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Heterogeneities in Axonal Structure and Transporter Distribution Lower Dopamine Reuptake Efficiency.ENeuro 2018Efficient clearance of dopamine (DA) from the synapse is key to regulating dopaminergic signaling. This role is fulfilled by DA transporters (DATs). Recent advances in...
Efficient clearance of dopamine (DA) from the synapse is key to regulating dopaminergic signaling. This role is fulfilled by DA transporters (DATs). Recent advances in the structural characterization of DAT from (dDAT) and in high-resolution imaging of DA neurons and the distribution of DATs in living cells now permit us to gain a mechanistic understanding of DA reuptake events . Using electron microscopy images and immunofluorescence of transgenic knock-in mouse brains that express hemagglutinin-tagged DAT in DA neurons, we reconstructed a realistic environment for MCell simulations of DA reuptake, wherein the identity, population and kinetics of homology-modeled human DAT (hDAT) substates were derived from molecular simulations. The complex morphology of axon terminals near active zones was observed to give rise to large variations in DA reuptake efficiency, and thereby in extracellular DA density. Comparison of the effect of different firing patterns showed that phasic firing would increase the probability of reaching local DA levels sufficiently high to activate low-affinity DA receptors, mainly owing to high DA levels transiently attained during the burst phase. The experimentally observed nonuniform surface distribution of DATs emerged as a major modulator of DA signaling: reuptake was slower, and the peaks/width of transient DA levels were sharper/wider under nonuniform distribution of DATs, compared with uniform. Overall, the study highlights the importance of accurate descriptions of extrasynaptic morphology, DAT distribution, and conformational kinetics for quantitative evaluation of dopaminergic transmission and for providing deeper understanding of the mechanisms that regulate DA transmission.
Topics: Action Potentials; Animals; Axons; Brain; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Humans; Mice, Transgenic; Molecular Dynamics Simulation; Protein Conformation; Synaptic Transmission; Tissue Culture Techniques
PubMed: 29430519
DOI: 10.1523/ENEURO.0298-17.2017 -
International Journal of Molecular... Oct 2021A major goal of current clinical research in Parkinson's disease (PD) is the validation and standardization of biomarkers enabling early diagnosis, predicting outcomes,... (Review)
Review
A major goal of current clinical research in Parkinson's disease (PD) is the validation and standardization of biomarkers enabling early diagnosis, predicting outcomes, understanding PD pathophysiology, and demonstrating target engagement in clinical trials. Molecular imaging with specific dopamine-related tracers offers a practical indirect imaging biomarker of PD, serving as a powerful tool to assess the status of presynaptic nigrostriatal terminals. In this review we provide an update on the dopamine transporter (DAT) imaging in PD and translate recent findings to potentially valuable clinical practice applications. The role of DAT imaging as diagnostic, preclinical and predictive biomarker is discussed, especially in view of recent evidence questioning the incontrovertible correlation between striatal DAT binding and nigral cell or axon counts.
Topics: Animals; Biomarkers; Dopamine Plasma Membrane Transport Proteins; Humans; Molecular Imaging; Parkinson Disease
PubMed: 34681899
DOI: 10.3390/ijms222011234 -
Addiction Biology Nov 2022As well known, cocaine induces stimulant effects and dopamine transporter (DAT) trafficking to the plasma membrane of dopaminergic neurons. In the present study, we...
As well known, cocaine induces stimulant effects and dopamine transporter (DAT) trafficking to the plasma membrane of dopaminergic neurons. In the present study, we examined cocaine-induced hyperactivity along with cocaine-induced DAT trafficking and the recovery rate of the dopaminergic system in female rats in comparison with male rats, demonstrating interesting gender differences. Female rats are initially more sensitive to cocaine than male rats in terms of both the DAT trafficking and hyperactivity induced by cocaine. Particularly, intraperitoneal (i.p.) administration of 5 mg/kg cocaine induced significant hyperactivity and DAT trafficking in female rats but not in male rats. After repeated cocaine exposures (i.e., i.p. administration of 20 mg/kg cocaine every other day from Day 0 to Day 32), cocaine-induced hyperactivity in female rats gradually became a clear pattern of two phases, with the first phase of the hyperactivity lasting for only a few minutes and the second phase lasting for over an hour beginning at ~30 min, which is clearly different from that of male rats. It has also been demonstrated that the striatal DAT distribution of female rats may recover faster than that of male rats after multiple cocaine exposures. Nevertheless, despite the remarkable gender differences, our recently developed long-acting cocaine hydrolase, known as CocH5-Fc(M6), can similarly and effectively block cocaine-induced DAT trafficking and hyperactivity in both male and female rats.
Topics: Male; Female; Rats; Animals; Dopamine Plasma Membrane Transport Proteins; Cocaine; Sex Factors; Dopamine Uptake Inhibitors; Cell Membrane
PubMed: 36301205
DOI: 10.1111/adb.13236 -
Neurochemistry International Jul 2014The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary...
The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.
Topics: Cloning, Molecular; Cocaine; Cysteine; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; HEK293 Cells; Humans; Protein Conformation; Protein Transport; Sulfhydryl Reagents; Tropanes; Tyramine; Zinc
PubMed: 24576496
DOI: 10.1016/j.neuint.2014.02.003 -
Parkinsonism & Related Disorders Sep 2023Glymphatic dysfunction can contribute to α-synucleinopathies. We examined glymphatic function in idiopathic Parkinson's disease (PD) utilizing Diffusion Tensor Image...
INTRODUCTION
Glymphatic dysfunction can contribute to α-synucleinopathies. We examined glymphatic function in idiopathic Parkinson's disease (PD) utilizing Diffusion Tensor Image Analysis aLong the Perivascular Space (DTI-ALPS).
METHODS
This study enrolled consecutive patients diagnosed with de novo PD between June 2017 and March 2019 who underwent brain DTI with concurrent I-2β-carbomethoxy-3β-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane (I-FP-CIT) SPECT, and age- and sex-matched controls. From DTI-ALPS, the ALPS-index was calculated as a ratio of diffusivities along the x-axis in the region of neural fibers passing vertically to the diffusivities perpendicular to them, which reflected perivascular water motion at the lateral ventricular body level. The ALPS-index of the PD and control groups was compared using Student's t-test; its correlations with clinical scores for motor and cognition (UPDRS-III, MMSE, and MoCA) and striatal dopamine transporter uptake measured by I-FP-CIT specific binding ratios (SBRs) were examined using a correlation coefficient.
RESULTS
In all, 54 patients in the de novo PD group (31 women, 23 men; mean age, 68.9 ± 9.4 years) and 54 in the control group (mean age, 69.0 ± 10.5 years) were included. The ALPS-index was lower in the PD group than in the controls (1.51 ± 0.22 versus 1.66 ± 0.20; P < 0.001). In the PD group, the ALPS-index negatively correlated with the UPDRS-III score (r = -0.526), and positively correlated with the MMSE (r = 0.377) and MoCA scores (r = 0.382) (all, P < 0.05). No correlation was observed between the ALPS-index and striatal I-FP-CIT SBRs (P > 0.05).
CONCLUSIONS
DTI-ALPS can reveal glymphatic dysfunction in patients with PD, whose severity correlated with motor and cognitive dysfunction, but not striatal dopamine transporter uptake.
Topics: Male; Humans; Female; Middle Aged; Aged; Parkinson Disease; Dopamine Plasma Membrane Transport Proteins; Tropanes
PubMed: 37523953
DOI: 10.1016/j.parkreldis.2023.105767 -
Biomolecules Jan 2020The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a...
The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a major role in the treatment of many central and peripheral nervous system diseases. In this study, we focused on the spatial structure of a NET constructed by homology modeling on dopamine transporter templates. We further examined molecular construction of primary binding pocket (S1) together with secondary binding site (S2) and extracellular loop 4 (EL4). The next stage involved docking of transporter inhibitors: Reboxetine, duloxetine, desipramine, and other commonly used drugs. The procedure revealed the molecular orientation of residues and disclosed ones that are the most important for ligand binding: Phenylalanine F72, aspartic acid D75, tyrosine Y152, and phenylalanine F317. Aspartic acid D75 plays a key role in recognition of the basic amino group present in monoamine transporter inhibitors and substrates. The study also presents a comparison of hNET models with other related proteins, which could provide new insights into their interaction with therapeutics and aid future development of novel bioactive compounds.
Topics: Animals; Binding Sites; Dopamine; Dopamine Plasma Membrane Transport Proteins; Drosophila Proteins; Drosophila melanogaster; Humans; Molecular Dynamics Simulation; Norepinephrine; Norepinephrine Plasma Membrane Transport Proteins; Protein Binding
PubMed: 31936154
DOI: 10.3390/biom10010102 -
The European Journal of Neuroscience Jan 2017Dopamine was first identified as a neurotransmitter localized to the midbrain over 50 years ago. The dopamine transporter (DAT; SLC6A3) and the vesicular monoamine... (Review)
Review
Dopamine was first identified as a neurotransmitter localized to the midbrain over 50 years ago. The dopamine transporter (DAT; SLC6A3) and the vesicular monoamine transporter 2 (VMAT2; SLC18A2) are regulators of dopamine homeostasis in the presynaptic neuron. DAT transports dopamine from the extracellular space into the cytosol of the presynaptic terminal. VMAT2 then packages this cytosolic dopamine into vesicular compartments for subsequent release upon neurotransmission. Thus, DAT and VMAT2 act in concert to move the transmitter efficiently throughout the neuron. Accumulation of dopamine in the neuronal cytosol can trigger oxidative stress and neurotoxicity, suggesting that the proper compartmentalization of dopamine is critical for neuron function and risk of disease. For decades, studies have examined the effects of reduced transporter function in mice (e.g. DAT-KO, VMAT2-KO, VMAT2-deficient). However, we have only recently been able to assess the effects of elevated transporter expression using BAC transgenic methods (DAT-tg, VMAT2-HI mice). Complemented with in vitro work and neurochemical techniques to assess dopamine compartmentalization, a new focus on the importance of transporter proteins as both models of human disease and potential drug targets has emerged. Here, we review the importance of DAT and VMAT2 function in the delicate balance of neuronal dopamine.
Topics: Animals; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Humans; Mesencephalon; Presynaptic Terminals; Synaptic Transmission
PubMed: 27520881
DOI: 10.1111/ejn.13357 -
Biomolecules Oct 2022Changes in dopaminergic and noradrenergic transmission are considered to be the underlying cause of attention deficit and hyperactivity disorder (ADHD). Atomoxetine...
Changes in dopaminergic and noradrenergic transmission are considered to be the underlying cause of attention deficit and hyperactivity disorder (ADHD). Atomoxetine (ATX) is a selective norepinephrine transporter (NET) inhibitor that is currently used for ADHD treatment. In this study, we aimed to evaluate the effect of atomoxetine on the behavior and brain activity of dopamine transporter knockout (DAT-KO) rats, which are characterized by an ADHD-like behavioral phenotype. Prepulse inhibition (PPI) was assessed in DAT-KO and wild type rats after saline and ATX injections, as well as behavioral parameters in the Hebb-Williams maze and power spectra and coherence of electrophysiological activity. DAT-KO rats demonstrated a pronounced behavioral and electrophysiological phenotype, characterized by hyperactivity, increased number of errors in the maze, repetitive behaviors and disrupted PPI, changes in cortical and striatal power spectra and interareal coherence. Atomoxetine significantly improved PPI and decreased repetitive behaviors in DAT-KO rats and influenced behavior of wild-type rats. ATX also led to significant changes in power spectra and coherence of DAT-KO and wild type rats. Assessment of noradrenergic modulation effects in DAT-KO provides insight into the intricate interplay of monoaminergic systems, although further research is still required to fully understand the complexity of this interaction.
Topics: Rats; Animals; Atomoxetine Hydrochloride; Dopamine Plasma Membrane Transport Proteins; Norepinephrine Plasma Membrane Transport Proteins; Cognition; Norepinephrine; Corpus Striatum
PubMed: 36291693
DOI: 10.3390/biom12101484 -
Toxicology Feb 2008The dopamine transporter has been shown to be the most relevant target site for the specificity of 1-methyl-4-phenylpyridinium ion (MPP+), a neurotoxin for dopaminergic...
The dopamine transporter has been shown to be the most relevant target site for the specificity of 1-methyl-4-phenylpyridinium ion (MPP+), a neurotoxin for dopaminergic neurons. In contrast, the mechanisms underlying the selective toxicity of manganese and rotenone, potentially toxic agents implicated in dopaminergic neuronal cell death, remain unknown. The aim of this study was to determine the cellular mechanisms of manganese or rotenone uptake in dopaminergic cells via the dopamine transporter. PC12 cells overexpressing the dopamine transporter, which were exposed to 10microM MPP+, showed extensive DNA fragmentation, a biochemical hallmark of apoptosis, whereas wild-type PC12 cells or vector-transfected PC12 cells, which were exposed to 5mM MPP+, did not show DNA fragmentation. In contrast, manganese and rotenone induced DNA fragmentation at slightly lower concentrations in PC12 cells overexpressing the dopamine transporter compared to control cells. Dopamine transporter inhibitors, such as mazindol, nomifensine, or GBR12909, inhibited MPP+-induced DNA fragmentation but did not affect manganese- and rotenone-induced DNA fragmentation in PC12 cells overexpressing the dopamine transporter. Finally, manganese accumulated to similar levels in PC12 cells overexpressing the dopamine transporter and control PC12 cells following incubation with manganese chloride. These results suggested that the dopamine transporter dose not confer cytotoxicity to manganese and rotenone.
Topics: 1-Methyl-4-phenylpyridinium; Animals; Blotting, Western; DNA Fragmentation; DNA, Complementary; Dopamine Plasma Membrane Transport Proteins; Humans; Manganese; Manganese Poisoning; PC12 Cells; Rats; Rotenone; Transfection; Uncoupling Agents
PubMed: 18206288
DOI: 10.1016/j.tox.2007.11.018 -
Neurochemical Research Jun 2020Dopamine (DA) is critical for motivation, reward, movement initiation, and learning. Mechanisms that control DA signaling have a profound impact on these important... (Review)
Review
Dopamine (DA) is critical for motivation, reward, movement initiation, and learning. Mechanisms that control DA signaling have a profound impact on these important behaviors, and additionally play a role in DA-related neuropathologies. The presynaptic SLC6 DA transporter (DAT) limits extracellular DA levels by clearing released DA, and is potently inhibited by addictive and therapeutic psychostimulants. Decades of evidence support that the DAT is subject to acute regulation by a number of signaling pathways, and that endocytic trafficking strongly regulates DAT availability and function. DAT trafficking studies have been performed in a variety of model systems, including both in vitro and ex vivo preparations. In this review, we focus on the breadth of DAT trafficking studies, with specific attention to, and comparison of, how context may influence DAT's response to different stimuli. In particular, this overview highlights that stimulated DAT trafficking not only differs between in vitro and ex vivo environments, but also is influenced by both sex and anatomical subregions.
Topics: Animals; Brain; Dopamine; Dopamine Plasma Membrane Transport Proteins; GABA Plasma Membrane Transport Proteins; Humans; Protein Transport; Signal Transduction
PubMed: 32146647
DOI: 10.1007/s11064-020-03001-6