DIETARY & GENETIC CONTROL OF ROS DAMAGE TO THE LENS

描述（由申请人提供）：活性氧（ROS）在年龄相关性白内障发展中的作用一直是白内障研究领域的主要研究课题。然而，活性氧的代谢释放的贡献的线粒体的透镜上皮细胞（LECs）尚未直接研究在线粒体的活LECs在完整的晶状体。私家侦探已经证明了正常小鼠和大鼠作为与年龄相关的人类白内障模型的价值，因为它们在中年后发展为进行性透镜混浊，通常导致成熟的白内障。所提出的研究将使用荧光染料技术的组合来直接测量个体年轻、中年和老年小鼠的LEC中的LEC线粒体膜电位、线粒体氧化周转和线粒体H2 O2释放，以它们的透镜不透明程度为背景。在可能的情况下，在相同的晶状体中，将确定线粒体和核DNA的累积氧化损伤程度以及透镜蛋白的变化程度。本研究将在3种小鼠模型中进行这些评估，所有小鼠均具有相同的遗传背景：1）热量限制（CR）的正常小鼠，2）额外过氧化氢酶仅易位至其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）敲除的小鼠（在所有情况下，小鼠将与适当的对照组匹配）。在另外的分组中，将对具有转基因的小鼠和具有敲除的小鼠施加CR，以确定它们各自的作用是以相加、协同还是对抗模式起作用。主要研究者已经在这3种模型中确定，CR显著延迟了白内障的发展，额外的过氧化氢酶的线粒体易位也是如此，GPx-1的敲除导致白内障发展更早和更晚期。此外，在两项初步研究中，显示老年小鼠LEC中线粒体的膜电位大幅降低。因此，背景已经为研究线粒体在LEC内自身造成的和更广泛的细胞氧化损伤中的作用、其在内部透镜蛋白质改变中的意义以及在白内障的发展中的作用做好了准备。这些研究有望阐明线粒体效率下降和由此产生的透镜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.