John S. Elam - US grants

The objective of the proposed research is to continue the characterization of glycoproteins and proteoglycans that undergo axonal transport to specific axonal and synaptic subfractions of the goldfish optic nerve. Synaptic fractions to be analyzed include soluble, synaptic vesicle, synaptic plasma membrane, and synaptic junctions. Methods of analysis of glycoproteins will include lectin affinity chromatography and one and two dimensional SDS gel electrophoresis. Glycopeptides derived from transported glycoproteins by proteolytic digestion will be evaluated by differential affinity to concanavalin A and susceptability to hydrolysis with alkaline borohydride. Soluble proteoglycans and particulate proteoglycans extracted with deoxycholate or guanidine will be separated by ion exchange chromatography, density gradient centrifugation and gel filtration and will be subjected to analysis of the type, size, and charge density of constituent glycosaminoglycan chains. Separate studies will evaluate the effect of physiological activity on transported glycoprotein turnover and will attempt to purify and immunocytochemically localize glycoproteins concentrated in isolated myelin. The significance of the proposed studies will be the clarification of the types of glycosylated macromolecules localized at functionally important regions of the axon and nerve terminal. Identification of such molecules and evaluation of their turnover should be a first step in elucidating their specific functions in neuron activity.

The overall objective of the proposed studies is to further characterize proteoglycans that undergo rapid axonal transport in the intact and regenerating goldfish optic nerve. Proteoglycans, as a glycoconjugate class, have become increasingly implicated in neuronal synaptic function and in the development and functional differentiation of neurons. As yet, however, there has been little information gained on the properties of proteoglycans undergoing axonal transport and on changes in proteoglycan transport that may accompany functional characterizing. The proposed studies will address the specific aim of characterizing axonally transporting proteoglycans that are soluble and that are affiliated with various membranous compartments, including synaptic vesicles, synaptic plasma membrane and synaptic junctions. An additional aim is to detect changes in the distribution or turnover of axonally transported proteoglycans that may be correlated with synpatic secretory activity. The final aim is to further characterize changes in axonally transported proteoglycans that have been preliminary shown to accompany regeneration of the optic nerve. All of these studies will employ labeling, ion exchange chromatography, gel filtration chromatography, density gradients, affinity chromatography and enzyme-chemical analysis of polysaccharide (glycosaminoglycan) chains. The results of these studies should clarify the role of proteoglycans in normal synaptic function and in the facilitation of CNS regeneration in the lower vertebrates.

Tritiated proline when injected into the nervous system of cats, is incorporated preferentially into glial cells. The unusual incorporation pattern suggested the existence of a previously unrecognized glial-neuronal communication system. This system involves the translocation of molecules from glial cells into neuronal soma followed by axonal transport and partial redistribution to adjacent glial processes. The long-term goals of the research are to characterize the molecules involved in the translocation sequence and to understand the operations and functions of the system. These studies will utilize anatomical and biochemical methods to characterize the preferential incorporation of proline into feline glia and to determine the translocation process to neurons. Other studies will document the suitability of the rat and determine the use of optimal neuronal pathways. Future studies will address questions regarding possible functions of the glial-neuronal communication system during neuronal activity and maturation. Although neuroscientists agree that brain function is carried out by combined actions of neurons and glia, the actual mechanisms of communication and cooperation are poorly understood. These studies are designed to improve this understanding.