bernice grafstein - US grants

It is proposed to investigate regeneration of goldfish optic axons with the following specific aims: I. To examine the role of phosphorylation of axonally transported proteins by investigating (a) the nature of the changes in protein phosphorylation that occur during regeneration; (b) the protein kinases responsible for these changes; (c) how agents that modulate protein phosphorylation affect regeneration, and (d) how alterations in the time course of regeneration affect the sequence of changes in phosphorylation. II. To determine how some axonally transported constituents involved in synaptic function change during regeneration, particularly with respect to whether or not the regenerating axons have formed retinotopically correct connections. The constituents to be investigated would be synapsin I, nicotinic acetylcholine receptors (defined by alpha-bungarotoxin binding), and acetylcholinesterase. III. To investigate the role of phosphorylation of proteins in the interaction between axons and glial cells, based on our observation that when the axons have degenerated the glial cells do not phosphorylate the same array of proteins as when the axons are intact. Major techniques in this study would be the analysis of phosphorylated proteins by 2-dimensional gel electrophoresis and HPLC, applied to studies of regenerating axons and studies of glial cells in vivo and in vitro. The overall objective is to analyses the properties of a successfully regenerating neuron, in order to further our understanding of the process of axonal regeneration and the mechanisms involved in its regulation. The ultimate application of this knowledge would be in developing techniques for promoting regeneration following damage to the central nervous system produced by conditions such as spinal cord injury, stroke or cerebral palsy, and would also have relevance for studies of Alzheimer's disease.

It is proposed to investigate a group of soluble glycoproteins that are prominent extracellular constituents of the goldfish CNS and show enhanced expression during regeneration of the goldfish optic nerve. We have found that these "exoglycoproteins" (XGPs) are associated with lipid and bind to heparin, properties that also occur in some molecules important in neuronal development and regeneration. Moreover, these properties suggest the possibility of some connection with the immune system. We have also detected XGPs or related proteins in cells of the optic nerve and brain of neonatal rats, which suggests that they may play a role in development of the central nervous system of mammals as well as in regeneration of neurons in goldfish. The objectives of the proposed experiments are to characterize the XGPs in the optic nerve and tectum of the goldfish, to determine their cellular source and distribution, and to explore their function. The specific aims would include developing molecular probes to these proteins, in order to determine their structural characteristics and their relationship to one another; characterizing the lipoprotein complexes that the proteins are associated with; identifying the cells that produce or take up the proteins; determining how the proteins change during goldfish optic nerve regeneration; and ascertaining whether they can influence growth and survival of goldfish and mammalian neurons. The goldfish visual system is the subject of this study because this pathway is capable of vigorous regeneration and hence serves as a powerful model for investigating the mechanisms that regulate regeneration. These investigations are expected to lead to techniques for promoting regeneration in the human central nervous system following damage caused by conditions such as spinal cord injury, head injury, stroke, cerebral palsy or neurodegenerative disease.