The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular Homeostasis.

ULK1 and ULK2 are thought to be essential for initiating autophagy, and Ulk1/2-deficient mice die perinatally of autophagy-related defects. Therefore, we used a conditional knockout approach to investigate the roles of ULK1/2 in the brain. Although the mice showed neuronal degeneration, the neurons showed no accumulation of P62(+)/ubiquitin(+) inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as an ULK1/2 interaction partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo, and did not require other autophagy proteins (e.g., ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis.

Post-translational modifications of serotonin transporter.

The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. Studies using in vivo and in vitro model systems demonstrated that diverse post-translational modifications, including phosphorylation, glycosylation, serotonylation, and disulfide bond formation, all favorably influences SERT conformation and allows the transporter to function most efficiently. This review discusses the post-translational modifications and their importance on the structure, maturation, and serotonin (5-HT) uptake ability of SERT. Finally, we discuss how these modifications are altered in diabetes mellitus and subsequently impairs the 5-HT uptake ability of SERT.

Discrepancy in Insulin Regulation between Gestational Diabetes Mellitus (GDM) Platelets and Placenta.

Earlier findings have identified the requirement of insulin signaling on maturation and the translocation of serotonin (5-HT) transporter, SERT to the plasma membrane of the trophoblast in placenta. Because of the defect on insulin receptor (IR) in the trophoblast of the gestational diabetes mellitus (GDM)-associated placenta, SERT is found entrapped in the cytoplasm of the GDM-trophoblast. SERT is encoded by the same gene expressed in trophoblast and platelets. Additionally, alteration in plasma 5-HT levels and the 5-HT uptake rates are associated with the aggregation rates of platelets. Therefore, here, we investigated a novel hypothesis that GDM-associated defects in platelet IR should change their 5-HT uptake rates, and this should be a leading factor for thrombosis in GDM maternal blood. The maternal blood and the placentas were obtained at the time of cesarean section from the GDM and non-diabetic subjects (n = 6 for each group), and the platelets and trophoblasts were isolated to determine the IR activity, surface level of SERT, and their 5-HT uptake rates.Interestingly, no significant differences were evident in IR tyrosine phosphorylation or the downstream elements, AKT and S6K in platelets and their aggregation rates in both groups. Furthermore, insulin stimulation up-regulated 5-HT uptake rates of GDM-platelets as it does in the control group. However, the phosphorylation of IR and the downstream elements were significantly lower in GDM-trophoblast and showed no response to the insulin stimulation while they showed 4-fold increase to insulin stimulation in control group. Similarly, the 5-HT uptake rates of GDM-trophoblast and the SERT expression on their surface were severalfold lower compared with control subjects. IR is expressed in all tissues, but it is not known if diabetes affects IR in all tissues equally. Here, for the first time, our findings with clinical samples show that in GDM-associated defect on IR is tissue type-dependent. While IR is impaired in GDM-placenta, it is unaffected in GDM-platelet.

Serotonin transporter protects the placental cells against apoptosis in caspase 3-independent pathway.

Serotonin (5-HT) and its specific transporter, SERT play important roles in pregnancy. Using placentas dissected from 18d gestational SERT-knock out (KO), peripheral 5-HT (TPH1)-KO, and wild-type (WT) mice, we explored the role of 5-HT and SERT in placental functions in detail. An abnormal thick band of fibrosis and necrosis under the giant cell layer in SERT-KO placentas appeared only moderately in TPH1-KO and minimally present in WT placentas. The majority of the changes were located at the junctional zone of the placentas in SERT. The etiology of these findings was tested with TUNEL assays. The placentas from SERT-KO and TPH1-KO showed 49- and 8-fold increase in TUNEL-positive cells without a concurrent change in the DNA repair or cell proliferation compared to WT placentas. While the proliferation rate in the embryos of TPH1-KO mice was 16-fold lower than the rate in gestational age matched embryos of WT or SERT-KO mice. These findings highlight an important role of continuous 5-HT signaling on trophoblast cell viability. SERT may contribute to protecting trophoblast cells against cell death via terminating the 5-HT signaling which changes cell death ratio in trophoblast as well as proliferation rate in embryos. However, the cell death in SERT-KO placentas is in caspase 3-independent pathway.

GDM-associated insulin deficiency hinders the dissociation of SERT from ERp44 and down-regulates placental 5-HT uptake.

Serotonin (5-HT) transporter (SERT) regulates the level of 5-HT in placenta. Initially, we found that in gestational diabetes mellitus (GDM), whereas free plasma 5-HT levels were elevated, the 5-HT uptake rates of trophoblast were significantly down-regulated, due to impairment in the translocation of SERT molecules to the cell surface. We sought to determine the factors mediating the down-regulation of SERT in GDM trophoblast. We previously reported that an endoplasmic reticulum chaperone, ERp44, binds to Cys200 and Cys209 residues of SERT to build a disulfide bond. Following this posttranslational modification, before trafficking to the plasma membrane, SERT must be dissociated from ERp44; and this process is facilitated by insulin signaling and reversed by the insulin receptor blocker AGL2263. However, the GDM-associated defect in insulin signaling hampers the dissociation of ERp44 from SERT. Furthermore, whereas ERp44 constitutively occupies Cys200/Cys209 residues, one of the SERT glycosylation sites, Asp208 located between the two Cys residues, cannot undergo proper glycosylation, which plays an important role in the uptake efficiency of SERT. Herein, we show that the decrease in 5-HT uptake rates of GDM trophoblast is the consequence of defective insulin signaling, which entraps SERT with ERp44 and impairs its glycosylation. In this regard, restoring the normal expression of SERT on the trophoblast surface may represent a novel approach to alleviating some GDM-associated complications.

Serotonin Signaling and the Hyperpermeable Endothelial Barrier in Sepsis: Clues to a Molecular Mechanism.

Sepsis is characterized by a severe systemic inflammatory response caused by hyperpermeability of the endothelial barrier resulting microvascular leakage, which is a leading factor to multiorgan failure. In sepsis, the hyperpermeable endothelial cells contribute to the activation of platelets, which release numerous mediators that affect coagulation, inflammatory response and are believed to directly or indirectly affect the integrity of the endothelial barrier. One such mediator is serotonin (5-hydroxytryptamine, 5-HT), a signaling molecule which mediates a number of cellular functions including regulation of cytoskeletal dynamics associated with barrier function of endothelial cells. The actions of 5-HT are mediated by different types of receptors and terminated via an uptake mechanism of a 5-HT transporter (SERT) on the platelet and endothelial cell. Earlier studies revealed unexpected discoveries concerning the impact of 5-HT signaling on the permeability of the endothelial barrier. These findings have been supported by the clinical reports on the anti-inflammatory property of 5-HT reuptake inhibitor, SSRIs in treating sepsis-related morbidity and mortality. This review focuses on a wide-range of literature to pinpoint cellular and molecular mechanisms that mediate 5-HT-induced microvascular injury in sepsis pathogenesis.

The role of ERp44 in maturation of serotonin transporter protein.

In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.

The nature of the binding between insulin receptor and serotonin transporter in placenta (review).

The interplay between the insulin receptor (IR) and serotonin transporter (SERT) allows reciprocal regulation of each other's physiological roles to ensure appropriate responses to specific environmental and developmental signals. The studies reported herein provided substantial evidence of how insulin signaling influences the modification and trafficking of SERT to the plasma membrane via enabling its association with specific endoplasmic reticulum (ER) proteins. While insulin signaling is important for the modifications of SERT proteins, the fact that phosphorylation of IR was significantly down-regulated in the placenta of SERT knock out (KO) mice suggests that SERT also regulates IR. Further suggestive of SERT functional regulation of IR, SERT-KO mice developed obesity and glucose intolerance with symptoms similar to those of type 2 diabetes. The picture emerging from those studies proposes that the interplay between IR and SERT maintains conditions supportive of IR phosphorylation and regulates insulin signaling in placenta which ultimately enables the trafficking of SERT to the plasma membrane. IR-SERT association thus appears to play a protective metabolic role in placenta and is impaired under diabetic conditions. This review focuses on recent findings describing the functional and physical associations between IR and SERT in placental cells, and the dysregulation of this process in diabetes.

Down-regulation of the serotonin transporter in hyperreactive platelets counteracts the pro-thrombotic effect of serotonin.

An elevated plasma concentration of serotonin ([5-HT]) is a common feature of cardiovascular disease often associated with enhanced platelet activation and thrombosis. Whether elevated in vivo plasma 5-HT per se represents an independent risk factor for platelet hyperreactivity or only is an epiphenomenon of cardiovascular disease is poorly understood. We examined in vitro and in vivo platelet function following a 24h elevation of plasma [5-HT] in mice. In vivo administration of 5-HT using osmotic minipumps increased plasma [5-HT] in treated mice compared to control mice instrumented with saline loaded pumps. 5-HT infusion did not increase systolic blood pressure, but markers of platelet activation including P-selectin and (PE)Jon/A staining were increased and these findings coincided with the enhanced aggregation of isolated platelets in response to type I fibrillar collagen. Tail bleeding times and the time to occlusion following chemical damage to the carotid artery were shortened in 5-HT-infused mice. 5-HT-infused mice were treated with paroxetine (Prx) to block 5-HT uptake via the serotonin transporter (SERT). Prx lowered platelet [5-HT] and attenuated platelet activation and aggregation. These results and our biochemical indices of enhanced 5-HT intracellular signaling in the platelets of 5-HT-infused mice reveal a mechanistic link between elevated plasma [5-HT], abnormal intracellular 5-HT signaling and accentuated platelet aggregation. Although a down-regulation of the serotonin transporter (SERT) on the platelet surface may counteract the pro-thrombotic influence of elevated plasma [5HT], this compensatory mechanism may fail to prevent the increased thrombotic risk caused by elevated plasma [5-HT].

Responses of Plasma Catecholamine, Serotonin, and the Platelet Serotonin Transporter to Cigarette Smoking.

Cigarette smoking is one of the major causes of coronary heart disease with a thirty percent mortality rate in the United States. Cigarette smoking acting on the central nervous system (CNS) to stimulate the sympathetic nervous system (SNS) through, which facilitates the secretion of serotonin (5-HT) and catecholamines to supraphysiological levels in blood. The enhanced levels of 5-HT and catecholamines in smokers' blood are associated with increases in G protein-coupled receptor signaling and serotonylation of small GTPases, which in turn lead to remodeling of cytoskeletal elements to enhance granule secretion and promote unique expression of sialylated N-glycan structures on smokers' platelets. These mechanisms enhance aggregation and adhesion of smokers' platelets relative to those of non-smokers. This review focuses on the known mechanisms by which 5-HT and SERT, in coordinated signaling with catecholamines, impacts cigarette smokers' platelet biology.

Association with serotonin transporter enables the phosphorylation of insulin receptor in placenta.

Upon binding to insulin, the ß-subunit of insulin receptor (IR) is phosphorylated and instantly activates intracellular signaling. A defect in this process causes the development of several metabolic disorders including non-insulin-dependent diabetes, such as type 2 and gestational diabetes mellitus (GDM). Under diabetic conditions the phosphorylation of IR in placenta, but not in platelets, is impaired. Interestingly the cellular distribution of the serotonin transporter (SERT), which utilizes the insulin signaling for posttranslational modification, shows tissue-type-dependent variation: SERT function is impaired in GDM-associated placenta, but not in platelets. In order to understand the correlation between IR, SERT and their tissue-type-dependent features, we tested an association between SERT and IR and whether this association affects the phosphorylation of IR. Using various approaches, we demonstrated a physical association between the Carboxyl terminal of SERT and the ß-subunit of IR. This association was found on the plasma membrane of the placenta and the platelets. Next, the contribution of the SERT-IR association to the phosphorylation of IR was analyzed in heterologous and endogenous expression systems following insulin-treatment. The in vivo impact of SERT-IR association on the phosphorylation of IR was explored in placenta and platelets of SERT gene knockout (KO) mice. The IR phosphorylation was significantly downregulated only in the placenta, but not in platelets of SERT-KO mice. These findings are supported by time course experiments, which demonstrate that the phosphorylation of IR occurs vis-a-vis IR-SERT association, and at least one of the IR binding domains is identified as the carboxyl-terminus of SERT. These findings suggest an important role for IR-SERT association in maintaining the phosphorylation of IR and regulating the insulin signaling in placenta.