This revised application makes use of the sea lamprey as a model system for axon regeneration in the CNS. Following spinal cord transection, efficient axon regeneration can occur for a select and sizable collection of affected neurons. The growth cones of these neurons apparently do not contain filopodia or lamellipodia during regeneration, and axon regeneration seems to be accomplished by pushing forces within the growth cone. In contrast, growth cones advance in embryonic CNS neurons by being pulled away from neuronal perikarya by their F-actin-rich filopodia and lamellipodia. Dr. Selzer's group has found a high correlation between expression of the 180 kD neurofilament protein (NF-180) and axon regeneration potential in lamprey neurons. Moreover, they have shown that lamprey growth cones that are able to advance following spinal cord transection contain densely packed neurofilaments and very few actin filaments. They thus hypothesize that transport of neurofilaments into the growth cone provides the driving force for its continued advance. To test this hypothesis, four Specific Aims are proposed. 1) The ability of axons to regenerate when NF-180 is underexpressed will be tested. Lampreys whose spinal cords have been transected will be injected four weeks later in the fourth ventricle with antisense DNA for NF-180, separated by an IRES (alternative ribosome entry site) from cDNA for a reporter molecule, like c-myc or b-galactosidase. A gene gun will be used to introduce the DNA. After an additional six weeks, axon regeneration will be evaluated by retrograde transport of HRP injected 5-15 mm caudal to the transection. Dr. Selzer's hypothesis predicts that regeneration will be inhibited by the antisense DNA. Semi-quantitative PCR, in situ hybridization, anti-NF-180 ELISA, and immunohistochemistry will be used collectively to determine how successfully the antisense DNA reduced expression of NF-180 mRNA and protein. 2) The effect on axon regeneration of overexpressing NF-180 will be assessed. With one major exception, the experimental details will be exactly the same as those used for Specific Aim 1. The exception is that the injected probe will include cDNA that encodes full length NF-180 to drive overexpression of the protein. Dr. Selzer's model predicts that overexpression of NF-180 will lead to accelerated regeneration. 3) Growth cone advance for transected axons will be observed in situ using confocal fluorescence microsocopy. At various times after spinal cord transection, the brain and rostral portion of the spinal cord will be dissected, and exposed Muller and Mauthner axons will be microinjected with 5,6-carboxyfluorescein. The specimens will then be mounted on a specially modified microscope stage, and a Z series of 20-30 images will be collected at regular intervals for periods as long as six hours. 4) The in situ studies will be extended to animals that either underexpress or overexpress NF-180, as described for Specific Aims 1 and 2.
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