Oxidative Stress

The central premise of this dissertation is that the small heat shock protein (sHSP), (Sa(BB-crystallin is essential for lens and retinal pigmented epithelial (RPE) cell function and oxidative stress defense. To date, the mechanism by which it confers protection is not known. We hypothesize that these functions could occur through its ability to protect mitochondrial function in lens and RPE cells. To test this hypothesis, we examined the expression of (Sa(BB-crystallin/sHSP in lens and RPE cells, we observed its localization in the cells, we examined translocation to the mitochondria in these cells upon oxidative stress treatment, we determined its ability to form complexes with and protect cytochrome c (cyt c) against damage, and we observed its ability to preserve mitochondrial function under oxidative stress conditions in lens and RPE cells. In addition to these studies, we examined the effect of mutations of (Sa(BB-crystallin/sHSP on its cellular localization and translocation patterns under oxidative stress, its in vivo and in vitro chaperone activity, and its ability to protect cyt c against oxidation. Our data demonstrated that (Sa(BB-crystallin/sHSP is expressed at high levels in the mitochondria of lens and RPE cells and specifically translocates to the mitochondria under oxidative stress conditions. We demonstrate that (Sa(BB-crystallin/sHSP complexes with cyt c and protects it against oxidative inactivation. Finally, we demonstrate that (Sa(BB-crystallin/sHSP directly protects mitochondria against oxidative inactivation in lens and RPE cells. Since oxidative stress is a key component of lens cataract formation and age-related macular degeneration (AMD), these data provide a new paradigm for understanding the etiology of these diseases.

Caloric restriction (CR), the reduction of nutrient intake short of malnutrition, extends the lifespan of various organisms and can improve measures of human health. Whether mechanisms of lifespan extension are conserved between humans and model organisms is unknown. In mammals, implementing CR is easily achieved by providing a restricted group with a fraction of the food consumed by an "ad libitum" fed group, which has unlimited food access. Due to the difficulty in directly controlling Drosophila food intake, caloric restriction, performed similarly to the mammalian paradigm, has never been tested in flies. Here, we demonstrate a system that allows measurement of food intake throughout life. This system will be used to measure fly lifespan under caloric restriction analogous to current mammalian studies. Our work will help tease apart the differences between the various caloric and dietary restriction paradigms in Drosophila, strengthening our understanding of how fly models relate to mammalian systems.