Zaugg, Jonas (2020). SLC transporter-mediated transfer of iron and amino acids across the placenta. (Thesis). Universität Bern, Bern

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Abstract

A successful pregnancy and the birth of a healthy baby depends to a great extent on the controlled supply of essential nutrients via the placenta. Inspired by clinical observations and in close collaborations with clinical obstetricians, we focused on the implementation of biochemical and cell biological in vitro models to mimick and investigate physiological relationships between the human placenta and gestational diseases. This work is structured into two clinically based sections studying solute carrier (SLC)-mediated materno-fetal transport of amino acids and iron across the placenta. The third methodologically oriented part describes the attempt to generate placental SLC knockout cells. The first clinically oriented part investigated the contribution of amino acid transporters of the SLC7 family in transplacental transfer of essential amino acids. Na+-independent placental leucine transport is mainly attributed to the System L-transporters LAT1/SLC7A5 and LAT2/SLC7A8. We determined physiological amino acid gradients of paired maternal and fetal serum samples by ion exchange chromatography. Subsequently, we assessed their importance by performing Spearman correlation analysis between materno-fetal gradients determined in vivo and anthropometric parameters. We found significant associations between reduced materno-fetal amino acid gradients with maternal gestational weight gain and maternal blood pressure suggesting a potential role of amino acid gradients in reduced intrauterine growth or even in hypertensive diseases like preeclampsia. Functional effects of a physiological gradient versus equimolar leucine concentrations were tested using in vitro uptake and Transwell®-based transfer assays. Interestingly, materno-fetal leucine transfer was significantly stimulated against a counter-directed gradient. These results suggest a currently underestimated effect of transplacental amino acid gradients on leucine transfer efficiency and underline their potential impact in pregnancy diseases associated with impaired fetal growth. Moreover, we investigated the relevance of SLC7 transporter in placental leucine transfer by applying the LAT1-specific inhibitor JPH203 and the System L-specific inhibitor JX009 synthesized by our collaborators in the NCCR TransCure network. These studies identified LAT1 as the major accumulative transporter in the placenta, but other System L-transporters like LAT2 as rate-limiting for leucine efflux to the fetus. The second clinical part of this thesis focused on investigating the molecular mechanisms of materno-fetal iron transport. Clinical studies suggest that pregnancies with elevated iron levels are more vulnerable to develop gestational diabetes mellitus (GDM), but the underlying mechanisms are unknown. We found differential regulation of iron transporters (DMT1/FPN1/ZIP8), receptors (TfR1), sensors (IRP1), regulators (HEPC) and oxidoreductases (HEPH/Zp) by RT-qPCR and by LC-MS/MS-based protein quantification in placental tissue from GDM compared to healthy pregnancies. Newly established hyperglycemic and hyperlipidemic trophoblast cell models, but also obesogenic HFHS diet in wildtype mice, mimicked to a great extent the expression results found in the clinical specimens of GDM patients. By expressional and functional assessment of normo¬glycemic, hyper¬glycemic and hyper¬glycemic-hyperlipidemic BeWo chorio¬carcinoma cells, we found altered cellular morphology, differential expression of iron transporters and reduced iron uptake confirming the impact of hyperglycemia on iron transport as observed in GDM patients. Iron depletion by the iron chelator deferoxamine (DFO) revealed a bidirectional relationship between placental glucose and iron homeostasis. Pathway analysis and rescue experiments indicated that dysregulated oxidative stress and autophagy contribute to reduced placental iron transport under hyperglycemic conditions. These adaptations are likely mediated by ferroptosis and could represent a protective mechanism preventing oxidative damage for both fetus and placenta caused by highly oxidative iron levels or hyperglycemia. Thus, in pregnancies with increased risk for GDM, antioxidant treatment and controlled iron supplementation could help to protect mother and fetus from impaired iron and glucose homeostasis. Within the last methodologically oriented part, we aimed to establish an efficient protocol to generate CRISPR/Cas9-mediated trophoblast knockout cell lines. Trophoblast cells lacking the function of single nutrient transporters would help to unravel their role in physiological nutrient transfer or in gestational diseases affecting fetal growth. To date, we were not able to successfully generate knockout cell lines by CRISPR/Cas9-mutagenesis. However, the achieved optimization of target site validation, BeWo transfection and characterization procedures of potential CRISPR/Cas9-mutant candidates created an advanced toolbox suitable to successfully continue the CRISPR/Cas9-mediated mutagenesis approach in future placental studies.

Item Type:	Thesis
Granting Institution:	Faculty of Medicine, University of Bern
Dissertation Type:	Single
Date of Defense:	17 November 2020
Subjects:	500 Science > 570 Life sciences; biology 600 Technology > 610 Medicine & health
Institute / Center:	04 Faculty of Medicine > Other Institutions > NCCR TransCure 04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine
Depositing User:	Jonas Zaugg
Date Deposited:	13 Sep 2021 11:54
Last Modified:	13 Sep 2021 12:03
URI:	https://boristheses.unibe.ch/id/eprint/307

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