GENE MANIPULATION &H202 DEGRADATION IN THE OCULAR LENS

DESCRIPTION: Maturity onset cataract results in more than one million operations per year utilizing more than 12% of the Medicare budget. Previous work has established that oxidative stress is an initiating or early event in the development of human maturity onset cataract. It also has been shown that H202 is a major oxidant present in elevated concentration in a significant fraction of cataract patients and experiments suggest that even where other potent oxidants such as superoxide and hydroxyl radical are present, removal of H2O2 eliminates the stress induced formation of cataract. This project proposes to examine the lens' ability to degrade H2O2 by preparing transgenic and knockout mice for a number of the key enzymes that are involved in metabolizing H2O2 including catalase, glutathione peroxidase and glutathione reductase. Such experiments will elucidate the contribution of each enzyme to the overall lens metabolism of H2O2 and indicate the effect of eliminating or enhancing the enzyme. Analyses will be carried out in organ culture with lenses from normal, transgenic and knockout lines. The effect of aging will be examined. The ability of the system to degrade H2O2 and resist H2O2 stress will be investigated utilizing well defined parameters such as the nonprotein and protein thiol levels, DNA integrity, cell viability, membrane transport, the activity of enzymes involved in H2O2 metabolism and changes in gene expression of certain components affected by oxidative stress. Since apoptosis has been shown to be involved in H2O2 induced cataract, lenses will be examined to see if the apoptotic process continues to be turned on. Crossbreeding of transgenics will present the opportunity to evaluate the effect of increasing the activity of two or more enzymes. Genomic DNAs or cDNAs are available for all the enzymes of interest and classical procedures can be used to prepare constructs for developing transgenics. Cell transfection will be employed to evaluate some of the constructs. Both lens specific and general promoters will be used to assess the effect of modification of enzyme levels either in the lens alone or throughout the organism. Work is also planned to utilize signal peptides to target catalase to an extracellular environment. If a transgenic is developed which has an enhanced ability to resist H202 stress based on the lens culture experiments mentioned above, animal model systems to evaluate the transgenics will be employed. The overall work will test the hypothesis that enhancing or weakening the anti-H202 defense system of the lens will have a profound effect on the ability of the lens to withstand both oxidative and nonoxidative stress. Preliminary data suggest that under oxidative stress, alpha crystallin may be phosphorylated in an atypical manner at tyrosine residues. This is the first finding of tyrosine phosphorylation of the crystallins. We intend to further confirm this observation and attempt to determine the effect of such phosphorylation.