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

项目2：定量评估氧化应激和1-C运输缺陷， a新台币风险的基础 从母体补充叶酸（FA）预防神经管缺陷的广泛疗效来看， （NTDs），以及四氢叶酸（THF）是细胞一碳（1-C） 在转移反应中，可以推断NTD很大程度上源于1-C运输反应中的FA可逆缺陷。 对这一观点的一个挑战是，有缺陷的1-C运输与>250个基因中的极少数相关。 其突变在鼠模型中引起NTD。FH 4也是一种有效的抗氧化分子， 活性氧/氮类（RONS）被认为是增加NTD的风险，我们假设，保护 对RONS是一个关键的贡献NTD预防行动的FA。过去供资期间生成的数据 循环为这一观点提供了证据，然而，迄今为止缺乏实验工具， 1-C运输缺陷与RONS过量作为基因的基本分子基础之间的区别 突变相关的NTDs。还需要这些工具来确定NTD预防措施在多大程度上 母体补充FA的效果来自于对1-C运输缺陷的保护， 异常为了克服这一发现的障碍，我们寻求建立和采用新的策略， 将首次允许在离体模型中对1-C通量和氧化还原状态进行无偏/非靶向调查 鼠NTDs的研究这些新工具将应用于完善的小鼠NTD模型，以及新的 在项目3中开发的NTD小鼠模型，最重要的是，研究1-C的贩运， WGS在项目1中鉴定的人类脊柱裂候选基因突变的细胞氧化还原后果 并被认为是NTD的潜在驱动因素。在项目更新中，我们提出以下目标：（1） 建立并采用一种新的非靶向稳定同位素示踪技术，以严格界定1-C的运输 小鼠NTD模型的缺陷。(2)建立并使用一种新氧化还原体方法来测量和量化 可能发生在NTD小鼠模型中的氧化还原活性分子水平的变化。(3)评估在多大程度 人类脊柱裂相关的罕见SNP（在项目1中鉴定）导致异常1-C 贩卖人口和RONS此外，我们试图确定引起NTD额外衰减的分子， 通过对抗氧化应激或克服1-C运输缺陷（添加或不添加5-C）来流行 离体NTD模型中的甲基-THF。我们的总体假设：1-C运输缺陷和RONS压力是 通过线粒体叶酸/甲酸循环相互联系，两者都有助于 FA可预防的NTD。对神经形成过程中这些相关过程的分子理解， 在此开发的新分析工具的推动下，有望对NTD的原因有新的见解， 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.