RESUMO
α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the predominant mediators of glutamate-induced excitatory neurotransmission. It is widely accepted that AMPA receptors are critical for the generation and spread of epileptic seizure activity. Dysfunction of AMPA receptors as a causal factor in patients with intractable epilepsy results in neurotransmission failure. Brain-specific serine/threonine-protein kinase 1 (SAD-B), a serine-threonine kinase specifically expressed in the brain, has been shown to regulate AMPA receptor-mediated neurotransmission through a presynaptic mechanism. In cultured rat hippocampal neurons, the overexpression of SAD-B significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Here, we showed that SAD-B downregulation exerted antiepileptic activity by regulating AMPA receptors in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We first used immunoblotting and immunohistochemistry analysis to demonstrate that SAD-B expression was increased in the epileptic rat brain. Subsequently, to explore the function of SAD-B in epilepsy, we used siRNA to knock down SAD-B protein and observed behavior after PTZ-induced seizures. We found that SAD-B downregulation attenuated seizure severity and susceptibility in the PTZ-induced epileptic model. Furthermore, we showed that the antiepileptic effect of SAD-B downregulation on PTZ-induced seizure was abolished by CNQX (an AMPA receptor inhibitor), suggesting that SAD-B modulated epileptic seizure by regulating AMPA receptors in the brain. Taken together, these findings suggest that SAD-B may be a potential and novel therapeutic target to limit epileptic seizures.
Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Epilepsia do Lobo Temporal/tratamento farmacológico , Agonistas de Aminoácidos Excitatórios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de AMPA/metabolismo , Adolescente , Adulto , Animais , Criança , Epilepsia do Lobo Temporal/induzido quimicamente , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pentilenotetrazol , Ratos Sprague-Dawley , Adulto JovemRESUMO
α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the predominant mediators of glutamate-induced excitatory neurotransmission. It is widely accepted that AMPA receptors are critical for the generation and spread of epileptic seizure activity. Dysfunction of AMPA receptors as a causal factor in patients with intractable epilepsy results in neurotransmission failure. Brain-specific serine/threonine-protein kinase 1 (SAD-B), a serine-threonine kinase specifically expressed in the brain, has been shown to regulate AMPA receptor-mediated neurotransmission through a presynaptic mechanism. In cultured rat hippocampal neurons, the overexpression of SAD-B significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Here, we showed that SAD-B downregulation exerted antiepileptic activity by regulating AMPA receptors in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We first used immunoblotting and immunohistochemistry analysis to demonstrate that SAD-B expression was increased in the epileptic rat brain. Subsequently, to explore the function of SAD-B in epilepsy, we used siRNA to knock down SAD-B protein and observed behavior after PTZ-induced seizures. We found that SAD-B downregulation attenuated seizure severity and susceptibility in the PTZ-induced epileptic model. Furthermore, we showed that the antiepileptic effect of SAD-B downregulation on PTZ-induced seizure was abolished by CNQX (an AMPA receptor inhibitor), suggesting that SAD-B modulated epileptic seizure by regulating AMPA receptors in the brain. Taken together, these findings suggest that SAD-B may be a potential and novel therapeutic target to limit epileptic seizures.
Assuntos
Humanos , Animais , Masculino , Feminino , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Adulto Jovem , Medicamentos de Ervas Chinesas/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de AMPA/metabolismo , Agonistas de Aminoácidos Excitatórios/metabolismo , Epilepsia do Lobo Temporal/tratamento farmacológico , Pentilenotetrazol , Ratos Sprague-Dawley , Epilepsia do Lobo Temporal/induzido quimicamenteRESUMO
Amyloid precursor protein (APP) shedding yields the Alzheimer's disease (AD)-related peptide amyloid ß (Aß) through ß- and γ-secretase cleavage. Alternatively, α-secretase cleavage generates a soluble and neuroprotective fragment (sAPPα) while precludes the production of Aß. Although metabotropic glutamate (mGlu) receptors were associated with induction of sAPPα production in astrocytes, there was no further evidence regarding the specific subtype receptor or the mechanisms involved in this action. In the present study, we used the dual mGlu2/3 receptor agonist LY379268, which in pure astrocyte cultures selectively activates mGlu3 receptor subtype since mGlu2 receptor subtype is not expressed by these cells. We showed that LY379268 incremented sAPPα release from cultured astrocytes by inducing α-secretases expression, whereas it decreased ß-secretase levels. LY379268-induced increase of PPAR-γ levels could be involved in the effect of the agonist on sAPPα release. Using the PDAPP-J20 murine model of AD we described a strong reduction in mGlu2/3 receptor expression in the hippocampus of 5- and 14-month-old transgenic mice compared to control littermates. Moreover, mGlu3 receptor expression is also decreased specifically in hippocampal astrocytes of these transgenic animals as a function of age. Therefore, diminished levels of hippocampal mGlu3 receptors might have implications in the development of the disease in these transgenic mice considering the anti-amyloidogenic action of mGlu3 receptors in astrocytes.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Astrócitos/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Envelhecimento , Aminoácidos/farmacologia , Precursor de Proteína beta-Amiloide/genética , Animais , Astrócitos/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células Cultivadas , Agonistas de Aminoácidos Excitatórios/metabolismo , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/agonistasRESUMO
In this study, we investigated the role of GABAergic and glutamatergic systems in the anticonvulsant action of 3-alkynyl selenophene (3-ASP) in a pilocarpine (PC) model of seizures. To this purpose, 21 day-old rats were administered with an anticonvulsant dose of 3-ASP (50 mg/kg, per oral, p.o.), and [(3)H]γ-aminobutyric acid (GABA) and [(3)H]glutamate uptakes were carried out in slices of cerebral cortex and hippocampus. [(3)H]GABA uptake was decreased in cerebral cortex (64%) and hippocampus (58%) slices of 21 day-old rats treated with 3-ASP. In contrast, no alteration was observed in [(3)H]glutamate uptake in cerebral cortex and hippocampus slices of 21 day-old rats that received 3-ASP. Considering the drugs that increase synaptic GABA levels, by inhibiting its uptake or catabolism, are effective anticonvulsants, we further investigated the possible interaction between sub-effective doses of 3-ASP and GABA uptake or GABA transaminase (GABA-T) inhibitors in PC-induced seizures in 21 day-old rats. For this end, sub-effective doses of 3-ASP (10 mg/kg, p.o.) and DL-2,4-diamino-n-butyric acid hydrochloride (DABA, an inhibitor of GABA uptake--2 mg/kg, intraperitoneally; i.p.) or aminooxyacetic acid hemihydrochloride (AOAA; a GABA-T inhibitor--10 mg/kg, i.p.) were co-administrated to 21 day-old rats before PC (400 mg/kg; i.p.) treatment, and the appearance of seizures was recorded. Results demonstrated that treatment with AOAA and 3-ASP or DABA and 3-ASP significantly abolished the number of convulsing animals induced by PC. The present study indicates that 3-ASP reduced [(3)H]GABA uptake, suggesting that its anticonvulsant action is related to an increase in inhibitory tonus.
Assuntos
Anticonvulsivantes/farmacologia , Neurônios GABAérgicos/metabolismo , Compostos Organosselênicos/farmacologia , Convulsões/tratamento farmacológico , Animais , Anticonvulsivantes/uso terapêutico , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Agonistas de Aminoácidos Excitatórios/metabolismo , Neurônios GABAérgicos/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Técnicas In Vitro , Masculino , Compostos Organosselênicos/uso terapêutico , Pilocarpina , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Ácido gama-Aminobutírico/metabolismoRESUMO
Medium spiny projection neurons (MSNs) are the main neuronal population in the neostriatum. MSNs are inhibitory and GABAergic. MSNs connect with other MSNs via local axon collaterals that produce lateral inhibition, which is thought to select cell assemblies for motor action. MSNs also receive inhibitory inputs from GABAergic local interneurons. This work shows, through the use of the paired pulse protocol, that somatostatin (SST) acts presynaptically to regulate GABA release from the terminals interconnecting MSNs. This SST action is reversible and not mediated through the release of dopamine. It is blocked by the SST receptor (SSTR) antagonist ciclosomatostatin (cicloSST). In contrast, SST does not regulate inhibition coming from interneurons. Because, SST is released by a class of local interneuron, it is concluded that this neuron helps to regulate the selection of motor acts.
Assuntos
Corpo Estriado/metabolismo , Potenciais Pós-Sinápticos Inibidores/fisiologia , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Somatostatina/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/metabolismo , Potenciais de Ação/fisiologia , Animais , Corpo Estriado/citologia , Agonistas de Aminoácidos Excitatórios/metabolismo , Antagonistas de Aminoácidos Excitatórios/metabolismo , N-Metilaspartato/metabolismo , Neurônios/citologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Valina/análogos & derivados , Valina/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismoRESUMO
In this short review we provide evidence that the branched-chain keto acids accumulating in the neurometabolic disorder maple syrup urine disease disturb rat cerebral cytoskeleton in a developmentally regulated manner. Alterations of protein phosphorylation leading to brain cytoskeletal misregulation and neural cell death caused by these metabolites are associated with energy deprivation, oxidative stress and excitotoxicity that may ultimately disrupt normal cell function and viability.
Assuntos
Encéfalo/metabolismo , Citoesqueleto/metabolismo , Doença da Urina de Xarope de Bordo/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Sobrevivência Celular , Citoesqueleto/patologia , Metabolismo Energético , Agonistas de Aminoácidos Excitatórios/metabolismo , Doença da Urina de Xarope de Bordo/enzimologia , Doença da Urina de Xarope de Bordo/patologia , Estresse Oxidativo , Fosforilação , Ratos , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The polyamines, spermine, spermidine, and putrescine, are a group of aliphatic amines that may act as physiological modulators of N-methyl-D-aspartate (NMDA) receptors. Although the modulatory role of polyamines in NMDA receptor function has long been known, the effects of polyamines on learning and memory only recently began to be unraveled. In the present study, we investigated the effect of bilateral infusions of spermidine (0.02-2 nmol), a polyamine agonist, into the CA1 region of the rat dorsal hippocampus on inhibitory avoidance learning 30 min pre-training, immediately post-training, 6 h post-training, or 10 min pre-test. Bilateral microinjections of 0.2 nmol spermidine prolonged step-down latencies compared to the respective control group when administered 30 min pre-training or immediately post-training. These results provide evidence that the modulatory effects of spermidine on the acquisition and/or early consolidation of memory of inhibitory avoidance tasks in the hippocampus occur within a limited time window.
Assuntos
Aprendizagem da Esquiva/fisiologia , Agonistas de Aminoácidos Excitatórios/metabolismo , Hipocampo/metabolismo , Memória/fisiologia , Espermidina/metabolismo , Análise de Variância , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/administração & dosagem , Hipocampo/efeitos dos fármacos , Inibição Psicológica , Masculino , Memória/efeitos dos fármacos , Microinjeções , Ratos , Ratos Wistar , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Espermidina/administração & dosagem , Fatores de TempoRESUMO
Radioligand binding assays evaluating directly the ability of a drug to interact with a defined molecular target is part of the drug discovery process. The need for a high throughput rate in screening drugs is actually leading to simplified experimental schemes that increase the probability of false negative results. Special concern involves voltage-gated ion channel drug discovery where a great care is required in designing assays because of frequent multiplicity of (interacting) binding sites. To clearly illustrate this situation, three different assays used in the academic drug discovery program of the authors were selected because they are rich of intrinsic artifacts: (I) (20 mmol/l caffeine almost duplicated [3H]ryanodine binding (89% higher than control) to rat heart microsomes at 0.3 mumol/l free calcium but did not exert any effect when using a high (107 mumol/l) free calcium, as mostly used in ryanodine binding assays; (II) An agonist for the ionotropic glutamate receptor of the kainate type can distinctly affect [3H]kainate binding to chicken cerebellum membranes depending on its concentration: unlabelled kainic acid per se either stimulated about 30% (at 50-100 nmol/l), had no effect (at 200 nmol/l) or even progressively decreased (at 0.3-2 mumol/l) the binding of 5 nmol/l [3H]kainate, emphasizing the risk of using a single concentration for screening a drug; (III) in a classical [3H]flunitrazepam binding assay, the stimulatory effect of a GABA (gamma-aminobutyric acid) agonist was only observed when using extensively washed rat brain synaptosomes (10 mumol/l GABA increased flunitrazepam binding by 90%). On the other hand, the inhibitory effect of a GABA antagonist was only observed when using crude synaptosomes (10 mumol/l bicuculine reduced [3H]flunitrazepam binding by 40%). It can be concluded that carefully designed radioligand assays which can be performed in an academic laboratory are appropriate for screening a small number of drugs, especially if these are potential hits because of their rational design. Therefore, the low throughput rate could be partially balanced by a higher performance when compared to what is done in a robotic high throughput screening where simplification of assay conditions can lead to false negative results.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Farmacologia/métodos , Ensaio Radioligante , Animais , Ligação Competitiva/efeitos dos fármacos , Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Agonistas de Aminoácidos Excitatórios/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Flunitrazepam/metabolismo , Técnicas In Vitro , Ácido Caínico/metabolismo , Ácido Caínico/farmacologia , Membranas/metabolismo , Microssomos/metabolismo , Miocárdio/metabolismo , Ratos , Ratos Wistar , Rianodina/metabolismo , Espermina/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 microM carbachol was used, the estimated IC50 value for kainate was 0.2 microM and the maximal inhibition of approximately 50% was obtained with 1 microM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 microM veratridine, but not 50 mM KCl, inhibited approximately 65% of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 +/- 38.0 to 2044.5 +/- 299.9 cpm/mg protein, retinal response decreased to 861.6 +/- 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.
Assuntos
Carbacol/antagonistas & inibidores , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Agonistas Muscarínicos/farmacologia , Fosfatidilinositóis/metabolismo , Receptores Muscarínicos/metabolismo , Retina/embriologia , Retina/metabolismo , Veratridina/farmacologia , Animais , Embrião de Galinha , Agonistas de Aminoácidos Excitatórios/metabolismo , Ácido Glutâmico/farmacologia , Ácido Caínico/metabolismo , Cloreto de Potássio , Receptores Muscarínicos/efeitos dos fármacos , Retina/efeitos dos fármacos , Canais de SódioRESUMO
In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 muM carbachol was used, the estimated IC50 value for kainate was 0.2 muM and the maximal inhibition of ~50 percent was obtained with 1 muM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 muM veratridine, but not 50 mM KCl, inhibited ~65 percent of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 + 38.0 to 2044.5 + 299.9 cpm/mg protein, retinal response decreased to 861.6 + 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.