of vertebrate neuromuscular junctions (NMJs), path-finding axonal growth cones of motoneurons differentiate into nerve terminals capable of releasing the neurotransmitter acetyIchoIine (ACh) contained within synaptic versicles (SVs) clustered at the active zone. ACh activates its receptors (AChRs) on target muscle cells to generate action potentials. Delineating the molecular signaling events that bring about the presynaptic clustering of SVs is therefore crucial for understanding how the NMJ develops. The presynaptic differentiation was studied through the following three aspects - tyrosine kinase signaling, the involvement of the cytoskeleton and the formation of the varicosities with the use of the Xenopus spinal motoneurons as the model system. Previously we have shown that in neurites derived from cultured Xenopus spinal motoneurons, polystyrene beads coated with the heparan sulfate proteoglycan (HSPG)-binding factor bFGF induce SV clustering at contact sites. In this study we used these beads and those coated with two other HSPG-associated growth factors, hepatocyte growth factor (HGF) and heparan-binding growth-associated molecule (HB-GAM), to investigate the signaling in presynaptic SV clustering. Clustering of SVs that occurred at bead-neurite contacts was visualized by immunofluorescent labeling with antibodies against the SV protein synaptotagmin.

We found that incubating neurons with the tyrosine kinase inhibitor tyrphostin RG50864 reduced SV clustering at bead-neurite contacts in a dose-dependent manner. Moreover, the addition of the tyrosine kinase inhibitor after overnight incubation of bFGF beads with neurons revealed that the inhibitor also dispersed SVs that had been clustered by the beads. These results suggest that tyrosine kinases are involved in both the formation and the maintenance of SV clusters. Varicosities are swellings along the neurite and contain SV clusters. Since SV clustering is the hallmark for presynaptic differentiation, we think that varicosities may represent hotspots of spontaneous presynaptic differentiation. Previously we have shown that neurotrophins promote neurite outgrowth and but not synaptogenesis. Using the varicosity as a marker for presynaptic differentiation, we plan to study the effect of neurotrophins, considered to be the growth stimulus, versus that of HSPG-binding factors, considered to be the synaptogenic signals, in the dispersal and the formation of presynaptic specializations. Since the cytoskeleton is crucial for the transport of molecules and structural differentiation within cells, it is worth investigating how this cellular specialization is involved in the trafficking of SVs. We found that at nanomolar concentrations of latrunculin A, a marine sponge toxin that binds to G-actin to prevent its polymerization into F-actin, inhibited the clustering of SVs that occurred at bead-neurite contacts. To further investigate the cytoskeleton involved in the development of presynaptic terminals, nocodazole, a toxin that depolymerizes microtubules, will be used to see if microtubules also play a role.