Dopamine Transporter Internalization Research Articles

A Conserved Intramolecular Ion-Pair Plays a Critical but Divergent Role in Regulation of Dimerization and Transport Function among the Monoamine Transporters.

The monoamine transporters, including the serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET), are the therapeutic targets for the treatment of many neuropsychiatric disorders. Despite significant progress in characterizing the structures and transport mechanisms of these transporters, the regulation of their transport functions through dimerization or oligomerization remains to be understood. In the present study, we identified a conserved intramolecular ion-pair at the third extracellular loop (EL3) connecting TM5 and TM6 that plays a critical but divergent role in the modulation of dimerization and transport functions among the monoamine transporters. The disruption of the ion-pair interactions by mutations induced a significant spontaneous cross-linking of a cysteine mutant of SERT and an increase in cell surface expression but with an impaired specific transport activity. On the other hand, similar mutations of the corresponding ion-pair residues in both DAT and NET resulted in an opposite effect on their oxidation-induced dimerization, cell surface expression, and transport function. Reversible biotinylation experiments indicated that the ion-pair mutations slowed down the internalization of SERT but stimulated the internalization of DAT. In addition, cysteine accessibility measurements for monitoring SERT conformational changes indicated that substitution of the ion-pair residues resulted in profound effects on the rate constants for cysteine modification in both the extracellular and cytoplasmatic substrate permeation pathways. Furthermore, molecular dynamics simulations showed that the ion-pair mutations increased the interfacial interactions in a SERT dimer but decreased it in a DAT dimer. Taken together, we propose that the transport function is modulated by the equilibrium between monomers and dimers on the cell surface, which is regulated by a potential compensatory mechanism but with different molecular solutions among the monoamine transporters. The present study provided new insights into the structural elements regulating the transport function of the monoamine transporters through their dimerization.

Cholesterol modulation of interactions between psychostimulants and dopamine transporters.

The dopamine transporter (DAT) is a key site of action for cocaine and amphetamines. Dysfunctional DAT is associated with aberrant synaptic dopamine transmission and enhanced drug-seeking and taking behavior. Studies in cultured cells and ex vivo suggest that DAT function is sensitive to membrane cholesterol content. Although it is largely unknown whether psychostimulants alter cholesterol metabolism in the brain, emerging evidence indicates that peripheral cholesterol metabolism is altered in patients with psychostimulant use disorder and circulating cholesterol levels are associated with vulnerability to relapse. Cholesterol interacts with sphingolipids forming lipid raft microdomains on the membrane. These cholesterol-rich lipid raft microdomains serve to recruit and assemble other lipids and proteins to initiate signal transduction. There are two spatially and functionally distinct populations of the DAT segregated by cholesterol-rich lipid raft microdomains and cholesterol-scarce non-raft microdomains on the plasma membrane. These two DAT populations are differentially regulated by DAT blockers (e.g. cocaine), substrates (e.g. amphetamine), and protein kinase C providing distinct cholesterol-dependent modulation of dopamine uptake and efflux. In this chapter, we summarize the impact of depletion and addition of membrane cholesterol on DAT conformational changes between the outward-facing and the inward-facing states, lipid raft-associated DAT localization, basal and induced DAT internalization, and DAT function. In particular, we focus on how the interactions of the DAT with cocaine and amphetamine are influenced by membrane cholesterol. Lastly, we discuss the therapeutic potential of cholesterol-modifying drugs as a new avenue to normalize DAT function and dopamine transmission in patients with psychostimulant use disorder.

Cocaine-regulated trafficking of dopamine transporters in cultured neurons revealed by a pH sensitive reporter

Cocaine acts by inhibiting plasma membrane dopamine transporter (DAT) function and altering its surface expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, arepoorly understood. In this study, we engineered and validated the use of DAT-pHluorin for studying DAT localization and its dynamic trafficking at neuronal processes of cultured mouse midbrain neurons. We demonstrate that unlike neuronal soma and dendrites, which contain a majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, whereas large varicosities contain abundant intracellular DAT within acidic intracellular structures. We also demonstrate that cocaine exposure leads to a Synaptojanin1-sensitive DAT internalization process followed by membrane reinsertion that lasts for days. Thus, our study reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.

Atorvastatin Modulates the Function and Trafficking of Dopamine Transporters

IntroductionDopaminergic signaling is involved in learning, memory, reward, movement, and emotion. The dopamine transporter (DAT) is the primary mechanism for terminating this signaling via DA uptake. A few lines of evidence indicate that DAT function is regulated by membrane cholesterol. Cholesterol is a critical component of plasma membranes and plays an essential role in regulating membrane fluidity, protein compartmentalization, and function. Statins are cholesterol‐lowering medications that have been linked to mild cognitive impairment. The goal of this project was to examine the effects of atorvastatin‐mediated reductions in cholesterol content on DAT function and trafficking.MethodsCultured cells & cholesterol measurement: HA‐tagged human DAT (hDAT) was stably expressed in neuroblastoma 2a (N2A) cells using G418 as selection. Cells were treated with atorvastatin (0.1, 0.3, 1.0, or 10 μM) and membrane cholesterol content was measured using Amplex Red.[3H]DA Uptake: After 24hr atorvastatin or vehicle treatment, cells were incubated with 15 nM [3H]DA in the presence of various concentrations of non‐labeled DA and [3H]DA uptake was measured to determine Vmax and Km.Cocaine inhibition of DA uptake & cholesterol replenishment: Cells were treated for 24hr with atorvastatin (1 μM) or vehicle and incubated with 15 nM [3H]DA and increasing concentrations of cocaine (100 nM‐10 μM). To determine if there was a causal effect of reduced cholesterol content and cocaine inhibition of DA uptake, atorvastatin‐treated cells were replenished with water soluble cholesterol (~40 mg per gram solid; MβCD) prior to cocaine inhibition.Amphetamine‐induced, DAT‐mediated DA efflux: Cells treated for 24hrs with either atorvastatin (1 μM) or vehicle were preloaded with 30 nM of [3H]DA. Then, cells were incubated with amphetamine (1 or 10 μM). AMPH‐induced DA efflux was determined by measuring DA content in the medium across time.Surface DAT Biotinylation: Basal surface levels of DAT in atorvastatin and vehicle treated cells, as well as DA‐induced DAT internalization, were measured by surface biotinylation using membrane non‐permeable sulfo‐NHS‐SS biotin.ImmunocytochemistryWe measured levels of F‐actin and G‐actin to determine the effects of atorvastatin treatment on actin dynamics using immunocytochemistry.ResultsWe found that atorvastatin dose dependently reduced membrane cholesterol content. This reduction was accompanied by decreases in the maximal rate of DA uptake and by decreases in the potency of cocaine to inhibit [3H]DA uptake. In contrast, atorvastatin treatment did not significantly alter AMPH‐induced DA efflux. Importantly, the reduced ability of cocaine to inhibit DA uptake was reversed by replenishing cholesterol in atorvastatin‐treated cells, suggesting a direct causal effect of reduced cholesterol content and cocaine inhibition. Lastly, we found that atorvastatin disrupted actin dynamics and abolished DA‐induced DAT internalization. The present study provides evidence for the first time that atorvastatin dysregulates DAT function and trafficking. Furthermore, this dysregulation may be associated with the cognitive deficits observed in patients receiving atorvastatin treatment.

Dopaminergic Ric GTPase activity impacts amphetamine sensitivity and sleep quality in a dopamine transporter-dependent manner in Drosophila melanogaster.

Dopamine (DA) is required for movement, sleep, and reward, and DA signaling is tightly controlled by the presynaptic DA transporter (DAT). Therapeutic and addictive psychostimulants, including methylphenidate (Ritalin; MPH), cocaine, and amphetamine (AMPH), markedly elevate extracellular DA via their actions as competitive DAT inhibitors (MPH, cocaine) and substrates (AMPH). DAT silencing in mice and invertebrates results in hyperactivity, reduced sleep, and blunted psychostimulant responses, highlighting DAT's essential role in DA-dependent behaviors. DAT surface expression is not static; rather it is dynamically regulated by endocytic trafficking. PKC-stimulated DAT endocytosis requires the neuronal GTPase, Rit2, and Rit2 silencing in mouse DA neurons impacts psychostimulant sensitivity. However, it is unknown whether or not Rit2-mediated changes in psychostimulant sensitivity are DAT-dependent. Here, we leveraged Drosophila melanogaster to test whether the Drosophila Rit2 ortholog, Ric, impacts dDAT function, trafficking, and DA-dependent behaviors. Orthologous to hDAT and Rit2, dDAT and Ric directly interact, and the constitutively active Ric mutant Q117L increased dDAT surface levels and function in cell lines and ex vivo Drosophila brains. Moreover, DAergic RicQ117L expression caused sleep fragmentation in a DAT-dependent manner but had no effect on total sleep and daily locomotor activity. Importantly, we found that Rit2 is required for AMPH-stimulated DAT internalization in mouse striatum, and that DAergic RicQ117L expression significantly increased Drosophila AMPH sensitivity in a DAT-dependent manner, suggesting a conserved impact of Ric-dependent DAT trafficking on AMPH sensitivity. These studies support that the DAT/Rit2 interaction impacts both baseline behaviors and AMPH sensitivity, potentially by regulating DAT trafficking.

Acetylcholine Receptor Stimulation Mediates Trafficking of the Dopamine and Glutamate Transporters through Activation of Protein Kinase C

The dopamine transporter (DAT) clears neurotransmitter from the extracellular space and serves as an important regulator of signal amplitude and duration at sites of dopamine release. Similarly, the neuronal excitatory amino acid transporter 3 (EAAT3) plays an important role in the control of glutamatergic tone. Several different intracellular signaling pathways have been observed to modulate activity of these neurotransmitter transporters through regulation of the trafficking of the carriers to and from the cell surface. In this study, we investigated how the activation of protein kinase C (PKC) facilitates internalization of DAT and insertion of EAAT3 by addressing the endogenous cell-surface receptors that activate PKC and regulate the DAT and EAAT3 in vivo. We report here that stimulation of M1/M5 muscarinic receptors in midbrain cultures decreases the ability of dopamine neurons to transport dopamine through DAT. The cholinergic agonist, carbachol decreases DAT activity in primary cultures while the M1/M5-specific antagonist, pirenzepine, blocks these effects. The M3 antagonist, DAU 5884, has no effect, but a positive modulator of M5, VU 0238429, enhances the loss of DAT function in response to carbachol and acetylcholine. These data implicate M1/M5 receptors on dopamine neurons in the modulation of DAT function. Bisindolylmaleimide, a PKC inhibitor, blocks the effects of carbachol stimulation on dopamine uptake, indicating a role for PKC in muscarinic receptor-mediated DAT internalization. We designed a specific Gq inhibitor peptide that also blocks the effects of carbachol on DAT in primary cultures, confirming Gq as the G-protein that couples M1/M5 receptors to PKC activation in these cells. It has been established that the activity of the neuronal glutamate transporter EAAT3 increases in response to PMA treatment in the C6 glioma cell line. By total internal reflection fluorescence (TIRF) microscopy of a GFP-tagged EAAT3 we were able to confirm that this was due to an increase in EAAT3 present at the cell surface following PMA treatment. Carbachol activation of endogenous muscarinic receptors in the C6 glioma line also resulted in increased cell-surface expression of EAAT3. This increase was inhibited by co-application of the Gq inhibitor peptide indicating that the increase in EAAT3 activity depends upon Gq-coupled PKC signaling through M1/M5 receptors. In acute midbrain slices, biotinylation of cell-surface proteins showed that the decreases in dopamine transport mediated by muscarinic receptor stimulation the result of decreases in DAT carriers a cell surface. In addition, increases in surface expression of EAAT3 in the same experiment also appear to be modulated by M1/M5 receptors. These data indicate that stimulation of cholinergic receptors can lead to downregulation and upregulation of dopamine and glutamate transporters, respectively through alterations in their membrane trafficking.

Acetylcholine Receptor Stimulation Activates Protein Kinase C Mediated Internalization of the Dopamine Transporter.

The dopamine transporter (DAT) clears neurotransmitters from the extracellular space and serves as an important regulator of signal amplitude and duration at sites of dopamine release. Several different intracellular signaling pathways have been observed to modulate DAT activity through the regulation of the trafficking of the carriers to and from the cell surface. Acute activation of protein kinase C (PKC) by phorbol esters facilitates clathrin-dependent internalization of the DAT in a variety of model systems; however, the physiological stimuli and cell-surface receptor systems that activate PKC and regulate the DAT in dopamine neurons remain elusive. We report here that stimulation of M1/M5 muscarinic receptors in midbrain cultures decreases the ability of dopamine neurons to transport dopamine through DAT. Application of the cholinomimetic drug carbachol leads to a decrease in DAT activity in primary cultures while the M1/M5-specific antagonist, pirenzepine, blocks these effects. The M3 antagonist, DAU 5884, does not affect, but a positive modulator of M5, VU 0238429, enhances the loss of DAT function in response to carbachol and acetylcholine. These data implicate M1/M5 receptors on dopamine neurons in the modulation of DAT function. Bisindolylmaleimide, a PKC inhibitor, blocks the effects of carbachol stimulation on dopamine uptake, supporting a role for PKC in muscarinic receptor-mediated DAT internalization. Furthermore, as shown previously for PKC-induced internalization, downregulation of the DAT is dependent on both clathrin and dynamin. A Gq-specific inhibitor peptide also blocks the effects of carbachol on DAT in primary cultures, confirming Gq as the G-protein that couples M1/M5 receptors to PKC activation in these cells. In acute midbrain slices, biotinylation of cell-surface proteins revealed the loss of dopamine transport mediated by muscarinic receptor stimulation was, indeed, due to loss of membrane expression of the DAT in endogenous tissue. These data indicate that stimulation of cholinergic pathways can lead to modulation of dopamine through internalization of the DAT.

Prolyl Oligopeptidase Regulates Dopamine Transporter Oligomerization and Phosphorylation in a PKC- and ERK-Independent Manner.

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.

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

Burst activation of dopamine neurons produces prolonged post-burst availability of actively released dopamine.

Both phasic and tonic modes of neurotransmission are implicated in critical functions assigned to dopamine. In learning, for example, sub-second phasic responses of ventral tegmental area (VTA) dopamine neurons to salient events serve as teaching signals, but learning is also interrupted by dopamine antagonists administered minutes after training. Our findings bridge the multiple timescales of dopamine neurotransmission by demonstrating that burst stimulation of VTA dopamine neurons produces a prolonged post-burst increase (>20 min) of extracellular dopamine in nucleus accumbens and prefrontal cortex. This elevation is not due to spillover from the stimulation surge but depends on impulse flow-mediated dopamine release. We identified Rho-mediated internalization of dopamine transporter as a mechanism responsible for prolonged availability of actively released dopamine. Thus, a critical consequence of burst activity of dopamine neurons may be post-burst sustained elevation of extracellular dopamine in terminal regions via an intracellular mechanism that promotes dopamine transporter internalization. These results demonstrate that phasic and tonic dopamine neurotransmission can be a continuum and may explain why both modes of signaling are critical for motivational and cognitive functions associated with dopamine.

The Dopamine Transporter Recycles via a Retromer-Dependent Postendocytic Mechanism: Tracking Studies Using a Novel Fluorophore-Coupling Approach.

Presynaptic reuptake, mediated by the dopamine (DA) transporter (DAT), terminates DAergic neurotransmission and constrains extracellular DA levels. Addictive and therapeutic psychostimulants inhibit DA reuptake and multiple DAT coding variants have been reported in patients with neuropsychiatric disorders. These findings underscore that DAT is critical for DA neurotransmission and homeostasis. DAT surface availability is regulated acutely by endocytic trafficking, and considerable effort has been directed toward understanding mechanisms that govern DAT's plasma membrane expression and postendocytic fate. Multiple studies have demonstrated DAT endocytic recycling and enhanced surface delivery in response to various stimuli. Paradoxically, imaging studies have not detected DAT targeting to classic recycling endosomes, suggesting that internalized DAT targets to either degradation or an undefined recycling compartment. Here, we leveraged PRIME (PRobe Incorporation Mediated by Enzyme) labeling to couple surface DAT directly to fluorophore, and tracked DAT's postendocytic itinerary in immortalized mesencephalic cells. Following internalization, DAT robustly targeted to retromer-positive endosomes, and DAT/retromer colocalization was observed in male mouse dopaminergic somatodendritic and terminal regions. Short hairpin RNA-mediated Vps35 knockdown revealed that DAT endocytic recycling requires intact retromer. DAT also targeted rab7-positive endosomes with slow, linear kinetics that were unaffected by either accelerating DAT internalization or binding a high-affinity cocaine analog. However, cocaine increased DAT exit from retromer-positive endosomes significantly. Finally, we found that the DAT carboxy-terminal PDZ-binding motif was required for DAT recycling and exit from retromer. These results define the DAT recycling mechanism and provide a unifying explanation for previous, seemingly disparate, DAT endocytic trafficking findings.SIGNIFICANCE STATEMENT The neuronal dopamine (DA) transporter (DAT) recaptures released DA and modulates DAergic neurotransmission, and a number of DAT coding variants have been reported in several DA-related disorders, including infantile parkinsonism, attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is also competitively inhibited by psychostimulants with high abuse potential. Therefore, mechanisms that acutely affect DAT availability will likely exert significant impact on both normal and pathological DAergic homeostasis. Here, we explore the cellular mechanisms that acutely control DAT surface expression. Our results reveal the intracellular mechanisms that mediate DAT endocytic recycling following constitutive and regulated internalization. In addition to shedding light on this critical process, these findings resolve conflict among multiple, seemingly disparate, previous reports on DAT's postendocytic fate.

Prolyl Oligopeptidase Regulates Dopamine Transporter Phosphorylation in the Nigrostriatal Pathway of Mouse.

Alpha-synuclein is the main component of Lewy bodies, a histopathological finding of Parkinson's disease. Prolyl oligopeptidase (PREP) is a serine protease that binds to α-synuclein and accelerates its aggregation in vitro. PREP enzyme inhibitors have been shown to block the α-synuclein aggregation process in vitro and in cellular models, and also to enhance the clearance of α-synuclein aggregates in transgenic mouse models. Moreover, PREP inhibitors have induced alterations in dopamine and metabolite levels, and dopamine transporter immunoreactivity in the nigrostriatal tissue. In this study, we characterized the role of PREP in the nigrostriatal dopaminergic and GABAergic systems of wild-type C57Bl/6 and PREP knockout mice, and the effects of PREP overexpression on these systems. Extracellular concentrations of dopamine and protein levels of phosphorylated dopamine transporter were increased and dopamine reuptake was decreased in the striatum of PREP knockout mice, suggesting increased internalization of dopamine transporter from the presynaptic membrane. Furthermore, PREP overexpression increased the level of dopamine transporters in the nigrostriatal tissue but decreased phosphorylated dopamine transporters in the striatum in wild-type mice. Our results suggest that PREP regulates the function of dopamine transporter, possibly by controlling the phosphorylation and transport of dopamine transporter into the striatum or synaptic membrane.

Membrane potential shapes regulation of dopamine transporter trafficking at the plasma membrane.

The dopaminergic system is essential for cognitive processes, including reward, attention and motor control. In addition to DA release and availability of synaptic DA receptors, timing and magnitude of DA neurotransmission depend on extracellular DA-level regulation by the dopamine transporter (DAT), the membrane expression and trafficking of which are highly dynamic. Data presented here from real-time TIRF (TIRFM) and confocal microscopy coupled with surface biotinylation and electrophysiology suggest that changes in the membrane potential alone, a universal yet dynamic cellular property, rapidly alter trafficking of DAT to and from the surface membrane. Broadly, these findings suggest that cell-surface DAT levels are sensitive to membrane potential changes, which can rapidly drive DAT internalization from and insertion into the cell membrane, thus having an impact on the capacity for DAT to regulate extracellular DA levels.

Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant

The dopamine (DA) transporter (DAT) facilitates high-affinity presynaptic DA reuptake that temporally and spatially constrains DA neurotransmission. Aberrant DAT function is implicated in attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target, and psychostimulant reward strictly requires binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKC-sensitive negative endocytic mechanism, or "endocytic brake," controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is unknown, and it is unknown whether this braking mechanism is unique to DAT or common to monoamine transporters. Here, we report that the cdc42-activated, nonreceptor tyrosine kinase, Ack1, is a DAT endocytic brake that stabilizes DAT at the plasma membrane and is released in response to PKC activation. Pharmacologic and shRNA-mediated Ack1 silencing enhanced basal DAT internalization and blocked PKC-stimulated DAT internalization, but had no effects on SERT endocytosis. Both cdc42 activation and PKC stimulation converge on Ack1 to control Ack1 activity and DAT endocytic capacity, and Ack1 inactivation is required for stimulated DAT internalization downstream of PKC activation. Moreover, constitutive Ack1 activation is sufficient to rescue the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT coding variants that exhibit trafficking dysregulation.

Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine

Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH's effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action.

The mechanism of amphetamine‐mediated trafficking of the dopamine transporter (1145.1)

Acute amphetamine (AMPH) exposure elevates extracellular dopamine by a variety of mechanisms including increasing the rate of internalization of the plasma membrane dopamine transporter (DAT). We report here that AMPH also activates the small GTPases, Rho and Rac. In recombinant expression systems, primary cultures and midbrain slices, activation of Rho and the downstream Rho‐kinase (ROCK) triggers endocytosis of DAT. Inhibition of RhoA activity blocks AMPH‐induced DAT internalization, while Rac1 inhibition has no significant effect on DAT trafficking. ROCK inhibitors also prevent AMPH‐induced DAT internalization. Intriguingly, we found that AMPH must enter the cell to initiate this cascade. Further, intracellular AMPH also increases cAMP, leading to the phosphorylation of Rho by protein kinase A (PKA) that inactivates Rho and serves as a break on DAT internalization, thus demonstrating an important interaction between PKA‐ and RhoA‐dependent signaling in mediating the actions of AMPH. In agreement with our observations in cell lines and brain slices, we have also shown that the activation of receptors that couple to PKA in dopamine neurons within the brain can antagonize the behavioral effects of AMPH in mice. These observations imply the existence of a novel intracellular target that mediates the effects of AMPH on Rho and cAMP signaling and suggest new pathways to target in order to disrupt AMPH action.Grant Funding Source: Supported by the National Institute of Mental Health Intramural Program.

Dopamine Transporter Endocytic Trafficking in Striatal Dopaminergic Neurons: Differential Dependence on Dynamin and the Actin Cytoskeleton

Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.

The Clathrin-Dependent Localization of Dopamine Transporter to Surface Membranes Is Affected by α-Synuclein

Parkinson's disease (PD) is a progressive age-dependent neurodegenerative disorder, predominantly affecting the dopamine-producing neurons residing at the substantia nigra. Abnormalities in α-synuclein (α-Syn) and dopamine transporter (DAT) are implicated in the pathogenesis of PD. We tested the hypothesis that α-Syn regulates surface DAT localization and DAT activity, in cultured cells co-expressing α-Syn and DAT, and in brains of mice modeling PD, transgenic for the mutant A53T α-Syn form. The results indicate that α-Syn expression affects the partitioning of DAT between the cell surface and intracellular compartments, resulting in lower surface DAT levels. Accordingly, lower uptake of tritiated dopamine was measured in synaptosomes of A53T α-Syn transgenic mouse brains. Importantly, we show that the effect of α-Syn on surface DAT is mediated by clathrin. Downregulation of clathrin by specific siRNAs directed against its heavy chain abolished the effect of α-Syn on phorbol 12-myristate 13-acetate-induced DAT internalization. These results suggest that α-Syn plays a role in regulating dopamine homeostasis through its involvement in clathrin-mediated endocytosis.

Differential targeting of the dopamine transporter to recycling or degradative pathways during amphetamine‐ or PKC‐regulated endocytosis in dopamine neurons

The dopamine transporter (DAT) clears the extracellular dopamine released during neurotransmission and is a major target for both therapeutic and addictive psychostimulant amphetamines. Amphetamine exposure or activation of protein kinase C (PKC) by the phorbol ester PMA has been shown to down-regulate cell surface DAT. However, in dopamine neurons, the trafficking itinerary and fate of internalized DAT has not been elucidated. By monitoring surface-labeled DAT in transfected dopamine neurons from embryonic rat mesencephalic cultures, we find distinct sorting and fates of internalized DAT after amphetamine or PMA treatment. Although both drugs promote DAT internalization above constitutive endocytosis in dopamine neurons, PMA induces ubiquitination of DAT and leads to accumulation of DAT on LAMP1-positive endosomes. In contrast, after amphetamine exposure DAT is sorted to recycling endosomes positive for Rab11 and the transferrin receptor. Furthermore, quantitative assessment of DAT recycling using an antibody-feeding assay reveals that significantly less DAT returns to the surface of dopamine neurons after internalization by PMA, compared with vehicle or amphetamine treatment. These results demonstrate that, in neurons, the DAT is sorted differentially to recycling and degradative pathways after psychostimulant exposure or PKC activation, which may allow for either the transient or sustained inhibition of DAT during dopamine neurotransmission.

Flotillins Regulate Membrane Mobility of the Dopamine Transporter but Are Not Required for Its Protein Kinase C Dependent Endocytosis

Flotillins were proposed to mediate clathrin-independent endocytosis, and recently, flotillin-1 was implicated in the protein kinase C (PKC)-triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin-mediated, we re-examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin-1, flotillin-2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC-dependent internalization and degradation of HA-DAT. In contrast, siRNAs to clathrin heavy chain and μ2 subunit of clathrin adaptor complex AP-2 as well as a dynamin inhibitor Dyngo-4A significantly decreased PKC-dependent endocytosis of HA-DAT. Similarly, endocytosis and degradation of DAT that is not epitope-tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co-localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA-DAT in the plasma membrane, suggesting that flotillin-organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin-mediated endocytosis is the major pathway of PKC-dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis.