Syntaxin 1 Ser14 phosphorylation is required for nonvesicular dopamine release.

Amphetamine (AMPH) is a psychostimulant that is commonly abused. The stimulant properties of AMPH are associated with its ability to increase dopamine (DA) neurotransmission. This increase is promoted by nonvesicular DA release mediated by reversal of DA transporter (DAT) function. Syntaxin 1 (Stx1) is a SNARE protein that is phosphorylated at Ser14 by casein kinase II. We show that Stx1 phosphorylation is critical for AMPH-induced nonvesicular DA release and, in Drosophila melanogaster, regulates the expression of AMPH-induced preference and sexual motivation. Our molecular dynamics simulations of the DAT/Stx1 complex demonstrate that phosphorylation of these proteins is pivotal for DAT to dwell in a DA releasing state. This state is characterized by the breakdown of two key salt bridges within the DAT intracellular gate, causing the opening and hydration of the DAT intracellular vestibule, allowing DA to bind from the cytosol, a mechanism that we hypothesize underlies nonvesicular DA release.

Autism-Associated Variant in the SLC6A3 Gene Alters the Oral Microbiome and Metabolism in a Murine Model.

Background: Altered dopamine (DA) signaling has been associated with autism spectrum disorder (ASD), a neurodevelopmental condition estimated to impact 1 in 54 children in the United States. There is growing evidence for alterations in both gastrointestinal function and oral microbiome composition in ASD. Recent work suggests that rare variants of the SLC6A3 gene encoding the DA transporter (DAT) identified in individuals with ASD result in structural and functional changes to the DAT. One such recently identified de novo mutation is a threonine to methionine substitution at position 356 of the DAT (DAT T356M). The DAT T356M variant is associated with ASD-like phenotypes in mice homozygous for the mutation (DAT T356M+/+), including social deficits, hyperactivity, and impaired DA signaling. Here, we determine the impact of this altered DA signaling as it relates to altered oral microbiota, and metabolic and gastrointestinal dysfunction. Methods: In the DAT T356M+/+ mouse, we determine the oral microbiota composition, metabolic function, and gastrointestinal (GI) function. We examined oral microbiota by 16S RNA sequencing. We measured metabolic function by examining glucose tolerance and we probed gastrointestinal parameters by measuring fecal dimensions and weight. Results: In the DAT T356M+/+ mouse, we evaluate how altered DA signaling relates to metabolic dysfunction and altered oral microbiota. We demonstrate that male DAT T356M+/+ mice weigh less (Wild type (WT) = 26.48 ± 0.6405 g, DAT T356M+/+ = 24.14 ± 0.4083 g) and have decreased body fat (WT = 14.89 ± 0.6206%, DAT T356M+/+ = 12.72 ± 0.4160%). These mice display improved glucose handling (WT = 32.60 ± 0.3298 kcal/g, DAT T356M+/+ = 36.97 ± 0.4910 kcal/g), and an altered oral microbiota. We found a significant decrease in Fusobacterium abundance. The abundance of Fusobacterium was associated with improved glucose handling and decreased body fat. Conclusions: Our findings provide new insights into how DAT dysfunction may alter gastrointestinal function, composition of the oral microbiota, and metabolism. Our data suggest that impaired DA signaling in ASD is associated with a number of metabolic and gastrointestinal changes which are common in individuals with ASD.

A network of phosphatidylinositol (4,5)-bisphosphate (PIP2) binding sites on the dopamine transporter regulates amphetamine behavior in Drosophila Melanogaster.

Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. Here, we developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). We have previously shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP2), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. In this study, we demonstrate that the interaction of PIP2 with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. We showed that PIP2 also interacts with intracellular loop 4 at R443. Further, we identified that R443 electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, we determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. We associated this inhibition with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, we showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. We present the first evidence linking PIP2, DAT, DA efflux, and phosphorylation processes with AMPH reward.

Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3.

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

Atypical dopamine efflux caused by 3,4-methylenedioxypyrovalerone (MDPV) via the human dopamine transporter.

Synthetic cathinones are similar in chemical structure to amphetamines, and their behavioral effects are associated with enhanced dopaminergic signaling. The past ten years of research on the common constituent of bath salts, MDPV (the synthetic cathinone 3,4-methylenedioxypyrovalerone), has aided the understanding of how synthetic cathinones act at the dopamine (DA) transporter (DAT). Several groups have described the ability of MDPV to block the DAT with high-affinity. In this study, we demonstrate for the first time a new mode of action of MDPV, namely its ability to promote DAT-mediated DA efflux. Using single cell amperometric assays, we determined that low concentrations of MDPV (1nM) can cause reverse transport of DA via DAT. Notably, administration of MDPV leads to hyperlocomotion in Drosophila melanogaster. These data describe further how MDPV acts at the DAT, possibly paving the way for novel treatment strategies for individuals who abuse bath salts.

Sodium-dependent vitamin C transporter-2 mediates vitamin C transport at the cortical nerve terminal.

It has been shown that vitamin C (VC) is transported at synaptic boutons, but how this occurs has not been elucidated. This study investigates the role of the sodium-dependent vitamin C transporter-2 (SVCT2) in transporting VC at the cortical nerve terminal. Immunostaining of cultured mouse superior cervical ganglion cells showed the SVCT2 to be expressed in presynaptic boutons, colocalizing with the vesicular monoamine transporter-2 and the norepinephrine transporter. Immunoblotting of enriched cortical synaptosomes demonstrated that the SVCT2 was enriched in presynaptic fractions, confirming a predominantly presynaptic location. In crude synaptosomes, known inhibitors of SVCT2 inhibited uptake of VC. Furthermore, the kinetic features of VC uptake were consistent with SVCT2-mediated function. VC was also found to efflux from synaptosomes by a mechanism not involving the SVCT2. Indeed, VC efflux was substantially offset by reuptake of VC on the SVCT2. The presence and function of the SVCT2 at the presynaptic nerve terminal suggest that it is the transporter responsible for recovery of VC released into the synaptic cleft.

mTORC2/rictor signaling disrupts dopamine-dependent behaviors via defects in striatal dopamine neurotransmission.

Disrupted neuronal protein kinase B (Akt) signaling has been associated with dopamine (DA)-related neuropsychiatric disorders, including schizophrenia, a devastating mental illness. We hypothesize that proper DA neurotransmission is therefore dependent upon intact neuronal Akt function. Akt is activated by phosphorylation of two key residues: Thr308 and Ser473. Blunted Akt phosphorylation at Ser473 (pAkt-473) has been observed in lymphocytes and postmortem brains of schizophrenia patients, and psychosis-prone normal individuals. Mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multiprotein complex that is responsible for phosphorylation of Akt at Ser473 (pAkt-473). We demonstrate that mice with disrupted mTORC2 signaling in brain exhibit altered striatal DA-dependent behaviors, such as increased basal locomotion, stereotypic counts, and exaggerated response to the psychomotor effects of amphetamine (AMPH). Combining in vivo and ex vivo pharmacological, electrophysiological, and biochemical techniques, we demonstrate that the changes in striatal DA neurotransmission and associated behaviors are caused, at least in part, by elevated D2 DA receptor (D2R) expression and upregulated ERK1/2 activation. Haloperidol, a typical antipsychotic and D2R blocker, reduced AMPH hypersensitivity and elevated pERK1/2 to the levels of control animals. By viral gene delivery, we downregulated mTORC2 solely in the dorsal striatum of adult wild-type mice, demonstrating that striatal mTORC2 regulates AMPH-stimulated behaviors. Our findings implicate mTORC2 signaling as a novel pathway regulating striatal DA tone and D2R signaling.

Rare autism-associated variants implicate syntaxin 1 (STX1 R26Q) phosphorylation and the dopamine transporter (hDAT R51W) in dopamine neurotransmission and behaviors.

BACKGROUND: Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA. METHODS: We adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD). OUTCOMES: Here, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms involve decreased phosphorylation of STX1 at Ser14 mediated by casein kinase 2 as well as a reduction in STX1/DAT interaction. These findings point to STX1/DAT interactions and STX1 phosphorylation as key regulators of DA homeostasis. INTERPRETATION: We determine the molecular identity and the impact of these variants with the intent of defining DA dysfunction and associated behaviors as possible complications of ASD.

PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein.

Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux without altering the hDAT physiological function of DA uptake. We evaluated the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present what is to our knowledge the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.

Neuronal ablation of p-Akt at Ser473 leads to altered 5-HT1A/2A receptor function.

The serotonergic system regulates a wide range of behavior, including mood and impulsivity, and its dysregulation has been associated with mood disorders, autism spectrum disorder, and addiction. Diabetes is a risk factor for these conditions. Insulin resistance in the brain is specifically associated with susceptibility to psychostimulant abuse. Here, we examined whether phosphorylation of Akt, a key regulator of the insulin signaling pathway, controls serotonin (5-HT) signaling. To explore how impairment in Akt function regulates 5-HT homeostasis, we used a brain-specific rictor knockout (KO) mouse model of impaired neuronal phosphorylation of Akt at Ser473. Cortical 5-HT1A and 5-HT2A receptor binding was significantly elevated in rictor KO mice. Concomitant with this elevated receptor expression, the 5-HT1A receptor agonist 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) led to an increased hypothermic response in rictor KO mice. The increased cortical 5-HT1A receptor density was associated with higher 5-HT1A receptor levels on the cortical cell surface. In contrast, rictor KO mice displayed significantly reduced head-twitch response (HTR) to the 5-HT2A/C agonist 2,5-dimethoxy-4-iodoamphetamine (DOI), with evidence of impaired 5-HT2A/C receptor signaling. In vitro, pharmacological inhibition of Akt significantly increased 5-HT1A receptor expression and attenuated DOI-induced 5-HT2A receptor signaling, thereby lending credence to the observed in vivo cross-talk between neuronal Akt signaling and 5-HT receptor regulation. These data reveal that defective central Akt function alters 5-HT signaling as well as 5-HT-associated behaviors, demonstrating a novel role for Akt in maintaining neuronal 5-HT receptor function.

Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate.

Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.

Yohimbine depresses excitatory transmission in BNST and impairs extinction of cocaine place preference through orexin-dependent, norepinephrine-independent processes.

The alpha2 adrenergic receptor (α(2)-AR) antagonist yohimbine is a widely used tool for the study of anxiogenesis and stress-induced drug-seeking behavior. We previously demonstrated that yohimbine paradoxically depresses excitatory transmission in the bed nucleus of the stria terminalis (BNST), a region critical to the integration of stress and reward pathways, and produces an impairment of extinction of cocaine-conditioned place preference (cocaine-CPP) independent of α(2)-AR signaling. Recent studies show yohimbine-induced drug-seeking behavior is attenuated by orexin receptor 1 (OX(1)R) antagonists. Moreover, yohimbine-induced cocaine-seeking behavior is BNST-dependent. Here, we investigated yohimbine-orexin interactions. Our results demonstrate yohimbine-induced depression of excitatory transmission in the BNST is unaffected by alpha1-AR and corticotropin-releasing factor receptor-1 (CRFR(1)) antagonists, but is (1) blocked by OxR antagonists and (2) absent in brain slices from orexin knockout mice. Although the actions of yohimbine were not mimicked by the norepinephrine transporter blocker reboxetine, they were by exogenously applied orexin A. We find that, as with yohimbine, orexin A depression of excitatory transmission in BNST is OX(1)R-dependent. Finally, we find these ex vivo effects are paralleled in vivo, as yohimbine-induced impairment of cocaine-CPP extinction is blocked by a systemically administered OX(1)R antagonist. These data highlight a new mechanism for orexin on excitatory anxiety circuits and demonstrate that some of the actions of yohimbine may be directly dependent upon orexin signaling and independent of norepinephrine and CRF in the BNST.

Impaired striatal Akt signaling disrupts dopamine homeostasis and increases feeding.

BACKGROUND: The 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 FINDINGS: We 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/SIGNIFICANCE: Acquired 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.

Gamma-tubulin is required for bipolar spindle assembly and for proper kinetochore microtubule attachments during prometaphase I in Drosophila oocytes.

In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the germline-specific γ-tubulin (γTub37C) in meiosis I spindles, and genetic studies have yielded conflicting data regarding the role of γTub37C in the formation of bipolar spindles at meiosis I. Our examination of living and fixed oocytes carrying either a null allele or strong missense mutation in the γtub37C gene demonstrates a role for γTub37C in the positioning of the oocyte nucleus during late prophase, as well as in the formation and maintenance of bipolar spindles in Drosophila oocytes. Prometaphase I spindles in γtub37C mutant oocytes showed wide, non-tapered spindle poles and disrupted positioning. Additionally, chromosomes failed to align properly on the spindle and showed morphological defects. The kinetochores failed to properly co-orient and often lacked proper attachments to the microtubule bundles, suggesting that γTub37C is required to stabilize kinetochore microtubule attachments in anastral spindles. Although spindle bipolarity was sometimes achieved by metaphase I in both γtub37C mutants, the resulting chromosome masses displayed highly disrupted chromosome alignment. Therefore, our data conclusively demonstrate a role for γTub37C in both the formation of the anastral meiosis I spindle and in the proper attachment of kinetochore microtubules. Finally, multispectral imaging demonstrates the presences of native γTub37C along the length of wild-type meiosis I spindles.

Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

Insulin reveals Akt signaling as a novel regulator of norepinephrine transporter trafficking and norepinephrine homeostasis.

Noradrenergic signaling in the CNS plays an essential role in circuits involving attention, mood, memory, and stress as well as providing pivotal support for autonomic function in the peripheral nervous system. The high-affinity norepinephrine (NE) transporter (NET) is the primary mechanism by which noradrenergic synaptic transmission is terminated. Data indicate that NET function is regulated by insulin, a hormone critical for the regulation of metabolism. Given the high comorbidity of metabolic disorders such as diabetes and obesity with mental disorders such as depression and schizophrenia, we sought to determine how insulin signaling regulates NET function and thus noradrenergic homeostasis. Here, we show that acute insulin treatment, through the downstream kinase protein kinase B (Akt), significantly decreases NET surface expression in mouse hippocampal slices and superior cervical ganglion neuron boutons (sites of synaptic NE release). In vivo manipulation of insulin/Akt signaling, with streptozotocin, a drug that induces a type 1-like diabetic state in mice, also results in aberrant NET function and NE homeostasis. Notably, we also demonstrate that Akt inhibition or stimulation, independent of insulin, is capable of altering NET surface availability. These data suggest that aberrant states of Akt signaling such as in diabetes and obesity have the potential to alter NET function and noradrenergic tone in the brain. Furthermore, they provide one potential molecular mechanism by which Akt, a candidate gene for mood disorders such as schizophrenia and depression, can impact brain monoamine homeostasis.

Rab11 supports amphetamine-stimulated norepinephrine transporter trafficking.

The norepinephrine transporter (NET) is a presynaptic plasma membrane protein that mediates reuptake of synaptically released norepinephrine. NET is also a major target for medications used for the treatment of depression, attention deficit/hyperactivity disorder, narcolepsy, and obesity. NET is regulated by numerous mechanisms, including catalytic activation and membrane trafficking. Amphetamine (AMPH), a psychostimulant and NET substrate, has also been shown to induce NET trafficking. However, neither the molecular basis nor the nature of the relevant membrane compartments of AMPH-modulated NET trafficking has been defined. Indeed, direct visualization of drug-modulated NET trafficking in neurons has yet to be demonstrated. In this study, we used a recently developed NET antibody and the presence of large presynaptic boutons in sympathetic neurons to examine basal and AMPH-modulated NET trafficking. Specifically, we establish a role for Rab11 in AMPH-induced NET trafficking. First, we found that, in cortical slices, AMPH induces a reduction in surface NET. Next, we observed AMPH-induced accumulation and colocalization of NET with Rab11a and Rab4 in presynaptic boutons of cultured neurons. Using tagged proteins, we demonstrated that NET and a truncated Rab11 effector (FIP2DeltaC2) do not redistribute in synchrony, whereas NET and wild-type Rab11a do. Analysis of various Rab11a/b mutants further demonstrates that Rab11 regulates NET trafficking. Expression of the truncated Rab11a effector (FIP2DeltaC2) attenuates endogenous Rab11 function and prevented AMPH-induced NET internalization as does GDP-locked Rab4 S22N. Our data demonstrate that AMPH leads to an increase of NET in endosomes of single boutons and varicosities in a Rab11-dependent manner.

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.

cGMP-dependent protein kinase Ialpha associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake.

BACKGROUND: The Na(+)/Cl(-)-dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear. RESULTS: In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIalpha specifically associates with hSERT. CONCLUSION: Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIalpha that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIalpha supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.

Subcellular localization of the antidepressant-sensitive norepinephrine transporter.

BACKGROUND: Reuptake of synaptic norepinephrine (NE) via the antidepressant-sensitive NE transporter (NET) supports efficient noradrenergic signaling and presynaptic NE homeostasis. Limited, and somewhat contradictory, information currently describes the axonal transport and localization of NET in neurons. RESULTS: We elucidate NET localization in brain and superior cervical ganglion (SCG) neurons, aided by a new NET monoclonal antibody, subcellular immunoisolation techniques and quantitative immunofluorescence approaches. We present evidence that axonal NET extensively colocalizes with syntaxin 1A, and to a limited degree with SCAMP2 and synaptophysin. Intracellular NET in SCG axons and boutons also quantitatively segregates from the vesicular monoamine transporter 2 (VMAT2), findings corroborated by organelle isolation studies. At the surface of SCG boutons, NET resides in both lipid raft and non-lipid raft subdomains and colocalizes with syntaxin 1A. CONCLUSION: Our findings support the hypothesis that SCG NET is segregated prior to transport from the cell body from proteins comprising large dense core vesicles. Once localized to presynaptic boutons, NET does not recycle via VMAT2-positive, small dense core vesicles. Finally, once NET reaches presynaptic plasma membranes, the transporter localizes to syntaxin 1A-rich plasma membrane domains, with a portion found in cholera toxin-demarcated lipid rafts. Our findings indicate that activity-dependent insertion of NET into the SCG plasma membrane derives from vesicles distinct from those that deliver NE. Moreover, NET is localized in presynaptic membranes in a manner that can take advantage of regulatory processes targeting lipid raft subdomains.