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The Journal of Biological Chemistry Aug 2023Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on...
Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on activation of Ca-dependent pathways, but the mechanism underlying reverse transport via endogenously expressed DAT is still unclear. Here, to enable concurrent visualization by live imaging of extracellular DA dynamics and cytosolic Ca levels, we employ the fluorescent Ca sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRAB expressed in cocultured "sniffer" cells. In the presence of the Na-channel blocker tetrodotoxin to prevent exocytotic DA release, AMPH induced in the cultured neurons a profound dose-dependent efflux of DA that was blocked both by inhibition of DAT with cocaine and by inhibition of the vesicular monoamine transporter-2 with Ro-4-1284 or reserpine. However, the AMPH-induced DA efflux was not accompanied by an increase in cytosolic Ca and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca. The independence of cytosolic Ca was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca in an autoreceptor-dependent manner regardless of the apparent independence of Ca for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT.
Topics: Amphetamine; Cocaine; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Vesicular Monoamine Transport Proteins; Humans; Cell Line, Tumor
PubMed: 37468107
DOI: 10.1016/j.jbc.2023.105063 -
Movement Disorders : Official Journal... Jun 2022The neurophysiological correlates of gastrointestinal symptoms (GISs) in Parkinson's disease (PD) are not well understood. It has been proposed that in patients with a...
BACKGROUND
The neurophysiological correlates of gastrointestinal symptoms (GISs) in Parkinson's disease (PD) are not well understood. It has been proposed that in patients with a gastrointestinal origin of PD dopaminergic neurodegeneration would be more symmetric.
OBJECTIVES
The aim is to assess the associations between GISs and asymmetry of nigrostriatal dopaminergic neurodegeneration in PD.
METHODS
Ninety PD patients were assessed using motor and GIS scales and I-FP-CIT SPECT. We calculated the asymmetry index and the predominant side of motor symptoms and dopamine transporter (DAT) imaging defect and assessed their association with GISs.
RESULTS
There were no significant differences in GISs between symmetric and asymmetric dopaminergic defect. Left predominant defect was related to more GIS and higher constipation scores.
CONCLUSIONS
GISs were associated with left predominant reduction in putaminal DAT binding but not asymmetry per se. It remains open whether left-sided DAT deficit is related to more pronounced GI involvement or symptom perception in PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.
Topics: Corpus Striatum; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Parkinson Disease; Tomography, Emission-Computed, Single-Photon; Tropanes
PubMed: 35274368
DOI: 10.1002/mds.28986 -
Journal of Neurochemistry Mar 2022Striatal dopamine release is key for learning and motivation and is composed of subregions including the dorsal striatum (DS), nucleus accumbens core, and the nucleus...
Striatal dopamine release is key for learning and motivation and is composed of subregions including the dorsal striatum (DS), nucleus accumbens core, and the nucleus accumbens shell. Spontaneously occurring dopamine release was compared across these subregions. Dopamine release/uptake dynamics differ across striatal subregions, with dopamine transient release amplitude and release frequency greatest in male mice, and the largest signals observed in the DS. Surprisingly, female mice exhibited little regional differences in dopamine release for DS and nucleus accumbens core regions, but lower release in the nucleus accumbens shell. Blocking voltage-gated K channel (Kv channels) with 4-aminopyridine enhanced dopamine detection without affecting reuptake. The 4-aminopyridine effects were greatest in ventral regions of female mice, suggesting regional differences in Kv channel expression. The dopamine transporter blocker cocaine also enhanced detection across subregions in both sexes, with greater overall increased release in females than males. Thus, sex differences in dopamine transmission are apparent and likely include differences in the Kv channel and dopamine transporter function. The lack of regional differences in dopamine release observed in females indicates differential regulation of spontaneous and evoked dopamine release.
Topics: 4-Aminopyridine; Animals; Cocaine; Corpus Striatum; Dopamine; Dopamine Antagonists; Dopamine Plasma Membrane Transport Proteins; Female; Male; Mice; Nucleus Accumbens; Sex Characteristics
PubMed: 34265080
DOI: 10.1111/jnc.15473 -
Cell Reports Sep 2022The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like...
The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show, by application of several super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the DA transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and DA D2 autoreceptor activation in a manner dependent on Ca influx via N-type voltage-gated Ca channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2 autoreceptor, syntaxin-1, and clathrin nanodomains. The data reveal a mechanism for rapid alterations of nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter.
Topics: Animals; Autoreceptors; Clathrin; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Mice; Phosphatidylinositols; Qa-SNARE Proteins
PubMed: 36170827
DOI: 10.1016/j.celrep.2022.111431 -
International Journal of Molecular... Feb 2021Prolyl oligopeptidase (PREP) is a serine protease that binds to alpha-synuclein (aSyn) and induces its aggregation. PREP inhibitors have been shown to have beneficial...
Prolyl oligopeptidase (PREP) is a serine protease that binds to alpha-synuclein (aSyn) and induces its aggregation. PREP inhibitors have been shown to have beneficial effects in Parkinson's disease models by enhancing the clearance of aSyn aggregates and modulating striatal dopamine. Additionally, we have shown that PREP regulates phosphorylation and internalization of dopamine transporter (DAT) in mice. In this study, we clarified the mechanism behind this by using HEK-293 and PREP knock-out HEK-293 cells with DAT transfection. We tested the effects of PREP, PREP inhibition, and alpha-synuclein on PREP-related DAT regulation by using Western blot analysis and a dopamine uptake assay, and characterized the impact of PREP on protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) by using PKC assay and Western blot, respectively, as these kinases regulate DAT phosphorylation. Our results confirmed our previous findings that a lack of PREP can increase phosphorylation and internalization of DAT and decrease uptake of dopamine. PREP inhibition had a variable impact on phosphorylation of ERK dependent on the metabolic state of cells, but did not have an effect on phosphorylation or function of DAT. PREP modifications did not affect PKC activity either. Additionally, a lack of PREP elevated a DAT oligomerization that is associated with intracellular trafficking of DAT. Our results suggest that PREP-mediated phosphorylation, oligomerization, and internalization of DAT is not dependent on PKC or ERK.
Topics: Dopamine Plasma Membrane Transport Proteins; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; Humans; Phosphorylation; Prolyl Oligopeptidases; Protein Kinase C; Protein Multimerization
PubMed: 33579026
DOI: 10.3390/ijms22041777 -
Molecules (Basel, Switzerland) Jun 2021Phenolic compounds are thought to be important to prevent neurodegenerative diseases (ND). Parkinson's Disease (PD) is a neurodegenerative disorder known for its typical...
Phenolic compounds are thought to be important to prevent neurodegenerative diseases (ND). Parkinson's Disease (PD) is a neurodegenerative disorder known for its typical motor features, the deposition of α-synuclein (αsyn)-positive inclusions in the brain, and for concomitant cellular pathologies that include oxidative stress and neuroinflammation. Neuroprotective activity of fisetin, a dietary flavonoid, was evaluated against main hallmarks of PD in relevant cellular models. At physiologically relevant concentrations, fisetin protected SH-SY5Y cells against oxidative stress overtaken by -butyl hydroperoxide (-BHP) and against methyl-4-phenylpyridinuim (MPP)-induced toxicity in dopaminergic neurons, the differentiated Lund human Mesencephalic (LUHMES) cells. In this cellular model, fisetin promotes the increase of the levels of dopamine transporter. Remarkably, fisetin reduced the percentage of cells containing αsyn inclusions as well as their size and subcellular localization in a yeast model of αsyn aggregation. Overall, our data show that fisetin exerts modulatory activities toward common cellular pathologies present in PD; remarkably, it modulates αsyn aggregation, supporting the idea that diets rich in this compound may prove beneficial.
Topics: Butyrates; Cell Line; Dopamine Plasma Membrane Transport Proteins; Flavonols; Humans; Models, Biological; Oxidative Stress; Parkinson Disease; Piperidines; Protein Aggregates; Saccharomyces cerevisiae; alpha-Synuclein; tert-Butylhydroperoxide
PubMed: 34199487
DOI: 10.3390/molecules26113353 -
Scientific Reports Jul 2021To evaluate the implication of F-fluorodeoxyglucose (FDG)- and dopamine transporter (DAT)-positron emission tomography (PET) in the diagnosis and clinical symptoms of...
To evaluate the implication of F-fluorodeoxyglucose (FDG)- and dopamine transporter (DAT)-positron emission tomography (PET) in the diagnosis and clinical symptoms of dementia with Lewy bodies (DLB), 55 DLB patients and 49 controls underwent neuropsychological evaluation and FDG-, DAT-, and F-Florbetaben (FBB) PET. DAT- and FDG-uptake and FDG/DAT ratio were measured in the anterior and posterior striatum. The first principal component (PC1) of FDG subject residual profiles was identified for each subject. Receiver operating characteristic curve analyses for the diagnosis of DLB were performed using FDG- and DAT-PET biomarkers as predictors, and general linear models for motor severity and cognitive scores were performed adding FBB standardized uptake value ratio as a predictor. Increased metabolism in the bilateral putamen, vermis, and somato-motor cortices, which characterized PC1, was observed in the DLB group, compared to the control group. A combination of posterior putamen FDG/DAT ratio and PC1 showed the highest diagnostic accuracy (91.8% sensitivity and 96.4% specificity), which was significantly greater than that obtained by DAT uptake alone. Striatal DAT uptake and PC1 independently contributed to motor severity and language, memory, frontal/executive, and general cognitive dysfunction in DLB patients, while only PC1 contributed to attention and visuospatial dysfunction.
Topics: Aged; Aged, 80 and over; Cognitive Dysfunction; Dopamine Plasma Membrane Transport Proteins; Humans; Lewy Body Disease; Male; Middle Aged
PubMed: 34257349
DOI: 10.1038/s41598-021-93442-y -
Molecular Psychiatry Jul 2023Concurrent cocaine and alcohol use is among the most frequent drug combination, and among the most dangerous in terms of deleterious outcomes. Cocaine increases...
Concurrent cocaine and alcohol use is among the most frequent drug combination, and among the most dangerous in terms of deleterious outcomes. Cocaine increases extracellular monoamines by blocking dopamine (DA), norepinephrine (NE) and serotonin (5-HT) transporters (DAT, NET and SERT, respectively). Likewise, ethanol also increases extracellular monoamines, however evidence suggests that ethanol does so independently of DAT, NET and SERT. Organic cation transporter 3 (OCT3) is an emergent key player in the regulation of monoamine signaling. Using a battery of in vitro, in vivo electrochemical, and behavioral approaches, as well as wild-type and constitutive OCT3 knockout mice, we show that ethanol's actions to inhibit monoamine uptake are dependent on OCT3. These findings provide a novel mechanistic basis whereby ethanol enhances the neurochemical and behavioral effects of cocaine and encourage further research into OCT3 as a target for therapeutic intervention in the treatment of ethanol and ethanol/cocaine use disorders.
Topics: Mice; Animals; Dopamine; Ethanol; Carrier Proteins; Cocaine-Related Disorders; Cocaine; Serotonin; Mice, Knockout; Cations; Dopamine Plasma Membrane Transport Proteins; Serotonin Plasma Membrane Transport Proteins
PubMed: 37308680
DOI: 10.1038/s41380-023-02064-5 -
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 -
Frontiers in Neuroscience 2023One of the hallmarks of Parkinson's disease (PD) is the progressive loss of dopaminergic neurons and associated dopamine depletion. Several mechanisms, previously... (Review)
Review
One of the hallmarks of Parkinson's disease (PD) is the progressive loss of dopaminergic neurons and associated dopamine depletion. Several mechanisms, previously considered in isolation, have been proposed to contribute to the pathophysiology of dopaminergic degeneration: dopamine oxidation-mediated neurotoxicity, high dopamine transporter (DAT) expression density per neuron, and autophagy-lysosome pathway (ALP) dysfunction. However, the interrelationships among these mechanisms remained unclear. Our recent research bridges this gap, recognizing autophagy as a novel dopamine homeostasis regulator, unifying these concepts. I propose that autophagy modulates dopamine reuptake by selectively degrading DAT. In PD, ALP dysfunction could increase DAT density per neuron, and enhance dopamine reuptake, oxidation, and neurotoxicity, potentially contributing to the progressive loss of dopaminergic neurons. This integrated understanding may provide a more comprehensive view of aspects of PD pathophysiology and opens new avenues for therapeutic interventions.
PubMed: 37694119
DOI: 10.3389/fnins.2023.1219441