RESUMO
Alcohol's deleterious effects on memory are well known. Acute alcohol-induced memory loss is thought to occur via inhibition of NMDA receptor (NMDAR)-dependent long-term potentiation in the hippocampus. We reported previously that ethanol inhibition of NMDAR function and long-term potentiation is correlated with a reduction in the phosphorylation of Tyr(1472) on the NR2B subunit and ethanol's inhibition of the NMDAR field excitatory postsynaptic potential was attenuated by a broad spectrum tyrosine phosphatase inhibitor. These data suggested that ethanol's inhibitory effect may involve protein tyrosine phosphatases. Here we demonstrate that the loss of striatal-enriched protein tyrosine phosphatase (STEP) renders NMDAR function, phosphorylation, and long-term potentiation, as well as fear conditioning, less sensitive to ethanol inhibition. Moreover, the ethanol inhibition was "rescued" when the active STEP protein was reintroduced into the cells. Taken together, our data suggest that STEP contributes to ethanol inhibition of NMDAR function via dephosphorylation of tyrosine sites on NR2B receptors and lend support to the hypothesis that STEP may be required for ethanol's amnesic effects.
Assuntos
Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Medo/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciais Sinápticos/efeitos dos fármacos , Amnésia/induzido quimicamente , Amnésia/enzimologia , Amnésia/genética , Animais , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Humanos , Potenciação de Longa Duração/genética , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Receptores de N-Metil-D-Aspartato/genética , Potenciais Sinápticos/genéticaRESUMO
Outside of Fragile X syndrome (FXS), the role of Fragile-X Mental Retardation Protein (FMRP) in mediating neuropsychological abnormalities is not clear. FMRP, p70-S6 kinase (S6K) and protein phosphatase 2A (PP2A) are thought to cooperate as a dynamic signaling complex. In our prior work, adult rats have enhanced CA1 hippocampal long-term depression (LTD) following an early life seizure (ELS). We now show that mGluR-mediated LTD (mLTD) is specifically enhanced following ELS, similar to FMRP knock-outs. Total FMRP expression is unchanged but S6K is hyperphosphorylated, consistent with S6K overactivation. We postulated that either disruption of the FMRP-S6K-PP2A complex and/or removal of this complex from synapses could explain our findings. Using subcellular fractionation, we were surprised to find that concentrations of FMRP and PP2A were undisturbed in the synaptosomal compartment but reduced in parallel in the cytosolic compartment. Following ELS FMRP phosphorylation was reduced in the cytosolic compartment and increased in the synaptic compartment, in parallel with the compartmentalization of S6K activation. Furthermore, FMRP and PP2A remain bound following ELS. In contrast, the interaction of S6K with FMRP is reduced by ELS. Blockade of PP2A results in enhanced mLTD; this is occluded by ELS. This suggests a critical role for the location and function of the FMRP-S6K-PP2A signaling complex in limiting the amount of mLTD. Specifically, non-synaptic targeting and the function of the complex may influence the "set-point" for regulating mLTD. Consistent with this, striatal-enriched protein tyrosine phosphatase (STEP), an FMRP "target" which regulates mLTD expression, is specifically increased in the synaptosomal compartment following ELS. Further, we provide behavioral data to suggest that FMRP complex dysfunction may underlie altered socialization, a symptom associated and observed in other rodent models of autism, including FXS.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Feminino , Hipocampo/patologia , Hipocampo/fisiopatologia , Técnicas In Vitro , Ácido Caínico/toxicidade , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Fosforilação , Gravidez , Proteína Fosfatase 2/metabolismo , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Convulsões/induzido quimicamente , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Frações Subcelulares/patologia , Tiazóis/farmacologiaRESUMO
The choice of an antibody for a protein-based research study is one of the most crucial steps in any project. Seemingly straightforward, the process is actually quite nuanced and filled with potential pitfalls. In this chapter, we will discuss five major topics that require consideration in the antibody selection process. These include overall study objectives and resources, details of both species and clonality, suitability in applications, and available detection methods. Each section will provide background information on the topic as well as specifics of antibody use in the laboratory. This chapter may be used as a guide to help vet antibody candidates for your project so you can stain with confidence.
Assuntos
AnticorposRESUMO
The hippocampal N-methyl-D-aspartate receptor (NMDAR) activity plays important roles in cognition and is a major substrate for ethanol-induced memory dysfunction. This receptor is a glutamate-gated ion channel, which is composed of NR1 and NR2 subunits in various brain areas. Although homomeric NR1 subunits form an active ion channel that conducts Na⺠and Ca²âº currents, the incorporation of NR2 subunits allows this channel to be modulated by the Src family of kinases, phosphatases, and by simple molecules such as ethanol. We have found that short-term ethanol application inhibits the NMDAR activity via striatal enriched protein tyrosine phosphatase (STEP)-regulated mechanisms. The genetic deletion of the active form of STEP, STEP61, leads to marked attenuation of ethanol inhibition of NMDAR currents. In addition, STEP61 negatively regulates Fyn and p38 mitogen-activated protein kinase (MAPK), and these proteins are members of the NMDAR super molecular complex. Here we demonstrate, using whole-cell electrophysiological recording, Western blot analysis, and pharmacological manipulations, that neurons exposed to a 3-h, 45 mM ethanol treatment develop an adaptive attenuation of short-term ethanol inhibition of NMDAR currents in brain slices. Our results suggest that this adaptation of NMDAR responses is associated with a partial inactivation of STEP61, an activation of p38 MAPK, and a requirement for NR2B activity. Together, these data indicate that altered STEP61 and p38 MAPK signaling contribute to the modulation of ethanol inhibition of NMDARs in brain neurons.
Assuntos
Adaptação Fisiológica , Corpo Estriado/efeitos dos fármacos , Etanol/farmacologia , Proteínas Tirosina Fosfatases/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Corpo Estriado/enzimologia , Corpo Estriado/metabolismo , Ativação Enzimática , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
Alcohol abuse leads to tolerance, dependence, and memory impairments that involve excitatory glutamatergic NMDA synaptic transmission. The NMDA receptor (NMDAR) is known to undergo activity-dependent adaptive functional changes. Since we observed that acute ethanol inhibition of the NMDAR was regulated by protein tyrosine phosphorylation, we investigated the role of protein tyrosine kinases and phosphatases on the NMDAR functions by chronic ethanol treatment. We carried out whole-cell recording, western blotting, and behavioral righting reflex measurements to assess the impact of chronic ethanol treatment on NMDAR function. Our results indicated that these receptors became resistant to the acute ethanol inhibition following chronic ethanol consumption. This resistance occurred without an increase in the NMDAR subunit expression but was associated with decreases in the levels of phospho-Y-1472 NR2B, increases in the levels of STEP33, increases in phospho-p38 mitogen-activated protein kinase (pp38 MAPK), and acquisition of tolerance to the sedative effects of ethanol. These data suggested that altered protein tyrosine phosphorylation of the NMDAR subunits significantly contributes to functional changes of this receptor by chronic ethanol ingestion. Therefore, preservation of the integrity of tyrosine phosphorylation mechanisms of the NMDAR may be important in controlling the progression of alcohol tolerance and dependence.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Benzilaminas/farmacologia , Bicuculina/análogos & derivados , Bicuculina/farmacologia , Depressores do Sistema Nervoso Central/administração & dosagem , Etanol/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Comportamento Alimentar/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/farmacologia , Hipocampo/citologia , Técnicas In Vitro , Masculino , Técnicas de Patch-Clamp/métodos , Ácidos Fosfínicos/farmacologia , Fosforilação/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Estatística como Assunto , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Calcium/calmodulin-dependent protein kinase II (CaMKII) has important roles in many processes in the central nervous system. It is enriched at the post-synaptic density (PSD), a localization which is thought to be critical for many of its proposed neuronal functions. In order to better understand the mechanisms that regulate association of CaMKII with the PSD, we compared the levels of autophosphorylation between PSD-associated kinase and kinase in other parts of the neuron. We were surprised to find that alphaCaMKII in a PSD-enriched fraction prepared from recovered hippocampal CA1-minislices had a relatively low level of threonine 286 (T286) phosphorylation and a relatively high level of threonine 305 (T305) phosphorylation. Furthermore, when the minislices were subjected to a treatment that mimics ischemic conditions, there was a significant translocation of alphaCaMKII to the PSD-enriched fraction accompanied with a dramatic reduction in T286 phosphorylation levels throughout the neuron. These findings have important implications for our understanding of the role of autophosphorylation in the localization of CaMKII.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Neurônios/ultraestrutura , Frações Subcelulares/enzimologia , Análise de Variância , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Técnicas In Vitro , Isquemia/metabolismo , Isquemia/fisiopatologia , Masculino , Neurônios/enzimologia , Fosforilação , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Treonina/metabolismoRESUMO
Aged rats are known to have deficits in spatial learning behavior in the Morris water maze. We have found that aged rats also have deficits in NR2B protein expression and that the protein expression deficit is correlated with their performance in the Morris water maze. To test whether this NR2B deficit was sufficient to account for the behavioral deficit, we used antisense oligonucleotides to specifically knock down NR2B subunit expression in the hippocampus of young rats. NR2B antisense treatment diminished NMDA receptor responses, abolished NMDA-dependent long-term potentiation (LTP), and impaired spatial learning. These data demonstrate the important role of NR2B in LTP and learning and memory and suggest a role for reduced NR2B expression in age-related cognitive decline.
Assuntos
Envelhecimento/metabolismo , Hipocampo/metabolismo , Ácido Cinurênico/análogos & derivados , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Receptores de N-Metil-D-Aspartato/deficiência , Potenciais de Ação/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Western Blotting , Bloqueadores dos Canais de Cálcio/farmacologia , Cateterismo , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Ácido Cinurênico/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Microinjeções , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacologia , Ratos , Ratos Endogâmicos F344 , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Espermina/farmacologiaRESUMO
N-Methyl-d-aspartate (NMDA)-type glutamate receptors in the hippocampus are important mediators of both memory formation and excitotoxicity. It is thought that glutamatergic neurons of the CA1, CA3 and dentate gyrus regions of the hippocampus contribute differentially to memory formation and are differentially sensitive to excitotoxicity. The subunit and/or splice variant composition of the NMDA receptor controls many aspects of receptor function such as ligand affinity, calcium permeability and channel kinetics, as well as interactions with intracellular anchoring and regulatory proteins. Thus, one possible explanation of the differences in NMDA receptor-dependent processes, such as synaptic plasticity and excitotoxicity, among the hippocampal sub-regions is that they differ in subunit and/or splice variant expression. Here we report that the NMDA receptor subunits NR1 and NR2B, along with the four splice variant cassettes of the NR1 subunit are differentially expressed in the CA1, CA3 and dentate gyrus of the hippocampus. Expression of the AMPA receptor subunits GluR1 and GluR2 also differ. These differences may contribute to functional differences, such as with excitotoxicity and synaptic plasticity, that exist between the sub-regions of the hippocampus.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Actinina/metabolismo , Animais , Western Blotting/métodos , Linhagem Celular , Hipocampo/anatomia & histologia , Humanos , Masculino , Proteínas do Tecido Nervoso/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Sinapsinas/metabolismo , Transfecção/métodosRESUMO
Hippocampal N-methyl-D-aspartate receptors (NMDARs) contribute to the expression of certain types of synaptic plasticity, such as long-term potentiation (LTP). It is well documented that tyrosine kinases increase NMDAR phosphorylation and potentiate NMDAR function. However, it is unclear how these phosphorylation changes result in enhanced NMDAR activity. We previously reported that NMDAR surface expression can be increased by LTP-inducing stimulation via tyrosine kinase-dependent mechanisms in the adult hippocampus [D.R. Grosshans, D.A. Clayton, S.J. Coultrap, M.D. Browning, Nat. Neurosci., 5 (2002) 27-33]. We therefore hypothesized that tyrosine phosphorylation of the NMDAR may enhance the trafficking of the receptor to the synaptic membrane. Here, we show that the stoichiometry of NR2A and NR2B tyrosine phosphorylation is significantly higher in synaptosomal membranes than intracellular microsomal/light membranes. Interestingly, NR2B tyrosine-1472, but not NR1 serine-896 or -897, phosphorylation is significantly higher in synaptosomal membranes than intracellular microsomal/light membranes. Furthermore, treatment of hippocampal slices with either a tyrosine phosphatase inhibitor or a tyrosine kinase inhibitor alters NMDAR tyrosine phosphorylation and produces a corresponding change in the concentration of NMDARs in the synaptosomal membrane fraction. Taken together, these data support the hypothesis that tyrosine phosphorylation may enhance NMDAR activity by increasing the number of NMDARs at the synaptic membrane.
Assuntos
Hipocampo/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Membranas Sinápticas/fisiologia , Tirosina/metabolismo , Animais , Western Blotting/métodos , Calnexina/metabolismo , Proteína 4 Homóloga a Disks-Large , Inibidores Enzimáticos/farmacologia , Imunoprecipitação/métodos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Compostos Organometálicos/farmacologia , Fenantrolinas/farmacologia , Fenóis/farmacologia , Fosforilação , Transporte Proteico/fisiologia , Proteínas Tirosina Fosfatases/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/química , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Inflammatory processes in the central nervous system are thought to contribute to Alzheimer's disease (AD). Chronic administration of nonsteroidal anti-inflammatory drugs (NSAIDs) decreases the incidence of Alzheimer's disease. There are very few studies, however, on the cognitive impact of chronic NSAID administration. The N-methyl-d-aspartate (NMDA) receptor is implicated in learning and memory, and age-related decreases in the NMDA NR2B subunit correlate with memory deficits. Sulindac, an NSAID that is a nonselective cyclooxygenase (COX) inhibitor was chronically administered to aged Fischer 344 rats for 2 months. Sulindac, but not its non-COX active metabolite, attenuated age-related deficits in learning and memory as assessed in the radial arm water maze and contextual fear conditioning tasks. Sulindac treatment also attenuated an age-related decrease in the NR1 and NR2B NMDA receptor subunits and prevented an age-related increase in the pro-inflammatory cytokine, interleukin 1beta (IL-1beta), in the hippocampus. These findings support the inflammation hypothesis of aging and have important implications for potential cognitive enhancing effects of NSAIDs in the elderly.
Assuntos
Envelhecimento/efeitos dos fármacos , Aldeído Desidrogenase/genética , Encefalite/tratamento farmacológico , Transtornos da Memória/tratamento farmacológico , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Sulindaco/farmacologia , Envelhecimento/metabolismo , Aldeído-Desidrogenase Mitocondrial , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Encefalite/fisiopatologia , Encefalite/prevenção & controle , Interleucina-1/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Nootrópicos/farmacologia , Nootrópicos/uso terapêutico , Ratos , Ratos Endogâmicos F344 , Receptores de N-Metil-D-Aspartato/metabolismo , Sulindaco/uso terapêutico , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Calcium currents through the L-type voltage-sensitive calcium channel (L-VSCC) are increased in neurons of area CA1 of the hippocampus in aged rats and rabbits. Furthermore, increases in mRNA for the pore forming subunit alpha(1D) (Ca(v)1.3) have been observed in the hippocampus of aged rats. We have studied the protein expression of the two pore forming subunits, alpha(1C) (Ca(v)1.2) and alpha(1D), of L-VSCCs in the hippocampus of young and aged rats. Here we report selective age-related changes in expression of alpha(1D) in the hippocampus. Specifically, we find that alpha(1D) protein is increased in area CA1 of aged rats while it is decreased in area CA3. Our data suggest that the altered calcium currents seen in aged animals may be due, at least in part, to alterations in the expression of the alpha(1D) subunit of the L-type calcium channel. These findings contribute to our understanding of the mechanisms responsible for changes in calcium homeostasis during aging.
Assuntos
Envelhecimento/metabolismo , Canais de Cálcio Tipo L/metabolismo , Hipocampo/metabolismo , Células Piramidais/metabolismo , Animais , Canais de Cálcio , Dendritos/metabolismo , Dendritos/ultraestrutura , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Imuno-Histoquímica , Plasticidade Neuronal/fisiologia , Células Piramidais/citologia , Ratos , Ratos Endogâmicos F344 , Transmissão Sináptica/fisiologia , Regulação para Cima/fisiologiaRESUMO
The hippocampus is critical for spatial memory formation in rodents. Calcium currents through L-type voltage-sensitive calcium channels (L-VSCCs) are increased in CA1 neurons of the hippocampus of aged rats. We have recently shown that expression of the calcium conducting L-VSCC subunit alpha(1D) (Ca(v)1.3) is selectively increased in area CA1 of aged rats. We and others have speculated that excessive Ca(2+) influx through L-VSCC may be detrimental to memory formation. Therefore, we investigated the relationship between age-related working memory decline and alpha(1D) protein expression in the hippocampus. In addition, we studied the effects of chronic treatment with the L-VSCC antagonist nimodipine (NIM) on age-related working memory deficits and alpha(1D) expression in the hippocampus. Here we report that age-related increases in alpha(1D) expression in area CA1 correlate with working memory impairment in Fischer 344 rats. Furthermore, we demonstrate that chronic NIM treatment ameliorates age-related working memory deficits and reduces expression of alpha(1D) protein in the hippocampus. The present results suggest that L-VSCCs participate in processes underlying memory formation and that increases in L-VSCC protein and currents observed with aging may play a role in age-related memory decline. Furthermore, the amelioration in age-related memory decline produced by NIM treatment may be mediated, at least in part, by reductions in the abnormally high levels of alpha(1D) protein in the aged hippocampus. These findings may have implications for patients with Alzheimer's disease, who show increased L-VSCC protein expression in the hippocampus, and for patients receiving chronic treatment with L-VSCC antagonists.
Assuntos
Envelhecimento/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Hipocampo/metabolismo , Transtornos da Memória/metabolismo , Nimodipina/farmacologia , Animais , Canais de Cálcio , Canais de Cálcio Tipo L/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Endogâmicos F344 , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Experimental, clinical, and epidemiologic studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) are beneficial in Alzheimer's disease and other neuroinflammatory processes. One possible mechanism is an interaction with peroxisome proliferator-activated receptors (PPARs). We examined the effect of a specific PPARgamma agonist, rosiglitazone, on contextual fear conditioning in aged rats. Male rats (20-months-old) were administered rosiglitazone in the diet for 2 months prior to behavioral testing. Young control and aged rats fed rosiglitazone froze significantly more than did the aged control rats in a hippocampal-dependent fear conditioning task. Rosiglitazone had no effect hippocampal interleukin-1beta levels, markers of oxidative damage, and NMDA receptor expression. Therefore, activation of PPARgamma prevented age-related deficits in hippocampal function.
Assuntos
Envelhecimento/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Medo/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Envelhecimento/fisiologia , Envelhecimento/psicologia , Animais , Condicionamento Psicológico/fisiologia , Medo/fisiologia , Medo/psicologia , Masculino , Ratos , Ratos Endogâmicos F344 , RosiglitazonaRESUMO
Phosphorylation of cardiac troponin I is a well established mechanism by which cardiac contractility is modulated. However, there are a number of phosphorylation sites on TnI which contribute singly or in combination to influence cardiac function. Accordingly, methods for accurately measuring site-specific TnI phosphorylation are needed. Currently, two strategies are employed: mass spectrometry, which is costly, difficult and has a low throughput; and Western blotting using phospho-specific antibodies, which is limited by the availability of reagents. In this report, we describe a cohort of new site-specific TnI phosphoantibodies, generated against physiologically relevant phosphorylation sites, that are superior to the current commercially available antibodies: to phospho-serine 22/23 which shows a >5-fold phospho-specificity for phosphorylated TnI; to phospho-serine 43, which has >3-fold phospho-specificity for phosphorylated TnI; and phospho-serine 150 which has >2-fold phospho-specificity for phosphorylated TnI. These new antibodies demonstrated greater sensitivity and specificity for the phosphorylated TnI than the most widely used commercially available reagents. For example, at a protein load of 20 µg of total cardiac extract, a commercially available antibody recognized both phosphorylated and dephosphorylated TnI to the same degree. At the same protein load our phospho-serine 22/23 antibody exhibited no cross-reactivity with dephosphorylated TnI. These new tools should allow a more accurate assessment and a better understanding of the role of TnI phosphorylation in the response of the heart to pathologic stress.
Assuntos
Anticorpos , Western Blotting/métodos , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional , Troponina I/metabolismo , Animais , Especificidade de Anticorpos , Biomarcadores/metabolismo , Bovinos , Modelos Animais de Doenças , Humanos , Camundongos , Infarto do Miocárdio/imunologia , Miocárdio/imunologia , Fosforilação , Valor Preditivo dos Testes , Ratos , Reprodutibilidade dos Testes , Suínos , Troponina I/imunologiaRESUMO
Every year, nearly 200,000 patients undergo radiation for brain tumors. For both patients and caregivers the most distressing adverse effect is impaired cognition. Efforts to protect against this debilitating effect have suffered from inadequate understanding of the cellular mechanisms of radiation damage. In the past it was accepted that radiation-induced normal tissue injury resulted from a progressive reduction in the survival of clonogenic cells. Moreover, because radiation-induced brain dysfunction is believed to evolve over months to years, most studies have focused on late changes in brain parenchyma. However, clinically, acute changes in cognition are also observed. Because neurons are fully differentiated post-mitotic cells, little information exists on the acute effects of radiation on synaptic function. The purpose of our study was to assess the potential acute effects of radiation on neuronal function utilizing ex vivo hippocampal brain slices. The cellular localization and functional status of excitatory and inhibitory neurotransmitter receptors was identified by immunoblotting. Electrophysiological recordings were obtained both for populations of neuronal cells and individual neurons. In the dentate gyrus region of isolated ex vivo slices, radiation led to early decreases in tyrosine phosphorylation and removal of excitatory N-methyl-D-aspartate receptors (NMDARs) from the cell surface while simultaneously increasing the surface expression of inhibitory gamma-aminobutyric acid receptors (GABA(A)Rs). These alterations in cellular localization corresponded with altered synaptic responses and inhibition of long-term potentiation. The non-competitive NMDAR antagonist memantine blocked these radiation-induced alterations in cellular distribution. These findings demonstrate acute effects of radiation on neuronal cells within isolated brain slices and open new avenues for study.
Assuntos
Neurônios/metabolismo , Neurônios/efeitos da radiação , Animais , Transporte Biológico/efeitos dos fármacos , Caspase 3/metabolismo , Ativação Enzimática/efeitos da radiação , Potenciação de Longa Duração/efeitos da radiação , Memantina/farmacologia , N-Metilaspartato/metabolismo , Fosforilação/efeitos da radiação , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/efeitos da radiaçãoRESUMO
Phosphoproteins are considered to be among the most important proteins in the body. They are the proteins that regulate almost all cell processes from cell division in cancer to neuronal signal transduction in learning and memory. This review will describe the development of a revolutionary immunochemical technique that produces antibodies that bind to target proteins only when the protein is in the phosphorylated state. These phospho-specific antibodies can thus be used to track the activity of a protein, not simply its level of expression. In this review, we will discuss both the design of the phosphopeptide immunogen and immunization. The affinity purification of the phospho-specific antibody as well as the methods most suitable for characterizing the phosphospecificity of the antibody will be described here. Taken together, these methods will cover the key procedures and protocols required to produce a phospho-specific antibody that works.
Assuntos
Anticorpos Fosfo-Específicos/imunologia , Anticorpos Fosfo-Específicos/isolamento & purificação , Western Blotting/métodos , Cromatografia de Afinidade/métodos , Imuno-Histoquímica/métodos , Fosfoproteínas/imunologia , Animais , HumanosRESUMO
NMDA receptor-dependent long-term potentiation (LTP) in the hippocampus is widely accepted as a cellular substrate for memory formation. Age-related declines in the expression of both NMDAR-dependent LTP and NMDAR subunit proteins in the CA1 region of the hippocampus have been well characterized and likely underlie age-related memory impairment. In the current study, we examined NMDAR-dependent LTP in young Fischer 344 rats (4 months old) and aged rats (24 months old) given either a control diet or a diet supplemented with blueberry extract for 6-8 weeks. NMDAR-dependent LTP was evoked by high-frequency stimulation (HFS) in the presence of nifedipine, to eliminate voltage-gated calcium channel LTP. Field excitatory postsynaptic potentials (fEPSPs) were increased by 57% 1 h after HFS in young animals, but this potentiation was reduced to 31% in aged animals. Supplementation of the diet with blueberry extract elevated LTP (63%) in aged animals to levels seen in young. The normalization of LTP may be due to the blueberry diet preventing a decline in synaptic strength, as measured by the slope of the fEPSP for a given fiber potential. The blueberry diet did not prevent age-related declines in NMDAR protein expression. However, phosphorylation of a key tyrosine residue on the NR2B subunit, important for increasing NMDAR function, was enhanced by the diet, suggesting that an increase in NMDAR function might overcome the loss in protein. This report provides evidence that dietary alterations later in life may prevent or postpone the cognitive declines associated with aging.
RESUMO
Long lasting changes in the strength of synaptic transmission in the hippocampus are thought to underlie certain forms of learning and memory. Accordingly, the molecular mechanisms that account for these changes are heavily studied. Postsynaptically, changes in synaptic strength can occur by altering the amount of neurotransmitter receptors at the synapse or by altering the functional properties of synaptic receptors. In this study, we examined the biochemical changes produced following chemically induced long term depression in acute hippocampal CA1 minislices. Using three independent methods, we found that this treatment did not lead to an internalization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Furthermore, when the plasma membrane was separated into synaptic membrane-enriched and extrasynaptic membrane-enriched fractions, we actually observed a significant increase in the concentration of AMPA receptors at the synapse. However, phosphorylation of Ser-845 on the AMPA receptor subunit GluR1 was significantly decreased throughout the neuron, including in the synaptic membrane-enriched fraction. In addition, phosphorylation of Ser-831 on GluR1 was decreased specifically in the synaptic membrane-enriched fraction. Phosphorylation of these residues has been demonstrated to control AMPA receptor function. From these data, we conclude that the decrease in synaptic strength is likely the result of a change in the functional properties of AMPA receptors at the synapse and not a decrease in the amount of synaptic receptors.
Assuntos
Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Membranas Sinápticas/metabolismo , Animais , Hipocampo/citologia , Masculino , Microdissecção , Neurônios/citologia , Fosforilação/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: The contribution of seizures to cognitive changes remains controversial. We tested the hypothesis that a single episode of neonatal seizures (sNS) on rat postnatal day (P) 7 permanently impairs hippocampal-dependent function in mature (P60) rats because of long-lasting changes at the synaptic level. METHODS: sNS was induced with subcutaneously injected kainate on P7. Learning, memory, mossy fiber sprouting, spine density, hippocampal synaptic plasticity, and glutamate receptor expression and subcellular distribution were measured at P60. RESULTS: sNS selectively impaired working memory in a hippocampal-dependent radial arm water-maze task without inducing mossy fiber sprouting or altering spine density. sNS impaired CA1 hippocampal long-term potentiation and enhanced long-term depression. Subcellular fractionation and cross-linking, used to determine whether glutamate receptor trafficking underlies the alterations of memory and synaptic plasticity, demonstrated that sNS induced a selective reduction in the membrane pool of glutamate receptor 1 subunits. sNS induced a decrease in the total amount of N-methyl-D-aspartate receptor 2A and an increase in the primary subsynaptic scaffold, PSD-95. INTERPRETATION: These molecular consequences are consistent with the alterations in plasticity and memory caused by sNS at the synaptic level. Our data demonstrate the cognitive impact of sNS and associate memory deficits with specific alterations in glutamatergic synaptic function.