The objective is to characterize the neuronal protein interactions that control axonal outgrowth, so as to develop strategies for promoting axonal regeneration in the human CNS following injury. When a nerve conditioning lesion precedes a more proximally located testing lesion by a conditioning interval of 2 weeks, axonal outgrowth occurs 25-100% faster than expected. Several lines of evidence suggest that this is due to the cell body response to axotomy, as reflected in an increased flux of proteins being carried by slow component b (SCb) of axonal transport.
The specific aims i nclude measurement of the changes in transport of SCb structural proteins (tubulin, actin, clathrin, fodrin) and regulatory proteins (calmodulin) within parent and daughter axons. The hypothesis that increased SCb transport causes an acceleration of outgrowth will be tested by shortening the conditioning interval (or increasing the distance to the testing lesion) to such an extent that the increase in SCb could not reach daughter axons in time to influence their outgrowth. The procedures include making conditioning lesions (or sham lesions) of sciatic nerves (in rats) and optic tracts (in goldfish), and testing lesions of the L4/L5 spinal nerves (in rats) and optic nerves (in goldfish). For SCb transport studies, 35-S methionine is injected near the appropriate spinal motor neurons and retinal ganglion cells one week before the testing lesion. Axonal outgrowth is allowed to proceed for 4-12 days before the animals are killed and nerves are removed for serial sectioning, homogenization, and protein separation by SDS polyacrylamide gel electrophoresis. Fluorograms are used to locate labeled proteins of interest for removal from gels and quantitation of radioactivity by liquid scintillation counting. To measure axonal outgrowth distances, growth cones are labeled by injecting a 1:1 mixture of 3-H lysine and 3-H proline into the spinal cord or eye 12-24 hours before killing the animal. Consecutive nerve segments are solubilized for liquid scintillation counting; the outgrowth distance is determined by analyzing the distribution of radioactivity.
