The role of reactive oxygen species (ROS) in the development of age-related cataract has been a major subject of interest and study in the field of cataract research. However, the contribution of the metabolic release of ROS by the mitochondria of the lens epithelial cells (LECs) has not been studied directly in the mitochondria of living LECs in intact lenses. The P.I. has demonstrated the value of normal mice and rats as models for age-related human cataract, as they develop progressive lens opacities after middle age, often leading to mature cataracts. The proposed study will use a combination of flurorescent dye techniques to directly measure the LEC mitochondrial membrane potential, mitochondrial oxidative turnover and mitochondrial H202 release in the LECs of individual young, middle aged and old mice, against the background of their degree of lens opacity. In the same lenses, where possible, the degree of accumulated oxidative damage to the mitochondrial and nuclear DNA, and of changes in the lens proteins, will be determined. The study will make these assessments in 3 mouse models, all on the same genetic background: 1) normal mice on caloric restriction (CR), 2) transgenic mice with additional catalase translocated only to their mitochondria, and 3) mice with the anti-oxidant enzyme glutathione peroxidase (Gpx-1) knocked out (in all cases the mice will be matched with appropriate controls). In additional groupings CR will be imposed on the mice with the transgene and on those with the knockout to determine whether their respective effects operate in additive, synergistic or oppositional modes. The principal investigator has already determined in these 3 models that CR significantly delays the development of cataracts, as does the mitochondrial translocation of additional catalase, and that the knockout of GPx-1 results in an earlier and more advanced degree of cataract development. Also, in two preliminary studies, a great reduction in the membrane potential of the mitochondria in LECs from old mice was shown. Thus, the background has been prepared for the studies of the role of mitochondria in both self-inflicted and wider cellular oxidative damage within the LECs, its implication in internal lens protein alterations, and in the development of cataract. These studies are expected to clarify the role of declining mitochondrial efficiency and the resultant oxidative damage to the lens as causal in age-related cataract.
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