MODELS FOR EXPLORING FREE RADICAL DAMAGE

在正常新陈代谢过程中会产生超氧化物和过氧化氢。 然而，当过量生产时，氧化剂物种可以转化为 通过与游离铁反应生成活性羟基自由基，这可以 降解DNA、蛋白质和膜脂，导致细胞 退化。大脑特别容易受到自由基的影响。 由于其独特的细胞膜成分造成的损害，以及它的高 氧代谢和游离铁的水平。脑细胞已经发育成熟 针对这些氧化剂物种的几种防御措施，包括生产 抗氧化酶和铁的储存在不会催化的形式 反应性自由基的形成。具体地说，抗氧化酶 超氧化物歧化酶将超氧化物还原为过氧化氢，这是 进而被谷胱甘肽过氧化物酶或过氧化氢酶还原为H20。 反过来，铁通常与铁蛋白结合，铁蛋白是主要的铁存储物质。 大脑中的分子。然而，当大脑的平衡被扰乱时 而自由基被允许积累，这会导致铁- 催化羟基自由基的产生和随后的细胞损伤。它 已经推测氧化应激可能在 帕金森病等神经退行性疾病的发病机制 以及正常衰老过程中的细胞退化。考察……的作用 抗氧化剂在防止神经细胞损伤方面，转基因小鼠将被 创造出过量表达铁结合蛋白铁蛋白或抗铁蛋白的 神经元中的氧化物酶谷胱甘肽过氧化物酶或过氧化氢酶 神经元特异性启动子。构造将被评估为其 Northern赋予培养细胞转基因活性的能力 分析、免疫细胞化学、酶分析以及对 铁、MPTP、6-OHDA和H202。在注射进小鼠胚胎后， 构建物的基因组插入将通过聚合酶链式反应和Southern 印迹分析。转基因小鼠将被培育成新的品系和 通过Northern分析检测转基因后代在脑中的表达， 原位杂交、免疫细胞化学和酶分析。 转基因品系将在正常老化过程中进行评估，以确定 丙二醛积聚引起的脂质过氧化，谷氨酰胺引起的蛋白质氧化 合成酶活性，通过GSH/GSSG比率评估的氧化应激，以及 对于细胞丢失或萎缩，以细胞数量和大小衡量 黑质酪氨酸羟化酶免疫细胞化学的比较 与正常对照组比较。纹状体多巴胺及其代谢物的水平， DOPAC，酪氨酸羟基酶活性，~3H-多巴胺结合，以及 将在转基因中评估多巴胺能细胞的数量和大小 与注射MPTP后的对照组进行比较。脑部的变化 在发育过程中的形态，以及在防止 大脑中的氧化应激，探索一种假设，一种基因 这些分子中的一种的增加可能与易感性有关 或防止帕金森病或老化过程中的神经元退化。

Superoxide and hydrogen peroxide are produced during normal metabolism. However, when produced in excess, the oxidant species can be converted by reaction with free iron to reactive hydroxyl radicals which can degrade DNA, proteins, and membrane lipids resulting in cellular degeneration. The brain is particularly susceptible to free-radical damage due to its unique cellular membrane composition, and its high levels of oxygen metabolism and free iron. Brain cells have developed several defense against these oxidant species including production of antioxidant enzymes and storage of iron in forms that will not catalyze formation of reactive radicals. Specifically, the antioxidant enzyme superoxide dismutase reduces superoxide to hydrogen peroxide, which is in turn reduced by either glutathione peroxidase or catalase to H20. Iron, in turn, is normally bound to ferritin, the major iron storage molecule in the brain. However, when balance in the brain is disturbed and free-radicals are allowed to accumulate, this can lead to iron- catalyzed production of hydroxyl radical and subsequent cell damage. It has been postulated that oxidative stress may play a role in the pathogenesis of neurodegenerative diseases like Parkinson Disease (PD) and cellular degeneration during normal aging. To examine the role of antiodixants in preventing neuronal cell damage, transgenic mice will be create which over-express the iron-binding protein ferritin, or the anti- oxidant enzymes glutathione peroxidase or catalase in neurons using neuron-specific promoters. Constructs will be evaluated for their ability to confer transgene activity on cultured cells by Northern analysis, immunocytochemistry, enzyme assays, and for sensitivity to iron, MPTP, 6-OHDA, and H202. Following injection into mouse embryos, genomic insertion of constructs will be confirmed by PCR and Southern blot analysis. Transgenic mice will be bred to create new lines and progeny examined for transgene expression in brain by Northern analysis, in situ hybridization, immunocytochemistry, and enzyme assays. Transgenic lines will be evaluated during normal aging for changes in lipid peroxidation by MDA accumulation, protein oxidation by glutamine synthetase activity, oxidative stress as assessed by GSH/GSSG ratios, and for cell loss or atrophy as measured by cell number and sizes in the substantia nigra using tyrosine hydroxylase immunocytochemistry compared with normal controls. Levels of striatal dopamine and its metabolite, DOPAC, tyrosine hydroxylase activity, 3H-dopamine binding, and dopaminergic cell numbers and sizes will be assessed in transgenic compared to controls following MPTP injection. Changes in brain morphology during development, and in protecting against the effects of oxidative stress in the brain, exploring the hypothesis that a genetic increase in one of these molecules may be involved in predisposition to or protection against PD or neuronal degeneration during aging.