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

开发诱导性滋养层特异性基因调节方法：滋养层 Lat1 在胎盘功能和胎儿生长调节中的作用

• 批准号: 10656023
• 负责人: Thomas L Brown
• 金额: $ 66.25万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656023
• 关键词: Adult; Affect; Amino Acid Transport System A; Amino Acid Transport System L; Amino Acid Transporter; Amino Acids; Animal Model; Animals; Biological Assay; Blastocyst Transfer; Blood Pressure; Cardiovascular Diseases; Cardiovascular Physiology; Cells; Child; Childhood; Complement; Defect; Development; Diabetes Mellitus; Disease; Doxycycline; Echocardiography; Embryo; Embryo Transfer; Essential Amino Acids; Experimental Models; FRAP1 gene; Family; Fetal Growth; Fetal Growth Retardation; Fetal Macrosomia; Folic Acid; Gene Expression; Gene Targeting; Gene Transfer; Gene Transfer Techniques; Genes; Gestational Diabetes; Human; Injury; Insulin Resistance; Knock-out; Labyrinth; Lentivirus; Life; Link; Measures; Mediating; Metabolic; Metabolic Diseases; Metabolism; Mitochondria; Modeling; Mus; Mutant Strains Mice; Nutrient; Nutritional; Obesity; Outcome; Perinatal mortality demographics; Placenta; Placentation; Pregnancy; Pregnancy Complications; Protein Biosynthesis; Protein Isoforms; Regulation; Resolution; Respiration; Risk; Role; Signal Transduction; Small Interfering RNA; Spirometry; System; Techniques; Telemetry; Testing; Thyroid Hormones; Time; Transduction Gene; Transfection; Transposase; Western Blotting; Woman; adenoviral mediated; blastocyst; cardiometabolism; effective therapy; glucose tolerance; in vivo; islet; knock-down; lentivirally transduced; member; novel; novel strategies; offspring; overexpression; perinatal morbidity; placental transfer; programs; promoter; solute; tool; trophoblast; uptake

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 总结 的 胎盘 引起胎儿生长异常的确切机制仍有待完全确定，然而， 氨基酸转运可能与IUGR和胎儿过度生长有关。大的中性氨基 酸性转运蛋白小亚基1（LAT 1）介导必需氨基酸和甲状腺激素的经胎盘转运 由转运系统L.重要的是，胎盘系统L氨基酸转运蛋白活性是 降低人类IUGR和增加胎儿过度生长的妇女。然而，目前尚不清楚， 胎盘LAT 1的表达/活性的变化与胎盘功能、胎儿发育和胎儿发育有关。 生长和后代心脏代谢结果。重要的是，整体Lat 1缺失导致胚胎死亡， 这使得很难确定LAT 1对胎盘功能的作用。其他基因， 虽然不是胚胎致死的，但可能影响早期胎盘发育和胎盘功能。 这需要工具在妊娠的特定时间点“关闭”或“开启”基因。因此，在本发明中， 迫切需要开发实现可诱导的滋养层特异性基因调节的方法。的 本研究的目的是开发和验证可诱导的滋养层细胞特异性基因的新方法， 在小鼠中的调节，并测试中心假设，恢复正常的滋养层Lat 1表达拯救 胚胎死亡率整体Lat 1缺失和滋养层特异性Lat 1敲低后， 胎盘的建立减少了胎盘对必需氨基酸的转运， mTOR，线粒体呼吸和蛋白质合成，限制胎儿生长和后代程序 代谢和心血管功能。我们提出了三个具体目标：目标1：开发和验证小鼠 具有可诱导的滋养层特异性Lat 1基因调节。我们的方法将是产生小鼠， 使用piggyBac在Lat 1-/-胚胎中进行强力霉素诱导的滋养层特异性Lat 1基因敲除或拯救 转座酶增强的转基因、慢病毒介导的囊胚和四倍体转导 互补试验。目的2：确定滋养层特异性Lat 1调节的作用 对胎盘功能、胎儿生长和后代长期结局的影响。我们的方法是（1）诱导 胎盘形成后Lat 1基因的敲低和（2）胎盘中滋养层Lat 1基因的拯救表达。 全Lat 1基因敲除（Lat 1-/-）胚胎。我们将测定氨基酸和甲状腺激素的经胎盘转运 激素，胎盘mTOR信号传导活性，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的3：确定LAT 1调节的作用 对原代人类滋养层细胞合胞化和功能的影响我们的方法是隔离 来自足月胎盘人滋养层（PHT）细胞，并测定合胞化、氨基酸和甲状腺 对照PHT细胞中的激素摄取、mTOR信号传导、线粒体呼吸和蛋白质合成， 细胞与siRNA介导的LAT 1敲低和PHT细胞与LAT 1过表达。