Mitochondrial nutrient transport and trophoblast differentiation

PROJECT SUMMARY Diabetes and obesity in pregnancy cause abnormalities in placental development that underlie adverse pregnancy outcomes such as spontaneous abortion, preeclampsia, and stillbirth. Central to healthy placental development is cytotrophoblast differentiation into syncytiotrophoblasts that can be disrupted by diabetes and

obesity by yet unknown mechanisms. Syncytiotrophoblasts facilitate nutrient transport, gas exchange, and hormone synthesis to support the pregnancy, and their differentiation depends on a highly coordinated progression of metabolic, epigenetic, and transcriptional reprogramming events. Mitochondrial flux of glucose

and glutamine has emerged as a major factor regulating cellular development, and our initial work shows that these metabolites play distinctive roles in cellular metabolism and gene expression during trophoblast differentiation. Glucose primarily fuels citrate and α-ketoglutarate production in rapidly dividing

cytotrophoblasts, while glutamine is the main source in terminally differentiated syncytiotrophoblasts. This shift is linked to reduced expression of the mitochondrial pyruvate carrier (MPC) in syncytiotrophoblasts, while mitochondrial glutamine transporter (SLC1A5var) expression remains stable. In this proposal, we will test the

central hypothesis that glucose-derived pyruvate and glutamine flux through the mitochondrial pyruvate (MPC) and glutamine (SLC1A5var) carriers, respectively, distinctly regulate energetic, epigenetic and transcriptional reprogramming necessary for trophoblast differentiation. For the proposed experiments, we will

use CRISPR-Cas9 genetic tools to manipulate expression of MPC (Aim 1) or SLC1A5var (Aim 2) in human trophoblast stem cell (TSC) models. In these lines, we will measure key indices of cellular metabolism using respirometry and state-of-the-art, high-performance liquid chromatography mass spectrometry (LCMS)-based

quantitative and 13C stable isotope tracing metabolomics methodologies. Moreover, we will examine genome- wide and locus-specific histone (H3) acetylation and methylation changes through integrative analysis of CUT&Tag- and RNAseq datasets. Both biochemical and morphologic differentiation will be characterized in

MPC or SLC1A5var loss of function or overexpressing TSCs. Additionally, in parallel experiments we will employ inhibitors of MPC and glutaminase in primary human trophoblasts to support key findings in TSCs. Successful completion of study aims will reveal novel, nutrient-responsive mechanisms controlling epigenetic

and transcriptional reprogramming events that are foundational to trophoblast differentiation. Defining these fundamental pathways is a necessary first step for developing new, evidence-based approaches that fine-tune placental fuel use and improve placental development and pregnancy outcomes for women with co-morbid

obesity and diabetes.