The Effects of Ethanol on Microtubules during Mouse Pre-implantation Development

乙醇对小鼠植入前发育过程中微管的影响

• 批准号: 8209221
• 负责人: Yixian Zheng
• 金额: $ 18.53万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8209221
• 关键词: Acetaldehyde; Acetylation; Affect; Alcohols; Arteries; Binding; Biological Assay; Biological Markers; Cell Adhesion; Cell Polarity; Cell Separation; Cell division; Cells; Deacetylation; Development; Developmental Gene; Diagnosis; Disease; Embryo; Endometrium; Ensure; Ethanol; Ethanol Metabolism; Event; Exposure to; Fetal Alcohol Spectrum Disorder; Fetal Growth; Fetus; Fibroblasts; HDAC6 gene; Histone Deacetylase; Image; Impairment; Implant; In Vitro; Invaded; Lead; Life; Link; Litter Size; Lysine; Microtubules; Modeling; Molecular; Molecular Target; Mus; NADH; Necrosis; Pathway interactions; Phenotype; Placenta; Placentation; Pregnancy; Production; Publishing; Reporting; Role; Staging; Therapeutic; Tubulin; Woman; adduct; alcohol effect; alcohol exposure; alcohol measurement; cell motility; cost; dietary supplements; drinking; embryonic stem cell; feeding; implantation; in vivo; migration; novel therapeutics; outcome forecast; pregnant; preimplantation; prevent; protein transport; public health relevance; research study; trophoblast

DESCRIPTION (provided by applicant): Fetal Alcohol Spectrum Disorders (FASD) affect as many as 9 out of 1,000 pregnancies in the US, and up to 40% of women report consuming some level of alcohol during their pregnancies. Numerous studies have been published characterizing the effect of ethanol on a developing fetus. However, the specific mechanisms have been proven more difficult to establish. The role of the placenta, for example, has been well established in FASD yet there is a lack of definitive biomarkers to define placental phenotypes resulting from ethanol exposure. We propose to study the earliest stages of placental formation, specifically at the level of trophectoderm differentiation, to establish what effect ethanol may have on this critical stage of development. In order to understand how alcohol could affect the motility and establishment of the trophectoderm, we will study the effect of ethanol on the cellular microtubule network. Comprised of 1- and 2-tubulin subunits, microtubule assembly are tightly regulated to ensure proper execution of intracellular organization, protein trafficking, cell migration, and cell division. Acetylation of 1-tubulin at its lysine-40 residue has been shown to affect microtubule assembly dynamics and cell motility. The NAD-dependent sirtuin SIRT2 and the histone deacetylase HDAC6 are responsible for regulating this acetylation. The metabolism of ethanol produces both NADH and acetaldehyde at the cost of NAD+. We propose that ethanol may directly interfere with the ability of SIRT2 and HDAC6 to deacetylate 1-tubulin via the production of acetaldehyde and cellular-depletion of NAD+, thus affecting the overall microtubule network. Using live-imaging, FRAP analysis, in vivo and in vitro mouse pre-implantation models, migration and cell-sorting assays, we will investigate how cell polarity and migration are affected by ethanol in mouse embryonic fibroblasts, mouse embryonic stem cells, and pre-implantation mouse embryos. Furthermore we will establish the effect of ethanol on the microtubule network as well as the mechanism of inhibition of SIRT2/HDAC6 deacetylation. We propose the following specific aims: 1) we will examine how cell polarity and migration are affected by the ethanol-induced disruption of the SIRT2/HDAC6-dependent deacetylation of 1-tubulin; 2) we will Investigate the impact of ethanol on the pre-implantation mouse embryo in regard to cell polarity, migration, and the expression of key developmental genes; 3) we will establish a direct mechanistic link between ethanol, SIRT2, HDAC6, and the deacetylation of 1-tubulin. These experiments will allow us to establish a specific mechanism for the ethanol-induced impairment of implantation and placentation. PUBLIC HEALTH RELEVANCE: Fetal Alcohol Spectrum Disorders affects as many as 9 out of 1,000 pregnancies in the US, and up to 40% of women report consuming some level of alcohol during their pregnancies. The proposed project studies the molecular mechanism for ethanol-induced impairment of fetal growth and will lead to better therapeutic strategies and advisory practices for women who drink while pregnant or while they may become pregnant.

描述（由申请人提供）：胎儿酒精谱系障碍（FASD）影响美国1,000例妊娠中的9例，高达40%的女性报告在怀孕期间摄入一定程度的酒精。已经发表了许多研究，描述了乙醇对发育中的胎儿的影响。然而，具体机制已被证明较难建立。例如，胎盘在FASD中的作用已经得到了很好的确立，但缺乏明确的生物标志物来定义乙醇暴露导致的胎盘表型。我们建议研究胎盘形成的最早阶段，特别是在滋养外胚层分化的水平，以确定乙醇可能对这个关键的发展阶段产生什么影响。 为了了解酒精如何影响滋养外胚层的运动和建立，我们将研究酒精对细胞微管网络的影响。由1-和2-微管蛋白亚基组成，微管组装受到严格调控，以确保细胞内组织、蛋白质运输、细胞迁移和细胞分裂的正确执行。1-微管蛋白在其赖氨酸-40残基的乙酰化已被证明会影响微管组装动力学和细胞运动性。NAD依赖性沉默调节蛋白SIRT 2和组蛋白脱乙酰酶HDAC 6负责调节这种乙酰化。乙醇的代谢以NAD+为代价产生NADH和乙醛。我们提出，乙醇可能直接干扰SIRT 2和HDAC 6的能力，通过生产乙醛和NAD+的细胞耗竭1-微管蛋白脱乙酰化，从而影响整个微管网络。使用实时成像，FRAP分析，在体内和体外小鼠植入前模型，迁移和细胞分选试验，我们将研究如何细胞极性和迁移的影响，乙醇在小鼠胚胎成纤维细胞，小鼠胚胎干细胞，和植入前小鼠胚胎。此外，我们还将建立乙醇对微管网络的影响以及抑制SIRT 2/HDAC 6脱乙酰化的机制。我们提出了以下具体目标：1）我们将研究乙醇诱导的1-微管蛋白SIRT 2/HDAC 6依赖性脱乙酰化的破坏如何影响细胞极性和迁移; 2）我们将研究乙醇对植入前小鼠胚胎细胞极性、迁移和关键发育基因表达的影响; 3）我们将在乙醇、SIRT 2、HDAC 6和1-微管蛋白的脱乙酰化之间建立直接的机制联系。这些实验将使我们能够建立一个特定的机制，乙醇诱导的损害植入和胎盘。 公共卫生相关性：在美国，胎儿酒精谱系障碍影响多达1，000例妊娠中的9例，高达40%的女性报告在妊娠期间摄入一定程度的酒精。拟议的项目研究了乙醇诱导的胎儿生长障碍的分子机制，并将为怀孕期间或可能怀孕期间饮酒的妇女提供更好的治疗策略和咨询实践。

项目成果

Generation and live imaging of an endogenous Cdx2 reporter mouse line.

• DOI: 10.1002/dvg.22049
• 发表时间: 2012-10
• 期刊: GENESIS
• 影响因子: 1.5
• 作者: McDole, Katie;Zheng, Yixian
• 通讯作者: Zheng, Yixian

A role for borg5 during trophectoderm differentiation.

• DOI: 10.1002/stem.428
• 发表时间: 2010-06
• 期刊: STEM CELLS
• 影响因子: 5.2
• 作者: Vong, Queenie P.;Liu, Zhonghua;Yoo, Jae Gyu;Chen, Rong;Xie, Wen;Sharov, Alexei A.;Fan, Chen-Ming;Liu, Chengyu;Ko, Minoru S. H.;Zheng, Yixian
• 通讯作者: Zheng, Yixian