RESUMEN
Glutamate is involved in fundamental functions, including neuronal plasticity and memory. Astrocytes are integral elements involved in synaptic function, and the GLT-1 transporter possesses a critical role in glutamate uptake. Here, we study the role of GLT-1, specifically located in astrocytes, in the consolidation, expression, reconsolidation and persistence of spatial object recognition memory in rats. Administration of dihydrokainic acid (DHK), a selective GLT-1 inhibitor, into the dorsal hippocampus around a weak training which only induces short-term memory, promotes long-term memory formation. This promotion is prevented by hippocampal administration of protein-synthesis translation inhibitor, blockade of Activity-regulated cytoskeleton-associated protein (Arc) translation or Brain-Derived Neurotrophic Factor (BDNF) action, which are plasticity related proteins necessary for memory consolidation. However, DHK around a strong training, which induces long-term memory, does not affect memory consolidation. Administration of DHK before the test session impairs the expression of long-term memory, and this effect is dependent of Arc translation. Furthermore, DHK impairs reconsolidation if applied before a reactivation session, and this effect is independent of Arc translation. These findings reveal specific consequences on spatial memory stages developed under hippocampal GLT-1 blockade, shedding light on the intricate molecular mechanisms, governed in part for the action of glia.
Asunto(s)
Astrocitos , Factor Neurotrófico Derivado del Encéfalo , Proteínas del Citoesqueleto , Ácido Glutámico , Hipocampo , Memoria Espacial , Animales , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Memoria Espacial/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Masculino , Ratas , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Ácido Glutámico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Ratas Wistar , Ácido Kaínico/farmacología , Ácido Kaínico/análogos & derivados , Consolidación de la Memoria/efectos de los fármacosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron degenerative disease that is associated with demyelination. The Wobbler (WR) mouse exhibits motoneuron degeneration, gliosis and myelin deterioration in the cervical spinal cord. Since male WRs display low testosterone (T) levels in the nervous system, we investigated if T modified myelin-relative parameters in WRs in the absence or presence of the aromatase inhibitor, anastrozole (A). We studied myelin by using luxol-fast-blue (LFB) staining, semithin sections, electron microscopy and myelin protein expression, density of IBA1+ microglia and mRNA expression of inflammatory factors, and the glutamatergic parameters glutamine synthetase (GS) and the transporter GLT1. Controls and WR + T showed higher LFB, MBP and PLP staining, lower g-ratios and compact myelin than WRs and WR + T + A, and groups showing the rupture of myelin lamellae. WRs showed increased IBA1+ cells and mRNA for CD11b and inflammatory factors (IL-18, TLR4, TNFαR1 and P2Y12R) vs. controls or WR + T. IBA1+ cells, and CD11b were not reduced in WR + T + A, but inflammatory factors' mRNA remained low. A reduction of GS+ cells and GLT-1 immunoreactivity was observed in WRs and WR + T + A vs. controls and WR + T. Clinically, WR + T but not WR + T + A showed enhanced muscle mass, grip strength and reduced paw abnormalities. Therefore, T effects involve myelin protection, a finding of potential clinical translation.
Asunto(s)
Esclerosis Amiotrófica Lateral , Modelos Animales de Enfermedad , Vaina de Mielina , Testosterona , Animales , Ratones , Vaina de Mielina/metabolismo , Vaina de Mielina/efectos de los fármacos , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Masculino , Testosterona/farmacología , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patologíaRESUMEN
Clavulanic acid (CLAV) is a non-antibiotic ß-lactam that has been used since the late 1970s as a ß-lactamase inhibitor in combination with amoxicillin, another ß-lactam with antibiotic activity. Its long-observed adverse reaction profile allows it to say that CLAV is a well-tolerated drug with mainly mild adverse reactions. Interestingly, in 2005, it was discovered that ß-lactams enhance the astrocytic expression of GLT-1, a glutamate transporter essential for maintaining synaptic glutamate homeostasis involved in several pathologies of the central nervous system (CNS). This finding, along with a favorable pharmacokinetic profile, prompted the appearance of several studies that intended to evaluate the effect of CLAV in preclinical disease models. Studies have revealed that CLAV can increase GLT-1 expression in the nucleus accumbens (NAcc), medial prefrontal cortex (PFC), and spinal cord of rodents, to affect glutamate and dopaminergic neurotransmission, and exert an anti-inflammatory effect by modulating the levels of the cytokines TNF-α and interleukin 10 (IL-10). CLAV has been tested with positive results in preclinical models of epilepsy, addiction, stroke, neuropathic and inflammatory pain, dementia, Parkinson's disease, and sexual and anxiety behavior. These properties make CLAV a potential therapeutic drug if repurposed. Therefore, this review aims to gather information on CLAV's effect on preclinical neurological disease models and to give some perspectives on its potential therapeutic use in some diseases of the CNS.
Asunto(s)
Antibacterianos , beta-Lactamas , Ácido Clavulánico/uso terapéutico , Ácido Clavulánico/metabolismo , Ácido Clavulánico/farmacología , Antibacterianos/uso terapéutico , beta-Lactamas/metabolismo , beta-Lactamas/farmacología , Núcleo Accumbens/metabolismo , Glutamatos/metabolismo , Glutamatos/farmacología , Transportador 2 de Aminoácidos Excitadores/metabolismoRESUMEN
Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. Once released, it binds to specific membrane receptors and transporters activating a wide variety of signal transduction cascades, as well as its removal from the synaptic cleft in order to avoid its extracellular accumulation and the overstimulation of extra-synaptic receptors that might result in neuronal death through a process known as excitotoxicity. Although neurodegenerative diseases are heterogenous in clinical phenotypes and genetic etiologies, a fundamental mechanism involved in neuronal degeneration is excitotoxicity. Glutamate homeostasis is critical for brain physiology and Glutamate transporters are key players in maintaining low extracellular Glutamate levels. Therefore, the characterization of Glutamate transporters has been an active area of glutamatergic research for the last 40 years. Transporter activity its regulated at different levels: transcriptional and translational control, transporter protein trafficking and membrane mobility, and through extensive post-translational modifications. The elucidation of these mechanisms has emerged as an important piece to shape our current understanding of glutamate actions in the nervous system.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/química , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Ácido Glutámico/metabolismo , Transmisión Sináptica/fisiología , Sistema de Transporte de Aminoácidos X-AG/genética , Animales , Transportador 1 de Aminoácidos Excitadores/química , Transportador 1 de Aminoácidos Excitadores/genética , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/química , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Humanos , Procesamiento Proteico-Postraduccional/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
During prolonged dehydration, body fluid homeostasis is challenged by extracellular fluid (ECF) hyperosmolality, which induce important functional changes in the hypothalamus, in parallel with other effector responses, such as the activation of the local renin-angiotensin system (RAS). Therefore, in the present study we investigated the role of sodium-driven ECF hyperosmolality on glial fibrillary acid protein (GFAP) immunoreactivity and protein expression, membrane capacitance, mRNA expression of RAS components and glutamate balance in cultured hypothalamic astrocytes. Our data show that hypothalamic astrocytes respond to increased hyperosmolality with a similar decrease in GFAP expression and membrane capacitance, indicative of reduced cellular area. Hyperosmolality also downregulates the transcript levels of angiotensinogen and both angiotensin-converting enzymes, whereas upregulates type 1a angiotensin II receptor mRNA. Incubation with hypertonic solution also decreases the immunoreactivity to the membrane glutamate/aspartate transporter (GLAST) as well as tritiated-aspartate uptake by astrocytes. This latter effect is completely restored to basal levels when astrocytes previously exposed to hypertonicity are incubated under isotonic conditions. Together with a direct effect on two important local signaling systems (glutamate and RAS), these synaptic rearrangements driven by astrocytes may accomplish for a coordinated increase in the excitatory drive onto the hypothalamic neurosecretory system, ultimately culminating with increased AVP release in response to hyperosmolality.
Asunto(s)
Astrocitos , Ácido Glutámico , Astrocitos/metabolismo , Células Cultivadas , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipotálamo/metabolismo , ARN MensajeroRESUMEN
Chronic alcohol intake leads to neuroinflammation and astrocyte dysfunction, proposed to perpetuate alcohol consumption and to promote conditioned relapse-like binge drinking. In the present study, human mesenchymal stem cells (MSCs) were cultured in 3D-conditions to generate MSC-spheroids, which greatly increased MSCs anti-inflammatory ability and reduced cell volume by 90% versus conventionally 2D-cultured MSCs, enabling their intravenous administration and access to the brain. It is shown, in an animal model of chronic ethanol intake and relapse-drinking, that both the intravenous and intra-cerebroventricular administration of a single dose of MSC-spheroids inhibited chronic ethanol intake and relapse-like drinking by 80-90%, displaying significant effects over 3-5 weeks. The MSC-spheroid administration fully normalized alcohol-induced neuroinflammation, as shown by a reduced astrocyte activation, and markedly increased the levels of the astrocyte Na-glutamate (GLT-1) transporter. This research suggests that the intravenous administration of MSC-spheroids may constitute an effective new approach for the treatment of alcohol-use disorders.
Asunto(s)
Consumo de Bebidas Alcohólicas/terapia , Alcoholismo/terapia , Consumo Excesivo de Bebidas Alcohólicas/terapia , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Esferoides Celulares/trasplante , Administración Intravenosa , Análisis de Varianza , Animales , Astrocitos/metabolismo , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Transportador 2 de Aminoácidos Excitadores/metabolismo , Femenino , Humanos , Infusiones Intraventriculares , Células Madre Mesenquimatosas/metabolismo , Ratas , Ratas Wistar , Recurrencia , Esferoides Celulares/metabolismo , Grasa Subcutánea/citologíaRESUMEN
This study was performed to evaluate the bilateral effects of focal permanent ischemia (FPI) on glial metabolism in the cerebral cortex. Two and 9 days after FPI induction, we analyze [18F]FDG metabolism by micro-PET, astrocyte morphology and reactivity by immunohistochemistry, cytokines and trophic factors by ELISA, glutamate transporters by RT-PCR, monocarboxylate transporters (MCTs) by western blot, and substrate uptake and oxidation by ex vivo slices model. The FPI was induced surgically by thermocoagulation of the blood in the pial vessels of the motor and sensorimotor cortices in adult (90 days old) male Wistar rats. Neurochemical analyses were performed separately on both ipsilateral and contralateral cortical hemispheres. In both cortical hemispheres, we observed an increase in tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and glutamate transporter 1 (GLT-1) mRNA levels; lactate oxidation; and glutamate uptake and a decrease in brain-derived neurotrophic factor (BDNF) after 2 days of FPI. Nine days after FPI, we observed an increase in TNF-α levels and a decrease in BDNF, GLT-1, and glutamate aspartate transporter (GLAST) mRNA levels in both hemispheres. Additionally, most of the unilateral alterations were found only in the ipsilateral hemisphere and persisted until 9 days post-FPI. They include diminished in vivo glucose uptake and GLAST expression, followed by increased glial fibrillary acidic protein (GFAP) gray values, astrocyte reactivity, and glutamate oxidation. Astrocytes presented signs of long-lasting reactivity, showing a radial morphology. In the intact hemisphere, there was a decrease in MCT2 levels, which did not persist. Our study shows the bilateralism of glial modifications following FPI, highlighting the role of energy metabolism adaptations on brain recovery post-ischemia.
Asunto(s)
Adaptación Fisiológica/fisiología , Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Neuroglía/metabolismo , Animales , Isquemia Encefálica/patología , Corteza Cerebral/patología , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Masculino , Neuroglía/patología , Ratas , Ratas WistarRESUMEN
This study aimed to evaluate and compare the immunoexpression of glucose transporter-1 (GLUT-1) and angiogenic index between pleomorphic adenomas (PAs), adenoid cystic carcinomas (ACCs), and mucoepidermoid carcinomas (MECs) of the salivary glands, and establish associations with the respective subtype/histological grade. Twenty PAs, 20 ACCs, and 10 MECs were submitted to morphological and immunohistochemical analysis. GLUT-1 expression was semi-quantitatively evaluated and angiogenic index was assessed by microvessel counts using anti-CD34 antibody. Higher GLUT-1 immunoexpression was observed in the MECs compared to PAs and ACCs (p = 0.022). Mean number of microvessels was 66.5 in MECs, 40.4 in PAs, and 21.2 in ACCs (p < 0.001). GLUT-1 expression and angiogenic index showed no significant correlation in the tumors studied. Results suggest that differences in biological behavior of the studied tumors are related to GLUT-1. Benign and malignant salivary gland tumors differ in the angiogenic index; however, angiogenesis may be independent of the tumor cell's metabolic demand.
Asunto(s)
Adenoma Pleomórfico/metabolismo , Carcinoma Adenoide Quístico/metabolismo , Carcinoma Mucoepidermoide/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Neovascularización Patológica , Neoplasias de las Glándulas Salivales/metabolismo , Adenoma Pleomórfico/irrigación sanguínea , Adenoma Pleomórfico/patología , Biomarcadores de Tumor/metabolismo , Carcinoma Adenoide Quístico/irrigación sanguínea , Carcinoma Adenoide Quístico/patología , Carcinoma Mucoepidermoide/irrigación sanguínea , Carcinoma Mucoepidermoide/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Microvasos/patología , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias de las Glándulas Salivales/irrigación sanguínea , Neoplasias de las Glándulas Salivales/patología , Glándulas Salivales/patologíaRESUMEN
Glutaric aciduria type I (GA I) is biochemically characterized by accumulation of glutaric and 3-hydroxyglutaric acids in body fluids and tissues, particularly in the brain. Affected patients show progressive cortical leukoencephalopathy and chronic degeneration of the basal ganglia whose pathogenesis is still unclear. In the present work we investigated parameters of bioenergetics and redox homeostasis in various cerebral structures (cerebral cortex, striatum and hippocampus) and heart of adult wild type (Gcdh(+/+)) and glutaryl-CoA dehydrogenase deficient knockout (Gcdh(-/-)) mice fed a baseline chow. Oxidative stress parameters were also measured after acute lysine overload. Finally, mRNA expression of NMDA subunits and GLT1 transporter was determined in cerebral cortex and striatum of these animals fed a baseline or high lysine (4.7%) chow. No significant alterations of bioenergetics or redox status were observed in these mice. In contrast, mRNA expression of the NR2B glutamate receptor subunit and of the GLT1 glutamate transporter was higher in cerebral cortex of Gcdh(-/-) mice. Furthermore, NR2B expression was markedly elevated in striatum of Gcdh(-/-) animals receiving chronic Lys overload. These data indicate higher susceptibility of Gcdh(-/-) mice to excitotoxic damage, implying that this pathomechanism may contribute to the cortical and striatum alterations observed in GA I patients.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/complicaciones , Encefalopatías Metabólicas/complicaciones , Lesiones Encefálicas/etiología , Regulación de la Expresión Génica/genética , Glutaril-CoA Deshidrogenasa/deficiencia , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Catalasa/metabolismo , Modelos Animales de Enfermedad , Transportador 2 de Aminoácidos Excitadores/metabolismo , Fluoresceínas/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Glutaril-CoA Deshidrogenasa/genética , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Malondialdehído/metabolismo , Ratones , Ratones Transgénicos , NAD/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Compuestos de Sulfhidrilo/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
N-methyl-d-aspartate (NMDA) preconditioning is induced by subtoxic doses of NMDA and it promotes a transient state of resistance against subsequent lethal insults. Interestingly, this mechanism of neuroprotection depends on adenosine A1 receptors (A1R), since blockade of A1R precludes this phenomenon. In this study we evaluated the consequences of NMDA preconditioning on the hippocampal A1R biology (i.e. expression, binding properties and functionality). Accordingly, we measured A1R expression in NMDA preconditioned mice (75mg/kg, i.p.; 24h) and showed that neither the total amount of receptor, nor the A1R levels in the synaptic fraction was altered. In addition, the A1R binding affinity to the antagonist [(3)H] DPCPX was slightly increased in total membrane extracts of hippocampus from preconditioned mice. Next, we evaluated the impact of NMDA preconditioning on A1R functioning by measuring the A1R-mediated regulation of glutamate uptake into hippocampal slices and on behavioral responses in the open field and hot plate tests. NMDA preconditioning increased glutamate uptake into hippocampal slices without altering the expression of glutamate transporter GLT-1. Interestingly, NMDA preconditioning also induced antinociception in the hot plate test and both effects were reversed by post-activation of A1R with the agonist CCPA (0.2mg/kg, i.p.). NMDA preconditioning or A1R modulation did not alter locomotor activity in the open field. Overall, the results described herein provide new evidence that post-activation of A1R modulates NMDA preconditioning-mediated responses, pointing to the importance of the cross-talk between glutamatergic and adenosinergic systems to neuroprotection.
Asunto(s)
Conducta Animal/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , N-Metilaspartato/farmacología , Receptor de Adenosina A1/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Animales , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos , Actividad Motora/efectos de los fármacos , Agonistas del Receptor Purinérgico P1/farmacología , Xantinas/farmacologíaRESUMEN
Congenital hypothyroidism is associated with delay in cell migration and proliferation in brain tissue, impairment of synapse formation, misregulation of neurotransmitters, hypomyelination and mental retardation. However, the mechanisms underlying the neuropsychological deficits observed in congenital hypothyroidism are not completely understood. In the present study we proposed a mechanism by which hypothyroidism leads to hippocampal neurotoxicity. Congenital hypothyroidism induces c-Jun-N-terminal kinase (JNK) pathway activation leading to hyperphosphorylation of the glial fibrillary acidic protein (GFAP), vimentin and neurofilament subunits from hippocampal astrocytes and neurons, respectively. Moreover, hyperphosphorylation of the cytoskeletal proteins was not reversed by T3 and poorly reversed by T4. In addition, congenital hypothyroidism is associated with downregulation of astrocyte glutamate transporters (GLAST and GLT-1) leading to decreased glutamate uptake and subsequent influx of Ca(2+) through N-methyl-D-aspartate (NMDA) receptors. The Na(+)-coupled (14)C-α-methyl-amino-isobutyric acid ((14)C-MeAIB) accumulation into hippocampal cells also might cause an increase in the intracellular Ca(2+) concentration by opening voltage-dependent calcium channels (VDCC). The excessive influx of Ca(2+) through NMDA receptors and VDCCs might lead to an overload of Ca(2+) within the cells, which set off glutamate excitotoxicity and oxidative stress. The inhibited acetylcholinesterase (AChE) activity might also induce Ca(2+) influx. The inhibited glucose-6-phosphate dehydrogenase (G6PD) and gamma-glutamyl transferase (GGT) activities, associated with altered glutamate and neutral amino acids uptake could somehow affect the GSH turnover, the antioxidant defense system, as well as the glutamate-glutamine cycle. Reduced levels of S100B and glial fibrillary acidic protein (GFAP) take part of the hypothyroid condition, suggesting a compromised astroglial/neuronal neurometabolic coupling which is probably related to the neurotoxic damage in hypothyroid brain.
Asunto(s)
Acetilcolinesterasa/metabolismo , Hipotiroidismo Congénito/enzimología , Hipocampo/enzimología , Estrés Oxidativo , Animales , Astrocitos/metabolismo , Recuento de Células , Hipotiroidismo Congénito/patología , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteínas Ligadas a GPI/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/crecimiento & desarrollo , Hipocampo/patología , Masculino , Neuronas/metabolismo , Ratas , Ratas Wistar , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Hormonas Tiroideas/sangreRESUMEN
Several types of animal models have been developed to investigate Alzheimer's disease (AD). Okadaic acid (OA), a potent inhibitor of phosphatases 1 and 2A, induces characteristics that resemble AD-like pathology. Memory impairment induced by intra-hippocampal injection of OA has been reported, accompanied by remarkable neuropathological changes including hippocampal neurodegeneration, a paired helical filament-like phosphorylation of tau protein, and formation of ß-amyloid containing plaque-like structures. Rats were submitted to bilateral intrahippocampal okadaic acid-injection (100 ng) and, 12 days after the surgery, behavioral and biochemical tests were performed. Using this model, we evaluated spatial cognitive deficit and neuroglial alterations, particularly astroglial protein markers such as glial fibrillary acidic protein (GFAP) and S100B, metabolism of glutamate, oxidative parameters and alterations in MAPKs. Our results indicate significant hippocampal changes, including increased GFAP, protein oxidation, and phosphorylation of p38(MAPK); and decreases in glutathione content, transporter EAAT2/GLT-1, and glutamine synthetase activity as well as a decrease in cerebrospinal fluid S100B. No alterations were observed in glutamate uptake activity and S100B content. In conclusion, the OA-induced model of dementia caused spatial cognitive deficit and oxidative stress in this model and, for the first time to our knowledge, specific astroglial alterations. Findings contribute to understanding diseases accompanied by cognitive deficits and the neural damage induced by AO administration.
Asunto(s)
Demencia/metabolismo , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Neuroglía/metabolismo , Animales , Trastornos del Conocimiento/líquido cefalorraquídeo , Trastornos del Conocimiento/complicaciones , Trastornos del Conocimiento/metabolismo , Demencia/líquido cefalorraquídeo , Demencia/inducido químicamente , Demencia/complicaciones , Demencia/psicología , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Ácido Glutámico/metabolismo , Glutatión/metabolismo , Hipocampo/metabolismo , Humanos , Masculino , Microinyecciones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Crecimiento Nervioso/líquido cefalorraquídeo , Factores de Crecimiento Nervioso/metabolismo , Ácido Ocadaico/administración & dosificación , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/líquido cefalorraquídeo , Proteínas S100/metabolismoRESUMEN
INTRODUCTION: Uptake of glutamate in the hippocampus by specialized transporters appears to be important for the prevention of glutamate-induced neurotoxicity. However, the role of these transporters in synaptic plasticity and learning is still unclear. We examined the expression pattern of glutamate transporters at different stages of spatial learning using a one-day (three blocks) version of the Morris Water Maze. METHODS: Male rats (Sprague Dawley, 3 months old) were divided into three groups (learner, swim control, or naïve control) and animals were sacrificed after the first, second, or third block of training. The hippocampi were immediately extracted and flash frozen for RNA analysis. Real time polymerase chain reaction was employed to examine the expression of glutamate transporter 1 (Glt-1), Glt1b, glutamate-aspartate transporter (GLAST) and excitatory amino acid carrier-1 (EAAC1) in whole hippocampi. RESULTS: EAAC1 and GLAST RNA were downregulated in the learner and swimmer groups (compared to naïve) after the first two blocks of training during the one-day protocol but EAAC1 returned to control levels by the end of the third block. GLAST levels were upregulated by the third block of training. Glt-1b expression was downregulated during the second block of training but returned to control by the third block. CONCLUSIONS: The observed decreases in glutamate transporter expression may be important during the early stages of spatial learning as a possible mechanism to enhance glutamatergic availability during critical stages of learning. However, similar decreases in glutamate transporter expression in both the learner and swimmer groups indicate that the observed differences may be task-induced. Additional experiments are currently underway to examine this possibility.
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Sistema de Transporte de Aminoácidos X-AG/metabolismo , Hipocampo/metabolismo , Aprendizaje por Laberinto/fisiología , Conducta Espacial/fisiología , Animales , Regulación hacia Abajo/fisiología , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 3 de Aminoácidos Excitadores/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-DawleyRESUMEN
Glutamate is an excitatory neurotransmitter involved in neuronal plasticity and neurotoxicity. Chronic stress produces several physiological changes on the spinal cord, many of them presenting sex-specific differences, which probably involve glutamatergic system alterations. The aim of the present study was to verify possible effects of exposure to chronic restraint stress and 17beta-estradiol replacement on [3H]-glutamate release and uptake in spinal cord synaptosomes of ovariectomized (OVX) rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided in controls and chronically stressed. Restraint stress or estradiol had no effect on [3H]-glutamate release. The chronic restraint stress promoted a decrease and 17beta-estradiol induced an increase on [3H]-glutamate uptake, but the uptake observed in the restraint stress +17beta-estradiol group was similar to control. Furthermore, 17beta-estradiol treatment caused a significant increase in the immunocontent of the three glutamate transporters present in spinal cord. Restraint stress had no effect on the expression of these transporters, but prevented the 17beta-estradiol effect. We suggest that changes in the glutamatergic system are likely to take part in the mechanisms involved in spinal cord plasticity following repeated stress exposure, and that 17beta-estradiol levels may affect chronic stress effects in this structure.
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Estradiol/farmacología , Ácido Glutámico/metabolismo , Restricción Física , Médula Espinal/efectos de los fármacos , Estrés Psicológico/metabolismo , Animales , Enfermedad Crónica , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 3 de Aminoácidos Excitadores/metabolismo , Femenino , Ovariectomía , Ratas , Ratas Wistar , Médula Espinal/metabolismo , Sinaptosomas/metabolismoRESUMEN
The role of excitotoxicity in the cerebral damage of glutaryl-CoA dehydrogenase deficiency (GDD) is under intense debate. We therefore investigated the in vitro effect of glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids, which accumulate in GDD, on [(3)H]glutamate uptake by slices and synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Glutamate uptake was significantly decreased by high concentrations of GA in cortical slices of 7-day-old rats, but not in cerebral cortex from 15- and 30-day-old rats and in striatum from all studied ages. Furthermore, this effect was not due to cellular death and was prevented by N-acetylcysteine preadministration, suggesting the involvement of oxidative damage. In contrast, glutamate uptake by brain slices was not affected by 3-OHGA exposure. Immunoblot analysis revealed that GLAST transporters were more abundant in the cerebral cortex compared to the striatum of 7-day-old rats. Moreover, the simultaneous addition of GA and dihydrokainate (DHK), a specific inhibitor of GLT1, resulted in a significantly higher inhibition of [(3)H]glutamate uptake by cortical slices of 7-day-old rats than that induced by the sole presence of DHK. We also observed that both GA and 3-OHGA exposure did not alter the incorporation of glutamate into synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Finally, GA in vivo administration did not alter glutamate uptake into cortical slices from 7-day-old rats. Our findings may explain at least in part why cortical neurons are more vulnerable to damage at birth as evidenced by the frontotemporal cortical atrophy observed in newborns affected by GDD.
Asunto(s)
Animales Recién Nacidos/metabolismo , Corteza Cerebral/metabolismo , Glutamatos/farmacocinética , Glutaratos/administración & dosificación , Glutaratos/metabolismo , Acetilcisteína/administración & dosificación , Acetilcisteína/metabolismo , Animales , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Glutamatos/metabolismo , Glutaril-CoA Deshidrogenasa/deficiencia , Técnicas In Vitro , Ácido Kaínico/análogos & derivados , Ácido Kaínico/metabolismo , Neostriado/metabolismo , Ratas , Ratas Wistar , Sinaptosomas/metabolismoRESUMEN
Analysis of the UGA3-GLT1 bidirectional promoter has indicated that its transcriptional activation is determined by the combined action of Gcn4p and Gln3p, and that its bidirectional character is influenced by chromatin organization, through the action of an Abf1p binding site and a polydAdTtract. Results presented in this paper show that lack of Gcn5p impairs histone acetylation and nucleosomal organization of the UGA3-GLT1 promoter, resulting in an asymmetrical transcriptional activation response of UGA3 and GLT1. The phenotype displayed by a double mutant impaired in GCN5 and in the Abf1p binding site indicates that the combined action of these two elements determines the bidirectional capacity of the UGA3-GLT1 intergenic region.
Asunto(s)
Proteínas de Unión al ADN/genética , Transportador 2 de Aminoácidos Excitadores/genética , Histona Acetiltransferasas/fisiología , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Acetilación , Sitios de Unión/genética , ADN Intergénico/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains unclear. In the present study, we investigated changes in expression of estrogen receptors (ERs) alpha and beta and excitatory amino acid transporters (EAAT) 1 and 2 in rat organotypic hippocampal slice cultures treated with estradiol and subsequently exposed to oxygen--glucose deprivation (OGD). Pretreatment with 17beta-estradiol (10 nM) for 7 days protected the CA1 area of hippocampus against OGD (60 min), reducing cellular injury by 46% compared to the vehicle control group. Levels of ERalpha protein were significantly reduced by 20% after OGD in both vehicle- and estradiol-treated cultures, whereas ERbeta was significantly up-regulated by 25% in the estradiol-treated cultures. In contrast, EAAT1 and EAAT2 levels were unchanged in response to estradiol treatment in this model of OGD. These findings suggest that estrogen-induced neuroprotection against ischemia might involve regulation of ERbeta and, consequently, of the genes influenced by this receptor.
Asunto(s)
Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Glucosa/metabolismo , Hipocampo/citología , Hipoxia , Animales , Células Cultivadas , Hipocampo/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
Exposure of the brain to a sublethal insult can protect against a subsequent brain injury. Hypoxic preconditioning induces tolerance to hypoxic--ischemic injury in neonatal rat brain and is associated with changes in gene and protein expression. To study the involvement of excitatory amino acid transporters (EAAT1 and EAAT2) and estrogen receptors (ERalpha and ERbeta) in neonatal hypoxia--induced ischemic tolerance, we examined changes in expression of these proteins in the cortex, hippocampus and striatum of newborn rats at different time points after exposure to sublethal hypoxia (8% O(2), 3h). Preconditioning with hypoxia 24h before hypoxia-ischemia afforded marked brain protection compared with littermate control animals as determined by morphological assessment. Immunoblot analysis showed that EAAT2 and ERalpha were significantly increased by 55% and 49%, respectively, in cortex at 24h after hypoxic-preconditioning. Surprisingly, at the same time point, a significant decrease of EAAT2 by 48% in striatum was observed. In contrast, hypoxic preconditioning had no effect on the levels of EAAT1 and ERbeta in any of the brain regions studied at any of the time points analyzed. The similar pattern of changes in EAAT2 and ERalpha levels suggests that ERalpha might interact with EAAT2 in producing preconditioning. The endogenous molecular mechanisms modulated by hypoxia preconditioning may contribute to the development of hypoxia-induced ischemic tolerance, and may provide novel therapeutic targets for the treatment of cerebral ischemia.