Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 27
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Curr Res Physiol ; 6: 100109, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38107787

RESUMO

High affinity methylaminoisobutyric acid(MeAIB)/glutamine(Gln) transport activity regulated by neuronal firing occurs at the plasma membrane in mature rat hippocampal neuron-enriched cultures. Spontaneous Ca2+-regulated transport activity was similarly inhibited by riluzole, a benzothiazole anticonvulsant agent, and by novel naphthalenyl substituted aminothiazole derivatives such as SKA-378. Here, we report that spontaneous transport activity is stimulated by 4-aminopyridine (4-AP) and that phorbol-myristate acetate (PMA) increases high K+ stimulated transport activity that is inhibited by staurosporine. 4-AP-stimulated spontaneous and PMA-stimulated high K+-induced transport is not present at 7 days in vitro (DIV) and is maximal by DIV∼21. The relative affinity for MeAIB is similar for spontaneous and high K+-stimulated transport (Km âˆ¼ 50 µM) suggesting that a single transporter is involved. While riluzole and SKA-378 inhibit spontaneous transport with equal potency (IC50 ∼ 1 µM), they exhibit decreased (∼3-5 X) potency for 4-AP-stimulated spontaneous transport. Interestingly, high K+-stimulated MeAIB transport displays lower and differential sensitivity to the two compounds. SKA-378-related halogenated derivatives of SKA-75 (SKA-219, SKA-377 and SKA-375) preferentially inhibit high K+-induced expression of MeAIB transport activity at the plasma membrane (IC50 < 25 µM), compared to SKA-75 and riluzole (IC50 > 100 µM). Ca2+-dependent spontaneous and high K+-stimulated MeAIB transport activity is blocked by ω-conotoxin MVIIC, ω-agatoxin IVA, ω-agatoxin TK (IC50 ∼ 500 nM) or cadmium ion (IC50 ∼ 20 µM) demonstrating that P/Q-type CaV channels that are required for activity-regulated presynaptic vesicular glutamate (Glu) release are also required for high-affinity MeAIB transport expression at the plasma membrane. We suggest that neural activity driven and Ca2+ dependent trafficking of the high affinity MeAIB transporter to the plasma membrane is a unique target to understand mechanisms of Glu/Gln recycling in synapses and acute neuroprotection against excitotoxic presynaptic Glu induced neural injury.

2.
Neuropharmacology ; 224: 109349, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36436594

RESUMO

Epileptogenic seizures, or status epilepticus (SE), leads to excitotoxic injury in hippocampal and limbic neurons in the kainic acid (KA) animal model of temporal lobe epilepsy (TLE). Here, we have further characterized neural activity regulated methylaminoisobutryic acid (MeAIB)/glutamine transport activity in mature rat hippocampal neurons in vitro that is inhibited by riluzole (IC50 = 1 µM), an anti-convulsant benzothiazole agent. We screened a library of riluzole derivatives and identified SKA-41 followed by a second screen and synthesized several novel chlorinated aminothiazoles (SKA-377, SKA-378, SKA-379) that are also potent MeAIB transport inhibitors in vitro, and brain penetrant following systemic administration. When administered before KA, SKA-378 did not prevent seizures but still protected the hippocampus and several other limbic areas against SE-induced neurodegeneration at 3d. When SKA-377 - 379, (30 mg/kg) were administered after KA-induced SE, acute neural injury in the CA3, CA1 and CA4/hilus was also largely attenuated. Riluzole (10 mg/kg) blocks acute neural injury. Kinetic analysis of SKA-378 and riluzoles' blockade of Ca2+-regulated MeAIB transport in neurons in vitro indicates that inhibition occurs via a non-competitive, indirect mechanism. Sodium channel NaV1.6 antagonism blocks neural activity regulated MeAIB/Gln transport in vitro (IC50 = 60 nM) and SKA-378 is the most potent inhibitor of NaV1.6 (IC50 = 28 µM) compared to NaV1.2 (IC50 = 118 µM) in heterologous cells. However, pharmacokinetic analysis suggests that sodium channel blockade may not be the predominant mechanism of neuroprotection here. Riluzole and our novel aminothiazoles are agents that attenuate acute neural hippocampal injury following KA-induced SE and may help to understand mechanisms involved in the progression of epileptic disease.


Assuntos
Epilepsia do Lobo Temporal , Estado Epiléptico , Ratos , Animais , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/tratamento farmacológico , Riluzol/farmacologia , Cinética , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/prevenção & controle , Hipocampo , Ácido Caínico/toxicidade , Modelos Animais de Doenças
3.
Mol Neurobiol ; 57(7): 3118-3142, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32474835

RESUMO

Vesicular glutamate transporters (VGLUTs) control quantal size of glutamatergic transmission and have been the center of numerous studies over the past two decades. VGLUTs contain two independent transport modes that facilitate glutamate packaging into synaptic vesicles and phosphate (Pi) ion transport into the synaptic terminal. While a transmembrane proton electrical gradient established by a vacuolar-type ATPase powers vesicular glutamate transport, recent studies indicate that binding sites and flux properties for chloride, potassium, and protons within VGLUTs themselves regulate VGLUT activity as well. These intrinsic ionic binding and flux properties of VGLUTs can therefore be modulated by neurophysiological conditions to affect levels of glutamate available for release from synapses. Despite their extraordinary importance, specific and high-affinity pharmacological compounds that interact with these sites and regulate VGLUT function, distinguish between the various modes of transport, and the different isoforms themselves, are lacking. In this review, we provide an overview of the physiologic sites for VGLUT regulation that could modulate glutamate release in an over-active synapse or in a disease state.


Assuntos
Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/genética
4.
J Neurochem ; 142(1): 29-40, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28423185

RESUMO

Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC50 1.3 ± 0.5 µM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K+ -stimulated MeAIB transport displays an affinity (Km ) for MeAIB of 37 ± 1.2 µM, saturates at ~ 200 µM, is dependent on extracellular Ca2+ , and is blocked by inhibition of voltage-gated Ca2+ channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca2+ and voltage-gated calcium channels, but is also blocked by the Na+ channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca2+ , but on Na+ ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K+ -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805.


Assuntos
Sistema X-AG de Transporte de Aminoácidos/efeitos dos fármacos , Hipocampo/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Riluzol/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Glutamina/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Potássio/farmacologia , Gravidez , Ratos , Ratos Sprague-Dawley , beta-Alanina/análogos & derivados , beta-Alanina/metabolismo
5.
J Neurosci ; 35(6): 2516-29, 2015 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-25673846

RESUMO

Rett syndrome (RTT) is an autism spectrum disorder caused by loss-of-function mutations in the gene encoding MeCP2, an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. Previously, we and others reported a role of microglia in the pathophysiology of RTT. To understand the mechanism of microglia dysfunction in RTT, we identified a MeCP2 target gene, SLC38A1, which encodes a major glutamine transporter (SNAT1), and characterized its role in microglia. We found that MeCP2 acts as a microglia-specific transcriptional repressor of SNAT1. Because glutamine is mainly metabolized in the mitochondria, where it is used as an energy substrate and a precursor for glutamate production, we hypothesize that SNAT1 overexpression in MeCP2-deficient microglia would impair the glutamine homeostasis, resulting in mitochondrial dysfunction as well as microglial neurotoxicity because of glutamate overproduction. Supporting this hypothesis, we found that MeCP2 downregulation or SNAT1 overexpression in microglia resulted in (1) glutamine-dependent decrease in microglial viability, which was corroborated by reduced microglia counts in the brains of MECP2 knock-out mice; (2) proliferation of mitochondria and enhanced mitochondrial production of reactive oxygen species; (3) increased oxygen consumption but decreased ATP production (an energy-wasting state); and (4) overproduction of glutamate that caused NMDA receptor-dependent neurotoxicity. The abnormalities could be rectified by mitochondria-targeted expression of catalase and a mitochondria-targeted peptide antioxidant, Szeto-Schiller 31. Our results reveal a novel mechanism via which MeCP2 regulates bioenergetic pathways in microglia and suggest a therapeutic potential of mitochondria-targeted antioxidants for RTT.


Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Microglia/metabolismo , Doenças Mitocondriais/metabolismo , Síndromes Neurotóxicas/metabolismo , Síndrome de Rett/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Consumo de Oxigênio/fisiologia , Cultura Primária de Células
6.
J Neurosci ; 32(45): 15886-901, 2012 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-23136427

RESUMO

The level and integrity of glutamate transmission during critical periods of postnatal development plays an important role in the refinement of pyramidal neuron dendritic arbor, synaptic plasticity, and cognition. Presently, it is not clear how excitatory transmission via the two predominant isoforms of the vesicular glutamate transporter (VGLUT1 and VGLUT2) participate in this process. To assess a neurodevelopmental role for VGLUT2 in pyramidal neuron maturation, we generated recombinant VGLUT2 knock-out mice and inactivated VGLUT2 throughout development using Emx1-Cre(+/+) knock-in mice. We show that VGLUT2 deficiency in corticolimbic circuits results in reduced evoked glutamate transmission, release probability, and LTD at hippocampal CA3-CA1 synapses during a formative developmental period (postnatal days 11-14). In adults, we find a marked reduction in the amount of dendritic arbor across the span of the dendritic tree of CA1 pyramidal neurons and reduced long-term potentiation and levels of synaptic markers spinophilin and VGLUT1. Loss of dendritic arbor is accompanied by corresponding reductions in the number of dendritic spines, suggesting widespread alterations in synaptic connectivity. Conditional VGLUT2 knock-out mice exhibit increased open-field exploratory activity yet impaired spatial learning and memory, endophenotypes similar to those of NMDA receptor knock-down mice. Remarkably, the impairment in learning can be partially restored by selectively increasing NMDA receptor-mediated glutamate transmission in adult mice by prolonged treatment with d-serine and a d-amino acid oxidase inhibitor. Our data indicate that VGLUT2 expression is pivotal to the proper development of mature pyramidal neuronal architecture and plasticity, and that such glutamatergic deficiency leads to cognitive malfunction as observed in several neurodevelopmental psychiatric disorders.


Assuntos
Dendritos/metabolismo , Aprendizagem/fisiologia , Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Percepção Espacial/fisiologia , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Hipocampo/fisiologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Comportamento Espacial/fisiologia , Sinapses/fisiologia , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/genética
7.
J Biol Chem ; 285(19): 14366-76, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20212045

RESUMO

Homeostatic scaling of glutamatergic and GABAergic transmission is triggered by prolonged alterations in synaptic neuronal activity. We have previously described a presynaptic mechanism for synaptic homeostasis and plasticity that involves scaling the level of vesicular glutamate (VGLUT1) and gamma-aminobutyric acid (GABA) (VGAT) transporter biosynthesis. These molecular determinants of vesicle filling and quantal size are regulated by neuronal activity in an opposite manner and bi-directionally. Here, we report that a striking induction of VGLUT2 mRNA and synaptic protein is triggered by a prolonged increase in glutamatergic synaptic activity in mature neocortical neuronal networks in vitro together with two determinants of inhibitory synaptic strength, the neuronal activity-regulated pentraxin (Narp), and glutamate decarboxylase (GAD65). Activity-dependent induction of VGLUT2 and Narp exhibits a similar intermediate-early gene response that is blocked by actinomycin D and tetrodotoxin, by inhibitors of ionotropic glutamate receptors and L-type voltage-gated calcium channels, and is dependent on downstream signaling via calmodulin, calcium/calmodulin-dependent protein kinase (CaMK) and extracellular signal-regulated kinase 1/2 (ERK1/2). The co-induction of VGLUT2 and Narp triggered by prolonged gamma-aminobutyric acid type A receptor blockade is independent of brain-derived nerve growth factor and TrkB receptor signaling. VGLUT2 protein induction occurs on a subset of cortically derived synaptic vesicles in excitatory synapses on somata and dendritic processes of multipolar GABAergic interneurons, recognized sites for the clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptors by Narp. We propose that VGLUT2 and Narp induction by excitation-transcription coupling leads to increased glutamatergic transmission at synapses on GABAergic inhibitory feedback neurons as part of a coordinated program of Ca(2+)-signal transcription involved in mechanisms of homeostatic plasticity after prolonged hyperactivity.


Assuntos
Proteína C-Reativa/metabolismo , Ácido Glutâmico/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transcrição Gênica , Proteína Vesicular 2 de Transporte de Glutamato/genética , Animais , Animais Recém-Nascidos , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cálcio/metabolismo , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Calmodulina/metabolismo , Células Cultivadas , Antagonistas GABAérgicos/farmacologia , Antagonistas de Receptores de GABA-A , Regulação da Expressão Gênica no Desenvolvimento , Glutamato Descarboxilase/metabolismo , Técnicas Imunoenzimáticas , Plasticidade Neuronal , Neurônios/citologia , Neurônios/efeitos dos fármacos , Piridazinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor trkA/metabolismo , Receptores de GABA-A/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Vesículas Sinápticas/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo
8.
J Biol Chem ; 284(17): 11224-36, 2009 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-19240036

RESUMO

System A transporters SNAT1 and SNAT2 mediate uptake of neutral alpha-amino acids (e.g. glutamine, alanine, and proline) and are expressed in central neurons. We tested the hypothesis that SNAT2 is required to support neurotransmitter glutamate synthesis by examining spontaneous excitatory activity after inducing or repressing SNAT2 expression for prolonged periods. We stimulated de novo synthesis of SNAT2 mRNA and increased SNAT2 mRNA stability and total SNAT2 protein and functional activity, whereas SNAT1 expression was unaffected. Increased endogenous SNAT2 expression did not affect spontaneous excitatory action-potential frequency over control. Long term glutamine exposure strongly repressed SNAT2 expression but increased excitatory action-potential frequency. Quantal size was not altered following SNAT2 induction or repression. These results suggest that spontaneous glutamatergic transmission in pyramidal neurons does not rely on SNAT2. To our surprise, repression of SNAT2 activity was not limited to System A substrates. Taurine, gamma-aminobutyric acid, and beta-alanine (substrates of the SLC6 gamma-aminobutyric acid transporter family) repressed SNAT2 expression more potently (10x) than did System A substrates; however, the responses to System A substrates were more rapid. Since ATF4 (activating transcription factor 4) and CCAAT/enhancer-binding protein are known to bind to an amino acid response element within the SNAT2 promoter and mediate induction of SNAT2 in peripheral cell lines, we tested whether either factor was similarly induced by amino acid deprivation in neurons. We found that glutamine and taurine repressed the induction of both transcription factors. Our data revealed that SNAT2 expression is constitutively low in neurons under physiological conditions but potently induced, together with the taurine transporter TauT, in response to depletion of neutral amino acids.


Assuntos
Sistemas de Transporte de Aminoácidos/fisiologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Regulação da Expressão Gênica , Glutamina/metabolismo , Neocórtex/citologia , Neurônios/metabolismo , Sistema A de Transporte de Aminoácidos , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Eletrofisiologia , Oócitos/metabolismo , Isoformas de Proteínas , Ratos , Xenopus laevis
9.
Neurochem Res ; 33(2): 238-47, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18080752

RESUMO

VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate--in contrast to plasma-membrane or mitochondrial glutamate transporters--and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of L-[(3)H]glutamate, but not D-[(3)H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of L-glutamate, but not L-aspartate, from intact oocytes preinjected with (3)H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H(+)/L-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated L-[(3)H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca(2+)-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca(2+)-dependent exocytosis.


Assuntos
Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Animais , Ratos , Proteínas Recombinantes/metabolismo , Trítio , Xenopus
10.
J Am Soc Nephrol ; 18(5): 1426-36, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17429052

RESUMO

Wasting of lean tissue as a consequence of metabolic acidosis is a serious problem in patients with chronic renal failure. A possible contributor is inhibition by low pH of the System A (SNAT2) transporter, which carries the amino acid L-glutamine (L-Gln) into muscle cells. The aim of this study was to determine the effect of selective SNAT2 inhibition on intracellular amino acid profiles and amino acid-dependent signaling through mammalian target of rapamycin in L6 skeletal muscle cells. Inhibition of SNAT2 with the selective competitive substrate methylaminoisobutyrate, metabolic acidosis (pH 7.1), or silencing SNAT2 expression with small interfering RNA all depleted intracellular L-Gln. SNAT2 inhibition also indirectly depleted other amino acids whose intracellular concentrations are maintained by the L-Gln gradient across the plasma membrane, notably the anabolic amino acid L-leucine. Consequently, SNAT2 inhibition strongly impaired signaling through mammalian target of rapamycin to ribosomal protein S6 kinase, ribosomal protein S6, and 4E-BP1, leading to impairment of protein synthesis comparable with that induced by rapamycin. It is concluded that even though SNAT2 is only one of several L-Gln transporters in muscle, it may determine intracellular anabolic amino acid levels, regulating the amino acid signaling that affects protein mass, nucleotide/nucleic acid metabolism, and cell growth.


Assuntos
Acidose/metabolismo , Sistemas de Transporte de Aminoácidos/fisiologia , Aminoácidos/metabolismo , Proteínas de Transporte/fisiologia , Células Musculares/metabolismo , Biossíntese de Proteínas/genética , Proteínas Quinases/fisiologia , Sistema A de Transporte de Aminoácidos , Sistemas de Transporte de Aminoácidos/antagonistas & inibidores , Sistemas de Transporte de Aminoácidos/genética , Animais , Células Cultivadas , Inativação Gênica , Concentração de Íons de Hidrogênio , Modelos Biológicos , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR , beta-Alanina/análogos & derivados , beta-Alanina/farmacologia
11.
J Biol Chem ; 282(8): 5152-9, 2007 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-17179157

RESUMO

Dendritic development is essential for the establishment of a functional nervous system. Among factors that control dendritic development, brain-derived neurotrophic factor (BDNF) has been shown to regulate dendritic length and complexity of cortical neurons. However, the cellular and molecular mechanisms that underlie these effects remain poorly understood. In this study, we examined the role of amino acid transport in mediating the effects of BDNF on dendritic development. We show that BDNF increases System A amino acid transport in cortical neurons by selective up-regulation of the sodium-coupled neutral amino acid transporter (SNAT)1. Up-regulation of SNAT1 expression and System A activity is required for the effects of BDNF on dendritic growth and branching of cortical neurons. Further analysis revealed that induction of SNAT1 expression and System A activity by BDNF is necessary in particular to enhance synthesis of tissue-type plasminogen activator, a protein that we demonstrate to be essential for the effects of BDNF on cortical dendritic morphology. Together, these data reveal that stimulation of neuronal differentiation by BDNF requires the up-regulation of SNAT1 expression and System A amino acid transport to meet the increased metabolic demand associated with the enhancement of dendritic growth and branching.


Assuntos
Sistema A de Transporte de Aminoácidos/biossíntese , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Diferenciação Celular/efeitos dos fármacos , Córtex Cerebral/fisiologia , Dendritos/fisiologia , Regulação para Cima/efeitos dos fármacos , Animais , Diferenciação Celular/fisiologia , Crescimento Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Camundongos
12.
Cell Mol Neurobiol ; 26(4-6): 679-93, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16710756

RESUMO

1. Selective protein-protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation. 2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1-endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3. 3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.


Assuntos
Aciltransferases/metabolismo , Neocórtex/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Vesículas Sinápticas/enzimologia , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos , Ácido Glutâmico/metabolismo , Modelos Biológicos , Neocórtex/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Vesículas Sinápticas/metabolismo , Distribuição Tecidual , Técnicas do Sistema de Duplo-Híbrido
13.
Neurochem Int ; 48(6-7): 643-9, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16546297

RESUMO

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.


Assuntos
Córtex Cerebral/fisiologia , Proteína Vesicular 1 de Transporte de Glutamato/biossíntese , Proteína Vesicular 2 de Transporte de Glutamato/biossíntese , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/biossíntese , Animais , Córtex Cerebral/metabolismo , Ácido Glutâmico/metabolismo , Homeostase , Humanos , Rede Nervosa/fisiologia , Plasticidade Neuronal , Neurônios/metabolismo , Sinapses/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/fisiologia , Proteína Vesicular 2 de Transporte de Glutamato/fisiologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/fisiologia
14.
J Mol Histol ; 36(4): 301-9, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-16200463

RESUMO

System A is a highly regulated, Na+-dependent transporter that accepts neutral amino acids containing short, polar side chains. System A plays an important role during rat development as decreased pup weights are observed in dams infused during gestation with a non-metabolizable System A substrate. Given the potential importance of SNAT1 during development in the rat brain, we examined whether SNAT1 would be present at an earlier gestation during organogenesis in multiple organs by immunohistochemistry and immunoblotting. SNAT1 protein was observed in the developing lungs, intestines, kidneys, heart, pancreas, and skeletal muscle of rats at prenatal days 14, 17, 19, 21, and postnatal day 2 rats. SNAT1 protein expression decreased in the liver and intestine shortly after birth and as the rat matured. SNAT1 expression was constant throughout development in the lungs and kidney and increased in the heart from prenatal day 19 to postnatal day 2. Highest levels of expression in older animals were seen in organs undergoing rapid cell division.


Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sistema A de Transporte de Aminoácidos/imunologia , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Feminino , Immunoblotting , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley
15.
J Neurosci ; 25(31): 7121-33, 2005 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-16079394

RESUMO

Homeostatic control of pyramidal neuron firing rate involves a functional balance of feedforward excitation and feedback inhibition in neocortical circuits. Here, we reveal a dynamic scaling in vesicular excitatory (vesicular glutamate transporters VGLUT1 and VGLUT2) and inhibitory (vesicular inhibitory amino acid transporter VIAAT) transporter mRNA and synaptic protein expression in rat neocortical neuronal cultures, using a well established in vitro protocol to induce homeostatic plasticity. During the second and third week of synaptic differentiation, the predominant vesicular transporters expressed in neocortical neurons, VGLUT1 and VIAAT, are both dramatically upregulated. In mature cultures, VGLUT1 and VIAAT exhibit bidirectional and opposite regulation by prolonged activity changes. Endogenous coregulation during development and homeostatic scaling of the expression of the transporters in functionally differentiated cultures may serve to control vesicular glutamate and GABA filling and adjust functional presynaptic excitatory/inhibitory balance. Unexpectedly, hyperexcitation in differentiated cultures triggers a striking increase in VGLUT2 mRNA and synaptic protein, whereas decreased excitation reduces levels. VGLUT2 mRNA and protein are expressed in subsets of VGLUT1-encoded neocortical neurons that we identify in primary cultures and in neocortex in situ and in vivo. After prolonged hyperexcitation, downregulation of VGLUT1/synaptophysin intensity ratios at most synapses is observed, whereas a subset of VGLUT1-containing boutons selectively increase the expression of VGLUT2. Bidirectional and opposite regulation of VGLUT1 and VGLUT2 by activity may serve as positive or negative feedback regulators for cortical synaptic transmission. Intracortical VGLUT1/VGLUT2 coexpressing neurons have the capacity to independently modulate the level of expression of either transporter at discrete synapses and therefore may serve as a plastic interface between subcortical thalamic input (VGLUT2) and cortical output (VGLUT1) neurons.


Assuntos
Homeostase , Neocórtex/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Células Cultivadas , Técnicas In Vitro , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Vias Neurais/citologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Neurônios/metabolismo , Células Piramidais/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo , Sinapses/fisiologia , Distribuição Tecidual , Regulação para Cima , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/genética
16.
J Neurosci ; 25(26): 6221-34, 2005 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-15987952

RESUMO

A fundamental question in synaptic physiology is whether the unitary strength of a synapse can be regulated by presynaptic characteristics and, if so, what those characteristics might be. Here, we characterize a newly proposed mechanism for altering the strength of glutamatergic synapses based on the recently identified vesicular glutamate transporter VGLUT1. We provide direct evidence that filling in isolated synaptic vesicles is subject to a dynamic equilibrium that is determined by both the concentration of available glutamate and the number of vesicular transporters participating in loading. We observe that changing the number of vesicular transporters expressed at hippocampal excitatory synapses results in enhanced evoked and miniature responses and verify biophysically that these changes correspond to an increase in the amount of glutamate released per vesicle into the synaptic cleft. In addition, we find that this modulation of synaptic strength by vesicular transporter expression is endogenously regulated, both across development to coincide with a maturational increase in vesicle cycling and quantal amplitude and by excitatory and inhibitory receptor activation in mature neurons to provide an activity-dependent scaling of quantal size via a presynaptic mechanism. Together, these findings underscore that vesicular transporter expression is used endogenously to directly regulate the extent of glutamate release, providing a concise presynaptic mechanism for controlling the quantal efficacy of excitatory transmission during synaptic refinement and plasticity.


Assuntos
Terminações Pré-Sinápticas/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Proteína Vesicular 1 de Transporte de Glutamato/fisiologia , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Potenciais Evocados/fisiologia , Ácido Glutâmico/metabolismo , Homeostase , Processamento de Imagem Assistida por Computador , Células PC12 , Técnicas de Patch-Clamp , Teoria Quântica , Ratos , Proteína Vesicular 1 de Transporte de Glutamato/genética
17.
Biochim Biophys Acta ; 1667(2): 157-66, 2004 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-15581851

RESUMO

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.


Assuntos
Sistema A de Transporte de Aminoácidos/síntese química , Sistema A de Transporte de Aminoácidos/metabolismo , Soluções Hipertônicas/farmacologia , Sistema A de Transporte de Aminoácidos/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Biotinilação , Western Blotting , Membrana Celular/química , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Diclororribofuranosilbenzimidazol/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Cinética , Peso Molecular , Fósforo/metabolismo , Prolina/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Radioisótopos , Especificidade por Substrato , Transcrição Gênica/efeitos dos fármacos
18.
Traffic ; 5(12): 1006-16, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15522101

RESUMO

Previous studies have shown that the vesicular monoamine transporter 2 (VMAT2) is localized to both large dense core vesicles and synaptic vesicles in vivo. However, when exogenously expressed in PC12 cells, VMAT2 localizes only to large dense core vesicles. This distribution is similar to that of the endogenous vesicular monoamine transporter 1 (VMAT1) in PC12 cells. When VMAT2 was expressed in a protein kinase A (PKA)-deficient PC12 cell line it localized to synaptic-like microvesicles. Expression of recombinant VMAT1 in the same cell line showed a heterogeneous distribution to both large dense core vesicles and synaptic-like microvesicles. Coexpression of the PKA catalytic subunit partially restored trafficking of both VMAT2 and VMAT1 to large dense core vesicles; treatment of wild-type PC12 cells with the PKA inhibitor H89 increased VMAT2 on synaptic-like microvesicles. The VMAT1 and VMAT2 in large dense core vesicles exhibit a larger molecular size than those located on synaptic-like microvesicles. This difference is due to differential N-linked glycosylation. In vitro phosphorylation experiments show that PKA does not phosphorylate VMAT2. A chimera containing the VMAT2 cytoplasmic C-terminus fused to vesicular acetylcholine transporter (VAChT) shows mislocalization to synaptic-like microvesicles and VAChT-like glycosylation in the PKA-deficient cell line. However, coexpression with PKA changes the chimera's trafficking to large dense core vesicles and increases the molecular size. These results suggest that protein kinase A affects the formation and/or composition of VMAT trafficking complexes.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Imunofluorescência , Glicosilação , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Células PC12 , Fosforilação , Isoformas de Proteínas/metabolismo , Transporte Proteico/fisiologia , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de Monoamina
19.
Cereb Cortex ; 14(5): 562-74, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15054072

RESUMO

SNAT1 mediates glutamine (Gln) influx into neurons and is believed to replenish the transmitters pools of glutamate (Glu) and gamma-aminobutyric acid (GABA). We investigated its distribution and cellular localization in the cerebral cortex and neighboring regions of rats and humans using light and electron microscopic immunocytochemical methods with specific antibodies. In the first somatic sensory cortex of rats and in areas 9, 10, 21 and 46 of the human cortex, numerous SNAT1-positive (+) cells were present in the cortical parenchyma and in the white matter; >95% of SNAT1+ cells were neurons, but some were astrocytes. Most SNAT1+ cells were pyramidal neurons, but numerous non-pyramidal neurons were also observed: SNAT1/GABA double-labeling studies showed that SNAT1 is expressed in all GABA+ neurons. SNAT1/synaptophysin studies showed that <0.1% of all synaptophysin+ puncta coexpressed SNAT1. SNAT1 immunoreactivity (ir) was also in leptomeninges, ependymal cells and choroid plexus. Electron microscopic studies showed that neuronal SNAT1 ir was almost exclusively observed in perikarya and dendritic profiles. SNAT1 ir was also in distal astrocytic processes, including end feet profiles, and in leptomeninges. These findings suggest that the major function of SNAT1 is not to replenish the transmitter pools of Glu and GABA.


Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Córtex Cerebral/ultraestrutura , Técnicas de Cultura , Humanos , Neurônios/ultraestrutura , Células Piramidais/citologia , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Distribuição Tecidual
20.
Pflugers Arch ; 447(5): 784-95, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12845534

RESUMO

The sodium-coupled neutral amino acid transporters (SNAT) of the SLC38 gene family resemble the classically-described System A and System N transport activities in terms of their functional properties and patterns of regulation. Transport of small, aliphatic amino acids by System A subtypes (SNAT1, SNAT2, and SNAT4) is rheogenic and pH sensitive. The System N subtypes SNAT3 and SNAT5 also countertransport H(+), which may be key to their operation in reverse, and have narrower substrate profiles than do the System A subtypes. Glutamine emerges as a favored substrate throughout the family, except for SNAT4. The SLC38 transporters undoubtedly play many physiological roles including the transfer of glutamine from astrocyte to neuron in the CNS, ammonia detoxification and gluconeogenesis in the liver, and the renal response to acidosis. Probing their regulation has revealed additional roles, and recent work has considered SLC38 transporters as therapeutic targets in neoplasia.


Assuntos
Sistema A de Transporte de Aminoácidos/fisiologia , Aminoácidos Neutros/metabolismo , Sódio/metabolismo , Sequência de Aminoácidos , Sistema A de Transporte de Aminoácidos/química , Sistema A de Transporte de Aminoácidos/genética , Animais , Transporte Biológico/fisiologia , Humanos , Dados de Sequência Molecular , Família Multigênica/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...