Mechanisms of Vitamin E Function Studied in Zebrafish

维生素E于1922年被发现是生殖和生殖所必需的脂溶因子。 名为-生育酚，源自希腊语意为“出生”，意为“携带”。迄今为止，- 生育酚在生殖中的作用一直不可能通过实验来接近，因为- 生育酚缺乏(E-)的母亲由于植入失败而不能产生胚胎组织。 为了绕过这一障碍，我们选择了使用顶级脊椎动物模型进行研究 斑马鱼的发育，因为它们产卵；因此，植入是不必要的。这个 斑马鱼模型是我们研究的理想模型，因为它和人类一样，对- 生育酚，表达ttp基因，需要维生素C，这是的重要决定因素- 生育酚在人体内的抗氧化功能。这一模式相对于传统模式的另一个优势 啮齿动物转基因模型是任何基因都可以在胚胎发生过程中被击倒，一种 我们计划利用的功能。我们已经成功地开发出了第一个明确的斑马鱼饮食。 使用我们的E定义饮食，我们能够生产E-鸡蛋。受精后48小时(HPF)， 许多E-胚胎表现出严重的发育畸形，这是第一次 -生育酚对胎儿神经和心血管发育所需的时间，而不仅仅是 植入。我们还发现-生育酚转运蛋白(-ttp)含量丰富。 在48HPF斑马鱼胚胎中表达，其表达随着氧化应激的增加而增加。 患有TTP的人出现严重的维生素E缺乏并进行性神经变性 基因缺陷，强调了这种蛋白质的关键重要性。-ttp在细胞中的表达 斑马鱼胚胎表明，在发育过程中，-ttp直接从蛋黄中产生-生育酚。 囊泡在发育中胚胎中特别需要的特定位置。我们建议 需要-生育酚来保护关键的氧化脂质介质免受进一步氧化 退化在特定发育阶段，尤其是神经系统中的退化 具体地说，谷胱甘肽过氧化物酶4(Gpx4)，磷脂氢过氧化物的解毒剂， 将氧化应激转化为由12/15-脂氧合酶介导的细胞死亡(12/15 -生育酚在体外可预防这些异常。 我们假设α-生育酚为特定的、关键的脂质提供抗氧化保护 胚胎发育所必需的介体，包括通过程序性细胞死亡造成的细胞损失。 为了解释-生育酚缺乏症的关键发育影响，我们提出了以下建议 目的1.明确氧化和抗氧化细胞凋亡调节因子在胚胎发育过程中的作用 目的2.明确-TTP在胚胎发育中的作用。完成 这些特定的目标将使我们能够确定特定的-生育酚分子功能和关键 抗氧化剂/氧化剂信号传导机制。定义由改变的分子靶点- 斑马鱼中缺乏生育酚将为理解为什么人类 生殖所需的-生育酚以及维持健康的神经系统

Vitamin E was discovered in 1922 as a lipid soluble factor necessary for reproduction and named -tocopherol from the Greek words for “birth” and for “to carry”. To date, - tocopherol’s role in reproduction has been impossible to approach experimentally because - tocopherol-deficient (E-) mothers do not produce embryonic tissues due to failed implantation. To circumvent this barrier, we have chosen to use the premier vertebrate model for studying development, the zebrafish, because they lay eggs; thus, implantation is not necessary. The zebrafish model is ideal for our studies because it, like humans, has a preference for - tocopherol, expresses the ttp gene and requires vitamin C, an important determinant in - tocopherol antioxidant function in humans. An additional strength of this model over traditional rodent transgenic models is that any gene can be knocked down during embryogenesis, a feature we plan to exploit. We have succeeded in developing the first defined zebrafish diet. Using our E- defined diet, we are able to produce E- eggs. By 48 hours post-fertilization (hpf), many E- embryos exhibit severe developmental malformations, which establishes for the first time that -tocopherol is required for fetal neurological and cardiovascular development, not just implantation. We also found that the -tocopherol transfer protein -TTP) is abundantly expressed by the 48 hpf zebrafish embryo and its expression increases with oxidative stress. Severe vitamin E deficiency with progressive neurologic degeneration occurs in humans with ttp gene defects, emphasizing the critical importance of this protein. -TTP expression in the zebrafish embryo suggests that during development -TTP directs -tocopherol from the yolk sac to specific sites in the developing embryo where it is especially required. We propose that -tocopherol is required to protect key oxidized lipid mediators from further oxidative degradation during specific developmental steps, especially those in the nervous system. Specifically, glutathione peroxidase 4 (GPx4), a detoxifier of phospholipid hydroperoxides, translates oxidative stress into cell death that is mediated by both 12/15-lipoxygenase (12/15 LOX) and apoptosis-inducing factor (AIF); -tocopherol prevents these abnormalities in vitro. We hypothesize that α-tocopherol provides antioxidant protection for specific, key lipid mediators necessary for embryonic development, including cell loss via programmed cell death. To explain the key developmental effects of -tocopherol deficiency, we propose the following Aim 1. Define the roles of oxidant and antioxidant apoptosis regulators during embryonic development; Aim 2. Define the role of -TTP during embryonic development. Completion of these specific aims will allow us to determine specific -tocopherol molecular functions and key antioxidant/oxidant signaling mechanisms. Defining the molecular targets altered by - tocopherol-deficiency in the zebrafish will provide a solid basis for understanding why humans require -tocopherol for reproduction as well as to maintain a healthy nervous system