Glaucoma is one of the leading causes of blindness in the world, and is particularly prevalent among African-Americans. It is the retinal ganglion cell (RGC) which is damaged and dies in glaucoma, causing loss of axons and an excavated appearance to the optic nerve head. Present treatment of glaucoma is directed at lowering the intraocular pressure. while elevated intraocular pressure is a proven risk factor for glaucoma, it is clearly not the only mechanism causing RGC loss. Understanding the basic mechanisms leading to RGC loss may lead to new avenues for the treatment of glaucoma. This application proposes several novel approaches to increase our understanding of the basic molecular biology of the RGC, and the genetic mechanisms of RGC damage and death in glaucoma. Efforts will be made to clone cDNAs representing genes that are preferentially expressed in the RGC. Rat RGCs will be purified using a two-step panning method. Differential display-PCR (DD-PCR) will be used to identify rat cDNAs that are expressed in RGCs but not in control tissues, such as liver and muscle. As a complementary approach, subtractive hybridization will be employed to construct a cDNA library enriched in RGC clones. DD-PCR will also be used to search for genes which are up- or downregulated in rat models of experimental glaucoma and in primate models of experimental glaucoma and optic nerve transection. Sequence analysis, in situ hybridization, and Northern analysis techniques will be used to characterize the differentially expressed RGC cDNAs. Because apoptosis has been implicated as an important mechanism of RGC death in glaucoma, the spatial and temporal patterns of expression of candidate apoptosis- related genes will be explored in RGCs using in situ hybridization and reverse transcription-PCR (RT-PCR). Increased understanding of RGC gene expression, and how it changes with glaucoma and apoptosis, may lead to neuro-protective strategies which will help prevent visual loss and blindness in glaucoma.
