During embryonic development neurons extend processes containing growth cones that locate, recognize, and contact the appropriate targets. A synapse forms, motility ceases and a signal is sent to the cell body that elicits the synthesis of proteins to consolidate and then maintain the synaptic ending. Neither the molecules involved in recognition of the target nor the nature of the communicating signal is known. Our goal is to define these two processes at the molecular level and we have preliminary evidence that glycoproteins are involved in both. Molecules that mediate recognition are present on the growth cone. We recently succeeded in isolating growth cones from a single population of Aplysia motorneurons. Analyses revealed a glycopeptide (GPwga) that is associated with a glycoprotein exposed on the surface of the growth cones. GPwga contain one or more oligosaccharide chains that bind to certain types of muscle cells. We hypothesize that these oligosaccharides are involved in target recognition. We intend to characterize the oligosaccharides and determine if they are able to interfere with the formation of neuromuscular junctions between identified motorneurons and their target muscles in vitro. Although almost nothing is known about the way in which axons and terminals communicate with the soma, it is clear that such communication exists since events such as axotomy, denenervation, etc. elicit changes in somatic protein synthesis. We have injected 3H-monosaccharides directly into the axon of the giant neuron R2 and found that five proteins are glycosylated in the axon. Some of these are subsequently transported toward the cell body. Partial characterization of these glycoproteins suggests the presence of single O-linked N-acetylglucosamine, a modification that is found on transcriptional factors in the nucleus. Consequently, we will test the hypothesis that proteins glycosylated in the axon are transported to the nucleus where they act as signals from the periphery. We have shown that axotomy of Aplysia neurons has affects on protein synthesis that are consistant and quantifiable. We will axotomize the R2 axon to see if it alters the glycosylation of the axonal species and will also modify the glycosylated species, by injecting galactosyl transferase and UDP-galactose into the axon, to see if we can interfere with the signal to the cell soma.
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