DIETARY & GENETIC CONTROL OF ROS DAMAGE TO THE LENS

描述(申请人提供)：在老年性白内障的发展过程中，活性氧物种(ROS)的作用一直是白内障研究领域的一个主要兴趣和研究课题。然而，晶状体上皮细胞(LECs)线粒体代谢释放ROS的作用尚未在完整晶状体中的活LECs线粒体中直接研究。P.I.已经证明了正常小鼠和大鼠作为年龄相关性人类白内障模型的价值，因为它们在中年后会出现渐进性晶状体混浊，通常会导致成熟的白内障。这项拟议的研究将使用荧光染料技术的组合来直接测量单个幼年、中年和老年小鼠晶状体上皮细胞的LEC线粒体膜电位、线粒体氧化转换率和线粒体H202的释放，并以它们的晶状体混浊程度为背景。在相同的晶状体中，如果可能，将确定对线粒体和核DNA的累积氧化损伤程度，以及晶状体蛋白质的变化程度。这项研究将在3个小鼠模型上进行这些评估，都是在相同的遗传背景下进行的：1)正常小鼠的卡路里限制(CR)，2)额外过氧化氢酶仅转移到线粒体的转基因小鼠，以及3)抗氧化酶谷胱甘肽过氧化物酶(GPX-1)被敲除的小鼠(在所有情况下，小鼠都将与适当的对照组相匹配)。在其他分组中，将对转基因小鼠和基因敲除小鼠施加CR，以确定它们各自的影响是以相加、协同还是相反的模式运作。首席研究人员已经在这3个模型中确定，CR显著延迟了白内障的发展，额外过氧化氢酶的线粒体易位也是如此，并且GPX-1的敲除导致了更早和更高级的白内障发展。此外，在两项初步研究中，老年小鼠晶状体上皮细胞的线粒体膜电位显著降低。因此，为研究线粒体在晶状体上皮细胞内自身和更广泛的细胞氧化损伤中的作用，其在晶状体内部蛋白质变化中的意义，以及在白内障的发展中的作用奠定了基础。这些研究有望阐明线粒体效率下降和由此导致的晶状体氧化损伤在年龄相关性白内障中的作用。

DESCRIPTION (provided by applicant): 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.