Project 2. Quantitative Assessment of Oxidative Stress and 1-C Trafficking Defects as a Basis for NTD Risk

摘要项目2：氧化应激和1-C转运缺陷的定量评估 NTD风险的基础 母体补充叶酸预防神经管畸形的广泛疗效 (NTDS)，以及四氢叶酸(THF)是细胞内一碳(1-C)的重要辅助因子 对于转移反应，人们可以推断，NTDS很大程度上源于1-C转运反应中FA可逆的缺陷。 对这一观点的挑战是，有缺陷的1-C交易与&gt；250基因中的极少数有关 在小鼠模型中，其突变会导致神经管畸形。FH4也是一种有效的抗氧化剂分子，因为 建议活性氧/氮物种(RON)增加NTD风险，我们假设保护 针对RONS是FA预防非TD行动的关键贡献者。过去资助期间产生的数据 循环为这一观点提供了证据，然而，迄今为止缺乏实验工具排除了严格的 作为基因基本分子基础的1-C转运缺陷与Rons过剩的区分 突变相关的NTDS。还需要这样的工具来确定NTD预防措施的程度 母体补充FA的效果来自对1-C转运缺陷和RONS诱发的保护作用 异常现象。为了克服这一发现的障碍，我们寻求建立和采用新的战略， 将首次允许对体外模型中的1-C通量和氧化还原状态进行无偏见/无针对性的调查 小鼠神经管畸形。这些新工具将应用于成熟的小鼠NTD模型，以及新的 在项目3中开发了NTD的小鼠模型，最重要的是，调查1-C贩运和 WGS在项目1中确定的人类脊柱裂候选基因突变的细胞氧化还原后果 并被牵连为NTDS的潜在驱动因素。在计划续签中，我们提出了以下目标：(1) 建立和应用一种新的非靶向稳定同位素示踪技术来严格定义1-C贩运 小鼠NTD模型中的缺陷。(2)建立和使用一种新的氧化还原组方法来测量和量化 NTD小鼠模型中可能发生的氧化还原活性分子水平的变化。(3)评估程度 与脊柱裂相关的人类罕见SNP(在项目1中确定)与异常的1-C有关 贩卖人口和罗恩。此外，我们试图确定在NTD中引起额外衰减的分子 通过对抗氧化应激或克服1-C转运缺陷而流行，添加或不添加5-C- 甲基四氢呋喃在体外NTD模型中的应用。我们的总体假设是：1-C交易缺陷和RONS应力是 由线粒体叶酸/甲酸循环相互联系，两者都有助于 FA可预防的NTDS。对神经形成过程中这些关联过程的分子理解， 在本文开发的新分析工具的推动下，承诺对NTD原因和 预防。

ABSTRACT PROJECT 2: Quantitiative Assessment of Oxidative Stress and 1-C Trafficking Defects as a Basis for NTD Risk From the broad efficacy of maternal folic acid (FA) supplementation for prevention of neural tube defects (NTDs), and knowledge that tetrahydrofolate (THF) is an essential cofactor for cellular one-carbon (1-C) transfer reactions, one may infer that NTDs largely stem from FA-reversible defects in 1-C trafficking reactions. A challenge to this view is that defective 1-C trafficking has been associated with very few of the >250 genes whose mutations cause NTD in murine models. FH4 is also an efficient antioxidant molecule, and since reactive oxygen/nitrogen species (RONS) are suggested to increase NTD risk, we hypothesized that protection against RONS is a key contributor to the NTD-preventing action of FA. Data generated during the past funding cycle provided evidence for this view, however a lack of experimental tools to date has precluded the rigorous discrimination between 1-C trafficking defects vs. RONS excess as fundamental molecular bases for gene mutation associated NTDs. Such tools are also needed to ascertain the extent to which the NTD-preventative effects of maternal FA supplementation arise from protection against 1-C trafficking defects vs. RONS-evoked abnormalities. To overcome this roadblock to discovery, we seek to establish and employ novel strategies that will allow for the first time, an unbiased/untargeted survey of both 1-C flux and redox status in ex vivo models of murine NTDs. These new tools will be applied to well-established murine NTD models, as well as new mouse models of NTD developed in Project 3, and most importantly, to investigate the 1-C trafficking and cellular redox consequences of human spina bifida candidate gene mutations, identified in Project 1 by WGS and implicated as potential drivers of NTDs. In the Program renewal, we propose the following goals: (1) establish and employ a novel untargeted stable isotope tracing technology to rigorously define 1-C trafficking defects in mouse NTD models. (2) establish and employ a novel redoxome approach to survey and quantify changes in the levels of redox-active molecules that may occur in mouse models of NTD. (3) Assess the extent to which spina bifida-associated rare SNPs in humans (identified in Project 1) contribute to aberrant 1-C trafficking and RONS. Further, we seek to identify molecules that elicit an additional attenuation in NTD prevalence by opposing oxidative stress or overcoming 1-C trafficking defects, with or without added 5- Methyl-THF in ex vivo NTD models. Our overall hypothesis: 1-C trafficking defects and RONS stress are interlinked by the mitochondrial folate/formate cycle and both contribute to the mechanistic basis for FA-preventable NTDs. A molecular understanding of these linked processes during neurulation, facilitated by new analytical tools developed herein, promise new insight into NTD causes and prevention.