Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pulldown assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the nonneuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the proteinprotein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.
Synaptic transmission depends on neurotransmitter pools stored within vesicles that undergo regulated exocytosis. In the brain, the vesicular monoamine transporter-2 (VMAT 2 ) is responsible for the loading of dopamine (DA) and other monoamines into synaptic vesicles. Prior to storage within vesicles, DA synthesis occurs at the synaptic terminal in a two-step enzymatic process. First, the rate-limiting enzyme tyrosine hydroxylase (TH) converts tyrosine to di-OH-phenylalanine. Aromatic amino acid decarboxylase (AADC) then converts di-OH-phenylalanine into DA. Here, we provide evidence that VMAT 2 physically and functionally interacts with the enzymes responsible for DA synthesis. In rat striata, TH and AADC co-immunoprecipitate with VMAT 2 , whereas in PC 12 cells, TH co-immunoprecipitates with the closely related VMAT 1 and with overexpressed VMAT 2 . GST pull-down assays further identified three cytosolic domains of VMAT 2 involved in the interaction with TH and AADC. Furthermore, in vitro binding assays demonstrated that TH directly interacts with VMAT 2 . Additionally, using fractionation and immunoisolation approaches, we demonstrate that TH and AADC associate with VMAT 2 -containing synaptic vesicles from rat brain. These vesicles exhibited specific TH activity. Finally, the coupling between synthesis and transport of DA into vesicles was impaired in the presence of fragments involved in the VMAT 2 /TH/AADC interaction. Taken together, our results indicate that DA synthesis can occur at the synaptic vesicle membrane, where it is physically and functionally coupled to VMAT 2 -mediated transport into vesicles.Monoamines, including dopamine (DA), 3 norepinephrine (NE), and serotonin (5-HT), are neurotransmitters that play major roles in a variety of brain functions, including emotion, reward, cognition, memory, attention, locomotion, and stress control (1-6). In neurons and neuroendocrine cells, monoamines are stored in large dense core vesicles (LDCVs) and small synaptic vesicles (SVs) (7-11) that undergo regulated exocytosis through a complex network of protein-protein interactions (12). Loading of monoamines into LDCVs and SVs of neurons and neuroendocrine cells is mediated by two vesicular monoamine transporter isoforms: VMAT 1 (13) and VMAT 2 (14). These transporters contain 12 putative transmembrane domains with both the N and C termini facing the cytosolic side of the vesicle membrane. VMAT 1 is mostly present in LDCVs of neuroendocrine cells, including chromaffin and PC12 cells, whereas VMAT 2 is primarily expressed by monoaminergic neurons of the central nervous system (15). In midbrain DA neurons, VMAT 2 is sorted to LDCVs and SVs in axon terminals and to LDCVs and tubulo-vesicular structures in the somatodendritic compartment (7)(8)(9)(10)(11)15).It is generally accepted that VMAT 2 transports DA that has been previously synthesized in the cytosolic compartment of the presynaptic terminal (16). DA synthesis requires two enzymatic reactions. First, tyrosine hydroxylase (TH) converts tyrosine into DOP...
Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral death in infants. Reduced CD8 T-cells and negligible interferon gamma (IFNγ) in the airway are associated with severe infant RSV disease, yet there is an abundance of alveolar macrophages (AM) and neutrophils. However, it is unclear, based on our current understanding of macrophage functional heterogeneity, if immature AM improve viral clearance or contribute to inflammation and airway obstruction in the IFNγ-deficient neonatal lung environment. The aim of the current study was to define the age-dependent AM phenotype during neonatal RSV infection and investigate their differentiation to classically activated macrophages (CAM) using i.n. IFNγ in the context of improving viral clearance. Neonatal and adult BALB/cJ mice were infected with 1×106 plaque forming units (PFU)/gram (g) RSV line 19 and their AM responses compared. Adult mice showed a rapid and robust CAM response, indicated by increases in major histocompatibility complex class II (MHC II), CD86, CCR7, and a reduction in mannose receptor (MR). Neonatal mice showed a delayed and reduced CAM response, likely due to undetectable IFNγ production. Intranasal (i.n.) treatment with recombinant mouse IFNγ (rIFNγ) increased the expression of CAM markers on neonatal AM, reduced viral lung titers, and improved weight gain compared to untreated controls with no detectable increase in CD4 or CD8 T-cell infiltration. In vitro infection of J774A.1 macrophages with RSV induced an alternatively activated macrophage (AAM) phenotype however, when macrophages were first primed with IFNγ, a CAM phenotype was induced and RSV spread to adjacent Hep-2 cells was reduced. These studies demonstrate that the neonatal AM response to RSV infection is abundant and immature, but can be exogenously stimulated to express the antimicrobial phenotype, CAM, with i.n. rIFNγ.
In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2 and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In the present study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B loop of Nox2, would inhibit ROS production by Nox2-, but not by Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O2•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance (EPR) confirmed that Nox2ds inhibits O2•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production in either Nox1 or Nox4 oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2 oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.
Our results demonstrate for the first time that extracellular H(2)O(2), at pathophysiological concentrations, stimulates rASMC Nox1-derived O(2)(•-), subsequent Ask1 activation and SMC hypertrophy. The data demonstrate a novel pathway by which H(2)O(2) enters vascular cells via aquaporins and activates Nox, leading to hypertrophy, and provide multiple novel targets for combinatorial therapeutics development targeting hypertrophy and vascular disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.