Dopamine Transporter Cell Surface Expression Research Articles

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.

Glucagon-like peptide-1 receptor regulation of basal dopamine transporter activity is species-dependent

IntroductionA solid body of preclinical evidence shows that glucagon-like peptide-1 receptor (GLP-1R) agonists attenuate the effects of substance use disorder related behaviors. The mechanisms underlying these effects remain elusive. In the present study, we hypothesized that GLP-1R activation modulates dopaminetransporter (DAT) and thus dopamine (DA) homeostasis in striatum. This was evaluated in three different experiments: two preclinical and one clinical. MethodsRat striatal DA uptake, DA clearance and DAT cell surface expression was assessed following GLP-1 (7-36)-amide exposure in vitro. DA uptake in mice was assesed ex vivo following systemic treatment with the GLP-1R agonist exenatide. In addition, DA uptake was measured in GLP-1R knockout mice and compared with DA-uptake in wild type mice. In healthy humans, changes in DAT availability was assessed during infusion of exenatide measured by single-photon emission computed tomography imaging. ResultsIn rats, GLP-1 (7-36)-amide increased DA uptake, DA clearance and DAT cell surface expression in striatum. In mice, exenatide did not change striatal DA uptake. In GLP-1R knockout mice, DA uptake was similar to what was measured in wildtype mice. In humans, systemic infusion of exenatide did not result in acute changes in striatal DAT availability. ConclusionsThe GLP-1R agonist-induced modulation of striatal DAT activity in vitro in rats could not be replicated ex vivo in mice and in vivo in humans. Therefore, the underlying mechanisms of action for the GLP-1R agonists-induced efficacy in varios addiction-like behavioural models still remain.

A peptide disrupting the D2R-DAT interaction protects against dopamine neurotoxicity

Dopamine reuptake from extracellular space to cytosol leads to accumulation of dopamine, which triggers neurotoxicity in dopaminergic neurons. Previous studies have shown that both dopamine D2 receptor (D2R) and dopamine transporter (DAT) are involved in dopamine neurotoxicity. However, blockade of either D2R or DAT causes side effects due to antagonism of other physiological functions of these two proteins. We previously found that DAT can form a protein complex with D2R and its cell surface expression is facilitated via D2R-DAT interaction, which regulates dopamine reuptake and intracellular dopamine levels. Here we found that an interfering peptide (DAT-S1) disrupting the D2R-DAT interaction protects neurons against dopamine neurotoxicity, and this effect is mediated by inhibiting DAT cell surface expression and inhibiting both caspase-3 and PARP-1 cleavage. This study demonstrates the role of the D2R-DAT complex in dopamine neurotoxicity and investigated the potential mechanisms, which might help better understand the mechanisms of dopamine neurotoxicity. The peptide may provide some insights to improve treatments for dopamine neurotoxicity and related diseases, such as Parkinson's disease, as well as methamphetamine- and 3,4-methsylenedioxy methamphetamine-induced neurotoxicity.

Identification of a Vav2-dependent mechanism for GDNF/Ret control of mesolimbic DAT trafficking.

Dopamine (DA) homeostasis is essential for a variety of brain activities. Dopamine transporter (DAT)-mediated DA reuptake is one of the most critical mechanisms for normal DA homeostasis. However, the molecular mechanisms underlying the regulation of DAT activity in the brain remain poorly understood. Here we show that the Rho-family guanine nucleotide exchange factor protein Vav2 is required for DAT cell surface expression and transporter activity modulated by glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor Ret. Mice deficient in either Vav2 or Ret displayed elevated DAT activity, which was accompanied by an increase in intracellular DA selectively in the nucleus accumbens. Vav2(-/-) mice exposed to cocaine showed reduced DAT activity and diminished behavioral cocaine response. Our data demonstrate that Vav2 is a determinant of DAT trafficking in vivo and contributes to the maintenance of DA homeostasis in limbic DA neuron terminals.

‘That DAT’ gene that causes dystonia-parkinsonism: broadening the phenotype

Dopamine transporter (DAT) deficiency, a recessive disorder due to mutations in the encoding SLC63 gene, was first described by Kurian et al. (2009, 2011). The typical phenotype originally reported was infantile dystonia-parkinsonism with a characteristic metabolic profile in the CSF, namely a raised homovanillic acid: 5-hydroxyindoleacetic acid (HVA:5-HIAA) ratio, usually in excess of 4.0. Presynaptic DAT, expressed by dopaminergic neurons, is responsible for reuptake of extracellular dopamine in a Na+/Cl−dependent manner (Torres et al., 2003). In this issue of Brain, Ng et al. (2014) describe a new cohort of patients with dopamine transporter deficiency syndrome (DTDS) and evaluate the functional consequences of SLC6A3 mutations using in vitro heterologous expression. The clinical significance of this paper is the new phenotypic associations. In addition to the classical presenation—infantile-onset progressive parkinsonism-dystonia—Ng et al. (2014) report cases who presented atypically, later in childhood and with a milder disease course. The new cohort consisted of eight cases with a median age at diagnosis of 13 years; one patient was diagnosed at 34 years. All eight patients had either homozygous or compound heterozygous missense or splice site mutations in the SLC63 gene. Three of eight cases, from one kindred, had juvenile onset progressive parkinsonism-dystonia. Notably, all three brothers had a normal birth history and neonatal course, and achieved normal early developmental milestones in infancy. At 10–11 years of age, they all developed a tremor affecting the head, with progression of symptoms in their 20 s. Another case, despite having infantile onset, survived into his fourth decade. Ng et al. (2014) also present evidence for dysfunction of the DAT gene in the new cases. In vitro studies of mutant transporter showed several defects in dopamine transporter function, including absent or reduced dopamine uptake, reduced DAT cell surface expression, and reduced affinity for dopamine. Loss of post-translational dopamine transporter glycosylation and failure of amphetamine-mediated dopamine efflux were also detected. The degree of dysfunction was greater for mutations causing infantile-onset than for those causing juvenile-onset disease. For example, the A314V mutation associated with atypical DTDS exhibited the highest residual dopamine uptake capacity compared with the mutations causing infantile DTDS. This report expands the phenotypic spectrum of DTDS, which includes presentation in infancy (early onset, rapidly-progressive disease), and in childhood/adolescence (later onset, slower disease progression). Genotype–phenotype analysis in this cohort, consistent with several other enzymatic recessive disorders, suggests that the higher residual dopamine transporter activity contributed to postponing disease presentation or slowing down progression in these late-onset cases. What is the significance of this report for clinicians, in particular paediatric neurologists and movement disorders specialists? DTDS is probably under-recognized. To date, apart from the original report (Kurian et al., 2009, 2011), there has been only one other study describing similar early-onset cases (Henriksen et al., 2012), and overall there are only a handful of cases in the literature. This study highlights the need to consider DTDS in the differential diagnosis for both infantile- and juvenile-onset movement disorders presenting with tremor and dystonia-parkinsonism. Some of these cases may be masquerading as cerebral palsy or other conditions that present with juvenile dystonia and parkinsonism (Schneider and Bhatia, 2010). Given that some of the patients with DTDS have oculogyric crises, other disorders in the dopamine synthesis pathway such as tyrosine hydroxylase deficiency and sepiapterin reductase deficiency enter into the differential diagnosis. Moreover, because tremor was common, juvenile parkinsonism needs to be kept in mind, particularly due to parkin gene mutations. However, in this regard, the DAT SPECT scan is normal in DTDS, whereas most cases of degenerative juvenile parkinsonism, including those arising from parkin gene mutations, will have an abnormal DAT scan. Hence the combination of the CSF analysis, showing a raised HVA/HIAA ratio, and a normal DAT scan, should be a clear pointer for DTDS, which of course can be confirmed on genetic testing. Finally, it is interesting to note that the cases described by Ng et al. (2014) are not only from an Asian background, but also include Italian and mixed European ancestry, and it is likely that these conditions are present in all countries. The message for clinicians is, when faced with a patient with infantile or adolescent dystonia-parkinsonsim, even if presenting in adulthood, don’t forget to consider ‘that DAT’ gene.

Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)-treated cells shows that G protein-coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

Role of Alpha‐Synuclein and it mutants on Dithiocarbamates Cytotoxicity

Mn‐containing dithiocarbamates, such as maneb (MB) and mancozeb (MZ), have been known to increase the toxicity of neurotoxin MPTP on dopaminergic neurons leading to cell death and Parkinson‐like symptoms. Dopamine transporter (DAT), a key protein in MPTP's toxicity, transports the active metabolite (MPP+) into dopaminergic neurons; increasing the cell surface expression of DAT increases uptake of MPTP. Alpha‐synuclein (both wt and A30P & A53T mutants) have been associated with Parkinson's, and are thought to regulate DAT trafficking by interacting with DAT.The aim of this study is elucidate the role of the alpha‐synuclein and its mutants on dithiocarbamate cytotoxicity. HEK cells transfected with DAT and alpha‐synuclein (either wt or mutant) were treated with dithiocarbamates. Lysates were used for co‐immunoprecipitation using anti‐DAT antibody. The precipitated proteins were subjected to Western blot analysis using alpha‐synuclein and DAT antibodies.We report roughly a 30% decrease in the DAT/alpha‐synuclein interaction in the presence of the A53T mutant versus wild‐type, but not in the present of A30P mutant. With dithiocarbamates the interaction of DAT and alpha‐synuclein (wt) was enhanced, with increases ranging from 50% to 200%. Pesticide treated A53T mutants respond more dramatically than A30P mutants in increasing the interaction between the DAT and the alpha‐synuclein.

Rescue of dopamine transporter function in hypoinsulinemic rats by a D2 receptor-ERK-dependent mechanism.

The dopamine (DA) transporter (DAT) is a major target for abused drugs and a key regulator of extracellular DA. A rapidly growing literature implicates insulin as an important regulator of DAT function. We showed previously that amphetamine (AMPH)-evoked DA release is markedly impaired in rats depleted of insulin with the diabetogenic agent streptozotocin (STZ). Similarly, functional magnetic resonance imaging experiments revealed that the blood oxygenation level-dependent signal following acute AMPH administration in STZ-treated rats is reduced. Here, we report that these deficits are restored by repeated, systemic administration of AMPH (1.78 mg/kg, every other day for 8 d). AMPH stimulates DA D(2) receptors indirectly by increasing extracellular DA. Supporting a role for D(2) receptors in mediating this "rescue," the effect was completely blocked by pre-treatment of STZ-treated rats with the D(2) receptor antagonist raclopride before systemic AMPH. D(2) receptors regulate DAT cell surface expression through ERK1/2 signaling. In ex vivo striatal preparations, repeated AMPH injections increased immunoreactivity of phosphorylated ERK1/2 (p-ERK1/2) in STZ-treated but not control rats. These data suggest that repeated exposure to AMPH can rescue, by activating D(2) receptors and p-ERK signaling, deficits in DAT function that result from hypoinsulinemia. Our data confirm the idea that disorders influencing insulin levels and/or signaling, such as diabetes and anorexia, can degrade DAT function and that insulin-independent pathways are present that may be exploited as potential therapeutic targets to restore normal DAT function.

Impaired Striatal Akt Signaling Disrupts Dopamine Homeostasis and Increases Feeding

BackgroundThe prevalence of obesity has increased dramatically worldwide. The obesity epidemic begs for novel concepts and therapeutic targets that cohesively address “food-abuse” disorders. We demonstrate a molecular link between impairment of a central kinase (Akt) involved in insulin signaling induced by exposure to a high-fat (HF) diet and dysregulation of higher order circuitry involved in feeding. Dopamine (DA) rich brain structures, such as striatum, provide motivation stimuli for feeding. In these central circuitries, DA dysfunction is posited to contribute to obesity pathogenesis. We identified a mechanistic link between metabolic dysregulation and the maladaptive behaviors that potentiate weight gain. Insulin, a hormone in the periphery, also acts centrally to regulate both homeostatic and reward-based HF feeding. It regulates DA homeostasis, in part, by controlling a key element in DA clearance, the DA transporter (DAT). Upon HF feeding, nigro-striatal neurons rapidly develop insulin signaling deficiencies, causing increased HF calorie intake.Methodology/Principal FindingsWe show that consumption of fat-rich food impairs striatal activation of the insulin-activated signaling kinase, Akt. HF-induced Akt impairment, in turn, reduces DAT cell surface expression and function, thereby decreasing DA homeostasis and amphetamine (AMPH)-induced DA efflux. In addition, HF-mediated dysregulation of Akt signaling impairs DA-related behaviors such as (AMPH)-induced locomotion and increased caloric intake. We restored nigro-striatal Akt phosphorylation using recombinant viral vector expression technology. We observed a rescue of DAT expression in HF fed rats, which was associated with a return of locomotor responses to AMPH and normalization of HF diet-induced hyperphagia.Conclusions/SignificanceAcquired disruption of brain insulin action may confer risk for and/or underlie “food-abuse” disorders and the recalcitrance of obesity. This molecular model, thus, explains how even short-term exposure to “the fast food lifestyle” creates a cycle of disordered eating that cements pathological changes in DA signaling leading to weight gain and obesity.

The Role of the Dopamine Transporter in Dopamine‐Induced DNA Damage

The neurotransmitter dopamine causes DNA damage, oxidative stress and is involved in the pathology of neurological diseases. To elucidate this potential link we investigated the mechanism of dopamine-induced DNA damage. We studied the role of the dopamine transporter (DAT) in MDCK and MDCK-DAT cells, containing the human DAT gene. After treatment with dopamine, only MDCK-DAT cells showed elevated chromosomal damage and dopamine uptake. Although stimulation of dopamine type 2 receptor (D(2)R) with quinpirole in the absence of dopamine did not induce genotoxicity in rat neuronal PC12 cells, interference with D(2)R signaling by inhibition of G-proteins, phosphoinositide 3 kinase and extracellular signal-regulated kinases reduced dopamine-induced genotoxicity and affected the ability of DAT to take up dopamine. Furthermore, the D(2)R antagonist sulpiride inhibited the dopamine-induced migration of DAT from cytosol to cell membrane. To determine whether oxidation of dopamine by monoamine oxidase (MAO) is relevant in its genotoxicity, we inhibited MAO, which reduced the formation of micronuclei and of the oxidative DNA adduct 8-oxodG. Overall, dopamine exerted its genotoxicity in vitro upon transport into the cells and oxidation by MAO. D(2)R signaling was involved in the genotoxicity of dopamine by affecting activation and cell surface expression of DAT and hence modulating dopamine uptake.

Akt-Dependent and Isoform-Specific Regulation of Dopamine Transporter Cell Surface Expression

Dopamine (DA) is a neurotransmitter implicated in multiple functions, including movement, cognition, motivation, and reward. The DA transporter (DAT) is responsible for clearing extracellular DA, thereby terminating DA neurotransmission. Previously, it has been shown that insulin signaling through protein kinase B/Akt regulates DAT function by fine-tuning DAT cell surface expression. Importantly, specific Akt isoforms (e.g., Akt1, Akt2) serve distinct physiological functions. Here, we demonstrate using isoform-specific Akt inhibitors that basal activity of Akt2, rather than Akt1, regulates DAT cell surface expression. Since Akt2 activation is mediated by insulin, these data further implicate insulin signaling as an important modulator of DAT function and dopaminergic tone.

Low and high cocaine locomotor responding male Sprague–Dawley rats differ in rapid cocaine-induced regulation of striatal dopamine transporter function

Adult outbred Sprague-Dawley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively). Importantly, LCRs and HCRs are distinguished by their differential responsiveness to acute cocaine-induced (but not baseline) locomotor activity, inhibition of the dopamine transporter (DAT) and resulting extracellular DA (HCR > LCR), as well as by repeated cocaine-induced locomotor sensitization and measures of cocaine's rewarding and reinforcing effects (LCR > HCR). Curiously, 30 min after acute cocaine HCRs exhibit greater DAT-mediated [(3)H]DA uptake into striatal synaptosomes than LCRs. To investigate this finding further, we measured locomotor activity, striatal [(3)H]DA uptake kinetics and DAT cell surface expression in LCRs and HCRs over an extended period (25-180 min) after a single relatively low-dose of cocaine (10 mg/kg, i.p.). HCRs exhibited the "predicted" locomotor response: a marked initial activation that returned to baseline by 120 min post-injection. While LCRs exhibited a >50% lower maximal locomotor response, this increase was sustained, lasting approximately 33% longer than in HCRs. At 25 min post-cocaine, maximal velocity (V(max)) of [(3)H]DA uptake was significantly higher by 25% in HCRs than LCRs, with no difference in affinity (K(m)). Despite the DAT V(max) difference, however, DAT surface expression did not differ between LCRs and HCRs. There was a similar trend (HCR > LCR) for DAT V(max) at 40 min, but not at 150 or 180 min. These findings suggest that, compared to LCRs, HCRs have an enhanced ability to rapidly up-regulate DAT function in response to acute cocaine, which may contribute to their more "normal" cocaine-induced locomotor activation.

Insulin regulation of dopamine transporter activity.

A growing body of literature points to insulin as an important regulator of dopamine transporter (DAT) function. Importantly, in vitro studies suggest that insulin regulation of DAT function might be mediated by the downstream effectors phosphotidylinositol 3‐kinase (PI3K), and its downstream effector protein kinase B (PKB, or Akt), which fine tunes DAT cell surface expression. Insulin status in rats was modified by administration of the diabetogenic agent streptozotocin (STZ), food restriction and high‐fat feeding (diet induced obesity (DIO)). A battery of biochemical and molecular approaches were used to measure DAT cell surface expression and activity of insulin downstream effectors. We found that Akt activity, as well as DAT cell surface expression, was significantly reduced in hypoinsulinemic rats and DIO rats. In addition, we have looked at the specific isoform of Akt involved in this pathway. We have available to us highly specific inhibitors of Akt1 (Akti‐1), Akt2 (Akti‐2) and of both (Akti‐1/2), allowing us to determine which Akt isoform regulates DA efflux and DAT trafficking. This work looks to further define the hypothesis that insulin signaling regulates DA clearance and AMPH actions.

Nicotinic receptor activation increases [3H]dopamine uptake and cell surface expression of dopamine transporters in rat prefrontal cortex.

Previous research shows that nicotine increases dopamine (DA) clearance in rat prefrontal cortex (PFC) and striatum via a nicotinic receptor (nAChR)-mediated mechanism. The present study investigated whether activation of nAChRs regulates DA transporter (DAT) function through a trafficking-dependent mechanism. After nicotine administration (0, 0.3, and 0.8 mg/kg s.c., 15-1440 min after injection), DAT function and trafficking in synaptosomes of PFC and striatum were determined. nAChR mediation of the effect of nicotine on DAT function and trafficking in PFC was determined by pretreatment with mecamylamine, dihydro-beta-erythroidine, or methyllycaconitine. Nicotine (0.8 mg/kg, 15 and 30 min after injection) increased the maximal velocity (V(max)) of [3H]DA uptake in PFC with no change in K(m), compared with control. Biotinylation and Western blot assays showed that nicotine (0.8 mg/kg; 30 min) increased DAT cell surface expression in PFC. In contrast, a lower dose of nicotine (0.3 mg/kg; 30 min) did not alter DAT function and trafficking in PFC. Pretreatment with mecamylamine, dihydro-beta-erythroidine, or methyllycaconitine (1.5, 8.0, and 10.0 mg/kg s.c., respectively) completely blocked the nicotine-induced increase in V(max) in PFC. In addition, mecamylamine completely blocked the nicotine-induced increase in DAT cell surface expression in PFC. Nicotine did not increase DAT function and cell surface expression in striatum, indicating that nicotine modulates DAT function in a brain region-specific manner. Thus, results from the present study suggest that the nicotine-induced increases in DAT function and cell surface expression in PFC may mediate some of the behavioral effects of nicotine.

PI3K signaling supports amphetamine-induced dopamine efflux

The dopamine (DA) transporter (DAT) is a major molecular target of the psychostimulant amphetamine (AMPH). AMPH, as a result of its ability to reverse DAT-mediated inward transport of DA, induces DA efflux thereby increasing extracellular DA levels. This increase is thought to underlie the behavioral effects of AMPH. We have demonstrated previously that insulin, through phosphatidylinositol 3-kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. PI3K signaling may represent a novel mechanism for regulating DA efflux evoked by AMPH, since only active DAT at the plasma membrane can efflux DA. Here, we show in both a heterologous expression system and DA neurons that inhibition of PI3K decreases DAT cell surface expression and, as a consequence, AMPH-induced DA efflux.

Rapid substrate‐induced down‐regulation of rat dopamine transporter function in vitro: dorsal striatum vs. nucleus accumbens

Dopamine (DA), d‐amphetamine (AMPH) and methamphetamine (METH) are DA transporter (DAT) substrates that also rapidly and transiently reduce cell surface expression of DATs in dorsal striatum (dSTR) and cells expressing cloned DATs. However, it is not clear if DATs are regulated similarly by substrates in nucleus accumbens (NAc). Here, we investigated whether brief in vitro exposure to DAT substrates differentially regulates DATs in synaptosomes prepared from dSTR or NAc of male Sprague Dawley rats. Synaptosomes were pre‐incubated with DA, AMPH, METH (3–30 μM) or buffer for 5–30 min at 37oC and washed twice at 4oC; specific uptake of 0.5 nM [3H]DA, defined with 100 μM cocaine, was measured for 3 min at 37oC. As expected, [3H]DA uptake was 54% lower in NAc than in dSTR, and substrate pre‐incubation significantly reduced [3H]DA uptake in dSTR in a time‐ and concentration‐dependent manner. Although in NAc [3H]DA uptake was also reduced by pre‐incubation with DA, AMPH or METH, statistical significance was not reached with any pre‐incubation time or concentration. Kinetic analyses after a 15 min pre‐incubation with 10 μM AMPH confirmed a lower maximal DAT velocity in dSTR, but not in NAc. Our results suggest that brief substrate exposure more efficaciously down‐regulates DATs in dSTR; however, surface biotinylation experiments are needed to confirm these observations. Supported by DA004216, DA014204, AA007464, DA015050.

Hypoinsulinemia regulates amphetamine-induced reverse transport of dopamine.

The behavioral effects of psychomotor stimulants such as amphetamine (AMPH) arise from their ability to elicit increases in extracellular dopamine (DA). These AMPH-induced increases are achieved by DA transporter (DAT)-mediated transmitter efflux. Recently, we have shown that AMPH self-administration is reduced in rats that have been depleted of insulin with the diabetogenic agent streptozotocin (STZ). In vitro studies suggest that hypoinsulinemia may regulate the actions of AMPH by inhibiting the insulin downstream effectors phosphotidylinositol 3-kinase (PI3K) and protein kinase B (PKB, or Akt), which we have previously shown are able to fine-tune DAT cell-surface expression. Here, we demonstrate that striatal Akt function, as well as DAT cell-surface expression, are significantly reduced by STZ. In addition, our data show that the release of DA, determined by high-speed chronoamperometry (HSCA) in the striatum, in response to AMPH, is severely impaired in these insulin-deficient rats. Importantly, selective inhibition of PI3K with LY294002 within the striatum results in a profound reduction in the subsequent potential for AMPH to evoke DA efflux. Consistent with our biochemical and in vivo electrochemical data, findings from functional magnetic resonance imaging experiments reveal that the ability of AMPH to elicit positive blood oxygen level–dependent signal changes in the striatum is significantly blunted in STZ-treated rats. Finally, local infusion of insulin into the striatum of STZ-treated animals significantly recovers the ability of AMPH to stimulate DA release as measured by high-speed chronoamperometry. The present studies establish that PI3K signaling regulates the neurochemical actions of AMPH-like psychomotor stimulants. These data suggest that insulin signaling pathways may represent a novel mechanism for regulating DA transmission, one which may be targeted for the treatment of AMPH abuse and potentially other dopaminergic disorders.

Individual differences in response to novelty predict prefrontal cortex dopamine transporter function and cell surface expression

Previous research has shown that individual differences in response to novelty predict self-administration and the locomotor response to psychostimulant drugs of abuse. The aim of the present study was to determine if individual differences in response to novelty based on inescapable or free-choice novelty tests predict dopamine transporter (DAT) function and trafficking as well as nicotine-induced modulation of DAT. Results show that the maximal velocity (Vmax) of [3H]dopamine uptake into prefrontal cortex (PFC) synaptosomes correlated negatively with the locomotor response to inescapable novelty. In contrast, Vmax correlated positively with novelty place preference in the free-choice novelty test. The divergent correlations between DAT and the two behavioral phenotypes suggest a differential contribution of DAT in these phenotypes, which are known not to be isomorphic. Furthermore, rats categorized as high responders to inescapable novelty had lower Vmax values, which were accompanied by less DAT expression at the cell surface in PFC compared with low responders, suggesting that inherent individual differences in DAT cellular localization may underlie the differential response to novelty. Compared with the saline control, nicotine increased Vmax and cell surface DAT immunoreactivity in PFC from high responders but not from low responders. Similarly, nicotine increased Vmax and cell surface DAT in PFC in rats classified as low in novelty place preference but not in rats classified as high in novelty place preference. Thus, despite the different behavioral phenotypes, the pharmacological effect of nicotine to increase DAT function and cell surface expression was apparent, such that rats with inherently lower DAT function show a greater sensitivity to the neurochemical effect of nicotine.

Parkin Disrupts the α-Synuclein/Dopamine Transporter Interaction: Consequences Toward Dopamine-induced Toxicity

Parkinson's disease is characterized by progressive neuronal degeneration of dopaminergic neurons in the substantia nigra. Many factors are thought to contribute to the neuronal cell death that occurs in Parkinson's disease, including alpha-synuclein-mediated toxicity. Previously, we have reported that alpha-synuclein directly couples to the carboxyl tail of the dopamine transporter (DAT) and that the alpha-synuclein/DAT protein complex formation accelerates DAT-mediated cellular dopamine (DA) uptake and DA-induced cellular apoptosis. In the present study, we report that parkin, an E2-dependent E3 protein ubiquitin ligase associated with recessive early onset Parkinson's disease, exerts a protective effect against DA-induced alpha-synuclein-dependent cell toxicity. Parkin impairs the alpha-synuclein/DAT coupling by interacting with the carboxyl-terminus of the DAT and blocks the alpha-synuclein-induced enhancement in both DAT cell surface expression and DAT-mediated DA uptake. Moreover, we have found that parkin protects against DA-induced cell toxicity in dopaminergic SK-N-SH cells. These findings will help identify the role of these proteins in the etiology and/or maintenance of Parkinson's disease.

Rapid regulation of dopamine transporters by tyrosine kinases in rat neuronal preparations

Termination of dopamine neurotransmission is primarily controlled by the plasma membrane-localized dopamine transporter. In this study, we investigated how this transporter is regulated by tyrosine kinases in neuronal preparations. In rat dorsal striatal synaptosomes, inhibition of tyrosine kinases by genistein or tyrphostin 23 resulted in a rapid (5-15 min), concentration-dependent decrease in [(3)H]dopamine uptake because of a reduction in maximal [(3)H]dopamine uptake velocity and dopamine transporter cell surface expression. The reduced transporter activity was associated with a decrease in phosphorylated p44/p42 mitogen-activated protein kinases. In primary rat mesencephalic neuronal cultures, the tyrosine kinase inhibitors similarly reduced [(3)H]dopamine uptake. When cultures were serum-deprived, acute activation of tyrosine kinase-coupled TrkB receptors by 100 ng/mL brain-derived neurotrophic factor significantly increased [(3)H]dopamine uptake; the effects were complex with increased maximal velocity but reduced affinity. The facilitatory effect of brain-derived neurotrophic factor on dopamine transporter activity depended on both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. Taken together, our results suggest that striatal dopamine transporter function and cell surface expression is constitutively up-regulated by tyrosine kinase activation and that brain-derived neurotrophic factor can mediate this type of rapid regulation.