Development of approaches for inducible trophoblast-specific gene modulation: the role of trophoblast Lat1 in the regulation of placental function and fetal growth

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PROJECT SUMMARY The placental precise mechanisms causing abnormal fetal growth remain to be fully established, however changes in amino acid transport may contribute to both IUGR and fetal overgrowth. The Large Neutral Amino Acid Transporter Small Subunit 1 (LAT1) mediates transplacental transfer of essential amino acids and thyroid hormones by the transporter System L. Importantly, placental System L amino acid transporter activity is decreased in human IUGR and increased in fetal overgrowth in women. However, it remains unknown if changes in the expression/activity of placental LAT1 are mechanistically linked to placental function, fetal growth and offspring cardiometabolic outcomes. Importantly, global Lat1 deletion leads to embryonic lethality in mid-gestation in mice, making it difficult to determine the role of LAT1 for placental function. Other genes, albeit not embryonically lethal, may influence both early placental development and the function of the established placenta, requiring tools to `turn-off' or `turn-on' genes at specific time points of gestation. Thus, there is an urgent need to develop approaches to achieve inducible, trophoblast-specific gene modulation. The objective of this proposal is to develop and validate novel approaches for inducible trophoblast-specific gene modulation in mice and to test the central hypothesis that restoring normal trophoblast Lat1 expression rescues the embryonic lethality of global Lat1 deletion and that trophoblast-specific Lat1 knockdown after the establishment of the placenta decreases placental transport of essential amino acids and inhibits placental mTOR, mitochondrial respiration and protein synthesis, restricts fetal growth and programs offspring metabolism and cardiovascular function. We propose three Specific Aims: Aim 1: Develop and validate mice with inducible trophoblast-specific Lat1 gene modulation. Our approach will be to generate mice with doxycycline-inducible, trophoblast-specific Lat1 gene knockdown or rescue in Lat1-/- embryos using piggyBac transposase-enhanced transgenesis, lentivirus-mediated transduction of blastocysts and tetraploid complementation assay, respectively. Aim 2: Determine the effect of trophoblast-specific Lat1 modulation on placental function, fetal growth and offspring long-term outcomes. Our approach will be to (1) induce Lat1 knockdown after the establishment of the placenta and (2) rescue trophoblast Lat1 gene expression in global Lat1 knockout (Lat1-/-) embryos. We will determine transplacental transport of amino acids and thyroid hormones, placental mTOR signaling activity, mitochondrial respiration and protein synthesis, fetal growth and offspring long-term metabolic and cardiovascular function Aim 3: Establish the effect of LAT1 modulation on primary human trophoblast syncytialization and function. Our approach will be to isolate primary human trophoblast (PHT) cells from term placentas and determine syncytialization, amino acid and thyroid hormone uptake, mTOR signaling, mitochondrial respiration and protein synthesis in control PHT cells, in PHT cell with siRNA mediated LAT1 knockdown and in PHT cells with LAT1 overexpression.