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

描述（由申请人提供）：在美国，胎儿酒精谱系障碍（FASD）影响了多达千分之九的孕妇，多达40%的女性报告在怀孕期间摄入了一定程度的酒精。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，具体的机制已被证明更难以建立。例如，胎盘在FASD中的作用已经得到了很好的证实，但目前还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上，以确定乙醇在这一关键发育阶段可能产生的影响。为了了解酒精如何影响滋养外胚层的运动和建立，我们将研究乙醇对细胞微管网络的影响。微管由1-微管蛋白和2-微管蛋白亚基组成，它们的组装受到严格调控，以确保细胞内组织、蛋白质运输、细胞迁移和细胞分裂的正常执行。1-微管蛋白赖氨酸-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。nad依赖性sirtuin SIRT2和组蛋白去乙酰化酶HDAC6负责调节这种乙酰化。乙醇的代谢产生NADH和乙醛，代价是NAD+。我们提出，乙醇可能直接干扰SIRT2和HDAC6通过乙醛的产生和NAD+的细胞消耗来使1-微管蛋白去乙酰化的能力，从而影响整个微管网络。通过实时成像、FRAP分析、体内和体外小鼠着床前模型、迁移和细胞分选分析，我们将研究乙醇如何影响小鼠胚胎成纤维细胞、小鼠胚胎干细胞和着床前小鼠胚胎中的细胞极性和迁移。此外，我们将建立乙醇对微管网络的影响以及抑制SIRT2/HDAC6去乙酰化的机制。我们提出以下具体目标：1)我们将研究细胞极性和迁移如何受到乙醇诱导的SIRT2/ hdac6依赖性1-微管蛋白去乙酰化的破坏的影响；2)研究乙醇对小鼠胚胎着床前细胞极性、迁移和关键发育基因表达的影响；3)我们将建立乙醇、SIRT2、HDAC6和1-微管蛋白去乙酰化之间的直接机制联系。这些实验将使我们能够建立乙醇诱导的植入和胎盘损伤的具体机制。

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.