Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling

Vesicular storage of ATP is one of the processes initiating purinergic chemical transmission. Although an active transport mechanism was postulated to be involved in the processes, a transporter(s) responsible for the vesicular storage of ATP remained unidentified for some time. In 2008, SLC17A9, the last identified member of the solute carrier 17 type I inorganic phosphate transporter family, was found to encode the vesicular nucleotide transporter (VNUT) that is responsible for the vesicular storage of ATP. VNUT transports various nucleotides in a membrane potential-dependent fashion and is expressed in the various ATP-secreting cells. Mice with knockout of the VNUT gene lose vesicular storage and release of ATP from neurons and neuroendocrine cells, resulting in blockage of the initiation of purinergic chemical transmission. Thus, VNUT plays an essential role in the vesicular storage and release of ATP. The VNUT knockout mice exhibit resistance for neuropathic pain and a therapeutic effect against diabetes by way of increased insulin sensitivity. Thus, VNUT inhibitors and suppression of VNUT gene expression may be used for therapeutic purposes through suppression of purinergic chemical transmission. This review summarizes the studies to date on VNUT and discusses what we have learned about the relevance of vesicular ATP release as a potential drug target.     

Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line

Mutations in alpha-synuclein have been linked to rare, autosomal dominant forms of Parkinson's disease. Despite its ubiquitous expression, mutant alpha-synuclein primarily leads to the loss of dopamine-producing neurons in the substantia nigra. alpha-Synuclein is a presynaptic nerve terminal protein of unknown function, although several studies suggest it is important for synaptic plasticity and maintenance. The present study utilized a new human mesencephalic cell line, MESC2.10, to study the effect of A53T mutant alpha-synuclein on dopamine homeostasis. In addition to expressing markers of mature dopamine neurons, differentiated MESC2.10 cells are electrically active, produce dopamine, and express wild-type human alpha-synuclein. Lentivirus-induced overexpression of A53T mutant alpha-synuclein in differentiated MESC2.10 cells resulted in down-regulation of the vesicular dopamine transporter (VMAT2), decreased potassium-induced and increased amphetamine-induced dopamine release, enhanced cytoplasmic dopamine immunofluorescence, and increased intracellular levels of superoxide. These results suggest that mutant alpha-synuclein leads to an impairment in vesicular dopamine storage and consequent accumulation of dopamine in the cytosol, a pathogenic mechanism that underlies the toxicity of the psychostimulant amphetamine and the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium. Interestingly, cells expressing A53T mutant alpha-synuclein were resistant to amphetamine-induced toxicity. Because extravesicular, cytoplasmic dopamine can be easily oxidized into reactive oxygen species and other toxic metabolites, mutations in alpha-synuclein might lead to Parkinson's disease by triggering protracted, low grade dopamine toxicity resulting in terminal degeneration and ultimately cell death.     

Vesicular neurotransmitter transporters

Neurotransmission depends on the regulated release of chemical transmitter molecules. This requires the packaging of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular transporters. The family of genes encoding the vesicular transporters for biogenic amines and acetylcholine have recently been cloned. Direct comparison of their transport characteristics and pharmacology provides information about vesicular transport bioenergetics, substrate feature recognition by each transporter, and the role of vesicular amine storage in the mechanism of action of psychopharmacologic and neurotoxic agents. Regulation of vesicular transport activity may affect levels of neurotransmitter available for neurosecretion and be an important site for the regulation of synaptic function. Gene knockout studies have determined vesicular transport function is critical for survival and have enabled further evaluation of the role of vesicular neurotransmitter transporters in behavior and neurotoxicity. Molecular analysis is beginning to reveal the sites involved in vesicular transporter function and the sites that determine substrate specificity. In addition, the molecular basis for the selective targeting of these transporters to specific vesicle populations and the biogenesis of monoaminergic and cholinergic synaptic vesicles are areas of research that are currently being explored. This information provides new insights into the pharmacology and physiology of biogenic amine and acetylcholine vesicular storage in cardiovascular, endocrine, and central nervous system function and has important implications for neurodegenerative disease.     

Adeno-associated virus-mediated gene transfer of human aromatic L-amino acid decarboxylase protects mixed striatal primary cultures from L-DOPA toxicity

Although L-DOPA is the drug of choice for Parkinson's disease, prolonged L-DOPA therapy results in decreased drug effectiveness and the appearance of motor complications. This may be due in part to the progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC). We have developed an adeno-associated virus vector (AAV-hAADC) that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats and monkeys improves L-DOPA responsiveness by improving AADC-mediated conversion of L-DOPA to dopamine. This is now the basis of a proposed therapy for advanced Parkinson's disease. A key concern has been that over-production of dopamine in striatal neurons could cause dopamine toxicity. To investigate this possibility in a controlled system, mixed striatal primary rat neuronal cultures were prepared. Exposure of cultures to high concentrations of L-DOPA induced the following changes: cell death in nigral and striatal neurons, aggregation of neurofilaments and focal axonal swellings, abnormal expression of DARPP-32, and activation of astroglia and microglial cells. Transduction of cultures with AAV-hAADC resulted in efficient and sustained neuronal expression of the AADC protein and prevented all the L-DOPA-induced toxicities. The protective effects were due primarily to AADC-dependent conversion of L-DOPA to dopamine and an increase in induction of vesicular monoamine transporter resulting in dopamine storage in cultured cells. These results suggest a neuroprotective role for AADC gene transfer against L-DOPA toxicity.     

Micro-chemical imaging of cesium distribution in Arabidopsis thaliana plant and its interaction with potassium and essential trace elements

Cesium as an alkali element exhibits a chemical reactivity similar to that of potassium, an essential element for plants. It has been suggested that Cs phytotoxicity might be due either to its competition with potassium to enter the plant, resulting in K starvation, or to its intracellular competition with K binding sites in cells. Such elemental interactions can be evidenced by chemical imaging, which determines the elemental distributions. In this study, the model plant Arabidopsis thaliana was exposed to 1 mM cesium in the presence (20 mM) or not of potassium. The quantitative imaging of Cs and endogenous elements (P, S, Cl, K, Ca, Mn, Fe, and Zn) was carried out using ion beam micro-chemical imaging with 5 microm spatial resolution. Chemical imaging was also evidenced by microfocused synchrotron-based X-ray fluorescence (microXRF) which presents a better lateral resolution (<1 microm) but is not quantitative. Cesium distribution was similar to potassium which suggests that Cs can compete with K binding sites in cells. Cesium and potassium were mainly concentrated in the vascular system of stems and leaves. Cs was also found in lower concentration in leaves mesophyll/epidermis. This late representing the larger proportion in mass, mesophyll/epidermis can be considered as the major storage site for cesium in A. thaliana. Trichomes were not found to accumulate cesium. Interestingly, increased Mn, Fe, and Zn concentrations were observed in leaves at high chlorosis. Mn and Fe increased more in the mesophyll than in veins, whereas zinc increased more in veins than in the mesophyll suggesting a tissue specific interaction of Cs with these trace elements homeostasis. This study illustrates the sensitivity of ion beam microprobe and microfocused synchrotron-based X-ray fluorescence to investigate concentrations and distributions of major and trace elements in plants. It also shows the suitability of these analytical imaging techniques to complement biochemical investigations of metallic stress in plants.     

Acid-sensing ion channels contribute to the increase in vesicular release from SH-SY5Y cells stimulated by extracellular protons

Acid-sensing ion channels (ASICs) have been reported to play a role in the neuronal dopamine pathway, but the exact role in neurotransmitter release remains elusive. Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line, which can release monoamine neurotransmitters. In this study, the expression of ASICs was identified in SH-SY5Y cells to further explore the role of ASICs in vesicular release stimulated by acid. We gathered evidence that ASICs could be detected in SH-SY5Y cells. In whole cell patch-clamp recording, a rapid decrease in extracellular pH evoked inward currents, which were reversibly inhibited by 100 μM amiloride. The currents were pH dependent, with a pH of half-maximal activation (pH(0.5)) of 6.01 ± 0.04. Furthermore, in calcium imaging and FM 1-43 dye labeling, it was shown that extracellular protons increased intracellular calcium levels and vesicular release in SH-SY5Y cells, which was attenuated by PcTx1 and amiloride. Interestingly, N-type calcium channel blockers inhibited the vesicular release induced by acidification. In conclusion, ASICs are functionally expressed in SH-SY5Y cells and involved in vesicular release stimulated by acidification. N-type calcium channels may be involved in the increase in vesicular release induced by acid. Our results provide a preliminary study on ASICs in SH-SY5Y cells and neurotransmitter release, which helps to further investigate the relationship between ASICs and dopaminergic neurons.     

Imaging and speciation of trace elements in biological environment

Mineral elements, often at the trace level, play a considerable role in physiology and pathology of biological systems. Metallogenomics, metalloproteomics, and metallomics are among the emerging disciplines which are critically dependent on spatially resolved concentration maps of trace elements in a cell or tissue, on information on chemical speciation, and on that on metal-binding coordination sites. The mini-review discusses recent progress in analytical techniques for element profiling on the genome scale, biological trace element imaging, and probing, identification and quantification of chemical species in the biological environment. Imaging of the element distribution in cells and tissue sections is becoming possible with sub-micrometer spatial resolution and picogram-level sensitivity owing to advances in laser ablation MS, ion beam and synchrotron radiation X-ray fluorescence microprobes. Progress in nanoflow chromatography and capillary electrophoresis coupled with element specific ICP MS and molecule-specific electrospray MS/MS and MALDI enables speciation of elements in microsamples in a complex biological environment. Laser ablation ICP MS, micro-SXRF, and micro-PIXE allow mapping of trace element distribution in 1D and 2D proteomics gels. The increasing sensitivity of EXAFS and XANES owing to the use of more intense synchrotron beams and efficient focusing optics provide information about oxidation state, fingerprint speciation of metal sites and metal-site structures.     

Proteomic characterization of Jurkat T leukemic cells after dopamine stimulation: A model of circulating dopamine-sensitive cells

T-cells are circulating dopamine-sensitive cells and may mirror, at the peripheral level, biochemical modifications occurring in dopaminergic neurons. The human CD4+ T leukemic Jurkat cell line has been thoroughly used and characterized as a suitable cell model to investigate T-cell signaling and apoptosis. Here, we describe their characterization as a model of circulating dopamine-sensitive cells and their response to a dopamine challenge by analyzing changes in the cell proteome. Jurkat cells express both D1- and D2-like dopamine receptors and both membrane and vesicular transporters, while they lack D4 receptor and tyrosine hydroxylase expression. A saturating, non-toxic, non-oxidant 50 μM dopamine challenge induces the upregulation of an interacting chaperone network known to protect specifically dopaminergic neurons, thus validating T-cells as a biomarker discovery source in Parkinson’s disease. Remodeling of the distribution pattern of β-actin and 14-3-3 isoforms is consistently observed upon dopamine treatment. As a whole, dopamine-specific alterations here observed might represent a biosensor for dopamine response at the peripheral level.     

Protection of intracellular dopamine cytotoxicity by dopamine disposition and metabolism factors

Dopamine has been hypothesized as a contributing factor for the selective degeneration of dopaminergic neurons in Parkinson's disease. However, the cytotoxic mechanisms of dopamine and its metabolites remain poorly understood. Using a stable aromatic amino acid decarboxylase (AADC) expressing a fibroblast cell line, we previously demonstrated a novel, non-oxidative cytotoxicity of intracellular dopamine. In this study, we further investigate the roles of dopamine metabolism and disposition proteins against intracellular dopamine cytotoxicity by co-expressing these factors in AADC-expressing cells. Our results indicate that overexpression of the vesicular monoamine transporter and monoamine oxidase A-induced protection against intracellular dopamine toxicity, and conversely that pharmacological inhibition of these pathways potentiated L-DOPA toxicity in catecholaminergic PC12 cells. Macrophage migration inhibitory factor and glutathione S-transferase (GST), factors that have recently been shown to be involved in dopamine metabolism, also exhibited a strong protective role against intracellular dopamine cytotoxicity. Our results support a potential role for non-oxidative cytoplasmic dopamine toxicity, and imply that disruption in dopamine disposition and/or metabolism could underlie the progressive degeneration of dopaminergic neurons in Parkinson's disease.     

Loaded dopamine is preferentially stored in the halo portion of PC12 cell dense core vesicles

Large dense core vesicles in rat pheochromocytoma cells are morphologically distinct from dense core vesicles in mast and chromaffin cells in that the dense core occupies a much smaller fraction of the vesicular volume, allowing for a much larger vesicular clear space, or halo. In this work, we present evidence indicating that upon treatment with L-DOPA the majority of the dopamine loaded into these vesicles is preferentially compartmentalized into the halo portion of the vesicle. Amperometry was used to monitor release of loaded neurotransmitter from cells in both isotonic and hypertonic extracellular conditions, with the latter condition causing inhibition of dense core dissociation. In combination with this we have used transmission electron microscopy to determine the morphological characteristics of dense core vesicles before and after treatment with L-DOPA in solutions of varied osmolarity. The results provide a more complete understanding of the complex interaction of molecules within dense core vesicles, suggesting that newly loaded dopamine is located in the halo of the vesicle. This finding has fundamental significance for studies of neurotransmitter release from dense core vesicles, as the core appears to have a function involving more than simple storage of neurotransmitter and associated molecules, and the often overlooked vesicular halo appears to be an important storage compartment for neurotransmitter.     

Selective vulnerability of dopaminergic neurons to microtubule depolymerization

Parkinson disease (PD) is characterized by the specific degeneration of dopaminergic (DA) neurons in substantia nigra and has been linked to a variety of environmental and genetic factors. Rotenone, an environmental PD toxin, exhibited much greater toxicity to DA neurons in midbrain neuronal cultures than to non-DA neurons. The effect was significantly decreased by the microtubule-stabilizing drug taxol and mimicked by microtubule-depolymerizing agents such as colchicine or nocodazole. Microtubule depolymerization disrupted vesicular transport along microtubules and caused the accumulation of dopamine vesicles in the soma. This led to increased oxidative stress due to oxidation of cytosolic dopamine leaked from vesicles. Inhibition of dopamine metabolism significantly reduced rotenone toxicity. Thus, our results suggest that microtubule depolymerization induced by PD toxins such as rotenone plays a key role in the selective death of dopaminergic neurons.     

Biomedical applications of the ESRF synchrotron-based microspectroscopy platform

Very little is known about the sub-cellular distribution of metal ions in cells. Some metals such as zinc, copper and iron are essential and play an important role in the cell metabolism. Dysfunctions in this delicate housekeeping may be at the origin of major diseases. There is also a prevalent use of metals in a wide range of diagnostic agents and drugs for the diagnosis or treatment of a variety of disorders. This is becoming more and more of a concern in the field of nanomedicine with the increasing development and use of nanoparticles, which are suspected of causing adverse effects on cells and organ tissues. Synchrotron-based X-ray and Fourier-transformed infrared microspectroscopies are developing into well-suited sub-micrometer analytical tools for addressing new problems when studying the role of metals in biology. As a complementary tool to optical and electron microscopes, developments and studies have demonstrated the unique capabilities of multi-keV microscopy: namely, an ultra-low detection limit, large penetration depth, chemical sensitivity and three-dimensional imaging capabilities. More recently, the capabilities have been extended towards sub-100nm lateral resolutions, thus enabling sub-cellular chemical imaging. Possibilities offered by these techniques in the biomedical field are described through examples of applications performed at the ESRF synchrotron-based microspectroscopy platform (ID21 and ID22 beamlines).     

Methamphetamine rapidly decreases vesicular dopamine uptake

Vesicular sequestration is important in the regulation of cytoplasmic concentrations of monoamines such as dopamine. Moreover, recent evidence suggests that increases in cytoplasmic dopamine levels, perhaps attributable to changes in vesicular monoamine transporter function, contribute to methamphetamine-induced dopaminergic deficits. Hence, we examined whether striatal vesicular uptake is altered following methamphetamine treatment. Multiple administrations of methamphetamine rapidly (within 1 h) decreased vesicular dopamine uptake and dihydrotetrabenazine binding, an effect that (a) persisted at least 24 h, (b) was associated with dopamine and not serotonin neurons, and (c) was unrelated to residual drug introduced by the original methamphetamine treatment. These data suggest that methamphetamine rapidly decreases vesicular monoamine transporter function in dopaminergic neurons, a phenomenon that may be associated with the long-term damage caused by this stimulant.     

Nonlinear‐optical stain‐free stereoimaging of astrocytes and gliovascular interfaces

Methods of nonlinear optics provide a vast arsenal of tools for label‐free brain imaging, offering a unique combination of chemical specificity, the ability to detect fine morphological features, and an unprecedentedly high, subdiffraction spatial resolution. While these techniques provide a rapidly growing platform for the microscopy of neurons and fine intraneural structures, optical imaging of astroglia still largely relies on filament‐protein‐antibody staining, subject to limitations and difficulties especially severe in live‐brain studies. Once viewed as an ancillary, inert brain scaffold, astroglia are being promoted, as a part of an ongoing paradigm shift in neurosciences, into the role of a key active agent of intercellular communication and information processing, playing a significant role in brain functioning under normal and pathological conditions. Here, we show that methods of nonlinear optics provide a unique resource to address long‐standing challenges in label‐free astroglia imaging. We demonstrate that, with a suitable beam‐focusing geometry and careful driver‐pulse compression, microscopy of second‐harmonic generation (SHG) can enable a high‐resolution label‐free imaging of fibrillar structures of astrocytes, most notably astrocyte processes and their endfeet. SHG microscopy of astrocytes is integrated in our approach with nonlinear‐optical imaging of red blood cells based on third‐harmonic generation (THG) enhanced by a three‐photon resonance with the Soret band of hemoglobin. With astroglia and red blood cells providing two physically distinct imaging contrasts in SHG and THG channels, a parallel detection of the second and third harmonics enables a high‐contrast, high‐resolution, stain‐free stereoimaging of gliovascular interfaces in the central nervous system. Transverse scans of the second and third harmonics are shown to resolve an ultrafine texture of blood‐vessel walls and astrocyte‐process endfeet on gliovascular interfaces with a spatial resolution within 1 μm at focusing depths up to 20 μm inside a brain.     

The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity

Uptake of the Parkinsonism-inducing toxin, 1-methyl-4-phenylpyridinium (MPP(+)), into dopaminergic terminals is thought to block Complex I activity leading to ATP loss and overproduction of reactive oxygen species (ROS). The present study indicates that MPP(+)-induced ROS formation is not mitochondrial in origin but results from intracellular dopamine (DA) oxidation. Although a mean lethal dose of MPP(+) led to ROS production in identified dopaminergic neurons, toxic doses of the Complex I inhibitor rotenone did not. Concurrent with ROS formation, MPP(+) redistributed vesicular DA to the cytoplasm prior to its extrusion from the cell by reverse transport via the DA transporter. MPP(+)-induced DA redistribution was also associated with cell death. Depleting cells of newly synthesized and/or stored DA significantly attenuated both superoxide production and cell death, whereas enhancing intracellular DA content exacerbated dopaminergic sensitivity to MPP(+). Lastly, depleting cells of DA in the presence of succinate completely abolished MPP(+)-induced cell death. Thus, MPP(+) neurotoxicity is a multi-component process involving both mitochondrial dysfunction and ROS generated by vesicular DA displacement. These results suggest that in the presence of a Complex I defect, misregulation of DA storage could lead to the loss of nigrostriatal neurons in Parkinson's disease.     

Reserpine sensitivity of catecholamine metabolism in murine neuroblastoma clone N1E-115.

—Neuroblastoma cells of clone NIE-115, originally obtained from the murine tumor C1300, resemble normal noradrenergic neurons in that they have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2; l -tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating)) and dopamine β-monooxygenase (EC 1.14.17.1; 3,4-dihydroxyphenylethylamine, ascorbate: oxygen oxidoreductase (β-hydroxylating)) activities, dense core versicles (100–300 nm in dia), long neurites and excitable membranes. These studies show that reserpine, a blocker of vesicular uptake in noradrenergic neurons, inhibits the accumulation and storage of catecholamines, as well as the conversion of dopamine to NE in neuroblastoma cells. Differentiated monolayer cultures took up [³H]dopamine [10⁻⁴] at a rate of 37 pmol/min per mg protein. Reserpine [5 × 10⁻⁵m ] did not affect the initial rate of uptake, but reduced the extent of uptake at saturation by 60%. Chromatographic examination of cell extracts showed that dopamine was converted to NE in control cultures, but not in reserpine treated cultures. Cells labelled with [³H]dopamine for 60 min and then exposed to release buffer without dopamine for an additional 60 min, retained approximately 40% of the label, 10% as dopamine and 30% as NE. Thirty-five per cent of the radioactivity retained was found, after homogenization and high speed centrifugation, to be associated with a particulate, subcellular fraction. Reserpine, present during release incubations, also reduced the ability of cells to store catecholamines. These results show that N1E-115 cells synthesize and store NE by reactions similar to those in normal noradrenergic neurons.