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.
Folate deficiency in pregnancy is associated with fetal malformations and restricted fetal growth and both low and high folate intake have been linked to cancer. In this work we will explore a previously unknown molecular link between folate availability and cell growth and proliferation that may help us better understand how these diseases develop. This research may generate unique information leading to novel treatments to alleviate restricted fetal growth and cancer.