Mammalian optic nerves, as with other mammalian central nerves do not normally regenerate when damaged. This general phenomenon remains a critical problem in the treatment of patients with central nerve damage. Our long-term goals are to understand the molecular and cellular mechanisms of nerve growth, and what conditions are required for nerves like the optic nerve to regenerate successfully.
The aim of this application is to address the nature of a possible intraaxonal molecular signal which may initiate and sustain nerve growth and regeneration. Such a signal may be impeded or missing in damaged central nerves. We have seen the appearance of novel polypeptide, designated A25, specifically within axons at crush sites in frog optic and sciatic nerves very shortly after damage. A25 is retrogradely transported from the site of damage, and probably arises as the result of processing of a fast orthogradely transported protein we have called A30. A25 is thus a good candidate for a signal molecule. We will study these proteins using biochemical and monoclonal antibody techniques. Crushed frog and rat optic nerves will be used to study these early molecular events. To determine if A25 is regeneration specific' or occurs during normal axonal outgrowth we intend to study development of the optic nerves in rats and frogs. We would also expect A25 to be produced in growing neurites of nerve cells in culture. To test this we will use retinal explants from rats and frogs. We can also usa the explant cultures to learn what conditions are necessary to produce A25. The synthesis, transport and post-translational processing of these proteins in retinal cells will be studied under a variety of conditions in vivo and in vitro using different labelled precursors, 2D-gels and fluorography. Initially, we intend to continue characterizing A25 and A30 biochemically, while we make monoclonal antibodies to these proteins. We can then use the antibodies in immunolocalization studies to determine the subcellular sites of A25 and A30, and also in experiments designed to test whether the antibodies inhibit neurite outgrowth in culture. These experiments will test whether A25 or A30 are involved in axonal growth. Using 20-gels will also allow us to follow changes in other growth associated proteins in these experiments.
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