The primary focus of this proposal is the investigation of alterations in gene expression associated with CNS regeneration in the goldfish and rat visual systems. Molecular biological tools will be employed to identify and examine the regulation of expression of proteins selectively synthesized during the axonal growth state. Comparisons between the axotomized goldfish retina, which does regenerate, and that of the adult rat retina, which does not regenerate, will aid in the identification of regeneration- associated mRNAs. Goldfish retina mRNAs will be screened for expression of known proteins likely to be involved in axonal growth and for the expression of novel proteins induced by axotomy. cDNAs for several proteins which may play an important role in development or regeneration are available, including GAP-43, laminin receptor, NGF receptor and oncogenes. The initial focus will be on the nicotinic acetyl-choline receptor, an optic nerve component postulated to play a role in synapse formation. The presence of the various subunits and subtypes of the nAChR will be assessed and their cellular localization within the retina will be investigated. In addition, novel mRNAs induced by axotomy will be identified by differential screening of goldfish retinal cDNA libraries and characterized as to their time course and cellular localization in the goldfish retina. Their expression in developing and control or axotomized adult rat retina will be assessed. Of particular interest are two proteins currently under investigation in this laboratory: a 68-70 kD doublet, and a 24-30 kD complex. The appearance of the latter constitutes the largest early response to axotomy yet reported. Effects of extrinsic agents on gene expression will be explored, using goldfish and rat retina explant cultures as in vitro models. Recent success in obtaining outgrowth from conditioned rat retina relies on the presence of laminin in the substratum. We will seek biochemical correlates of the neuronal response to its presence, in order to further test the hypothesis that neurite contact with laminin facilitates regeneration in the mammalian CNS. We will also investigate the effects on gene expression of putative signaling agents known to enhance neurite outgrowth in vitro. These studies should increase our understanding of the molecular events underlying axonal regeneration, and will form the basis of future studies exploring the regulation of gene expression during axon outgrowth.
