Richard T. Ambron - US grants

DESCRIPTION (from applicant's abstract) Nerve injury triggers long-term alterations that require changes in protein synthesis and which may result in the restoration of function. Often, however, regeneration fails, resulting in sensory deficits, chronic pain, and paralysis. Efforts to promote growth and minimize sensory defects would be facilitated if we knew the identity of the signals that inform the cell soma that its axon has been injured and how these signals regulated the transcriptional programs that are responsible for successful regeneration. Using the nervous system of Aplysia californica as a model the applicants found that positive injury signals activated at the site of axon injury are retrogradely transported to the cell nucleus. When axoplasm containing these signals is injected into non-injured neurons, it induces the same growth and hyperexcitability that appears when the axons of these cells are injured. A similar hyperexcitability occurs after axotomy in mammalian neurons and is thought to be responsible for chronic pain. To identify the signals responsible for these changes, they analyzed the axoplasm and found it to be enriched in 2 kinases, ERK and SAPK. Most of the ERK is in the phosphorylated (active) form and the applicants hypothesize that activation occurs when an influx of calcium at the lesion site activates phosphokinase C. They will manipulate calcium levels using an ionophore to see whether PKC is affected. How ERK is retrogradely transported is not known. They will inject recombinant ERK directly into the axon to monitor its transport and will use specific antibodies and subcellular fractionation of axoplasm to see if it occurs in association with an organelle. Once ERK reaches the nucleus it phosphorylates the transcription factor C/EBP. This could increase the affinity of C/EBP for DNA, alter transcription, or regulate its entry into the nucleus. Each possibility will be assessed using recombinant wild type and mutated C/EBP. Interestingly ERK is also activated by nerve inflammation, which also induces hyperexcitability. This suggests that hyperexcitability is due to ERK acting on C/EBP. They will attempt to interfere with this process by microinjecting antibodies and oligonucleotides and by using mutated ERK and C/EBP. In contrast, retrogradely transported SAPK is constitutively active, although its activity increases after injury, and it may be involved in growth through c-Jun. The investigators have antibodies and recombinant proteins to investigate this possibility and will use strategies similar to those employed for ERK.

During embryonic development, neurons extend growth cones that seek out and synapse on the appropriate target. The culmination of such contacts is the neuronal circuitry that underlies behaviors. The goal of our research is to determine how neurons recognize, and are recognized, by their targets. Any attempt to define recognition at the molecular level requires the ability to isolate and characterize molecules on the growth cone surface and the means to assess the function of putative recognition ligands isolated from the growth cones. These prerequisites are difficult to attain in the complex and heterogenous vertebrate system. They can be attained, however, by using the nervous system of Aplysia california where many neurons can be recognized as individuals and are large enough to study using biochemical, immunological and electrophysio- logical approaches. Growth cones, for example, can be isolated in pure form RUQ neurons growing in vitro and their polypeptide composition can be examined. Also, neuron L7 synapses on auricle and gill vein muscles to elicit initiation of the heartbeat and gill withdrawl, respectively. This simple circuit has been assembled in vitro and synaptogenesis between L7 and its target muscles has been quantitated, thereby providing a statistically meaningful way to assay components of the growth cone that block synapse formation. One of the constituents enriched at RUQ growth cones is a 75kd membrane glycoprotein that is exposed on the surface. This glycoprotein is one member of a class of similar glycoproteins (GP-75) that are rapidly transported to the terminals of many Aplysia neurons, including L7. GP-75 is heterogenous since it contains a glycopeptide composed of several oligosaccharides that bind to wheat germ agglutinin. It is significant, therefore, that one or more of these oligosacch-arides bind specifically to the muscle cells that are targets of L7. WE will test two hypotheses: first, that the GP-75 found ont eh growth cones of motor neurons has oligosaccharides that participate in recognition of muscle cells during synaptogenesis. Second, that muscle cells have receptors on their surface that recognize GP-75 oligosaccharide. These ideas will be investigated by: 1) isolating and characterizing the GP-75 oligosaccharides and identifying the constituents that block the binding of GP-75 to isolated muscle membranes; 2) generating monoclonal antibodies against the various species of GP-75 to determine their distribution among identified neurons; 3) identifying the antibodies and GP-75 oligosaccharides that prevent synaptogenesis between L7 and its targets; and 4), using the oligosaccharides as affinity ligands to isolate the receptors from each muscle type. In this way we will define the ligands and receptors involved in the formation of simple circuit.

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.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

DESCRIPTION: Studies on regeneration and retrograde transport in Aplysia neurons identified a set of retrogradely transported proteins which contain a nuclear import signal. This signal peptide sequence is homologous to the nuclear import sequences found in a variety of other proteins, such as the S40 large T-antigen and exogenous proteins with this sequence attached are subject to both retrograde transport and nuclear import when introduced into the axon. Proteins containing such sequences have now been obtained and identified in rat. These proteins are thought to represent prototypes for the elusive nerve injury signal which is thought to mediate the cell body response following nerve injury. The experiments in this application are intended to characterize endogenous proteins containing these sequences and their associated polypeptides.The biology of these proteins in Aplysia and rat nerve will be explored in the hopes of providing insights into the cell body response after nerve injury and identifying the underlying molecular mechanisms.