Dopamine Transporter Function Research Articles

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.

Amphetamine Induces Sex-Dependent Loss of the Striatal Dopamine Transporter in Sensitized Mice.

Dopamine transporter (DAT) controls dopamine signaling in the brain through the reuptake of synaptically released dopamine. DAT is a target of abused psychostimulants such as amphetamine (Amph). Acute Amph administration induces transient DAT endocytosis, which, among other Amph effects on dopaminergic neurons, elevates extracellular dopamine. However, the effects of repeated Amph abuse, leading to behavioral sensitization and drug addiction, on DAT are unknown. Hence, we developed a 14 d Amph-sensitization protocol in knock-in mice expressing HA-epitope-tagged DAT (HA-DAT) and investigated the effects of Amph challenge on sensitized HA-DAT animals. The Amph challenge resulted in the highest locomotor activity on Day 14 in both sexes, which was sustained for 1 h in male but not female mice. Strikingly, significant (by 30-60%) loss of the HA-DAT protein in the striatum was caused by the Amph challenge of sensitized males but not females. Amph also reduced V max of dopamine transport in the striatal synaptosomes of males without changing K m values. Consistently, immunofluorescence microscopy revealed a significant increase of HA-DAT colocalization with the endosomal protein VPS35 only in Amph-challenged males. Amph-induced loss of striatal HA-DAT in sensitized mice was blocked by chloroquine, vacuolin-1, and inhibitor of Rho-associated kinases ROCK1/2, indicative of the involvement of endocytic trafficking in the DAT protein loss. Interestingly, an apparent degradation of HA-DAT protein was observed in the nucleus accumbens and not in the dorsal striatum. We propose that Amph challenge in sensitized mice triggers Rho-mediated endocytosis and post-endocytic trafficking of DAT in a brain-region-specific and sex-dependent manner.

Inhibition of LRRK2 kinase activity rescues deficits in striatal dopamine physiology in VPS35 p.D620N knock-in mice

Dysregulation of dopamine neurotransmission profoundly affects motor, motivation and learning behaviors, and can be observed during the prodromal phase of Parkinson’s disease (PD). However, the mechanism underlying these pathophysiological changes remains to be elucidated. Mutations in vacuolar protein sorting 35 (VPS35) and leucine-rich repeat kinase 2 (LRRK2) both lead to autosomal dominant PD, and VPS35 and LRRK2 may physically interact to govern the trafficking of synaptic cargos within the endo-lysosomal network in a kinase-dependent manner. To better understand the functional role of VPS35 and LRRK2 on dopamine physiology, we examined Vps35 haploinsufficient (Haplo) and Vps35 p.D620N knock-in (VKI) mice and how their behavior, dopamine kinetics and biochemistry are influenced by LRRK2 kinase inhibitors. We found Vps35 p.D620N significantly elevates LRRK2-mediated phosphorylation of Rab10, Rab12 and Rab29. In contrast, Vps35 haploinsufficiency reduces phosphorylation of Rab12. While striatal dopamine transporter (DAT) expression and function is similarly impaired in both VKI and Haplo mice, that physiology is normalized in VKI by treatment with the LRRK2 kinase inhibitor, MLi-2. As a corollary, VKI animals show a significant increase in amphetamine induced hyperlocomotion, compared to Haplo mice, that is also abolished by MLi-2. Taken together, these data show Vps35 p.D620N confers a gain-of-function with respect to LRRK2 kinase activity, and that VPS35 and LRRK2 functionally interact to regulate DAT function and striatal dopamine transmission.

Dopamine Transporter Deficiency Syndrome (DTDS): Expanding the Clinical Phenotype and Precision Medicine Approaches.

Infantile parkinsonism-dystonia due to dopamine transporter deficiency syndrome (DTDS) is an ultrarare childhood movement disorder caused by biallelic loss-of-function mutations in the SLC6A3 gene. Advances in genomic analysis have revealed an evolving spectrum of SLC6A3-related neurological and neuropsychiatric disorders. Since the initial clinical and genetic characterisation of DTDS in 2009, there have been thirty-one published cases with a variety of protein-truncating variants (nonsense variants, splice-site changes, and deletions) and missense changes. Amino acid substitutions result in mutant proteins with impaired dopamine transporter function due to reduced transporter activity, impaired dopamine binding, reduced cell-surface expression, and aberrant posttranslational protein modification with impaired glycosylation. In this review, we provide an overview of the expanding clinical phenotype of DTDS and the precision therapies in development, including pharmacochaperones and gene therapy.

The Alkylamine Stimulant 1,3-Dimethylamylamine Exhibits Substrate-Like Regulation of Dopamine Transporter Function and Localization.

The alkylamine stimulant 1,3-dimethylamylamine (DMAA) is used non-medically as an appetite suppressant and exercise performance enhancer despite adverse cardiovascular effects that have limited its legal status. There is scant research describing the mechanism of action of DMAA, making it difficult to gauge risks or therapeutic potential. An important molecular target of structurally related phenethylamines, such as amphetamine, for regulating mood, cognition, movement, and the development of substance use disorder is the dopamine transporter, which limits the range and magnitude of dopamine signaling via re-uptake from the extracellular space. The present studies were therefore initiated to characterize the effects of DMAA on dopamine transporter function. Specifically, we tested the hypothesis that DMAA exhibits substrate-like effects on dopamine transporter function and trafficking. In transport assays in human embryonic kidney cells, DMAA inhibited dopamine uptake by the human dopamine transporter in a competitive manner. Docking analysis and molecular dynamics simulations supported these findings, revealing that DMAA binds to the S1 substrate binding site and induces a conformational change from outward-facing open to outward-facing closed states, similar to the known substrates. Further supporting substrate-like effects of DMAA, the drug stimulated dopamine transporter endocytosis in a heterologous expression system via cocaine- and protein kinase A-sensitive mechanisms, mirroring findings with amphetamine. Together, these data indicate that DMAA elicits neurological effects by binding to and regulating function of the dopamine transporter. Furthermore, pharmacologic distinctions from amphetamine reveal structural determinants for regulating transporter conformation and add mechanistic insight for the regulation of dopamine transporter endocytosis. Significance Statement The alkylamine stimulant 1,3-dimethylamylamine (DMAA) is used as an appetite suppressant and exercise performance enhancer and is structurally similar to amphetamine, but there is scant research describing its mechanism of action. Characterizing the effects of DMAA on dopamine transporter function supports evaluation of potential risks and therapeutic potential, while also revealing mechanistic details of dynamic transporter-substrate interactions.

Dopamine Transporter Deficient Rodents: Perspectives and Limitations for Neuroscience.

The key element of dopamine (DA) neurotransmission is undoubtedly DA transporter (DAT), a transmembrane protein responsible for the synaptic reuptake of the mediator. Changes in DAT's function can be a key mechanism of pathological conditions associated with hyperdopaminergia. The first strain of gene-modified rodents with a lack of DAT were created more than 25 years ago. Such animals are characterized by increased levels of striatal DA, resulting in locomotor hyperactivity, increased levels of motor stereotypes, cognitive deficits, and other behavioral abnormalities. The administration of dopaminergic and pharmacological agents affecting other neurotransmitter systems can mitigate those abnormalities. The main purpose of this review is to systematize and analyze (1) known data on the consequences of changes in DAT expression in experimental animals, (2) results of pharmacological studies in these animals, and (3) to estimate the validity of animals lacking DAT as models for discovering new treatments of DA-related disorders.

Assessing the Location, Relative Expression and Subclass of Dopamine Receptors in the Cerebellum of Hemi-Parkinsonian Rats

Parkinson’s Disease (PD) is a neurodegenerative disease with loss of dopaminergic neurons in the nigrostriatal pathway resulting in basal ganglia (BG) dysfunction. This is largely why much of the preclinical and clinical research has focused on pathophysiological changes in these brain areas in PD. The cerebellum is another motor area of the brain. Yet, if and how this brain area responds to PD therapy and contributes to maintaining motor function fidelity in the face of diminished BG function remains largely unanswered. Limited research suggests that dopaminergic signaling exists in the cerebellum with functional dopamine receptors, tyrosine hydroxylase (TH) and dopamine transporters (DATs); however, much of this information is largely derived from healthy animals and humans. Here, we identified the location and relative expression of dopamine 1 receptors (D1R) and dopamine 2 receptors (D2R) in the cerebellum of a hemi-parkinsonian male rat model of PD. D1R expression was higher in PD animals compared to sham animals in both hemispheres in the purkinje cell layer (PCL) and granule cell layer (GCL) of the cerebellar cortex. Interestingly, D2R expression was higher in PD animals than sham animals mostly in the posterior lobe of the PCL, but no discernible pattern of D2R expression was seen in the GCL between PD and sham animals. To our knowledge, we are the first to report these findings, which may lay the foundation for further interrogation of the role of the cerebellum in PD therapy and/or pathophysiology.

Neurotoxic effects of exposure to glyphosate in rat striatum: Effects and mechanisms of action on dopaminergic neurotransmission

The main objective of this study was to evaluate the effects and possible mechanisms of action of glyphosate and a glyphosate-based herbicide (GBH) on dopaminergic neurotransmission in the rat striatum. Acute exposure to glyphosate or GBH, administered by systemic (75 or 150 mg/kg, i.p.) or intrastriatal (1, 5, or 10 mM for 1 h) routes, produced significant concentration-dependent increases in dopamine release measured in vivo by cerebral microdialysis coupled to HPLC with electrochemical detection. Systemic administration of glyphosate also significantly impaired motor control and decreased striatal acetylcholinesterase activity and antioxidant capacity. At least two mechanisms can be proposed to explain the glyphosate-induced increases in extracellular dopamine levels: increased exocytotic dopamine release from synaptic vesicles or inhibition of dopamine transporter (DAT). Thus, we investigated the effects of intrastriatal administration of glyphosate (5 mM) in animals pretreated with tetrodotoxin (TTX) or reserpine. It was observed that TTX (10 or 20 μM) had no significant effect on glyphosate-induced dopamine release, while reserpine (10 mg/kg i.p) partially but significantly reduced the dopamine release. When glyphosate was coinfused with nomifensine (50 μM), the increase in dopamine levels was significantly higher than that observed with glyphosate or nomifensine alone. So, two possible hypotheses could explain this additive effect: both glyphosate and nomifensine act through different mechanisms at the dopaminergic terminals to increase dopamine levels; or both nomifensine and glyphosate act on DAT, with glyphosate simultaneously inhibiting reuptake and stimulating dopamine release by reversing the DAT function. Future research is needed to determine the effects of this pesticide at environmentally relevant doses.

Nilotinib: from animal-based studies to clinical investigation in Alzheimer's disease patients.

Nilotinib: from animal-based studies to clinical investigation in Alzheimer's disease patients.

Atorvastatin differentially regulates the interactions of cocaine and amphetamine with dopamine transporters

The function of the dopamine transporter (DAT) is regulated by membrane cholesterol content. A direct, acute removal of membrane cholesterol by methyl-β-cyclodextrin (MβCD) has been shown to reduce dopamine (DA) uptake and release mediated by the DAT. This is of particular interest because a few widely prescribed statins that lower peripheral cholesterol levels are blood-brain barrier (BBB) penetrants, and therefore could alter DAT function through brain cholesterol modulation. The goal of this study was to investigate the effects of prolonged atorvastatin treatment (24 h) on DAT function in neuroblastoma 2A cells stably expressing DAT. We found that atorvastatin treatment effectively lowered membrane cholesterol content in a concentration-dependent manner. Moreover, atorvastatin treatment markedly reduced DA uptake and abolished cocaine inhibition of DA uptake, independent of surface DAT levels. These deficits induced by atorvastatin treatment were reversed by cholesterol replenishment. However, atorvastatin treatment did not change amphetamine (AMPH)-induced DA efflux. This is in contrast to a small but significant reduction in DA efflux induced by acute depletion of membrane cholesterol using MβCD. This discrepancy may involve differential changes in membrane lipid composition resulting from chronic and acute cholesterol depletion. Our data suggest that the outward-facing conformation of DAT, which favors the binding of DAT blockers such as cocaine, is more sensitive to atorvastatin-induced cholesterol depletion than the inward-facing conformation, which favors the binding of DAT substrates such as AMPH. Our study on statin-DAT interactions may have clinical implications in our understanding of neurological side effects associated with chronic use of BBB penetrant statins.

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.

ITat transgenic mice exhibit hyper-locomotion in the behavioral pattern monitor after chronic exposure to methamphetamine but are unaffected by Tat expression

Although antiretroviral therapy (ART) has increased the quality of life and lifespan in people living with HIV (PWH), millions continue to suffer from the neurobehavioral effects of the virus. Additionally, the abuse of illicit drugs (methamphetamine in particular) is significantly higher in PWH compared to the general population, which may further impact their neurological functions. The HIV regulatory protein, Tat, has been implicated in the neurobehavioral impacts of HIV and is purported to inhibit dopamine transporter (DAT) function in a way similar to methamphetamine. Thus, we hypothesized that a combination of Tat expression and methamphetamine would exert synergistic deleterious effects on behavior and DAT expression. We examined the impact of chronic methamphetamine exposure on exploration in transgenic mice expressing human Tat (iTat) vs. their wildtype littermates using the behavioral pattern monitor (BPM).During baseline, mice exhibited sex-dependent differences in BPM behavior, which persisted through methamphetamine exposure, and Tat activation with doxycycline. We observed a main effect of methamphetamine, wherein exposure, irrespective of genotype, increased locomotor activity and decreased specific exploration. After doxycycline treatment, mice continued to exhibit drug-dependent alterations in locomotion, with no effect of Tat, or methamphetamine interactions. DAT levels were higher in wildtype, saline-exposed males compared to all other groups.These data support stimulant-induced changes of locomotor activity and exploration, and suggest that viral Tat and methamphetamine do not synergistically interact to alter these behaviors in mice. These findings are important for future studies attempting to disentangle the effect of substances that impact DAT on HAND-relevant behaviors using such transgenic animals.

Dopaminergic nuclei in the chick midbrain express serotonin receptor subfamily genes.

Serotonin (5-hydroxytryptamine, 5-HT) is a phylogenetically conserved modulator of numerous aspects of neural functions. Serotonergic neurons in the dorsal and median raphe nucleus provide ascending innervation to the entire forebrain and midbrain. Another important neural modulatory system exists in the midbrain, the dopaminergic system, which is associated to reward processing and motivation control. Dopaminergic neurons are distributed and clustered in the brain, classically designated as groups A8-A16. Among them, groups A8-A10 associated with reward processing and motivation control are located in the midbrain and projected to the forebrain. Recently, midbrain dopaminergic neurons were shown to be innervated by serotonergic neurons and modulated by 5-HT, with the crosstalk between serotonergic and dopaminergic systems attracting increased attention. In birds, previous studies revealed that midbrain dopaminergic neurons are located in the A8-A10 homologous clusters. However, the detailed distribution of dopaminergic neurons and the crosstalk between serotonergic and dopaminergic systems in the bird are poorly understood. To improve the understanding of the regulation of the dopaminergic by the serotonergic system, we performed in situ hybridization in the chick brainstem. We prepared RNA probes for chick orthologues of dopaminergic neuron-related genes; tyrosine hydroxylase (TH) and dopa decarboxylase (DDC), noradrenaline related genes; noradrenaline transporter (NAT) and dopamine beta-hydroxylase (DBH), and serotonin receptor genes; 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1E, 5-HTR1F, 5-HTR2A, 5-HTR2B, 5-HTR2C, 5-HTR3A, 5-HTR4, 5-HTR5A, and 5-HTR7. We confirmed that the expression of tyrosine hydroxylase (TH) and NAT was well matched in all chick dopaminergic nuclei examined. This supported that the compensation of the function of dopamine transporter (DAT) by NAT is a general property of avian dopaminergic neurons. Furthermore, we showed that 5-HTR1A and 5-HTR1B were expressed in midbrain dopaminergic nuclei, suggesting the serotonergic regulation of the dopaminergic system via these receptors in chicks. Our findings will help us understand the interactions between the dopaminergic and serotonergic systems in birds at the molecular level.

Effective R2 relaxation rate, derived from dual-contrast fast-spin-echo MRI, enables detection of hemisphere differences in iron level and dopamine function in Parkinson’s disease and healthy individuals

BackgroundClinical estimates of brain iron concentration are achievable with quantitative transverse relaxation rate R2, via time-consuming multiple spin-echo (SE) sequences. The objective of this study was to investigate whether quantitative iron-sensitive information may be derived from 3.0 T dual-contrast fast-spin-echo (FSE) sequences (typically employed in anatomical non-quantitative evaluations), as a routinely-collected alternative to evaluate iron levels in healthy (HC) and Parkinson’s disease (PD) brains. New methodMRI 3.0 T FSE data from the Parkinson’s Progression Markers Initiative (PPMI) (12 PD, 12 age- and gender-matched HC subjects) were cross-sectionally and longitudinally evaluated. A new measure, ‘effective R2’, was calculated for bilateral subcortical grey matter (caudate nucleus, putamen, globus pallidus, red nucleus, substantia nigra). Linear regression analysis was performed to correlate ‘effective R2’ with models of age-dependent brain iron concentration and striatal dopamine transporter (DaT) receptor binding ratio. ResultsEffective R2 was strongly correlated with estimated brain iron concentration. In PD, putaminal effective R2 difference was observed between the hemispheres contra-/ipsi-lateral to the predominantly symptomatic side at onset. This hemispheric difference was correlated with the putaminal DaT binding ratios in PD. Comparison with existing method(s)Effective R2, derived from rapid dual-contrast FSE sequences, showed viability as an alternative to R2 from SE sequences. Linear correlation of effective R2 with estimated iron concentration was comparable to documented iron-dependent R2. The effective R2 correlation coefficient was consistent with theoretical R2 iron-dependence at 3.0 T. ConclusionsEffective R2 has clinical potential as a fast quantitative method, as an alternative to R2, to aid evaluation of brain iron levels and DaT function.

Mutant VPS35-D620N induces motor dysfunction and impairs DAT-mediated dopamine recycling pathway.

The D620N mutation in vacuolar protein sorting protein 35 (VPS35) gene has been identified to be linked to late onset familial Parkinson disease (PD). However, the pathophysiological roles of VPS35-D620N in PD remain unclear. Here, we generated the transgenic Caenorhabditis elegans overexpressing either human wild type or PD-linked mutant VPS35-D620N in neurons. C. elegans expressing VPS35-D620N, compared with non-transgenic controls, showed movement disorders and dopaminergic neuron loss. VPS35-D620N worms displayed more swimming induced paralysis but showed no defects in BSR assays, thus indicating the disruption of dopamine (DA) recycling back inside neurons. Moreover, VPS35 formed a protein interaction complex with DA transporter (DAT), RAB5, RAB11 and FAM21. In contrast, the VPS35-D620N mutant destabilized these interactions, thus disrupting DAT transport from early endosomes to recycling endosomes, and decreasing DAT at the cell surface. These effects together increased DA in synaptic clefts, and led to dopaminergic neuron degeneration and motor dysfunction. Treatment with reserpine significantly decreased the swimming induced paralysis in VPS35-D620N worms, as compared with vehicle treated VPS35-D620N worms. Our studies not only provide novel insights into the mechanisms of VPS35-D620N-induced dopaminergic neuron degeneration and motor dysfunction via disruption of DAT function and the DA signaling pathway but also indicate a potential strategy to treat VPS35-D620N-related PD and other disorders.

Structure-Activity Relationships of Dopamine Transporter Pharmacological Chaperones.

Mutations in the dopamine transporter gene (SLC6A3) have been implicated in many human diseases. Among these is the infantile parkinsonism-dystonia known as Dopamine Transporter Deficiency Syndrome (DTDS). Afflicted individuals have minimal to no functional dopamine transporter protein. This is primarily due to retention of misfolded disease-causing dopamine transporter variants. This results in a variety of severe motor symptoms in patients and the disease ultimately leads to death in adolescence or young adulthood. Though no treatment is currently available, pharmacological chaperones targeting the dopamine transporter have been shown to rescue select DTDS disease-causing variants. Previous work has identified two DAT pharmacological chaperones with moderate potency and efficacy: bupropion and ibogaine. In this study, we carried out structure-activity relationships (SARs) for bupropion and ibogaine with the goal of identifying the chemical features required for pharmacological chaperone activity. Our results show that the isoquinuclidine substituent of ibogaine and its analogs is an important feature for pharmacological chaperone efficacy. For bupropion, the secondary amine group is essential for pharmacological chaperone activity. Lastly, we describe additional ibogaine and bupropion analogs with varying chemical modifications and variable pharmacological chaperone efficacies at the dopamine transporter. Our results contribute to the design and refinement of future dopamine transporter pharmacological chaperones with improved efficacies and potencies.

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.

Allosteric Dopamine transporter modulator inhibits cocaine‐induced behavior

The dopamine transporter (DAT) is a transmembrane protein responsible for reuptake the neurotransmitter dopamine back into the presynaptic neurons. Dopamine (DA) is involved in driving motivation, it is increased when people expect a reward, and it is also involved in many central nervous systems (CNS) disorders. The inhibition of the reuptake process prevents dopamine in the synaptic cleft from getting back into the presynaptic neuron. Cocaine and amphetamine both inhibit DA reuptake by binding to the orthosteric site but through different mechanisms. Cocaine is a classical inhibitor that blocks the transporter through competitive inhibition. On the other hand, amphetamine is a substrate of DAT and causes competitive inhibition of DA reuptake and reversal of the transporter leading to the release of DA. As a result of the DA reuptake inhibition, the DA in the synaptic cleft increases, leading to amplified downstream dopaminergic signaling primarily in the mesolimbic pathway. Because DAT is a primary target of psychostimulants, compounds acting on the DAT in novel ways could potentially treat psychostimulant use disorders. We previously found a novel compound—KM822 and characterized it as an allosteric modulator of DAT function that significantly decreases cocaine‐induced locomotive response in planarians. To test if KM822 has similar effects in mammals, we administered KM822 and cocaine through intracranial infusions into Long Evans rats' nucleus accumbens (NAc) and measured locomotion. We targeted the NAc as it is a part of the brain that plays a crucial role in the mesolimbic dopaminergic pathway and has been recognized by its high density of DAT. Our results showed that KM822 significantly decreased hyper‐locomotion induced by cocaine and notably did not cause any increase in locomotion by itself. KM822's ability to block hyper‐locomotion induced by amphetamine, another psychostimulant with a different mechanism of action, was also examined. Finally, we examined KM822's ability to interfere with cocaine's rewarding effect using the conditioned place preference (CPP) assay. In this assay, animals are tested for their preference for a cocaine‐associated environment. Unlike locomotion assay, this assay is more relevant to behaviors associated with cocaine addiction and therefore has high translational value. Overall, these studies demonstrated KM822's ability to block DAT inhibitor‐induced behaviors in rats and provided strong evidence that the novel allosteric DAT modulator KM822 has significant potential for treating psychostimulant addiction.

MKP3 Overexpression and ERK1/2 Phosphorylation in Dopaminergic Neurons Regulate DAT Trafficking and Function

Dopamine is a neurotransmitter that plays a critical role in movement, motivation, and reward. Dopamine neurotransmission is regulated by the dopamine transporter (DAT), which translocates dopamine from the extracellular space to the presynaptic neuron. DAT is a target for psychostimulants such as cocaine, giving DAT a key role in psychostimulant‐use disorders. DAT cell‐surface expression regulated by trafficking to and from the plasma membrane is an important mechanism for controlling DAT function. Two signaling pathways that contribute to DAT trafficking are the protein kinase C (PKC) and mitogen activated protein kinases (MAPKs), including the extracellular signal‐regulated kinase (ERK1/2) and p38 MAPK. ERK1/2 is inactivated by the MAP kinase phosphatase MKP3, which dephosphorylates and inactivates ERK1/2. Previous data from the lab demonstrated that activation of PKC by administration of phorbol 12‐myristate 13‐acetate (PMA) in an in vitro system results in decreased DAT cell‐surface expression, and overexpression of MKP3 attenuates this effect. Furthermore, other studies have shown that ERK1/2 activation, downstream of PKC activation, may lead to phosphorylation of DAT on residue threonine 53 (Thr53), however the significance of this phosphorylation is not completely understood. In order to characterize the contributions of MKP3 and ERK1/2 to DAT phosphorylation and trafficking in vivo, we have developed two viral constructs based on adeno‐associated viral (AAV) vectors that enable cre recombinase (cre)‐dependent expression. One vector cre‐dependently expresses MKP3. The other vector is based on a genetic tool named OptoSOS. This vector enables cre‐dependent, blue‐light induced activation of ERK1/2, and as a result, can spatiotemporally activate intracellular ERK1/2 signaling. Both viral vectors are employed with transgenic rats that express cre in tyrosine hydroxylase‐positive cells (TH‐Cre rats) to drive expression in dopaminergic neurons. From these studies, we find that MKP3 overexpression and resulting ERK1/2 inactivation increases DAT surface expression by blocking the internalization and degradation of the transporter leading to an accumulation of DAT on the cell‐surface. Interestingly, MKP3 overexpression also decreases phosphorylation at Thr53. While we have shown that ERK inactivation results in increased DAT cell‐surface expression, we have employed the OptoSOS system to activate ERK1/2 and assess resulting changes in DAT trafficking. We have confirmed that ERK1/2 is activated by blue‐light in a cre‐dependent manner, and similar to what we observe with MKP3 overexpression, ERK1/2 activation results in changes in DAT cell‐surface expression and phosphorylation. Taken together, our results indicate that MKP3 and ERK1/2 serve critical physiological roles in DAT regulation and can be utilized to investigate the mechanistic relationship between DAT trafficking, phosphorylation, and transporter activity.

Possible Mechanisms Underlying the Effects of Glucagon-Like Peptide-1 Receptor Agonist on Cocaine Use Disorder.

Cocaine use disorder (CUD) is a major public health challenge with a high relapse rate and lack of effective pharmacotherapies; therefore, there is a substantial need to identify novel medications to treat this epidemic. Since the advent of glucagon-like peptide-1 (GLP-1) receptors (GLP-1Rs) agonists (GLP-1RAs), their potential has been extensively explored and expanded. In this review, we first summarized the biological effects of GLP-1, GLP-1Rs, and GLP-1RAs. Subsequently, the recent literature examining the behavioral effects and the possible pharmacological mechanisms of GLP-1RAs on CUD was reviewed. Increasing preclinical evidence suggests that GLP-1RAs are promising in regulating dopamine release, dopamine transporter (DAT) surface expression and function, mesolimbic reward system and GABAergic neurons, and maladaptive behaviors in animal models of self-administration and conditioned place preference. In addition, the emerging role of GLP-1RAs in inhibiting inflammatory cytokines was reported. These findings indicate that GLP-1RAs perform essential functions in the modulation of cocaine-seeking and cocaine-taking behaviors likely through multifaceted mechanisms. Although the current preclinical evidence provides convincing evidence to support GLP-1RA as a promising pharmacotherapy for CUD, other questions concerning clinical availability, impact and specific mechanisms remain to be addressed in further studies.