Role of Siah1 in ethanol-induced apoptosis and teratogenesis

描述(由申请人提供)：产前酒精暴露是导致智力低下的主要已知原因。越来越多的证据表明，细胞过度死亡是酒精所致出生缺陷发病机制的主要组成部分。然而，在理解乙醇如何导致胚胎细胞凋亡方面存在着根本性的差距。这个缺口的持续存在代表着一个重要的问题，因为在这个缺口被填补之前，对酒精诱导的导致畸形的细胞凋亡的理解在很大程度上仍然是不可理解的。我们的长期目标是开发有效的策略来对抗乙醇的致畸作用；这些策略的基础是通过靶向参与细胞凋亡的特定蛋白来预防酒精诱导的细胞凋亡。这项特殊建议的总体目标是建立七个缺席同源家族的成员Siah1，作为预防乙醇诱导的细胞凋亡和致畸的可行靶点。有待检验的中心假设是，Siah1通过激活P53途径介导乙醇诱导的神经脊细胞(NCC)的凋亡，抑制Siah1介导的凋亡可以防止乙醇诱导的畸形发生。我们的假设是基于我们实验室提供的强有力的初步数据而提出的。为了验证我们的假设，将解决以下具体目标：Aim1：表征Siah1在乙醇诱导的NCCs凋亡中的作用。我们将确定乙醇对Siah1基因和蛋白表达的影响，并探讨乙醇促进Siah1核转位和诱导细胞凋亡的可能性。目的：验证Siah1通过激活P53通路介导乙醇诱导神经干细胞凋亡的假说。这将通过确定Siah1在乙醇诱导的P53信号通路的激活以及在诱导P53下游促凋亡蛋白和细胞凋亡中的作用来实现。目的：明确抑制Siah1功能在乙醇致畸保护中的作用。这将通过使用全胚胎培养、Siah1基因敲除小鼠和口服摄入FASD模型来完成。我们将确定敲除Siah1是否能减少培养的小鼠胚胎中乙醇诱导的畸形，以及Siah1-/-小鼠胚胎中Siah1缺乏是否可以在体内对乙醇诱导的畸形产生保护作用。这项拟议的工作具有创新性，因为它专注于一种新的方法，针对直接参与细胞凋亡途径的特定蛋白，以防止乙醇诱导的细胞凋亡和致畸。申请中描述的理论概念也具有很高的创新性，因为这是第一次试图通过新发现的Siah1在细胞凋亡中的作用来防止乙醇诱导的细胞凋亡和致畸作用。这项研究的结果将是重要的，因为这些目标的实现所获得的洞察力将有助于阐明Siah1在介导乙醇诱导的畸形发生中的作用。预计他们还将提出预防乙醇致畸的策略，并从根本上推进FASD研究领域。

DESCRIPTION (provided by applicant): Prenatal ethanol exposure is the leading known cause of mental retardation. Growing evidence suggests that excessive cell death is a major component of the pathogenesis of ethanol-induced birth defects. However, there is a fundamental gap in understanding how ethanol leads to apoptotic cell death in embryos. Continued existence of this gap represents an important problem because, until it is filled, understanding of ethanol- induced apoptosis that leads to teratogenesis will remain largely incomprehensible. Our long-term goal is directed toward the development of effective strategies against ethanol's teratogenesis; strategies based on prevention of ethanol-induced apoptosis through targeting specific proteins involved in apoptosis. The overall objective of this particular proposal is to establish Siah1, a member of the seven in absentia homology family, as a feasible target for the prevention of ethanol-induced apoptosis and teratogenesis. The central hypothesis to be tested is that Siah1 mediates ethanol-induced apoptosis in neural crest cells (NCCs) by activating the p53 pathway and that the inhibition of Siah1-mediated apoptosis can prevent ethanol-induced teratogenesis. Our hypothesis has been formulated on the basis of strong preliminary data produced in our laboratory. To test our hypothesis, the following specific aims will be addressed: Aim1: To characterize the role of Siah1 in ethanol-induced apoptosis in NCCs. We will determine the effects of ethanol on Siah1 mRNA and protein expression, and investigate the potential of ethanol to promote Siah1 nuclear translocation and induce apoptosis. Aim2: To test the hypothesis that Siah1 mediates ethanol-induced apoptosis in NCCs by activating the p53 pathway. This will be accomplished by determining the role of Siah1 in ethanol-induced activation of p53 signaling pathway and in the induction of p53 downstream proapoptotic proteins and apoptosis. Aim3: To define the role of the inhibition of Siah1 function in conferring protection against ethanol-induced teratogenesis. This will be accomplished by using whole embryo culture, Siah1 knockout mice and an oral intake FASD model. We will determine whether knocking down Siah1 diminishes ethanol-induced malformations in cultured mouse embryos and whether Siah1 deficiency in Siah1-/- mouse embryos can confer in vivo protection against ethanol-induced teratogenesis. The proposed work is innovative, because it focuses on a novel approach, targeting specific proteins directly involved in the apoptotic pathway, to preventing ethanol-induced apoptosis and teratogenesis. The theoretical concept described in the application is also highly innovative because this is the first study attempting to prevent ethanol-induced apoptosis and teratogenesis specifically through the newly recognized actions of Siah1 in apoptosis. The results from this study will be significant, because the insights gained by the accomplishment of these aims will help in elucidating the role of Siah1 in mediating ethanol- induced teratogenesis. They are also expected to yield strategies for the prevention of ethanol's teratogenesis and to fundamentally advance the field of FASD research.