Inhibition of mechanistic target of rapamycin signaling decreases levels of O-GlcNAc transferase and increases serotonin release in the human placenta

Abstract

Changes in placental function, in particular down-regulation of placental O-GlcNAc transferase (OGT) in response to maternal stress and increased placental secretion of serotonin into the fetal circulation following maternal infection, have been mechanistically linked to adverse neurodevelopment in mice. We hypothesized that mTOR signaling is a key regulator of trophoblast serotonin synthesis and OGT protein expression and that serotonin is secreted by the human placenta into the fetal circulation. Placental homogenates (n=46) from elective terminations at 8-22 weeks of gestation and from healthy term women were sexed and the protein levels of OGT and enzymes involved in serotonin synthesis was determined. Primary human trophoblast (PHT) cells were isolated from normal term placenta (n=27), cultured and transfected (n=8) with siRNA targeting a scramble sequence (control), raptor (inhibits mTOR Complex 1), or rictor (inhibits mTOR Complex 2). Subsequently, conditioned media and PHT cell lysates were collected. Free serotonin concentration was measured using ELISA in cell culture media and in platelet-depleted normal term umbilical vein and artery plasma (n=38). Both mTORC1 and mTORC2 inhibition down-regulated OGT levels in PHT cells. The level of serotonin synthesis enzyme TPH-1 was higher in early gestation female placentas and at term serotonin concentration was 3-fold higher in the umbilical vein than in the umbilical artery. Inhibition of mTORC2, but not mTORC1, increased cultured PHT cell serotonin secretion. Our data is consistent with the model that mTOR signaling is a key regulator of trophoblast serotonin synthesis and OGT protein expression.

Mechanistic Target of Rapamycin Complex 2 Regulation of the Primary Human Trophoblast Cell Transcriptome

“…Specifically, both mTORC1 and 2 are positive regulators of trophoblast amino acid and folate transport ( Rosario et al, 2015a , 2016a , b ) and O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression ( Kelly et al, 2020 ), whereas mTORC1 activation promotes placental mitochondrial biogenesis/respiration ( Rosario et al, 2013 ). We also demonstrated that mTORC 2 signaling is a negative regulator of trophoblast serotonin synthesis ( Kelly et al, 2020 ). However, mTORC2 regulation of other trophoblast functions remains largely unknown.…”

Maternal serotonin: implications for the use of selective serotonin reuptake inhibitors during gestation

“…Furthermore, fetal platelets in late pregnancy express functional SERT [35] so that both fetal platelet SERT and the placenta regulate free serotonin concentrations in the fetal circulation [16, 33, 36]. Similarly, placental synthesis of serotonin in humans appears to decrease later in pregnancy as demonstrated by the downregulation of the endocrine machinery for the synthesis of serotonin in the third trimester compared to the first trimester [33, 37].…”

Oleic acid stimulation of amino acid uptake in primary human trophoblast cells is mediated by phosphatidic acid and mTOR signaling

“…An equalizer sample was used between each plate to correct for variations, and human cerebellum (Novus Biologicals, catalog number NB820-59180) and mouse brain were used as positive controls to confirm the presence of each target. Antibody details are found in Table 2 Traditional PAGE western blot was performed as previously described 24,25 using a pre-cast gel system (Biorad). Twenty microgram total protein was loaded and separated on Bis-Tris gels (4%-20%).…”