mTOR as a trophoblast folate sensor

描述（由申请人提供）：叶酸对胎儿的正常发育和生长至关重要。围孕期叶酸缺乏与神经管缺陷有关，母体叶酸水平低与胎儿生长受限有关。此外，改变叶酸的可利用性已被牵连在胎儿编程。叶酸是合成嘌呤和胸苷核苷酸所必需的，这是DNA复制和修复所必需的。此外，叶酸是DNA甲基化的关键甲基供体，DNA甲基化是表观遗传调控的关键机制。虽然受损的DNA合成/修复和基因甲基化通常被认为可以解释叶酸缺乏和不良妊娠结局之间的联系，但机制联系仍然难以捉摸。此外，介导和调节胎盘叶酸转运的机制知之甚少。雷帕霉素靶蛋白（mTOR）信号通路通过调节细胞内营养物质和生长因子的水平来调控细胞的生长、增殖和代谢。mTOR存在于两种复合物中，mTOR复合物1（mTORC 1）和mTORC 2，其具有不同的上游调节剂和下游靶点。mTORC 1是一种氨基酸传感器，我们最近报道滋养层mTOR信号是氨基酸转运的正调控因子。在这个项目中，我们将探索一种新的机制，它提供了叶酸的可用性和细胞生长和功能之间的新联系。核心假设是mTOR调节滋养层叶酸摄取，并作为质子偶联叶酸转运蛋白（PCFT）介导的叶酸传感器发挥作用。具体目标：（1）确定mTOR信号传导在调节滋养层叶酸摄取中的作用，（2）建立mTOR信号传导在滋养层叶酸传感中的作用，和（3）鉴定将叶酸可用性与滋养层mTOR信号传导联系起来的机制。方法：我们将在原代人滋养层细胞中使用药理学和基因沉默方法来确定mTORC 1和2的抑制/激活对细胞叶酸摄取的影响，并确定相关机制。我们将进一步探讨叶酸缺乏和重新引入对对照细胞和PCFT沉默细胞中mTORC 1和mTORC 2信号传导的影响。将使用成像方法和邻近连接测定法来确定mTOR、PCFT和其他蛋白质（如已显示参与mTOR的氨基酸感测的Rag和Ragulator）的亚细胞定位和共定位。此外，我们将研究妊娠小鼠母体叶酸缺乏对胎盘mTOR信号传导、叶酸和氨基酸转运以及胎儿生长的影响。重要性：这项工作解决了一个主要的知识差距，并将导致识别的机制，通过介导和调节经胎盘的叶酸转运和叶酸的可用性如何调节滋养层细胞的生长和功能。此外，拟议的研究将帮助我们更好地了解母体叶酸状态与胎儿生长发育之间的分子联系。创新：我们的中心假设在概念上是新颖和创新的，因为在任何细胞类型或组织中尚未报道mTOR和/或PCFT在细胞叶酸传感中的作用。

DESCRIPTION (provided by applicant): Folate is critical for normal fetal development and growth. Periconceptional folate deficiency is associated with neural tube defects and low maternal folate levels are linked to restricted fetal growth. In addition, altered folate availabilty has been implicated in fetal programming. Folate is essential for the synthesis of purine and thymidine nucleotides, which are needed for DNA replication and repair. In addition, folate is a critical methyl donor for DNA methylation, which is a key mechanism of epigenetic regulation. Although impaired DNA synthesis/repair and gene methylation are generally believed to explain the association between folate deficiency and poor pregnancy outcomes, the mechanistic links remain elusive. In addition, the mechanisms mediating and regulating placental folate transport are poorly understood. The mechanistic target of rapamycin (mTOR) signaling pathway responds to changes in nutrient availability and growth factor signaling to control cell growth, proliferation and metabolism. mTOR exists in two complexes, mTOR Complex 1 (mTORC1) and mTORC2, which have distinct upstream regulators and downstream targets. mTORC1 is an amino acid sensor and we recently reported that trophoblast mTOR signaling is a positive regulator of amino acid transport. In this project we will explore a new mechanism, which provides a novel link between folate availability and cell growth and function. The central hypothesis is that mTOR regulates trophoblast folate uptake and functions as a folate sensor mediated by the proton-coupled folate transporter (PCFT). Specific Aims: (1) Determine the role of mTOR signaling in regulating trophoblast folate uptake, (2) Establish the role of mTOR signaling in trophoblast folate sensing and (3) Identify the mechanism linking folate availability o trophoblast mTOR signaling. Approach: We will use pharmacological and gene-silencing approaches in primary human trophoblast cells to determine the effect of inhibition/activation of mTORC1 and 2 on cellular folate uptake, and identify the mechanisms involved. We will further explore the effects of folate deficiency and re-introduction on mTORC1 and mTORC2 signaling in control cells and in PCFT silenced cells. Subcellular localization and co- localization of mTOR, PCFT and other proteins, such as Rag and Ragulator that have been shown to participate in amino acid sensing by mTOR, will be determined using imaging approaches and proximity ligation assay. In addition, we will study the effect of maternal folate deficiency in pregnant mice on placental mTOR signaling, folate and amino acid transport and fetal growth. Significance: This work addresses a major gap in knowledge and will lead to the identification of mechanisms by which transplacental folate transport is mediated and regulated and how folate availability modulates trophoblast growth and function. Moreover, the proposed research will help us better understand the molecular links between maternal folate status and fetal growth and development. Innovation: Our central hypothesis is conceptually novel and innovative because a role of mTOR and/or PCFT in cellular folate sensing has not been reported in any cell type or tissue.