Genetic Regulation of Glutathione Redox Balance in Mice

描述(申请人提供)：谷胱甘肽是一种三肽分子，是细胞内主要的内源性抗氧化剂。它的含量丰富，并参与了广泛的保护性抗氧化和解毒机制。动态平衡机制维持谷胱甘肽主要处于还原状态(GSH)；GSH的氧化导致其氧化二聚体积累形成GSSG。因此，检测GSH/GSSG的比率是氧化应激和氧化还原维持的一个很好的指标。许多慢性衰老疾病都有氧化应激成分，会导致GSH：GSSG显著下降，使细胞和组织更容易受到进一步的损害。尽管调节谷胱甘肽平衡的生化途径是明确的，但这个参数的遗传调节还没有，尽管研究结果表明哺乳动物的谷胱甘肽平衡是由遗传学调节的。目前的项目确定了调节小鼠谷胱甘肽平衡的基因。这项工作可能会揭示维持疾病中谷胱甘肽平衡的治疗的新靶点，这将有可能在人类身上改善患者的治疗、预后和生活质量。我们的实验策略是通过量化GSH和GSSG的浓度并计算几种组织中的GSH：GSSG来证实C57BL/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B6×D2杂交(BXD小鼠)产生的重组近交系小鼠品系，以便进行QTL分析，并找到与GSH：GSSG相关的基因座。同时，我们将对另外10个近交系进行品系调查，其中包括4个野生来源的品系，这些品系将对这项研究产生重大的遗传变异。菌株具有 非常不同的GSH：GSSG将进行杂交，产生F2后代；然后进行QTL分析，以确认与BXD小鼠识别的基因座。目的1检测至少10个重组近交系BXD小鼠肝脏、肾脏、心脏和脑中GSH/GSSG比值(以及[GSH]和[GSSG]绝对值)的遗传调控是否导致显著差异。如果这一假设是正确的，我们将在另外60个BXD菌株中使用完整的RI线分析来识别基因座并建议调节谷胱甘肽平衡的候选基因。目的研究12个近交系4-6月龄小鼠肝脏、肾脏、心脏和脑中谷胱甘肽的平衡。这套12个菌株包括4个野生来源的菌株，以最大限度地增加遗传变异和在小鼠中找到谷胱甘肽平衡的遗传调节器的机会。我们将用高GSH：GSSG和低GSH：GSSG的小鼠品系杂交产生F2后代，并鉴定调节基因座。目的3比较14~15月龄中年近交系小鼠肝、肾、心脏和脑中谷胱甘肽的平衡，以及12个不同遗传类型近交系幼鼠3~4月龄对照小鼠肝、肾、心、脑中谷胱甘肽的平衡。我们将在年龄上有重要差异的菌株之间进行杂交，以确定造成这种差异的基因座。在所有目标中，基因识别将建议临床治疗。 与公共健康相关：谷胱甘肽是一种内源性细胞抗氧化剂，在多种慢性疾病中调节失调，例如糖尿病和阿尔茨海默病；保持适当的谷胱甘肽平衡可以防止或减缓这些疾病的进展。谷胱甘肽的状态似乎受到遗传学的影响，但过去的研究主要集中在生化工具上，而不是遗传工具上。该项目将首次确定基因变异如何影响哺乳动物的谷胱甘肽平衡，这是一项长期努力的一部分，目的是帮助确定各种慢性病的遗传易感性，并指导未来更有效的临床干预措施的设计。

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. PUBLIC HEALTH RELEVANCE: Glutathione is an endogenous cellular antioxidant that becomes dysregulated in a wide variety of chronic disease, for example, diabetes and Alzheimer's disease; maintaining the proper balance of glutathione may prevent or slow progression of these diseases. Glutathione status appears to be affected by genetics, but past research has focused on biochemical rather than genetic tools. This project will, for the first time, identify how genetic variation influences glutathione balance in mammals as part of a long-term effort to help determine the genetic susceptibility to a wide variety of chronic disease and guide future design of more effective clinical interventions.