This is a proposal to determine if regeneration of spinal cord axons is guided or otherwise modulated by the actions of netrins and semaphorins, molecules that guide axons during development and are present in the CNS of mature vertebrates. Following spinal cord transection, mammalian axons ordinarily do not regenerate, which complicates the study of molecular mechanisms of regeneration. By contrast, lampreys recover from complete spinal transection and axons regenerate selectively in their correct paths. Other advantages of the lamprey for regeneration research include: 1) Identified giant reticulospinal neurons differ from one another in their regenerative abilities, which have been determined previously. 2) These neurons can be visualized in vivo and in CNS wholemounts. 3) Molecular expression patterns can be correlated with regenerative abilities in individual neurons and identified neuron types. 4) In vivo transfection of neurons with Gene Gun results in long-lasting transgene expression. We already have partial sequences for two semaphorins, one netrin and two netrin receptors in lamprey. We have localized expression of three of these by in situ hybridization and have evidence that their expression is modulated by spinal cord transection. We now propose to determine whether overexpression or underexpression of netrin and semaphorin receptors in reticulospinal neurons alters the probability and/or pathway specificity of their regeneration. We will complete the full length cloning of the proposed guidance molecules and their receptors, and localize their expression in uninjured and spinal cord transected animals. The expression of receptors will then be enhanced or inhibited in reticulospinal neurons by Gene Gun transfection with the sense or antisense (alternatively, a truncated dominant negative) cDNA for netrin and semaphorin receptors. Cotransfection with the reporter Green Fluorescent Protein will distinguish transfected from untransfected cells. The probability of regeneration will be determined by retrograde transport of HRP, while the directional specificity of regeneration will be determined by intracellular injection of HRP and observation of the regenerated axons in spinal cord wholemounts. If developmental guidance molecules influence the effectiveness of axon regeneration in CNS, their manipulation could form the basis for therapies to improve regeneration of interrupted axons in patients with spinal cord injuries, traumatic brain injuries and stroke.
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