The mammalian central nervous system (CNS) does not support nerve regeneration. On the other hand, cold-blooded vertebrates show successful nerve regeneration in their CNS. The study of the biochemistry of nerve regeneration in the CNS of cold-blooded vertebrates is of interest in basic research, as well as for the development of treatments for human conditions derived from pathological or traumatic damage to nervous tissues. Studies with the optic nerve system of teleost fish have identified several proteins related to nerve regeneration. The relevance of some of these proteins in axonal growth and pathfinding has already been reported in the scientific literature. Among the proteins induced during optic nerve regeneration, RICH proteins (Regeneration Induced CNPase Homotog proteins) were recently cloned (corresponding cDNAs) and showed to be homologous to mammalian CNPases. CNPases are enzymes (phosphodiesterases) present in the myelin sheath of mammals that are proposed to interact with membranes and the cytosketeton, but their specific function is not well defined yet. RICH proteins share similar catalytic properties with mammalian CNPases, but our knowledge of these proteins is still limited. In the current funding period of the proposal, the participation of three subdomains of RICH proteins in catalysis and intracellular localization has been studied by site directed mutagenesis. Currently, the effect of the deletion of individual subdomains in subcellular localization as well as in the morphology of eukaryotic ceils overexpressing these proteins is being investigated. This proposal is aimed at the continuation and extension of these studies. Cell lines of proneuronal phenotype and zebrafish retina explants will be transfected with constructs that express wild type RICH protein and the deletion mutants. Neurite growth properties will be monitored in the transfectants. Efforts at finding target proteins that interact with RICH in neuronal cells will be continued by multiple approaches, including overlay and two-hybrid library screenings. Finally, the participation of RICH protein in the development of the nervous system will be analyzed with studies in zebrafish embryos, using microinjection and RNA interference approaches. The proposed experhnents should extend our knowledge of RICH proteins, particularly in the understanding of their role in nerve regeneration and the development of the nervous system.