Fusun Kilic, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Hyperglycemia is one of the leading causes of congenital malformations affecting the neuronal development of embryos. Serotonin (5HT) is a vasoconstrictor compound that also acts as a developmental signal early in rodent embryogenesis. Genetic and pharmacological disruption of 5HT-signaling causes maternal and prenatal morbidity and mortality via mediating high blood pressure and neuroanatomical abnormalities, respectively. Although neurons producing 5HT are among the first to develop in mammalian central nervous system, the biosynthesis of 5HT within the embryo occurs after the serotonergic neurons are first detectable. Before that time, the initial 5HT is derived from the maternal-embryonic circulation by the high-affinity 5HT transport system (SERT) on placenta. SERT on the membranes of the trophoblast cells regulates extracellular 5HT levels and prevents the vasoconstriction in the placental vascular bed and thereby secures a stable blood flow to the embryo. Also, Therefore, SERT plays a major role during the pregnancy for mother and the developing embryo. Our preliminary experiments in rat placenta demonstrated that hyperglycemia associated with diabetes induced mRNA expression of SERT, with no change at protein level. Then, we investigated the effect of sustained hyperglycemia in vitro on the 5HT transport system of HRP-1, rat placenta cells. 5HT uptake rates of cells in the presence of D-glucose at diabetes-like concentrations (30 mmol/l) were decreased significantly as compared to normoglycemic conditions (5.5 mmol/l), and this effect is not cell-type specific, but is sugar-type specific. We next tested the self-association ability of SERT in the presence of diabetic glucose because SERT is an oligomeric protein in the membrane of living cells and that is the preferred conformation. Our preliminary data indicate that at high 30 mM glucose partially diminishes the appearance of oligomeric SERT. The goal of this research project is to identify the underlying mechanism by which high glucose alters the functional expression of SERT. The results of the proposed research will lead to a better understanding of the impact of diabetes on the function and structure of placental SERT, and the impact of placental SERT on the regulation of 5HT levels, which have a central role in neuronal development of embryos. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The serotonin transporter (SERT) on the platelet surface is the major mechanism for the uptake of plasma serotonin (5-hydroxytryptamine;5HT), which subsequently is stored in platelet dense granules. The 5HT uptake capacity of the platelets depends on the number of SERT molecules on the plasma membrane. Earlier studies established that surface SERT expression in platelets shows a biphasic relationship to extracellular 5HT concentrations. Specifically, in platelets, plasma membrane SERT levels and 5HT uptake by SERT initially rise as plasma 5HT levels are increased, but then fall below normal as the plasma 5HT level continues to rise. Indeed, our in vivo and in vitro studies confirm a dynamic relationship between extracellular 5HT elevations, loss of surface SERT, and depletion of platelet 5HT. Additionally, we show for the first time that 5HT-depleted platelets appear to aggregate more readily, and elevating plasma 5HT in vivo appears to trigger platelet 5HT depletion and platelet aggregation. This project by a new investigator is designed to define the biochemical mechanisms by which elevated 5HT down-regulates the expression of SERT on the platelet membrane, and to examine the impact of this abnormality on platelet aggregation. The latter findings represent the future direction of the P.I., who is a protein biochemist transitioning to physiological models. The preliminary studies support the hypothesis that high extracellular 5HT leads to abnormalities in the platelet trafficking of SERT, which reduces the density of SERT molecules on the plasma membrane to deplete 5HT content. These events appear to promote platelet aggregation, a new finding just uncovered. Mechanistically, our studies will investigate the link between elevated extracellular 5HT and loss of surface SERT by focusing on the intracellular tethering of SERT by Rab4, and the 5HT-mediated phosphorylation of vimentin that promotes SERT internalization in platelets. Two model systems will be used: (a) 5HT-pretreated platelets isolated from untreated C57BL/6J mice, and (b) platelets isolated from C57BL/6J mice in which plasma 5HT will be elevated for 24 hours by osmotic minipump to ensure that our finding of SERT down-regulation by high extracellular 5HT in isolated platelets occurs in vivo and impacts platelet function. Overall, this project will provide the first detailed information on the 5HT-mediated biochemical pathways that regulate the number of functional SERT molecules on the platelet surface. The importance of understanding the structure, function, and regulation of SERT is underscored by the observations that plasma 5HT may be elevated in the plasma, either locally or globally, during atherosclerosis, hypertension, stroke, and other cardiovascular diseases. PUBLIC HEALTH RELEVANCE: In preliminary studies we demonstrated that plasma serotonin level at high concentration alters the characteristics of its specific transporter, serotonin transporter protein. We also showed that the number of transporter on the platelet membrane decreased through an alteration in its recycling dynamic. Furthermore, we identified two proteins that play major roles in the trafficking process of serotonin transporter. Based on our findings and others'published data we hypothesize that elevation in the plasma serotonin level alters the trafficking rate of the serotonin transporter from intracellular locations to the platelet membrane which in turn reduces the uptake rate of platelet. These studies are important because they will elucidate the mechanism by which plasma 5HT levels influence the number of functionally active SERT molecules on the platelet membrane. In specific, our studies will provide critical data that could be essential in strategies aimed at regulating SERT function in the treatment of diseases and disorders related to abnormal 5HT levels, such as neurological disorders and cardiovascular diseases.

DESCRIPTION (provided by applicant): Serotonin (5HT) is a vasoconstrictor that also acts as a developmental signal early in rodent embryogenesis. Genetic and pharmacological disruption of 5HT-signaling causes maternal and prenatal morbidity and mortality via mediating high blood pressure and neuroanatomical abnormalities, respectively. As a mitogen, the initial action of 5HT is important for the development of the embryo. However, the embryo does not synthesis 5HT but receives it from the maternal-embryonic circulation via a specific 5HT transporter (SERT) on the membranes of the trophoblast cells. Therefore, regulation of the transporter's activity constitutes a key mechanism for the flow of 5HT from mother to the developing embryo. SERT assembles into a number of higher-order complexes and functions as an oligomer and not simply as individual monomers in an oligomeric complex. SERT functions most favorably in the oligomeric form. However, the mediators that facilitate the oligomerization of SERT proteins have not been identified yet. The only study of this subject shows that glucose, at diabetes-like concentrations, down-regulates 5HT uptake rates of placental SERT through inhibiting its oligomerization. In human placental trophoblastoma (JAR) cells, our preliminary assays showed that SERT proteins colocalized with and bound to ERp44, endoplasmic reticulum (ER) located chaperone. ERp44 plays a critical role in the release of proteins from ER via binding to Ero1-L1. Our next preliminary studies showed that glucose, at diabetes-like concentrations, down regulates the mRNA level expression of ERp44. Therefore, these findings strengthen our confidence on the involvement of ERp44 in oligomerization of SERT proteins. Further, we showed that two mutant forms of SERT could neither associate with each others nor with ERp44. Based on our studies, we hypothesize that ERp44 has a defined role in the assembly of SERT proteins by forming disulfide bridges between SERT monomers. Once the oligomerization of SERT is completed, transporter in the matured form dissociate from the ERp44-Ero1-L1 complex and leave ER to enter the membrane trafficking. The proposed study will utilized pulse-chase experiments and immunoprecipitation assays to monitor the biogenesis of SERT proteins in (i) JAR cells, and in (ii) ERp44 or Ero1-L1 knock-down JAR cells. Also introducing Cys residues at different positions on the second external loop of SERT and then cross-linking them, we will assess the impact of additional intramolecular disulfide bonds on Ero1-L1- ERp44-SERT association. Overall, this project will provide the first detailed information on the involvement of ERp44-Ero1-L1 in the oligomerization of placental SERT proteins and shed light on the mechanism in which glucose prevents the oligomerization of placental SERT. The importance of understanding the structure, function, and regulation of SERT is underscored by the observations that placental SERT may be not functionally active during diabetes, hypertension, stroke, and other cardiovascular diseases during pregnancy. PUBLIC HEALTH RELEVANCE: As a mitogen, the initial action of serotonin (5HT) is important for the development of embryo however embryo does not synthesis 5HT but receives it from the maternal- embryonic circulation via a specific 5HT transporter (SERT) on the membranes of the trophoblast cells. Therefore, regulation of the transporter's activity constitutes a key mechanism for the flow of 5HT from mother to the developing embryo. SERT assembles into a number of higher-order complex and function as oligomers and not simply as individual monomers in an oligomeric complex. SERT functions most favorable in the oligomeric form however, the mediators which facilitates the oligomerization of SERT proteins have not been identified yet. This is the first study that identified two ER chaperones, ERp44 and Ero1-L1, in facilitating the oligomeric expression of SERT in placental cells. The importance of understanding the structure, function, and regulation of placental SERT is underscored by the many clinical and experimental studies which have implicated abnormal or inadequate 5HT accumulation in placenta and placental vascular bed. Our findings will provide data that could facilitate novel strategies aimed at regulating the functional efficiency of placental SERT and subsequently control 5HT-mediated embryonic development.