Transmembrane topology of vesicular glutamate transporter 2

Vesicular glutamate transporter (VGLUT) plays an essential role in L-glutamate signaling in neurons and some peripheral tissues through vesicular storage of L-glutamate in secretory vesicles. To investigate the topology of VGLUT in membranes, we prepared site-directed antibodies against the amino-terminal (anti-N), 1st putative loop (anti-L), and carboxyl terminal (anti-C) regions. None of the antibodies reacted with VGLUT2 expressed in COS cells because they could not gain access to the antigen. However, both the anti-N and anti-C antibodies recognized VGLUT2 when the cells were permeabilized with digitonin, while the anti-L antibodies did not. Immunological reactivity to anti-L-antibodies appeared when the cells were permeabilized with Triton X-100. These results suggest that both the amino-terminal and carboxyl-terminal regions of VGLUT2 in membranes face the cytoplasm while the 1st loop faces the lumen.     

囊泡谷氨酸转运体与神经系统疾病

囊泡谷氨酸转运体(vesicular glutamate transporters,VGLUTs)能特异地装载谷氨酸进入突触囊泡并促进释放,它包括3个成员,其中VGLUT1和VGLUT2是谷氨酸能神经元和它们轴突末端高度特异的标志,同时VGLUT1标志着皮质-皮质投射,VGLUT2标志着丘脑-皮层投射。而VGLUT3则会出现在胆碱能中间神经元、5-羟色胺能神经元、海马和皮层中GABA能中间神经元中。VGLUTs的异常会导致兴奋性神经递质谷氨酸的异常,从而诱发多种神经系统疾病。该文综述了VGLUTs的功能障碍与阿尔采末病(Alzheimer’sdisease,AD)、帕金森病(Parkinson’s disease,PD)、精神分裂症、抑郁症、癫痫、耳聋发病的关系的研究进展,为这些疾病的防治提供新的线索。     

Inhibitor of the glutamate vesicular transporter (VGLUT)

The vesicular glutamate transporter (VGLUT) is responsible for the uptake of the excitatory amino acid, L-glutamate, into synaptic vesicles. VGLUT activity is coupled to an electrochemical gradient driven by a vacuolar ATPase and stimulated by low Cl-. VGLUT has relatively low affinity (K(m) = 1-3 mM) for glutamate and is pharmacologically and structurally distinct from the Na+-dependent, excitatory amino acid transporters (EAATs) found on the plasma membrane. Because glutamatergic neurotransmission begins with vesicular release, compounds that block the uptake of glutamate into the vesicle may reduce excitotoxic events. Several classes of competitive VGLUT inhibitors have emerged including amino acids and amino acid analogs, fatty acids, azo dyes, quinolines and alkaloids. The potency with which these agents inhibit VGLUT varies from millimolar (amino acids) to nanomolar (azo dyes) concentrations. These inhibitors represent highly diverse structures and have collectively begun to reveal key pharmacophore elements that may elucidate the key interactions important to binding VGLUT. Using known inhibitor structures and preliminary molecular modeling, a VGLUT pharmacophore is presented that will aid in the design of new, highly potent and selective agents.     

Synthesis and in vitro pharmacology of substituted quinoline-2,4-dicarboxylic acids as inhibitors of vesicular glutamate transport

The vesicular glutamate transport (VGLUT) system selectively mediates the uptake of L-glutamate into synaptic vesicles. Uptake is linked to an H+-ATPase that provides coupling among ATP hydrolysis, an electrochemical proton gradient, and glutamate transport. Substituted quinoline-2,4-dicarboxylic acids (QDCs), prepared by condensation of dimethyl ketoglutaconate (DKG) with substituted anilines and subsequent hydrolysis, were investigated as potential VGLUT inhibitors in synaptic vesicles. A brief panel of substituted QDCs was previously reported (Carrigan et al. Bioorg. Med. Chem. Lett. 1999, 9, 2607-2612)(1) and showed that certain substituents led to more potent competitive inhibitors of VGLUT. Using these compounds as leads, an expanded series of QDC analogues were prepared either by condensation of DKG with novel anilines or via aryl-coupling (Suzuki or Heck) to dimethyl 6-bromoquinolinedicarboxylate. From the panel of almost 50 substituted QDCs tested as inhibitors of the VGLUT system, the 6-PhCH=CH-QDC (K(i) = 167 microM), 6-PhCH2CH2-QDC (K(i) = 143 microM), 6-(4'-phenylstyryl)-QDC (K(i) = 64 microM), and 6-biphenyl-4-yl-QDC (K(i) = 41 microM) were found to be the most potent blockers. A preliminary assessment of the key elements needed for binding to the VGLUT protein based on the structure-activity relationships for the panel of substituted QDCs is discussed herein. The substituted QDCs represent the first synthetically derived VGLUT inhibitors and are promising templates for the development of selective transporter inhibitors.     

Lentiviral Delivery of a Vesicular Glutamate Transporter 1 (VGLUT1)-Targeting Short Hairpin RNA Vector Into the Mouse Hippocampus Impairs Cognition

Glutamate is the principle excitatory neurotransmitter in the mammalian brain, and dysregulation of glutamatergic neurotransmission is implicated in the pathophysiology of several psychiatric and neurological diseases. This study utilized novel lentiviral short hairpin RNA (shRNA) vectors to target expression of the vesicular glutamate transporter 1 (VGLUT1) following injection into the dorsal hippocampus of adult mice, as partial reductions in VGLUT1 expression should attenuate glutamatergic signaling and similar reductions have been reported in schizophrenia. The VGLUT1-targeting vector attenuated tonic glutamate release in the dorsal hippocampus without affecting GABA, and selectively impaired novel object discrimination (NOD) and retention (but not acquisition) in the Morris water maze, without influencing contextual fear-motivated learning or causing any adverse locomotor or central immune effects. This pattern of cognitive impairment is consistent with the accumulating evidence for functional differentiation along the dorsoventral axis of the hippocampus, and supports the involvement of dorsal hippocampal glutamatergic neurotransmission in both spatial and nonspatial memory. Future use of this nonpharmacological VGLUT1 knockdown mouse model could improve our understanding of glutamatergic neurobiology and aid assessment of novel therapies for cognitive deficits such as those seen in schizophrenia.     

Developmentally regulated expression of VGLUT3 during early post-natal life

Three subtypes of vesicular glutamate transporters, named VGLUT1-3, accumulate glutamate into synaptic vesicles. In this study, the post-natal expression of VGLUT3 was determined with specific probes and antiserums in the rat brain and compared with that of VGLUT1 and VGLUT2. The expression of VGLUT1 and VGLUT2 increases linearly during post-natal development. In contrast, VGLUT3 developmental pattern appears to have a more or less biphasic profile. A first peak of expression is centered around post-natal day 10 (P10) while the second one is reached in the adult brain. Between P1 and P15, VGLUT3 is observed in the frontal brain (striatum, accumbens, and hippocampus) and in the caudal brain (colliculi, pons and cerebellum). During a second phase extending from P15 to adulthood, the labeling of the caudal brain fades away. The adult pattern is reached at P21. We further analyzed the transient expression of VGLUT3 in the cerebellum and found it to correspond to a temporary expression in Purkinje cells. At P10 VGLUT3 immunoreactivity was present both in the soma and terminals of Purkinje cells (PC), where it colocalized with the vesicular inhibitory amino acid transporter (VIAAT). In agreement with data from the literature [Gillespie, D.C., Kim, G., Kandler, K., 2005. Inhibitory synapses in the developing auditory system are glutamatergic. Nat. Neurosci. 8, 332-338], our results suggest that during the first 2 weeks of post-natal life PC may have the potential to transiently release simultaneously GABA and glutamate.     

Regulation of vesicular monoamine and glutamate transporters by vesicle-associated trimeric G proteins: new jobs for long-known signal transduction molecules

Neurotransmitters of neurons and neuroendocrine cells are concentrated first in the cytosol and then in either small synaptic vesicles ofpresynaptic terminals or in secretory vesicles by the activity of specific transporters of the plasma and the vesicular membrane, respectively. In the central nervous system the postsynaptic response depends--amongst other parameters-on the amount of neurotransmitter stored in a given vesicle. Neurotransmitter packets (quanta) vary over a wide range which may be also due to a regulation of vesicular neurotransmitter filling. Vesicular filling is regulated by the availability of transmitter molecules in the cytoplasm, the amount of transporter molecules and an electrochemical proton-mediated gradient over the vesicular membrane. In addition, it is modulated by vesicle-associated heterotrimeric G proteins, Galphao2 and Galphaq. Galphao2 and Galphaq regulate vesicular monoamine transporter (VMAT) activities in brain and platelets, respectively. Galphao2 also regulates vesicular glutamate transporter (VGLUT) activity by changing its chloride dependence. It appears that the vesicular content activates the G protein, suggesting a signal transduction from the luminal site which might be mediated by a vesicular G protein-coupled receptor or as an alternative possibility by the transporter itself. Thus, G proteins control transmitter storage and thereby probablylink the regulation of the vesicular content to intracellular signal cascades.     

Pharmacology of excitatory amino acid transporters (EAATs and VGLUTs)

L-Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system (CNS). It contributes not only to fast synaptic neurotransmission but also to complex physiological processes like plasticity, learning, and memory. Glutamate is synthesized in the cytoplasm and stored in synaptic vesicles by a proton gradient-dependent uptake system (VGLUTs). Following its exocytotic release, glutamate activates different kinds of glutamate receptors and mediates excitatory neurotransmission. To terminate the action of glutamate and maintain its extracellular concentration below excitotoxic levels, glutamate is quickly removed by Na(+)-dependent glutamate transporters (EAATs). Recently, three vesicular glutamate transporters (VGLUT1-3) and five Na(+)-dependent glutamate transporters (EAAT1-5) were identified. VGLUTs and EAATs are thought to play important roles in neuronal disorders, such as amyotrophic lateral sclerosis, Alzheimer's disease, cerebral ischemia, and Huntington's disease. In this review, the development of new compounds to regulate the function of VGLUTs and EAATs will be described.     

Antipsychotics increase vesicular glutamate transporter 2 (VGLUT2) expression in thalamolimbic pathways

Recently the two vesicular-glutamate-transporters VGLUT1 and VGLUT2 have been cloned and characterized. VGLUT1 and VGLUT2 together label all glutamatergic neurons, but because of their distinct expression patterns in the brain they facilitate our ability to define between a VGLUT1-positive cortical and a VGLUT2-positive subcortical glutamatergic systems. We have previously demonstrated an increased cortical VGLUT1 expression as marker of antidepressant activity. Here, we assessed the effects of different psychotropic drugs on brain VGLUT2 mRNA and protein expression. The typical antipsychotic haloperidol, and the atypicals clozapine and risperidone increased VGLUT2 mRNA selectively in the central medial/medial parafascicular, paraventricular and intermediodorsal thalamic nuclei; VGLUT2 protein was accordingly amplified in paraventricular and ventral striatum and in prefrontal cortex. The antidepressants fluoxetine and desipramine and the sedative anxiolytic diazepam had no effect. These results highlight the implication of thalamo-limbic glutamatergic pathways in the action of antipsychotics. Increased VGLUT2 expression in these neurons might constitute a marker for antipsychotic activity and subcortical glutamate neurotransmission might be a possible novel target for future generation antipsychotics.     

Release of endogenous glutamate by AMPA receptors expressed in cultured rat costal chondrocytes

We have previously demonstrated the release of endogenous glutamate by activation of DL-alpha-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors expressed by bone, while there is no information available on the possible functional expression of glutamatergic signaling molecules in cartilage to date. In rat costal chondrocytes cultured for 4 to 28 d, expression of mRNA was seen for several chondral marker genes including sox9, runt-related gene 2/core binding factor alpha-1 (Runx-2/Cbfa-1), type II collagen and aggrecan, but not for the adipocyte marker gene peroxisome proliferator-activated receptor gamma (PPARgamma). Expression of mRNA was drastically increased for Runx-2/Cbfa-1 during culturing from 7 to 14 d with a gradual increase thereafter up to 28 d, while a transient increase was seen in mRNA expression for both type-II collagen and sox-9 on 14 d and for aggrecan on 7 d respectively, in chondrocytes cultured for a period up to 28 d. Irrespective of the culture period up to 21 d, marked expression was seen by cultured chondrocytes with mRNA for GluR3 subunit of AMPA receptors, in addition to vesicular glutamate transporter-1 (VGLUT1) required for the condensation and subsequent exocytotic release of glutamate in the glutamatergic neurotransmission in the brain. Cultured rat costal chondrocytes underwent spontaneous release of endogenous glutamate, while an inhibitor of AMPA receptor desensitization significantly prolonged the duration of endogenous glutamate release stimulated by AMPA. These results suggest that endogenous glutamate could be released from intracellular vesicular constituents associated with VGLUT1 through activation of AMPA receptors expressed by cultured rat costal chondrocytes.     

头孢曲松对甲基苯丙胺致大鼠伏隔核神经元和谷氨酸转运体改变的影响

目的观察头孢曲松对甲基苯丙胺(METH)急性、亚急性处理时致神经损伤情况的影响,以及对伏隔核中谷氨酸转运体1(GLT1)与囊泡型谷氨酸转运体1(VGLUT1)的变化的影响。方法建立METH急性毒性模型,同时利用谷氨酸转运体调节剂头孢曲松调控谷氨酸转运体的表达,尼氏染色实验观测神经元中尼氏小体的变化,利用Western blot实验检测其谷氨酸转运体蛋白表达的变化。结果 METH急性给药组与盐水对照组相比,刻板行为明显增加(P<0.01),尼氏小体明显减少;伏隔核中GLT1和VGLUT1的表达增加分别为53.7%和102%(P<0.05);头孢曲松预防给药组与METH组相比,大鼠刻板行为明显减少,伏隔核中GLT1的表达增加36%(P<0.05);VGLUT1的表达下调56%(P<0.05);METH亚急性处理后,与盐水对照组相比,伏隔核中GLT1的蛋白表达增加40.9%,VGLUT1蛋白表达增加52.9%;预防给予头孢曲松后,头孢曲松预防给药组与METH组相比,GLT1和VGLUT1蛋白表达差异没有显著性。结论 METH处理导致神经损伤,引起伏隔核中谷氨酸转运体的表达变化;头孢曲松能激活谷氨酸转运体的表达,缓解METH引起的神经损伤。     

Selective cortical VGLUT1 increase as a marker for antidepressant activity

The two recently characterized vesicular glutamate transporters (VGLUT) presynaptically mark and differentiate two distinct excitatory neuronal populations and thus define a cortical and a subcortical glutamatergic system (VGLUT1 and VGLUT2 positive, respectively). These two systems might be differentially implicated in brain neuropathology. Still, little is known on the modalities of VGLUT1 and VGLUT2 regulations in response to pharmacological or physiological stimuli. Given the importance of cortical neuronal activity in psychosis we investigated VGLUT1 mRNA and protein expression in response to chronic treatment with commonly prescribed psychotropic medications. We show that agents with antidepressant activity, namely the antidepressants fluoxetine and desipramine, the atypical antipsychotic clozapine, and the mood stabilizer lithium increased VGLUT1 mRNA expression in neurons of the cerebral cortex and the hippocampus and in concert enhanced VGLUT1 protein expression in their projection fields. In contrast the typical antipsychotic haloperidol, the cognitive enhancers memantine and tacrine, and the anxiolytic diazepam were without effect. We suggest that VGLUT1 could be a useful marker for antidepressant activity. Furthermore, adaptive changes in VGLUT1 positive neurons could constitute a common functional endpoint for structurally unrelated antidepressants, representing promising antidepressant targets in tracking specificity, mechanism, and onset at action.     

The diverse roles of vesicular glutamate transporter 3

The expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the ability of most traditionally accepted excitatory neurons to release glutamate by exocytosis. However, several cell populations (serotonin and dopamine neurons) have been demonstrated to release glutamate in vitro and do not obviously express these transporters. Rather, these neurons express a novel, third isoform that in fact appears confined to neurons generally associated with a transmitter other than glutamate. They include serotonin and possibly dopamine neurons, cholinergic interneurons in the striatum, and GABAergic interneurons of the hippocampus and cortex. Although the physiological role of VGLUT3 remains largely conjectural, several observations in vivo suggest that the glutamate release mediated by VGLUT3 has an important role in synaptic transmission, plasticity, and development.     

甲基苯丙胺急性处理对大鼠纹状体中神经元和谷氨酸转运体的影响

目的:观察甲基苯丙胺(METH)急性处理致神经损伤情况,以及纹状体中谷氨酸转运体1(GLT1)与囊泡型谷氨酸转运体1(VGLUT1)的变化。方法:建立METH急性毒性模型,同时利用谷氨酸转运体激动剂头孢曲松调控谷氨酸转运体的表达,尼氏染色实验观测神经元中尼氏小体的变化,利用Western blot实验检测其谷氨酸转运体蛋白表达的变化。结果:与盐水对照组相比,METH给药组刻板行为明显增加(P<0.01),尼氏小体显著减少,纹状体中GLT1和VGLUT1的表达增加分别为23.1%和66.1%(P<0.05);与METH组相比,头孢曲松预防给药组大鼠刻板行为明显减少,纹状体中GLT1的表达增加15.2%(P<0.05),VGLUT1的表达降低,但没有统计学意义(P>0.05)。结论:METH急性处理能导致神经损伤,引起纹状体中谷氨酸转运体的表达变化;头孢曲松能激活谷氨酸转运体的表达,缓解METH引起的神经损伤。     

Secretory organelles in ECL cells: Effects of pharmacological blockade of the gastrin/CCK2 receptor versus its elimination by gene targeting

Histamine-producing ECL cells are numerous in the stomach. They express gastrin/CCK₂ receptors and respond to gastrin by releasing histamine. Ultrastructurally, they display numerous and very characteristic secretory organelles: granules, secretory vesicles and microvesicles. This paper focuses on the impact of the gastrin/CCK₂ receptor on the ultrastructure of the ECL cells. The effects of pharmacological blockade of the receptor are compared with the effects of receptor elimination following selective gene targeting. Long-term administration of powerful gastrin/CCK₂ receptor antagonists was found to induce hypotrophy of rat stomach ECL cells with reduced number of granules, secretory vesicles and microvesicles. In gastrin/CCK₂ receptor knockout mice ECL cells, i.e., histamine-storing cells with the characteristic ultrastructure of ECL cells, had disappeared from the oxyntic mucosa and been replaced by a novel population of endocrine-like cells. These cells harbored granules and microvesicles, but were devoid of histamine and secretory vesicles. We suggest that the gastrin/CCK₂ receptor is important for the proper differentiation of the ECL cells and for maintaining their characteristic ultrastructure.     

Valproic acid reduces enhanced vesicular glutamate transporter immunoreactivities in the dentate gyrus of the seizure prone gerbil

To elucidate the relationship between glutamatergic current and vesicular glutamate transporter (VGLUT) expressions, we performed the comparative analyses of evoked potentials and VGLUT immunoreactivities in the dentate gyrus, and its response to antiepileptic drug treatments in a gerbil model. The EPSP slope that could be evoked in seizure sensitive (SS) gerbils was significantly greater than in seizure resistant (SR) gerbils. There was also a strong trend towards the larger population spike amplitude in SS gerbils. In addition, VGLUT immunoreactivities were markedly enhanced in the dentate gyrus of SS gerbils, as compared with the SR gerbils. Following valproic acid (VPA, 30 mg/kg), the population spike amplitude and the EPSP slope in response to the stimulus were markedly reduced in the dentate gyri both of SR and of SS gerbils, although this dosage of VPA had no effect in low stimulus currents in SS gerbils. Vigabatrin (VGB) and low dosage of VPA treatment did not affect the evoked responses. Similarly, VPA treatment reduced enhanced VGLUT immunoreactivities in the dentate gyrus of SS gerbils, whilst VGB did not. These findings suggest that up-regulation of VGLUT immunoreactivities may be related to the hyperexcitability of granule cells in SS gerbils, and altered VGLUT immunoreactivity in the dentate gyrus may be independent of GABAergic transmission.     

Functional significance of co-localization of GABA and Glu in nerve terminals: a hypothesis

Salient features of the co-transmission by GABA and Glu in neural signaling are summarized. Experimental data have been accumulating which demonstrate; i) GABA-immunoreactivity in and GABA-release from constitutively Gluergic hippocampal mossy fibre terminals, ii) plasticity of the GABAergic phenotype of constitutively Gluergic granule cells of the Dentate Gyrus, iii) expression of GABA(A) receptor gamma(3) subunit in the mossy fibre termination zone in the CA3 subfield, iv) co-labeling of terminals for GABA and Glu in the retina, brain stem and spinal cord, and v) functional compatibility of vesicular Glu (VGLUT3) and GABA (VIAAT) transporters. It is not clear, however, whether or not Glu and GABA are released from the same terminals, and packaged in the same vesicles. Using multiple transmitters neurons may serve to reduce the metabolic cost and errors of signaling.