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

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.

项目 概括 这 胎盘 导致异常胎儿生长的精确机制仍有待完全确定，但是变化 氨基酸的转运可能会导致IUGR和胎儿过度生长。大中性氨基 酸转运蛋白小亚基1（LAT1）介导了必需氨基酸和甲状腺的移植转移 转运蛋白系统的激素L。重要的是，胎盘系统L氨基酸转运蛋白活性是 人类IUGR的减少，女性的胎儿过度生长增加。但是，是否尚不清楚是否 胎盘LAT1的表达/活性的变化在机械上与胎盘功能，胎儿相关 生长和后代心脏代谢结果。重要的是，全球LAT1删除导致胚胎杀伤力 小鼠妊娠中期，使得难以确定LAT1在胎盘功能中的作用。其他基因， 尽管不是胚胎致死，但可能会影响早期胎盘发育和 已建立的胎盘，需要在妊娠的特定时间点上“关闭”或“转交”基因的工具。因此， 迫切需要开发方法来实现诱导型滋养细胞特异性基因调节。这 该建议的目的是开发和验证诱导滋养细胞特异性基因的新颖方法 小鼠的调节并测试中心假设，即恢复正常滋养细胞LAT1表达救援 全球LAT1删除的胚胎致死性以及滋养细胞特定的LAT1敲低 胎盘的建立减少必需氨基酸的胎盘运输并抑制胎盘 MTOR，线粒体呼吸和蛋白质合成，限制了胎儿的生长和后代 代谢和心血管功能。我们提出了三个特定目标：目标1：开发和验证小鼠 具有诱导型滋养细胞特异性LAT1基因调制。我们的方法是与 使用PiggyBac在LAT1 - / - 胚胎中的多西环素诱导，滋养细胞特异性的LAT1基因敲低或营救 转座酶增强的转基因，慢病毒介导的胚泡和四倍体的转导 互补测定。 AIM 2：确定滋养细胞特异性LAT1调制的效果 关于胎盘功能，胎儿生长和后代长期结局。我们的方法是（1）诱导 建立胎盘后的LAT1敲低和（2）拯救滋养滋养层的LAT1基因表达 全局LAT1敲除（LAT1 - / - ）胚胎。我们将确定氨基酸和甲状腺的移植运输 激素，胎盘MTOR信号活性，线粒体呼吸和蛋白质合成，胎儿生长和 后代长期代谢和心血管功能目标3：确定LAT1调制的效果 关于原发性人类滋养细胞合胞体化和功能。我们的方法是隔离主要 来自期限胎盘的人滋养细胞（PHT）细胞，并确定合成化，氨基酸和甲状腺 对照PHT细胞中的激素摄取，MTOR信号，线粒体呼吸和蛋白质合成，PHT中 siRNA介导的LAT1敲低的细胞和具有LAT1过表达的PHT细胞。