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 nonmedically 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 reuptake 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 neurologic 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 athletic 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 Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments.

Dopamine transporter (DAT) controls dopamine neurotransmission by clearing synaptically released dopamine. However, trafficking itineraries of DAT, which determine its cell-surface concentration near synapses, are poorly characterized. It is especially unknown how DAT is transported between spatially distant midbrain somatodendritic and striatal axonal compartments. To examine this "long-range" trafficking, the localization and membrane diffusion of HA-epitope tagged DAT in the medial forebrain bundle (MFB) of a knock-in mouse (both sexes) were analyzed using confocal, super-resolution and EM in intact brain and acute brain slices. HA-DAT was abundant in the plasma membrane of MFB axons, similar to the striatum, although the intracellular fraction of HA-DAT in MFB was more substantial. Intracellular HA-DAT colocalized with VPS35, a subunit of the retromer complex mediating recycling from endosomes, in a subset of axons. Late endosomes, lysosomes, and endoplasmic reticulum were abundant in the soma but minimally present in MFB axons, suggesting that biosynthesis and lysosomal degradation of DAT are confined to soma. Together, the data suggest that membrane diffusion is the main mode of long-range DAT transport through MFB, although the contribution of vesicular traffic can be significant in a population of MFB axons. Based on HA-DAT diffusion rates, plasma membrane DAT in MFB axons turns over with a halftime of ∼20 d, which explains the extremely slow turnover of DAT protein in the brain. Unexpectedly, the mean diameter of DAT-labeled MFB axons was observed to be twice larger than reported for striatum. The implications of this finding for dopamine neuron physiology are discussed.SIGNIFICANCE STATEMENT The dopamine transporter (DAT) is a key regulator of dopamine neurotransmission and a target of abused psychostimulants. In the present study, we examined, for the first time, mechanisms of the long-range traffic of DAT in intact brain and acute brain slices from the knock-in mouse expressing epitope-tagged DAT. Using a combination of confocal, super-resolution and EM, we defined DAT localization and its membrane diffusion parameters in medial forebrain bundle axonal tracts connecting midbrain somatodendritic and striatal axonal compartments of dopaminergic neurons. In contrast to the widely accepted model of long-range axonal transport, our studies suggest that DAT traffics between midbrain and striatum, mainly by lateral diffusion in the plasma membrane with only a limited contribution of vesicular transport in recycling endosomes.

Heterogeneities in Axonal Structure and Transporter Distribution Lower Dopamine Reuptake Efficiency.

Efficient clearance of dopamine (DA) from the synapse is key to regulating dopaminergic signaling. This role is fulfilled by DA transporters (DATs). Recent advances in the structural characterization of DAT from Drosophila (dDAT) and in high-resolution imaging of DA neurons and the distribution of DATs in living cells now permit us to gain a mechanistic understanding of DA reuptake events in silico. Using electron microscopy images and immunofluorescence of transgenic knock-in mouse brains that express hemagglutinin-tagged DAT in DA neurons, we reconstructed a realistic environment for MCell simulations of DA reuptake, wherein the identity, population and kinetics of homology-modeled human DAT (hDAT) substates were derived from molecular simulations. The complex morphology of axon terminals near active zones was observed to give rise to large variations in DA reuptake efficiency, and thereby in extracellular DA density. Comparison of the effect of different firing patterns showed that phasic firing would increase the probability of reaching local DA levels sufficiently high to activate low-affinity DA receptors, mainly owing to high DA levels transiently attained during the burst phase. The experimentally observed nonuniform surface distribution of DATs emerged as a major modulator of DA signaling: reuptake was slower, and the peaks/width of transient DA levels were sharper/wider under nonuniform distribution of DATs, compared with uniform. Overall, the study highlights the importance of accurate descriptions of extrasynaptic morphology, DAT distribution, and conformational kinetics for quantitative evaluation of dopaminergic transmission and for providing deeper understanding of the mechanisms that regulate DA transmission.

Brain Region-Specific Trafficking of the Dopamine Transporter.

The dopamine (DA) transporter (DAT) controls dopaminergic neurotransmission by removing extracellular DA. Although DA reuptake is proposed to be regulated by DAT traffic to and from the cell surface, the membrane trafficking system involved in the endocytic cycling of DAT in the intact mammalian brain has not been characterized. Hence, we performed immunolabeling and quantitative analysis of the subcellular and regional distribution of DAT using the transgenic knock-in mouse expressing hemagglutinin (HA) epitope-tagged DAT (HA-DAT) and by using a combination of electron microscopy and a novel method for immunofluorescence labeling of HA-DAT in acute sagittal brain slices. Both approaches demonstrated that, in midbrain somatodendritic regions, HA-DAT was present in the plasma membrane, endoplasmic reticulum, and Golgi complex, with a small fraction in early and recycling endosomes and an even smaller fraction in late endosomes and lysosomes. In the striatum and in axonal tracts between the midbrain and striatum, HA-DAT was detected predominantly in the plasma membrane, and quantitative analysis revealed increased DAT density in striatal compared with midbrain plasma membranes. Endosomes were strikingly rare and lysosomes were absent in striatal axons, in which there was little intracellular HA-DAT. Acute administration of amphetamine in vivo (60 min) or to slices ex vivo (10-60 min) did not result in detectable changes in DAT distribution. Altogether, these data provide evidence for regional differences in DAT plasma membrane targeting and retention and suggest a surprisingly low level of endocytic trafficking of DAT in the striatum along with limited DAT endocytic activity in somatodendritic areas. SIGNIFICANCE STATEMENT: The dopamine transporter (DAT) is the key regulator of the dopamine neurotransmission in the CNS. In the present study, we developed a new approach for studying DAT localization and dynamics in intact neurons in acute sagittal brain slices from the knock-in mouse expressing epitope-tagged DAT. For the first time, the fluorescence imaging analysis of DAT was combined with the immunogold labeling of DAT and quantitative electron microscopy. In contrast to numerous studies of DAT trafficking in heterologous expression systems and dissociated cultured neurons, studies in intact neurons revealed a surprisingly low amount of endocytic trafficking of DAT at steady state and after acute amphetamine treatment and suggested that non-vesicular transport could be the main mechanism establishing DAT distribution within the dopaminergic neuron.

Action potentials and amphetamine release antipsychotic drug from dopamine neuron synaptic VMAT vesicles.

Based on lysotracker red imaging in cultured hippocampal neurons, antipsychotic drugs (APDs) were proposed to accumulate in synaptic vesicles by acidic trapping and to be released in response to action potentials. Because many APDs are dopamine (DA) D2 receptor (D2R) antagonists, such a mechanism would be particularly interesting if it operated in midbrain DA neurons. Here, the APD cyamemazine (CYAM) is visualized directly by two-photon microscopy in substantia nigra and striatum brain slices. CYAM accumulated slowly into puncta based on vacuolar H(+)-ATPase activity and dispersed rapidly upon dissipating organelle pH gradients. Thus, CYAM is subject to acidic trapping and released upon deprotonation. In the striatum, Ca(2+)-dependent reduction of the CYAM punctate signal was induced by depolarization or action potentials. Striatal CYAM overlapped with the dopamine transporter (DAT). Furthermore, parachloroamphetamine (pCA), acting via vesicular monoamine transporter (VMAT), and a charged VMAT, substrate 1-methyl-4-phenylpyridinium (MPP(+)), reduced striatal CYAM. In vivo CYAM administration and in vitro experiments confirmed that clinically relevant CYAM concentrations result in vesicular accumulation and pCA-dependent release. These results show that some CYAM is in DA neuron VMAT vesicles and suggests a new drug interaction in which amphetamine induces CYAM deprotonation and release as a consequence of the H(+) countertransport by VMAT that accompanies vesicular uptake, but not by inducing exchange or acting as a weak base. Therefore, in the striatum, APDs are released with DA in response to action potentials and an amphetamine. This synaptic corelease is expected to enhance APD antagonism of D2Rs where and when dopaminergic transmission occurs.

Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor.

Wounding usually causes considerable cell damage, and released ATP promotes migration of nearby epithelium. ATP binds to purinergic receptors on the cell surface and induces transactivation of the EGF receptor through signaling by the Src family kinases (SFKs). Here we tested whether ATP activates these kinases through Pyk2, a member of the focal adhesion kinase family. Pyk2 was rapidly and potently activated by treating corneal epithelial cells with ATP, and physical interaction of Pyk2 with the SFKs was enhanced. Disruption of Pyk2 signaling either by siRNA or by expression of a dominant-negative mutant led to inhibition of ATP-induced activation of the SFKs and the EGF receptor. Inhibiting Pyk2 activity also blocked ATP stimulation of healing of wounds in epithelial cell sheets. These data suggest that ATP stimulates sequential activation of Pyk2, SFKs, and the EGF receptor to induce cell migration.

Brief treatment with heparin-binding EGF-like growth factor, but not with EGF, is sufficient to accelerate epithelial wound healing.

BACKGROUND: Heparin-binding EGF-like growth factor (HB-EGF) contains, in contrast to EGF, a domain that binds to negatively charged glycans on cell surfaces and in extracellular matrix. We speculated that a short exposure to HB-EGF induces prolonged biological effects such as healing of wounds after immobilization in tissues. METHODS: Epithelial cell sheets in tissue and corneas in organ culture were treated briefly with HB-EGF or EGF and binding of the growth factors, time course of activation of the EGF receptor, and healing of wounds were compared. RESULTS: Treating human corneal epithelial cells for 2 min with HB-EGF resulted in 8h of detectable activation of the EGF receptor, but activation was much shorter after EGF treatment. A brief treatment with HB-EGF, but not with EGF, induced significant acceleration of healing in wounds in epithelial sheets in tissue and organ culture. Bound HB-EGF was detectable up to 16 h after brief treatments. Neutralizing antibodies added after HB-EGF treatment blocked acceleration of healing, demonstrating the role of bound HB-EGF in accelerating healing. CONCLUSIONS: A brief exposure to HB-EGF, but not to EGF, is sufficient to induce prolonged activation of the EGF receptor and to enhance healing. GENERAL SIGNIFICANCE: Bound HB-EGF can serve as a pool that induces prolonged activation of the EGF receptor. EGF has been used experimentally to treat poorly healing wounds, but the frequent applications that are necessary have hampered its use clinically. The findings imply that HB-EGF may be a useful long-acting alternative to EGF.

Free edges in epithelia as cues for motility.

One of the primary functions of any epithelium is to act as a barrier. To maintain integrity, epithelia migrate rapidly to cover wounds, and there is intense interest in understanding how wounds are detected. Numerous soluble factors are present in the wound environment and epithelia can sense the presence of adjacent denuded extracellular matrix. However, the presence of such cues is expected to be highly variable, and here we focus on the presence of edges in the epithelial sheets as a stimulus, since they are universally and continuously present in wounds. Using a novel tissue culture model, free edges in the absence of any other identifiable cues were found to trigger activation of the epidermal growth factor receptor and increase cell motility. Edges bordered by inert physical barriers do not activate the receptor, indicating that activation is related to mechanical factors rather than to specific cell cell interactions.

Free edges in epithelial cell sheets stimulate epidermal growth factor receptor signaling.

The ability of epithelia to migrate and cover wounds is essential to maintaining their functions as physical barriers. Wounding induces many cues that may affect the transition to motility, including the immediate mechanical perturbation, release of material from broken cells, new interactions with adjacent extracellular matrix, and breakdown of physical separation of ligands from their receptors. Depending on the exact nature of wounds, some cues may be present only transiently or insignificantly. In many epithelia, activation of the epidermal growth factor receptor (EGFR) is a central event in induction of motility, and we find that its continuous activation is required for progression of healing of wounds in sheets of corneal epithelial cells. Here, we examine the hypothesis that edges, which are universally and continuously present in wounds, are a cue. Using a novel culture model we find that their presence is sufficient to cause activation of the EGFR and increased motility of cells in the absence of other cues. Edges that are bordered by agarose do not induce activation of the EGFR, indicating that activation is not due to loss of any specific type of cell-cell interaction but rather due to loss of physical constraints.

Pyk2 activation triggers epidermal growth factor receptor signaling and cell motility after wounding sheets of epithelial cells.

Activation of the epidermal growth factor receptor (EGFR) is a key signaling event that promotes cells to move and cover wounds in many epithelia. We have previously shown that wounding activates the EGFR through activation of the Src family kinases (SFKs), which induce proteolytic shedding of epidermal growth factor-like ligands from the cell surface. A major goal in wound healing research is to identify early signals that promote motility, and here we examined the hypothesis that members of the focal adhesion kinase family are upstream activators of the SFKs after wounding. We found that focal adhesion kinase is not activated by wounding but that a different family member, Pyk2 (PTK2B/RAFTK/CAKbeta), is activated rapidly and potently. Pyk2 interaction with c-Src is increased after wounding, as determined by co-immunoprecipitation experiments. Disruption of Pyk2 signaling either by small interfering RNA or by expression of a dominant negative mutant led to inhibition of wound-induced activation of the SFKs and the EGFR, and conversely, overexpression of wild-type Pyk2 stimulated SFK and EGFR kinase activities in cells. In wound healing studies, Pyk2 small interfering RNA or dominant negative inhibited cell migration. These results show that activation of Pyk2 is an early signal that promotes wound healing by stimulating the SFK/EGFR signaling pathway.

Wounding sheets of epithelial cells activates the epidermal growth factor receptor through distinct short- and long-range mechanisms.

Wounding epithelia induces activation of the epidermal growth factor receptor (EGFR), which is absolutely required for induction of motility. ATP is released from cells after wounding; it binds to purinergic receptors on the cell surface, and the EGFR is subsequently activated. Exogenous ATP activates phospholipase D, and we show here that ATP activates the EGFR through the phospholipase D2 isoform. The EGFR is activated in cells far (>0.3 cm) from wounds, which is mediated by diffusion of extracellular ATP because activation at a distance from wounds is abrogated by eliminating ATP in the medium with apyrase. In sharp contrast, activation of the EGFR near wounds is not sensitive to apyrase. Time-lapse microscopy revealed that cells exhibit increased motilities near edges of wounds; this increase in motility is not sensitive to apyrase, and apyrase does not detectably inhibit healing of wounds in epithelial sheets. This novel ATP/PLD2-independent pathway activates the EGFR by a transactivation process through ligand release, and it involves signaling by a member of the Src family of kinases. We conclude that wounding activates two distinct signaling pathways that induce EGFR activation and promote healing of wounds in epithelial cells. One pathway signals at a distance from wounds through release of ATP, and another pathway acts locally and is independent on ATP signaling.

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor.

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.

Epithelial cell motility is triggered by activation of the EGF receptor through phosphatidic acid signaling.

Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid, and there is currently much interest in elucidating messenger functions for this molecule. We report here that wounding sheets of corneal epithelial and Madin Darby canine kidney cells induces strong activation of phospholipase D, and we provide evidence that activation is amplified through a positive feed-back loop. Short-chain analogues of phosphatidic acid induce motility robustly in corneal and other epithelial cell types. The effects of these analogues were not the result of their conversion to the corresponding diacylglycerol or lysophosphatidic acid, implying that phosphatidic acid acts directly on one or more cellular targets. Strikingly, phosphatidic acid signaling was found to stimulate the epidermal growth factor receptor (EGFR) through a transactivation process. Healing of wounds in sheets of corneal epithelial cells is absolutely dependent on epidermal growth factor receptor signaling, and the present data suggest that its activation is a result of wound-induced phospholipase D activation.

Wounding induces motility in sheets of corneal epithelial cells through loss of spatial constraints: role of heparin-binding epidermal growth factor-like growth factor signaling.

Cellular responses to wounding have often been studied at a molecular level after disrupting cell layers by mechanical means. This invariably results in damage to cells at the edges of the wounds, which has been suggested to be instrumental for initiating wound healing. To test this, we devised an alternative procedure to introduce gaps in layers of corneal epithelial cells by casting agarose strips on tissue culture plates. In contrast to mechanical wounding, removal of the strips did not lead to detectable membrane leakage or to activation of the stress-activated kinase JNK. Nonetheless, cells at the edge underwent the typical morphological transition to a highly motile phenotype, and the gaps closed at rates similar to those of mechanically induced wounds. To allow biochemical analysis of cell extracts, a procedure was devised that makes cell-free surface area acutely available to a large proportion of cells in culture. Rapid activation of the epidermal growth factor receptor (EGFR) was detected by immunoblotting, and the addition of an EGFR-blocking antibody completely abolished wound healing. In addition, wound healing was inhibited by agents that block signaling by the heparin-binding epidermal growth factor-like growth factor (HB-EGF). Cells stimulated with cell-free tissue culture surface released a soluble factor that induced activation of the EGFR, which was distinct from HB-EGF. These studies suggest that the triggering event for the induction of motility in corneal epithelial cells is related to the sudden availability of permissive surface area rather than to mechanical damage, and they demonstrate a central role of signaling through HB-EGF.