The long-term objective of this research plan is to elucidate the molecular mechanisms underlying nerve growth and regeneration. It is generally believed that neurons in the brain and retina of mammals are unable to regenerate nerve fibers, which presents a great challenge for the treatment of retinal degenerative disorders and damage. Recent data have established that retinal ganglion cells (RGCs) are intrinsically regulated in their ability to extend nerve fibers, and that the proto-oncogene Bcl-2 is a key regulator supporting the mechanism for nerve elongation. This growth-stimulating activity of Bcl-2 does not seem to be a consequence of its well-known anti-apoptotic function. The central question here is the mechanisms by which Bcl-2 promotes the regeneration of retinal nerve fibers. It has long been recognized that neural differentiation impacts nerve growth. It is now evident that overexpression of Bcl-2 suppresses the function of a differentiation signal, p53-mediated transcriptional activation, and expression of terminal differentiation markers in neurons. Therefore, it is hypothesized that Bcl-2 may function as a repressor of the p53-p21WAF1 pathway inhibiting neural terminal differentiation and thus, maintaining the ability of RGCs to grow nerve fibers. Using genetically-engineered mouse models as well as novel retinotectal co-cultures, this research plan is aimed at elucidating functional pathway(s) through which Bcl-2 may support optic nerve regeneration. Our goals are: (1) to investigate whether Bcl-2 inhibits the terminal differentiation of RGCs by determining levels and timing of RGC markers and differentiation-related protein expression in retinas of wild type and Bcl-2 mutant mice; (2) to test whether Bcl-2 supports nerve regeneration via suppression of the differentiation signal, p53-p21WAF1 pathway. Here, functional interactions between Bcl-2 and p53 will be assessed by comparing transcriptional activity, subcellular localization, and phosphorylation of p53 in wild type, Bcl-2 mutant, and Bcl-2/p53 double mutant mice; and (3) to determine whether Bcl-2 acts on a common cellular pathway to support nerve regeneration and neuronal survival by comparing the ability of RGCs from Bcl-x, and Bcl-2 transgenic mice to survive and regenerate nerve fibers. Given the functional importance of Bcl-2 in neuronal survival and nerve regeneration, these studies should provide valuable information for the development of neuroprotective drugs and therapeutic strategies for numerous diseases mediated by dysregulated cell death and nerve growth, including glaucoma and macular degeneration.
