Sammanda Ramamoorthy

A Role for p38 Mitogen-Activated Protein Kinase in the Regulation of the Serotonin Transporter: Evidence for Distinct Cellular Mechanisms Involved in Transporter Surface Expression

The serotonin transporter (SERT) is regulated by various signaling mechanisms that may operate to maintain appropriate levels of synaptic serotonin (5-HT). We demonstrate that one of the mitogen-activated protein kinases (MAPKs), p38 MAPK, regulates SERT. Treatment of rat midbrain synaptosomes with p38 MAPK-specific inhibitors, PD169316 [4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole] or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], reduced 5-HT uptake. An additive SERT inhibition by PD169316 and β-phorbol 12-myristate 13-acetate (β-PMA) indicated the involvement of a protein kinase C (PKC)-independent MAPK pathway. Kinetic studies indicated a significant decrease in the transport capacity (Vmax) after PD169316 treatment of synaptosomes. Biotinylation studies showed reduced SERT proteins in the plasma membrane of synaptosomes after p38 MAPK inhibition and PKC activation. Phosphorylation studies using synaptosomes revealed decreased SERT phosphorylation by PD169316 but increased phosphorylation by β-PMA. d-Amphetamine enhanced SERT basal phosphorylation and PD169316 blocked this effect. SERT interaction with protein phosphatase 2A catalytic subunit and syntaxin 1A decreased after PD169316 or β-PMA treatment of synaptosomes. In synaptosomes, PKC activation but not p38 MAPK inhibition resulted in SERT redistribution from cholesterolrich lipid raft fractions to nonlipid raft fractions. The presence of phospho-p38 MAPK in synaptosomes and human embryonic kidney 293 (HEK-293) cells suggested the presence of constitutively active p38 MAPK in these preparations. Cotransfection of HEK-293 cells with SERT and a constitutively active form of MAP kinase kinase 3b(E) [MKK3b(E)] increased 5-HT transport, and RNA interference targeted to p38 MAPK inhibited 5-HT uptake, confirming the involvement of active p38 MAPK in SERT expression. Although PD169316 inhibited SERT insertion to the plasma membrane, β-PMA increased SERT internalization in HEK-293 cells. Together, these results indicate a distinct role of p38 MAPK in SERT regulation.

D2 Receptors Regulate Dopamine Transporter Function via an Extracellular Signal-Regulated Kinases 1 and 2-Dependent and Phosphoinositide 3 Kinase-Independent Mechanism

The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D2 dopamine receptors (D2R) has been reported. The high affinity of DA and other DAT substrates for the D2R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP+) with live cell imaging techniques to identify the role of two D2R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D2R regulation of DAT. Addition of the D2/D3 receptor agonist quinpirole (0.1–10 μM) to human embryonic kidney cells coexpressing human DAT and D2 receptor (short splice variant, D2SR) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP+ accumulation. The D2/D3 agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP+ accumulation. D2SR activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP+ accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D2R stimulation. These experiments demonstrate that D2SR stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D2R. Together, these results suggest a novel mechanism by which D2SRautoreceptors may regulate DAT in the central nervous system.

Regulation of monoamine transporters: Influence of psychostimulants and therapeutic antidepressants

Synaptic neurotransmission in the central nervous system (CNS) requires the precise control of the duration and the magnitude of neurotransmitter action at specific molecular targets. At the molecular level, neurotransmitter signaling is dynamically regulated by a diverse set of macromolecules including biosynthetic enzymes, secretory proteins, ion channels, pre- and postsynaptic receptors and transporters. Monoamines, 5-hydroxytryptamine or serotonin (5-HT), norepinephrine (NE), and dopamine (DA) play an important modulatory role in the CNS and are involved in numerous physiological functions and pathological conditions. Presynaptic plasma membrane transporters for 5-HT (SERT), NE (NET), and DA (DAT), respectively, control synaptic actions of these monoamines by rapidly clearing the released amine. Monoamine transporters are the sites of action for widely used antidepressants and are high affinity molecular targets for drugs of abuse including cocaine, amphetamine, and 3,4-methylenedioxymetamphetamine (MDMA) “Ecstasy”. Monoamine transporters also serve as molecular gateways for neurotoxins. Emerging evidence indicates that regulation of transporter function and surface expression can be rapidly modulated by “intrinsic” transporter activity itself, and antidepressant and psychostimulant drugs that block monoamine transport have a profound effect on transporter regulation. Therefore, disregulations in the functioning of monoamine transporters may underlie many disorders of transmitter imbalance such as depression, attention deficit hyperactivity disorder, and schizophrenia. This review integrates recent progress in understanding the molecular mechanisms of monoamine transporter regulation, in particular, posttranscriptional regulation by phosphorylation and trafficking linked to cellular protein kinases, protein phosphatases, and transporter interacting proteins. The review also discusses the possible role of psychostimulants and antidepressants in influencing monoamine transport regulation.

Regulation of monoamine transporters: Role of transporter phosphorylation

Presynaptic biogenic amine transporters mediate reuptake of released amines from the synapse, thus regulating serotonin, dopamine and norepinephrine neurotransmission. Medications utilized in the treatment of depression, attention deficit-hyperactivity disorder and other psychiatric disorders possess high affinity for amine transporters. In addition, amine transporters are targets for psychostimulants. Altered expression of biogenic amine transporters has long been implicated in several psychiatric and degenerative disorders. Therefore, appropriate regulation and maintenance of biogenic amine transporter activity is critical for the maintenance of normal amine homoeostasis. Accumulating evidence suggests that cellular protein kinases and phosphatases regulate amine transporter expression, activity, trafficking and degradation. Amine transporters are phosphoproteins that undergo dynamic control under the influence of various kinase and phosphatase activities. This review presents a brief overview of the role of amine transporter phosphorylation in the regulation of amine transport in the normal and diseased brain. Understanding the molecular mechanisms by which phosphorylation events affect amine transporter activity is essential for understanding the contribution of transporter phosphorylation to the regulation of monoamine neurotransmission and for identifying potential new targets for the treatment of various brain diseases.

Regulation of Dopamine Transporter Function and Cell Surface Expression by D3 Dopamine Receptors

D3 dopamine receptors are expressed by dopamine neurons and are implicated in the modulation of presynaptic dopamine neurotransmission. The mechanisms underlying this modulation remain ill defined. The dopamine transporter, which terminates dopamine transmission via reuptake of released neurotransmitter, is regulated by receptor- and second messenger-linked signaling pathways. Whether D3 receptors regulate dopamine transporter function is unknown. We addressed this issue using a fluorescent imaging technique that permits real time quantification of dopamine transporter function in living single cells. Accumulation of the fluorescent dopamine transporter substrate trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium (ASP+) in human embryonic kidney cells expressing human dopamine transporter was saturable and temperature-dependent. In cells co-expressing dopamine transporter and D3 receptors, the D2/D3 agonist quinpirole produced a rapid, concentration-dependent, and pertussis toxin-sensitive increase of ASP+ uptake. Similar agonist effects were observed in Neuro2A cells and replicated in human embryonic kidney cells using a radioligand uptake assay in which binding to and activation of D3 receptors by [3H]dopamine was prevented. D3 receptor stimulation activated phosphoinositide 3-kinase and MAPK. Inhibition of either kinase prevented the quinpirole-induced increase in uptake. D3 receptor activation differentially affected dopamine transporter function and subcellular distribution depending on the duration of agonist exposure. Biotinylation experiments revealed that the rapid increase of uptake was associated with increased cell surface and decreased intracellular expression and increased dopamine transporter exocytosis. In contrast, prolonged agonist exposure reduced uptake and transporter cell surface expression. These results demonstrate that D3 receptors regulate dopamine transporter function and identify a novel mechanism by which D3 receptors regulate extracellular dopamine concentrations.

Phosphorylation of threonine residue 276 is required for acute regulation of serotonin transporter by cyclic GMP.

Cellular protein kinases, phosphatases, and other serotonin transporter (SERT) interacting proteins participate in several signaling mechanisms regulating SERT activity. The molecular mechanisms of protein kinase G (PKG)-mediated SERT regulation and the site of transporter phosphorylation were investigated. Treatment of rat midbrain synaptosomes with 8-bromo-cGMP increased SERT activity, and the increase was selectively blocked by PKG inhibitors. The Vmax value for serotonin (5-HT) transport increased following cGMP treatment. However, surface biotinylation studies showed no change in SERT surface abundance following PKG activation. 32P metabolic labeling experiments showed increased SERT phosphorylation in the presence of cGMP that was abolished by selectively inhibiting PKG. Phosphoamino acid analysis revealed that cGMP-stimulated native SERT phosphorylation occurred only on threonine residues. When added to CHO-1 cells expressing SERT, 8-bromo-cGMP stimulated 5-HT transport and SERT phosphorylation. Mutation of SERT threonine 276 to alanine completely abolished cGMP-mediated stimulation of 5-HT transport and SERT phosphorylation. Although the T276A mutation had no significant effect on 5-HT transport or SERT protein expression, mutation to aspartate (T276D) increased the level of 5-HT uptake to that of cGMP-stimulated 5-HT uptake in wild-type SERT-expressing cells and was no longer sensitive to cGMP. These findings provide the first identification of a phosphorylation site in SERT and demonstrate that phosphorylation of Thr-276 is required for cGMP-mediated SERT regulation. They also constitute the first evidence that in the central nervous system PKG activation stimulates endogenous SERT activity by a trafficking-independent mechanism.

Phosphorylation of the Norepinephrine Transporter at Threonine 258 and Serine 259 Is Linked to Protein Kinase C-mediated Transporter Internalization

Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315–19326). Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32P-labeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (β-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. β-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the Gαq-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to β-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by β-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by β-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following β-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization.

Long-Term Consequences of Methamphetamine Exposure in Young Adults Are Exacerbated in Glial Cell Line-Derived Neurotrophic Factor Heterozygous Mice

Methamphetamine abuse in young adults has long-term deleterious effects on brain function that are associated with damage to monoaminergic neurons. Administration of glial cell line-derived neurotrophic factor (GDNF) protects dopamine neurons from the toxic effects of methamphetamine in animal models. Therefore, we hypothesized that a partial GDNF gene deletion would increase the susceptibility of mice to methamphetamine neurotoxicity during young adulthood and possibly increase age-related deterioration of behavior and dopamine function. Two weeks after a methamphetamine binge (4 × 10 mg/kg, i.p., at 2 h intervals), GDNF+/− mice had a significantly greater reduction of tyrosine hydroxylase immunoreactivity in the medial striatum, a proportionally greater depletion of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum, and a greater increase in activated microglia in the substantia nigra than wild-type mice. At 12 months of age, methamphetamine-treated GDNF+/− mice exhibited less motor activity and lower levels of tyrosine hydroxylase-immunoreactivity, dopamine, DOPAC, and serotonin than wild-type mice. Greater striatal dopamine transporter activity in GDNF+/− mice may underlie their differential response to methamphetamine. These data suggest the possibility that methamphetamine use in young adults, when combined with lower levels of GDNF throughout life, may precipitate the appearance of parkinsonian-like behaviors during aging.

Prefrontal Cortical Kappa-Opioid Receptor Modulation of Local Neurotransmission and Conditioned Place Aversion

Kappa-opioid receptors (KORs) are important for motivation and other medial prefrontal cortex (mPFC)-dependent behaviors. Although KORs are present in the mPFC, their role in regulating transmission in this brain region and their contribution to KOR-mediated aversion are not known. Using in vivo microdialysis in rats and mice, we demonstrate that intra-mPFC administration of the selective KOR agonist U69,593 decreased local dopamine (DA) overflow, while reverse dialysis of the KOR antagonist nor-Binaltorphimine (nor-BNI) enhanced mPFC DA overflow. Extracellular amino-acid levels were also affected by KORs, as U69,593 reduced glutamate and GABA levels driven by the glutamate reuptake blocker, l-trans-pyrrolidine-2,4-dicarboxylate. Whole-cell recordings from mPFC layer V pyramidal neurons revealed that U69,593 decreased the frequency, but not amplitude, of glutamatergic mini EPSPs. To determine whether KOR regulation of mPFC DA overflow was mediated by KOR on DA terminals, we utilized a Cre recombinase-driven mouse line lacking KOR in DA neurons. In these mice, basal DA release or uptake was unaltered relative to controls, but attenuation of mPFC DA overflow by local U69,593 was not observed, indicating KOR acts directly on mPFC DA terminals to locally inhibit DA levels. Conditioning procedures were then used to determine whether mPFC KOR signaling was necessary for KOR-mediated aversion. U69,593-mediated conditioned place aversion was blocked by intra-mPFC nor-BNI microinjection. These findings demonstrate that mPFC KORs negatively regulate DA and amino-acid neurotransmission, and are necessary for KOR-mediated aversion.

Regulation of Antidepressant-Sensitive Serotonin Transporters

The indoleamine serotonin (5-hydroxytryptamine [5HT]) plays an important role as a chemical messenger in the adult central nervous system (CNS) and periphery, regulating a host of diverse processes, including mood, sleep, sexual drive, gastrointestinal motility, thyroid function, and vasoconstriction (38,55). Though more than a half dozen distinct 5HT receptor subtypes exist to confer the specific actions of 5HT on target cells (45), a single gene product (11,51,71,102) encoding the serotonin transporter (SERT), appears to be exclusively responsible for the inactivation of extracellular 5HT. SERTs are members of a large gene family of Na+ and Cl− coupled, plasma membrane transporters (1,10),most closely related in sequence to the 1-norepinephrine (NE) transporter (NET) ((73,96) and the dopamine (DA) transporter (DAT) (44,60,113,119). SERT expression in the adult CNS appears to be predominantly, if not exclusively, neuronal (5,11,20,100), restricted to serotonergic neurons of the raphe complex and their projections. Clearance of synaptic and extrasynaptic 5HT appears to be the principal role for SERTs; however, certain cells, notably platelets (100,116), utilize SERTs to acquire 5HT from the environment for subsequent release, since they lack the essential biosynthetic machinery for 5HT synthesis. SERTs have also been identified in intestinal crypt epithelia (121), adrenal chromaffin cells (13,118), mast cells (51), medullary thyroid carcinoma cells (21), thyroid follicular cells (117), and placenta (8).