RESUMO
The cystine/glutamate antiporter system xc- has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT-/-) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT-/- mice led to the hypothesis that system xc- deletion would negatively affect life- and healthspan. Still, till now the role of system xc- in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT-/- mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT+/+ mice, was attenuated in xCT-/- mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT-/- mice. Targeting system xc- is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging.
Assuntos
Sistema y+ de Transporte de Aminoácidos , Cistina , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Cisteína , Cistina/metabolismo , Ácido Glutâmico , Hipocampo/metabolismo , Longevidade , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: The astroglial enzyme glutamine synthetase (GS) is deficient in small loci in the brain in adult patients with different types of focal epilepsy; however, the role of this deficiency in the pathogenesis of epilepsy has been difficult to assess due to a lack of sufficiently sensitive and specific animal models. The aim of this study was to develop an in vivo approach for precise and specific deletions of the GS gene in the postnatal brain. METHODS: We stereotaxically injected various adeno-associated virus (AAV)-Cre recombinase constructs into the hippocampal formation and neocortex in 22-70-week-old GSflox/flox mice to knock out the GS gene in a specific and focal manner. The mice were subjected to seizure threshold determination, continuous video-electroencephalographic recordings, advanced in vivo neuroimaging, and immunocytochemistry for GS. RESULTS: The construct AAV8-glial fibrillary acidic protein-green fluorescent protein-Cre eliminated GS in >99% of astrocytes in the injection center with a gradual return to full GS expression toward the periphery. Such focal GS deletion reduced seizure threshold, caused spontaneous recurrent seizures, and diminished functional connectivity. SIGNIFICANCE: These results suggest that small loci of GS deficiency in the postnatal brain are sufficient to cause epilepsy and impaired functional connectivity. Additionally, given the high specificity and precise spatial resolution of our GS knockdown approach, we anticipate that this model will be extremely useful for rigorous in vivo and ex vivo studies of astroglial GS function at the brain-region and single-cell levels.
Assuntos
Epilepsia , Doenças Metabólicas , Animais , Astrócitos/metabolismo , Encéfalo/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Amônia Ligase/genética , Glutamina , Humanos , Camundongos , Convulsões/patologiaRESUMO
Objectives: The cystine/glutamate antiporter (system xc-) is believed to contribute to nonvesicular glutamate release from glial cells in various brain areas. Although recent investigations implicate system xc- in mood disorders, unambiguous evidence has not yet been established. Therefore, we evaluated the possible role of system xc- in the depressive state. Methods: We conducted a protein expression analysis of the specific subunit of system xc- (xCT) in brain regions of the corticosterone mouse model, Flinders Sensitive Line rat model and post-mortem tissue of depressed patients. We next subjected system xc- deficient mice to the corticosterone model and analysed their behaviour in several tests. Lastly, we subjected additional cohorts of xCT-deficient and wild-type mice to N-acetylcysteine treatment to unveil whether the previously reported antidepressant-like effects are dependent upon system xc-. Results: We did not detect any changes in xCT expression levels in the animal models or patients compared to proper controls. Furthermore, loss of system xc- had no effect on depression- and anxiety-like behaviour. Finally, the antidepressant-like effects of N-acetylcysteine are not mediated via system xc-. Conclusions: xCT protein expression is not altered in the depressed brain and system xc- deficiency does not affect depression-associated behaviour in the corticosterone mouse model.
Assuntos
Sistema y+ de Transporte de Aminoácidos/deficiência , Encéfalo/metabolismo , Depressão/genética , Depressão/fisiopatologia , Idoso de 80 Anos ou mais , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Anti-Inflamatórios , Encéfalo/patologia , Corticosterona , Modelos Animais de Doenças , Comportamento Exploratório , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , RatosRESUMO
The communication between the immune and central nervous system (CNS) is affected in many neurological disorders. Peripheral injections of the endotoxin lipopolysaccharide (LPS) are widely used to study this communication: an LPS challenge leads to a biphasic syndrome that starts with acute sickness and is followed by persistent brain inflammation and chronic behavioral alterations such as depressive-like symptoms. In vitro, the response to LPS treatment has been shown to involve enhanced expression of system x c - . This cystine-glutamate antiporter, with xCT as specific subunit, represents the main glial provider of extracellular glutamate in mouse hippocampus. Here we injected male xCT knockout and wildtype mice with a single intraperitoneal dose of 5 mg/kg LPS. LPS-injection increased hippocampal xCT expression but did not alter the mainly astroglial localization of the xCT protein. Peripheral and central inflammation (as defined by cytokine levels and morphological activation of microglia) as well as LPS-induced sickness and depressive-like behavior were significantly attenuated in xCT-deficient mice compared with wildtype mice. Our study is the first to demonstrate the involvement of system x c - in peripheral and central inflammation in vivo and the potential therapeutic relevance of its inhibition in brain disorders characterized by peripheral and central inflammation, such as depression.
Assuntos
Sistema y+ de Transporte de Aminoácidos/deficiência , Depressão/metabolismo , Comportamento de Doença/fisiologia , Inflamação/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Citocinas/metabolismo , Depressão/patologia , Transportador 2 de Aminoácido Excitatório/metabolismo , Deleção de Genes , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Inflamação/patologia , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , RNA Mensageiro/metabolismoRESUMO
Little is known about olfactory glands' regulation despite their presumed importance for normal functioning of the cilia of olfactory neurons. The aim of this study was to establish an assay for olfactory gland activation by using large-scale quantitative electron microscopy (EM). In addition we wanted to test the hypothesis that cholinergic drugs activate the olfactory glands, by using our newly established EM assay. In total, over 70 000 secretory gland vesicles were quantified in over 3000 cells. Olfactory gland cell size (40.8 µm2 ± 2.0 SD), vesicle diameter (812 nm ± 57 SD) and vesicles per cell (21.6 ± 4.2 SD) were also quantified. The vesicle percentage of the cell area varied between 24% and 30%. In a blinded study we found no significant effects of cholinergic agents on parameters of vesicle number or vesicle diameter. Unexpectedly, pilocarpine treatment increased olfactory gland size, probably by inducing cell swelling. In conclusion, we have established a quantitative EM assay for olfactory gland activation and provided new data on basic olfactory gland cell characteristics. By using the EM assay, olfactory glands are shown not to be activated by cholinergic agents, which indicates an alternative regulation pathway or constitutive secretion from olfactory glands.
Assuntos
Acetilcolina/farmacologia , Agonistas Colinérgicos/farmacologia , Microscopia Eletrônica/métodos , Agonistas Muscarínicos/farmacologia , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/ultraestrutura , Pilocarpina/farmacologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nervo Olfatório/fisiologia , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/fisiologiaRESUMO
The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-)) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges.
Assuntos
Sistema y+ de Transporte de Aminoácidos/biossíntese , Sistema y+ de Transporte de Aminoácidos/genética , Autoanticorpos/genética , Autoanticorpos/metabolismo , Encéfalo/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Coelhos , Ratos , Análise de Sequência de Proteína/métodos , Homologia de SequênciaRESUMO
GLT-1 (EAAT2; slc1a2) is the major glutamate transporter in the brain, and is predominantly expressed in astrocytes, but at lower levels also in excitatory terminals. We generated a conditional GLT-1 knock-out mouse to uncover cell-type-specific functional roles of GLT-1. Inactivation of the GLT-1 gene was achieved in either neurons or astrocytes by expression of synapsin-Cre or inducible human GFAP-CreERT2. Elimination of GLT-1 from astrocytes resulted in loss of â¼80% of GLT-1 protein and of glutamate uptake activity that could be solubilized and reconstituted in liposomes. This loss was accompanied by excess mortality, lower body weight, and seizures suggesting that astrocytic GLT-1 is of major importance. However, there was only a small (15%) reduction that did not reach significance of glutamate uptake into crude forebrain synaptosomes. In contrast, when GLT-1 was deleted in neurons, both the GLT-1 protein and glutamate uptake activity that could be solubilized and reconstituted in liposomes were virtually unaffected. These mice showed normal survival, weight gain, and no seizures. However, the synaptosomal glutamate uptake capacity (Vmax) was reduced significantly (40%). In conclusion, astrocytic GLT-1 performs critical functions required for normal weight gain, resistance to epilepsy, and survival. However, the contribution of astrocytic GLT-1 to glutamate uptake into synaptosomes is less than expected, and the contribution of neuronal GLT-1 to synaptosomal glutamate uptake is greater than expected based on their relative protein expression. These results have important implications for the interpretation of the many previous studies assessing glutamate uptake capacity by measuring synaptosomal uptake.
Assuntos
Astrócitos/metabolismo , Epilepsia/metabolismo , Epilepsia/prevenção & controle , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Sinaptossomos/metabolismo , Animais , Astrócitos/ultraestrutura , Peso Corporal , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Eletroencefalografia , Epilepsia/mortalidade , Transportador 2 de Aminoácido Excitatório/genética , Feminino , Lipossomos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/metabolismoRESUMO
Epilepsy is one of the most common chronic neurologic diseases, yet approximately one-third of affected patients do not respond to anticonvulsive drugs that target neurons or neuronal circuits. Reactive astrocytes are commonly found in putative epileptic foci and have been hypothesized to be disease contributors because they lose essential homeostatic capabilities. However, since brain pathology induces astrocytes to become reactive, it is difficult to distinguish whether astrogliosis is a cause or a consequence of epileptogenesis. We now present a mouse model of genetically induced, widespread chronic astrogliosis after conditional deletion of ß1-integrin (Itgß1). In these mice, astrogliosis occurs in the absence of other pathologies and without BBB breach or significant inflammation. Electroencephalography with simultaneous video recording revealed that these mice develop spontaneous seizures during the first six postnatal weeks of life and brain slices show neuronal hyperexcitability. This was not observed in mice with neuronal-targeted ß1-integrin deletion, supporting the hypothesis that astrogliosis is sufficient to induce epileptic seizures. Whole-cell patch-clamp recordings from astrocytes further suggest that the heightened excitability was associated with impaired astrocytic glutamate uptake. Moreover, the relative expression of the cation-chloride cotransporters (CCC) NKCC1 (Slc12a2) and KCC2 (Slc12a5), which are responsible for establishing the neuronal Cl(-) gradient that governs GABAergic inhibition were altered and the NKCC1 inhibitor bumetanide eliminated seizures in a subgroup of mice. These data suggest that a shift in the relative expression of neuronal NKCC1 and KCC2, similar to that observed in immature neurons during development, may contribute to astrogliosis-associated seizures.
Assuntos
Astrócitos/metabolismo , Gliose/metabolismo , Integrina beta1/metabolismo , Convulsões/metabolismo , Potenciais de Ação , Animais , Astrócitos/fisiologia , Barreira Hematoencefálica/metabolismo , Células Cultivadas , Gliose/fisiopatologia , Ácido Glutâmico/metabolismo , Integrina beta1/genética , Camundongos , Neurônios/metabolismo , Neurônios/fisiologia , Convulsões/etiologia , Convulsões/patologia , Convulsões/fisiopatologia , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/genética , Simportadores/metabolismo , Cotransportadores de K e Cl-RESUMO
The EAAT2 glutamate transporter, accounts for >90% of hippocampal glutamate uptake. Although EAAT2 is predominantly expressed in astrocytes, â¼10% of EAAT2 molecules are found in axon terminals. Despite the lower level of EAAT2 expression in glutamatergic terminals, when hippocampal slices are incubated with low concentration of d-aspartate (an EAAT2 substrate), axon terminals accumulate d-aspartate as quickly as astroglia. This implies an unexplained mismatch between the distribution of EAAT2 protein and of EAAT2-mediated transport activity. One hypothesis is that (1) heteroexchange of internal substrate with external substrate is considerably faster than net uptake and (2) terminals favor heteroexchange because of high levels of internal glutamate. However, it is currently unknown whether heteroexchange and uptake have similar or different rates. To address this issue, we used a reconstituted system to compare the relative rates of the two processes in rat and mice. Net uptake was sensitive to changes in the membrane potential and was stimulated by external permeable anions in agreement with the existence of an uncoupled anion conductance. By using the latter, we also demonstrate that the rate of heteroexchange also depends on the membrane potential. Additionally, our data further suggest the presence of a sodium leak in EAAT2. By incorporating the new findings in our previous model of glutamate uptake by EAAT2, we predict that the voltage sensitivity of exchange is caused by the voltage-dependent third Na(+) binding. Further, both our experiments and simulations suggest that the relative rates of net uptake and heteroexchange are comparable in EAAT2.
Assuntos
Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Lipossomos/metabolismo , Aminoácidos/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/ultraestrutura , Bovinos , Simulação por Computador , Transportador 2 de Aminoácido Excitatório/genética , Técnicas In Vitro , Ionóforos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Potássio/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Valinomicina/farmacologiaRESUMO
The oxidation resistance gene 1 (OXR1) prevents oxidative stress-induced cell death by an unknown pathway. Here, depletion of human OXR1 (hOXR1) sensitized several human cell lines to hydrogen peroxide-induced oxidative stress, reduced mtDNA integrity, and increased apoptosis. In contrast, depletion of hOXR1 in cells lacking mtDNA showed no significant change in ROS or viability, suggesting that OXR1 prevents intracellular hydrogen peroxide-induced increase in oxidative stress levels to avoid a vicious cycle of increased oxidative mtDNA damage and ROS formation. Furthermore, expression of p21 and the antioxidant genes GPX2 and HO-1 was reduced in hOXR1-depleted cells. In sum, these data reveal that human OXR1 upregulates the expression of antioxidant genes via the p21 signaling pathway to suppress hydrogen peroxide-induced oxidative stress and maintain mtDNA integrity.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA Mitocondrial/genética , Proteínas/fisiologia , Antioxidantes/metabolismo , Apoptose , Dosagem de Genes , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismo , Proteínas Mitocondriais , Estresse Oxidativo , Transdução de Sinais , Regulação para CimaRESUMO
The physiological roles of the betaine/GABA transporter (BGT1; slc6a12) are still being debated. BGT1 is a member of the solute carrier family 6 (the neurotransmitter, sodium symporter transporter family) and mediates cellular uptake of betaine and GABA in a sodium- and chloride-dependent process. Most of the studies of BGT1 concern its function and regulation in the kidney medulla where its role is best understood. The conditions here are hostile due to hyperosmolarity and significant concentrations of NH4Cl and urea. To withstand the hyperosmolarity, cells trigger osmotic adaptation, involving concentration of a transcriptional factor TonEBP/NFAT5 in the nucleus, and accumulate betaine and other osmolytes. Data from renal cells in culture, primarily MDCK, revealed that transcriptional regulation of BGT1 by TonEBP/NFAT5 is relatively slow. To allow more acute control of the abundance of BGT1 protein in the plasma membrane, there is also post-translation regulation of BGT1 protein trafficking which is dependent on intracellular calcium and ATP. Further, betaine may be important in liver metabolism as a methyl donor. In fact, in the mouse the liver is the organ with the highest content of BGT1. Hepatocytes express high levels of both BGT1 and the only enzyme that can metabolize betaine, namely betaine:homocysteine -S-methyltransferase (BHMT1). The BHMT1 enzyme removes a methyl group from betaine and transfers it to homocysteine, a potential risk factor for cardiovascular disease. Finally, BGT1 has been proposed to play a role in controlling brain excitability and thereby represents a target for anticonvulsive drug development. The latter hypothesis is controversial due to very low expression levels of BGT1 relative to other GABA transporters in brain, and also the primary location of BGT1 at the surface of the brain in the leptomeninges. These issues are discussed in detail.
RESUMO
Glutamate is the major excitatory neurotransmitter, and is inactivated by cellular uptake catalyzed mostly by the glutamate transporter subtypes GLT-1 (EAAT2) and GLAST (EAAT1). Astrocytes express both GLT-1 and GLAST, while axon terminals in the neocortex only express GLT-1. To evaluate the role of GLT-1 in glutamate homeostasis, we injected GLT-1 knockout (KO) mice and wild-type littermates with [1-(13)C]glucose and [1,2-(13)C]acetate 15 min before euthanization. Metabolite levels were analyzed in extracts from neocortex and cerebellum and (13)C labeling in neocortex. Whereas the cerebellum in GLT-1-deficient mice had normal levels of glutamate, glutamine, and (13)C labeling of metabolites, glutamate level was decreased but labeling from [1-(13)C] glucose was unchanged in the neocortex. The contribution from pyruvate carboxylation toward labeling of these metabolites was unchanged. Labeling from [1,2-(13)C] acetate, originating in astrocytes, was decreased in glutamate and glutamine in the neocortex indicating reduced mitochondrial metabolism in astrocytes. The decreased amount of glutamate in the cortex indicates that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT-1 plays a role in glutamate homeostasis in the cortex. Glutamate is the major excitatory neurotransmitter, and is inactivated by uptake via GLT-1 (EAAT2) and GLAST (EAAT1) transporters, while axon terminals in the neocortex only express GLT-1. To evaluate the role of GLT-1 in glutamate homeostasis, we used [1-(13)C]glucose and [1,2-(13)C]acetate injection and NMR spectroscopy. The results indicate that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT-1 plays a role in glutamate homeostasis in the neocortex.
Assuntos
Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Homeostase/fisiologia , Neocórtex/fisiologia , Aminoácidos/metabolismo , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Cromatografia Líquida de Alta Pressão , Interpretação Estatística de Dados , Metabolismo Energético/fisiologia , Transportador 2 de Aminoácido Excitatório/genética , Feminino , Glucose/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Knockout , Ácido Pirúvico/metabolismoRESUMO
Islet function is incompletely understood in part because key steps in glutamate handling remain undetermined. The glutamate (excitatory amino acid) transporter 2 (EAAT2; Slc1a2) has been hypothesized to (a) provide islet cells with glutamate, (b) protect islet cells against high extracellular glutamate concentrations, (c) mediate glutamate release, or (d) control the pH inside insulin secretory granules. Here we floxed the EAAT2 gene to produce the first conditional EAAT2 knock-out mice. Crossing with Nestin-cyclization recombinase (Cre) eliminated EAAT2 from the brain, resulting in epilepsy and premature death, confirming the importance of EAAT2 for brain function and validating the genetic construction. Crossing with insulin-Cre lines (RIP-Cre and IPF1-Cre) to obtain pancreas-selective deletion did not appear to affect survival, growth, glucose tolerance, or ß-cell number. We found (using TaqMan RT-PCR, immunoblotting, immunocytochemistry, and proteome analysis) that the EAAT2 levels were too low to support any of the four hypothesized functions. The proteome analysis detected more than 7,000 islet proteins of which more than 100 were transporters. Although mitochondrial glutamate transporters and transporters for neutral amino acids were present at high levels, all other transporters with known ability to transport glutamate were strikingly absent. Glutamate-metabolizing enzymes were abundant. The level of glutamine synthetase was 2 orders of magnitude higher than that of glutaminase. Taken together this suggests that the uptake of glutamate by islets from the extracellular fluid is insignificant and that glutamate is intracellularly produced. Glutamine synthetase may be more important for islets than assumed previously.
Assuntos
Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteoma/metabolismo , Animais , Cruzamentos Genéticos , Transportador 2 de Aminoácido Excitatório/genética , Deleção de Genes , Ácido Glutâmico/genética , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Knockout , Proteoma/genéticaRESUMO
The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [(3)H]taurine. Furthermore, most of the uptake of [(3)H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.
Assuntos
Encéfalo/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Fígado/metabolismo , Taurina/metabolismo , Animais , Encéfalo/citologia , Química Encefálica , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Córtex Renal/citologia , Córtex Renal/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Fígado/citologia , Masculino , Camundongos , Camundongos Knockout , Coelhos , Ratos , Ratos Wistar , Taurina/genéticaRESUMO
The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was â¼0.013 mg/g tissue (â¼130 molecules µm⻳), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-D-aspartate (4-methoxy-7-nitroindolinyl-D-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and D-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.
Assuntos
Sistema Nervoso Central/citologia , Transportador 3 de Aminoácido Excitatório/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Ácido Aspártico/farmacologia , Sistema Nervoso Central/anatomia & histologia , Ácido D-Aspártico/metabolismo , Dendritos/metabolismo , Dendritos/ultraestrutura , Inibidores Enzimáticos/farmacologia , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Transportador 2 de Aminoácido Excitatório/deficiência , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/deficiência , Transportador 3 de Aminoácido Excitatório/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato Descarboxilase/metabolismo , Técnicas In Vitro , Rim/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Imunoeletrônica , Proteína Básica da Mielina/metabolismo , Neurônios/efeitos dos fármacos , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Proteolipídeos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Frações Subcelulares/metabolismo , Sinaptofisina/metabolismo , Proteína Vesicular 1 de Transporte de GlutamatoRESUMO
Human studies of intrasex variability have shown that males are intellectually more variable. Here we have performed retrospective statistical analysis of human intrasex variability in several different properties and performances that are unrelated or indirectly related to intelligence: (a) birth weights of nearly 48,000 babies (Medical Birth Registry of Norway); (b) adult weight, height, body mass index and blood parameters of more than 2,700 adults aged 18-90 (NORIP); (c) physical performance in the 60 meter dash event of 575 junior high school students; and (d) psychological performance reflected by the results of more than 222,000 undergraduate university examination grades (LIST). For all characteristics, the data were analyzed using cumulative distribution functions and the resultant intrasex variability for males was compared with that for females. The principal finding is that human intrasex variability is significantly higher in males, and consequently constitutes a fundamental sex difference.
Assuntos
Logro , Fenótipo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Peso ao Nascer , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Psicológicos , Fatores Sexuais , Adulto JovemRESUMO
Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.
Assuntos
Transportador 2 de Aminoácido Excitatório/metabolismo , Lipopolissacarídeos/farmacologia , Neuroglia/efeitos dos fármacos , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Ácido Aspártico/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Biotinilação , Western Blotting , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Transportador 1 de Aminoácido Excitatório/biossíntese , Transportador 2 de Aminoácido Excitatório/biossíntese , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microscopia Confocal , Neuroglia/citologia , Neuroglia/metabolismo , Fenótipo , Transporte Proteico , Regulação para CimaRESUMO
Glutamate functions as a neurotransmitter in the central nervous system (CNS) and neuromuscular junctions in insects. High-affinity glutamate transporters are responsible for keeping the resting levels of excitatory amino acids below the synaptic activation threshold by removing them from the extracellular fluid, thereby preventing them from reaching toxic levels. Peptides representing the N- and C-terminal regions of a glutamate transporter cloned from the cabbage looper caterpillar (Trichoplusia ni) were synthesized and used to generate polyclonal antibodies. The antibodies produced immunohistochemical staining in both muscular and nervous system T. ni tissues. Neuromuscular junctions in the skeletal muscles produced the most intense labelling, but no visceral muscle or sensory nerves were labelled. In the CNS, the neuropile of the ganglia, but not the connectives, gave a diffuse staining. Electron microscopical examination of ganglia and neuromuscular junctions showed that the plasma membrane of glial cells, but not that of neurons was labelled, in agreement with the notion that most of the glutamate uptake sites in this insect are in glial cells.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Sistema X-AG de Transporte de Aminoácidos/imunologia , Animais , Especificidade de Anticorpos , Imuno-Histoquímica , Proteínas de Insetos/imunologia , Microscopia Imunoeletrônica , Mariposas/ultraestrutura , Sistema Nervoso/metabolismo , Junção Neuromuscular/metabolismoRESUMO
Removal of L-glutamate (Glu) from the synapse is critical to maintain normal transmission and to prevent excitotoxicity, and is performed exclusively by excitatory amino acid transporters (EAATs). We investigated the effects of substrates and blockers of EAATs on extracellular Glu and cellular viability in organotypic cultures of rat hippocampus. Seven-day treatment with a range of drugs (L-trans-pyrrolidine-2,4-dicarboxylate, (2S,4R)-4-methyl-glutamate, (+/-)-threo-3-methylglutamate and DL-threo-beta-benzyloxyaspartate), in the presence of 300 microM added Glu, resulted in increased extracellular Glu and a significant correlation between Glu concentration and cellular injury (as indicated by lactate dehydrogenase release). In contrast, (2S,3s,4R)-2-(carboxycyclopropyl)glycine (L-CCG-III) exerted a novel neuroprotection against this toxicity, and elevations in extracellular Glu were not toxic in the presence of this compound. Similar results were obtained following two-week treatment of cultures without added Glu. Whilst blockade of GLT-1 alone was relatively ineffective in producing excitotoxic injury, heteroexchange of Glu by EAAT substrates may exacerbate excitotoxicity.