Genetic Regulation of Glutathione Redox Balance in Mice

描述（由申请方提供）：谷氨酰胺是一种三肽分子，是细胞中主要的内源性抗氧化剂。它是丰富的，并参与了广泛的保护性抗氧化和解毒机制。稳态机制使谷胱甘肽主要保持在其还原态（GSH）; GSH的氧化导致其氧化二聚体形式GSSG的积累。因此，检查GSH：GSSG的比率是氧化应激和氧化还原维持的良好指标。许多慢性衰老疾病都有氧化应激成分，导致GSH：GSSG显著下降，使细胞和组织更容易受到进一步的损害。虽然调节谷胱甘肽平衡的生化途径是明确的，但该参数的遗传调节不是，尽管研究结果表明谷胱甘肽平衡在哺乳动物中受遗传学调节。目前的项目确定了调节小鼠谷胱甘肽平衡的基因。这项工作可能揭示了在疾病中维持谷胱甘肽平衡的疗法的新靶点，这将有可能改善人类患者的治疗，预后和生活质量。 我们的实验策略是通过定量GSH和GSSG浓度并计算几种组织中GSH：GSSG来证实C57 BL/6（B6）和DBA/2（D2）小鼠之间存在谷胱甘肽平衡的差异。然后，我们将分析从B6 x D2杂交（BXD小鼠）产生的重组近交系小鼠品系，以进行QTL分析，并在小鼠中找到负责GSH：GSSG的基因座。同时，我们将进行一项涉及10个额外近交系的菌株调查，包括4个将为本研究提供显著遗传变异的野生衍生菌株。菌株 将非常不同的GSH：GSSG杂交并产生F2后代;然后进行QTL分析以确认用BXD小鼠鉴定的基因座。 目的1测试肝脏、肾脏、心脏和大脑中GSH：GSSG比率（以及[GSH]和[GSSG]的绝对值）的遗传调节是否导致至少10个重组近交系BXD小鼠之间的显著变化。如果这一假设是正确的，我们将使用完整的RI线分析在60个额外的BXD菌株，以确定基因座，并提出候选基因调节谷胱甘肽平衡。 目的2定义了4-6月龄的12个近交系小鼠的肝、肾、心和脑中谷胱甘肽的平衡。这组12种菌株包括4种野生来源的菌株，以最大限度地提高遗传变异和在小鼠中找到谷胱甘肽平衡的遗传调节因子的机会。我们将与高和低GSH：GSSG的小鼠品系杂交以产生F2子代并鉴定调控基因座。 目的3比较了来自12个遗传多样性近交系的中年、14- 15月龄小鼠和3-4月龄对照小鼠的肝脏、肾脏、心脏和大脑中的谷胱甘肽平衡。我们将对在衰老方面具有重要差异的菌株进行交叉，以确定导致这种差异的基因座。在所有目标中，基因鉴定将建议临床治疗。

DESCRIPTION (provided by applicant): Glutathione, a tripeptide molecule, is the primary endogenous antioxidant in cells. It is abundant and participates in a wide array of protective antioxidant and detoxification mechanisms. Homeostatic mechanisms maintain glutathione predominantly in its reduced state (GSH); oxidation of GSH causes the accumulation of its oxidized dimer form GSSG. Thus, examining the ratio of GSH:GSSG is a good indicator of oxidative stress and redox maintenance. Many chronic diseases of aging have oxidative stress components that cause significant declines in GSH:GSSG, rendering cells and tissues more susceptible to further damage. Although the biochemical pathways that regulate glutathione balance are well-defined, the genetic regulation of this parameter is not, despite findings suggesting that glutathione balance is regulated by genetics in mammals. The current project identifies genes regulating glutathione balance in mice. This work may reveal novel targets for therapies that maintain glutathione balance in disease, which would have the potential in humans to improve patient treatment, prognosis, and quality of life. Our experimental strategy is to confirm that differences in glutathione balance exist between C57BL/6 (B6) and DBA/2 (D2) mice by quantifying GSH and GSSG concentrations and calculating GSH:GSSG in several tissues. We will then analyze recombinant inbred mouse strains produced from B6 x D2 crosses (BXD mice) in order to perform QTL analysis and find loci responsible for GSH:GSSG in mice. Concurrently, we will perform a strain survey involving 10 additional inbred strains, including 4 wild-derived strains that will contribute significant genetic variation to this study. Strains with very different GSH:GSSG will be crossed and F2 progeny will be produced; QTL analysis will then proceed to confirm the loci identified with the BXD mice. Aim 1 tests if genetic regulation o GSH:GSSG ratios (as well as absolute values of [GSH] and [GSSG]) in liver, kidney, heart, and brain causes significant variation among at least 10 recombinant inbred BXD lines of mice. If this hypothesis is correct, we will use complete RI line analysis in 60 additional BXD strains to identify loci and suggest candidate genes regulating glutathione balance. Aim 2 defines glutathione balance in liver, kidney, heart, and brain in each of 12 inbred strains of mice aged 4-6 months. The set of 12 strains includes 4 wild-derived strains to maximize genetic variance and the chance of finding genetic regulators of glutathione balance in mice. We will cross mouse strains with higher and lower GSH:GSSG to produce F2 progeny and identify regulatory loci. Aim 3 compares glutathione balance in liver, kidney, heart, and brain of inbred mice in middle-aged, 14- 15 month old mice, and in young adult 3-4 month old control mice from 12 genetically diverse inbred strains. We will cross strains with important differences in aging to identify loci responsible for this difference. In all aims, gene identification will suggest clinical treatments.