RESUMO
Intrinsic antioxidant defenses are important for neuronal longevity. We found that in rat neurons, synaptic activity, acting via NMDA receptor (NMDAR) signaling, boosted antioxidant defenses by making changes to the thioredoxin-peroxiredoxin (Prx) system. Synaptic activity enhanced thioredoxin activity, facilitated the reduction of overoxidized Prxs and promoted resistance to oxidative stress. Resistance was mediated by coordinated transcriptional changes; synaptic NMDAR activity inactivated a previously unknown Forkhead box O target gene, the thioredoxin inhibitor Txnip. Conversely, NMDAR blockade upregulated Txnip in vivo and in vitro, where it bound thioredoxin and promoted vulnerability to oxidative damage. Synaptic activity also upregulated the Prx reactivating genes Sesn2 (sestrin 2) and Srxn1 (sulfiredoxin), via C/EBPbeta and AP-1, respectively. Mimicking these expression changes was sufficient to strengthen antioxidant defenses. Trans-synaptic stimulation of synaptic NMDARs was crucial for boosting antioxidant defenses; chronic bath activation of all (synaptic and extrasynaptic) NMDARs induced no antioxidative effects. Thus, synaptic NMDAR activity may influence the progression of pathological processes associated with oxidative damage.
Assuntos
Antioxidantes/metabolismo , Estresse Oxidativo/fisiologia , Peroxirredoxinas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Tiorredoxinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Regulação da Expressão Gênica/fisiologia , Camundongos , Neurônios/metabolismo , Proteínas Nucleares , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Peroxidases , Proteínas/metabolismo , Ratos , Transdução de Sinais/fisiologia , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Transcrição Gênica/fisiologiaRESUMO
Synaptic activity promotes resistance to diverse apoptotic insults, the mechanism behind which is incompletely understood. We show here that a coordinated downregulation of core components of the intrinsic apoptosis pathway by neuronal activity forms a key part of the underlying mechanism. Activity-dependent protection against apoptotic insults is associated with inhibition of cytochrome c release in most but not all neurons, indicative of anti-apoptotic signaling both upstream and downstream of this step. We find that enhanced firing activity suppresses expression of the proapoptotic BH3-only member gene Puma in a NMDA receptor-dependent, p53-independent manner. Puma expression is sufficient to induce cytochrome c loss and neuronal apoptosis. Puma deficiency protects neurons against apoptosis and also occludes the protective effect of synaptic activity, while blockade of physiological NMDA receptor activity in the developing mouse brain induces neuronal apoptosis that is preceded by upregulation of Puma. However, enhanced activity can also confer resistance to Puma-induced apoptosis, acting downstream of cytochrome c release. This mechanism is mediated by transcriptional suppression of apoptosome components Apaf-1 and procaspase-9, and limiting caspase-9 activity, since overexpression of procaspase-9 accelerates the rate of apoptosis in active neurons back to control levels. Synaptic activity does not exert further significant anti-apoptotic effects downstream of caspase-9 activation, since an inducible form of caspase-9 overrides the protective effect of synaptic activity, despite activity-induced transcriptional suppression of caspase-3. Thus, suppression of apoptotic gene expression may synergize with other activity-dependent events such as enhancement of antioxidant defenses to promote neuronal survival.
Assuntos
Apoptose/fisiologia , Inibição Neural/fisiologia , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia , 4-Aminopiridina/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Bicuculina/farmacologia , Caspase 9/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Citocromos c/metabolismo , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Antagonistas GABAérgicos/farmacologia , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Bloqueadores dos Canais de Potássio , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Sinapses/efeitos dos fármacos , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Fatores de Tempo , Transfecção/métodos , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Peroxiredoxins are an important family of cysteine-based antioxidant enzymes that exert a neuroprotective effect in several models of neurodegeneration. However, under oxidative stress they are vulnerable to inactivation through hyperoxidation of their active site cysteine residues. We show that in cortical neurons, the chemopreventive inducer 3H-1,2-dithiole-3-thione (D3T), that activates the transcription factor Nuclear factor erythroid 2-related factor (Nrf2), inhibits the formation of inactivated, hyperoxidized peroxiredoxins following oxidative trauma, and protects neurons against oxidative stress. In both neurons and glia, Nrf2 expression and treatment with chemopreventive Nrf2 activators, including D3T and sulforaphane, up-regulates sulfiredoxin, an enzyme responsible for reducing hyperoxidized peroxiredoxins. Induction of sulfiredoxin expression is mediated by Nrf2, acting via a cis-acting antioxidant response element (ARE) in its promoter. The ARE element in Srxn1 contains an embedded activator protein-1 (AP-1) site which directs induction of Srxn1 by synaptic activity. Thus, raising Nrf2 activity in neurons prevents peroxiredoxin hyperoxidation and induces a new member of the ARE-gene family, whose enzymatic function of reducing hyperoxidized peroxiredoxins may contribute to the neuroprotective effects of Nrf2 activators.
Assuntos
Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Peroxirredoxinas/metabolismo , Tionas/farmacologia , Tiofenos/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Córtex Cerebral/citologia , Interações Medicamentosas , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Hidroquinonas/farmacologia , Indóis , Camundongos , Mutação/fisiologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/metabolismo , Peroxirredoxinas/genética , RNA Mensageiro/metabolismo , Ratos , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transfecção/métodosRESUMO
In previously proposed models of A beta soluble oligomers, the N-terminal domain A beta(1-16), which is missing in p3 peptides, protects the hydrophobic core of the oligomers from the solvent. Without this N-terminal part, oligomers of p3 peptides would likely expose hydrophobic residues to water and would consequently be less stable. We thus suggest, based on theoretical and experimental results, that p3 peptides would have a low propensity to assemble into stable oligomers, evolving then directly to fibrillar aggregates. These properties may explain why p3 would be devoid of any impact on synaptic function and moreover, strengthen the hypothesis that A beta oligomers are the principal synaptotoxic forms of A beta peptides in Alzheimer disease.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Modelos Moleculares , Peptídeos beta-Amiloides/genética , Humanos , Conformação ProteicaRESUMO
Tissue-type plasminogen activator (tPA) is available for the treatment of thromboembolic stroke in humans. However, adverse effects of tPA have been observed in animal models of ischemic brain injuries. In the present study, we have used a synthetic tPA inhibitor, named 2,7-bis-(4-amidino-benzylidene)-cycloheptan-1-one dihydrochloride (tPA stop), to investigate the role of endogenous tPA in the cerebral parenchyma. In mouse cortical cell cultures, we observed that although tPA stop reduced N-methyl-D-aspartic acid (NMDA)-mediated excitotoxic neuronal death, it failed to modulate alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate-mediated necrosis. In addition, we found that tPA stop could prevent the deleterious effects of both endogenous and exogenous tPA during NMDA exposure. At the functional level, tPA stop was found to prevent tPA-dependent potentiation of NMDA receptor-evoked calcium influx. The relevance of those findings was strengthened by the observation of a massive reduction of NMDA-induced excitotoxic lesion in rats when tPA stop was co-injected. Altogether, these data demonstrate that the blockade of the endogenous proteolytic activity of tPA in the cerebral parenchyma could be a powerful neuroprotective strategy raised against brain pathologies associated with excitotoxicity.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Inibidores de Serina Proteinase/farmacologia , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Cicloeptanos , Agonistas de Aminoácidos Excitatórios/toxicidade , Técnicas In Vitro , Masculino , Camundongos , N-Metilaspartato/toxicidade , Neurônios/citologia , Neurotoxinas/toxicidade , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Neuronal death after ischemia-induced brain damage depends largely upon the activation of the N-methyl-D-aspartate (NMDA) excitatory glutamate receptor that is a target for many putative neuroprotective agents. Whereas the NMDA receptors mediate ischemic brain damage, blocking them is deleterious in humans. Here, the authors investigated whether nitrous oxide or xenon, which are gaseous anesthetics with a remarkably safe clinical profile that have been recently demonstrated as effective inhibitors of the NMDA receptor, may reduce the following: (1) ischemia-induced brain damage in vivo, when given after occlusion of the middle cerebral artery (MCAO), a condition needed to make these potentially neuroprotective agents therapeutically valuable; or (2) NMDA-induced Ca2+ influx in cortical cell cultures, a major critical event involved in excitotoxic neuronal death. The authors have shown that both nitrous oxide at 75 vol% and xenon at 50 vol% reduce ischemic neuronal death in the cortex by 70% and further decrease NMDA-induced Ca2+ influx by 30%. In addition, xenon at 50%, but not nitrous oxide at 75 vol%, further decreases ischemic brain damage in the striatum (a subcortical structure that is known to be resistant to neuroprotective interventions). However, at a higher concentration (75 vol%), xenon exhibits potentially neurotoxic effects. The mechanisms of the neuroprotective and potentially neurotoxic effects of nitrous oxide and xenon, as well as the possible therapeutic implications in humans, are discussed.
Assuntos
Anestésicos Inalatórios/farmacologia , Isquemia Encefálica/tratamento farmacológico , Óxido Nitroso/farmacologia , Xenônio/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Agonistas de Aminoácidos Excitatórios , Masculino , Atividade Motora/efeitos dos fármacos , N-Metilaspartato , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Equilíbrio Postural/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
Alzheimer's disease is characterized by the loss of memory and synaptic damage. Evidence is accumulating for a causal role of soluble oligomeric species of amyloid-ß peptide (Aßo) in the impairment of synaptic plasticity and cognition but the precise mechanisms underlying these effects are still not clear. Synaptic plasticity such as long-term potentiation is thought to underlie learning and memory. While the effect of Aß on long-term potentiation is well documented, a more general understanding of Aß action on various aspects of plasticity involving synaptic and extrasynaptic receptors and the nature of the mechanisms involved in its effects are lacking. Using a combination of electrophysiological and biochemical techniques in mouse hippocampal slices, we show here that Aßo drastically affects synaptic plasticities induced by high stimulation frequencies through the involvement of extrasynaptic glutamate receptors. Experiments on hippocampal slices as well as on cultured cortical neurons show that Aßo potentiates extrasynaptic NMDA receptors-mediated responses. Pharmacological characterization indicates that GluN2B-containing NMDARs are involved in these responses. When synaptic and extrasynaptic glutamate receptor-mediated effects are dissociated using cortical neurons in culture, it appears that Aßo has differential effects on these two receptors types. We conclude that the pool of extrasynaptic GluN2B-containing NMDARs is a major target of Aßo in the hippocampus. During high frequency stimulation, Aßo dramatically impairs long-term neuronal responses.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Hipocampo/patologia , Plasticidade Neuronal/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Peptídeos beta-Amiloides/química , Animais , Sinalização do Cálcio/fisiologia , Células Cultivadas , Interpretação Estatística de Dados , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Transcriptional coactivators and corepressors often have multiple targets and can have opposing actions on transcription and downstream physiological events. The coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is under-expressed in Huntington's disease and is a regulator of antioxidant defenses and mitochondrial biogenesis. We show that in primary cortical neurons, expression of PGC-1α strongly promotes resistance to excitotoxic and oxidative stress in a cell autonomous manner, whereas knockdown increases sensitivity. In contrast, the transcriptional corepressor silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) specifically antagonizes PGC-1α-mediated antioxidant effects. The antagonistic balance between PGC-1α and SMRT is upset in favor of PGC-1α by synaptic activity. Synaptic activity triggers nuclear export of SMRT reliant on multiple regions of the protein. Concomitantly, synaptic activity post-translationally enhances the transactivating potential of PGC-1α in a p38-dependent manner, as well as upregulating cyclic-AMP response element binding protein-dependent PGC-1α transcription. Activity-dependent targeting of PGC-1α results in enhanced gene expression mediated by the thyroid hormone receptor, a prototypical transcription factor coactivated by PGC-1α and repressed by SMRT. As a consequence of these events, SMRT is unable to antagonize PGC-1α-mediated resistance to oxidative stress in synaptically active neurons. Thus, PGC-1α and SMRT are antagonistic regulators of neuronal vulnerability to oxidative stress. Further, this coactivator-corepressor antagonism is regulated by the activity status of the cell, with implications for neuronal viability.
Assuntos
Antioxidantes/metabolismo , Neurônios/metabolismo , Correpressor 2 de Receptor Nuclear/antagonistas & inibidores , Correpressor 2 de Receptor Nuclear/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Animais , Neurônios/patologia , Correpressor 2 de Receptor Nuclear/genética , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Post-mitotic neurons must have strong antioxidant defenses to survive the lifespan of the organism. We recently showed that neuronal antioxidant defenses are boosted by synaptic activity. Elevated synaptic activity, acting via the N-methyl-D-aspartate (NMDA) receptor, enhances thioredoxin activity, facilitates the reduction of hyperoxidized peroxiredoxins, and promotes resistance to oxidative stress. In contrast, blockade of spontaneous synaptic NMDA receptor activity renders neurons highly sensitive to hyperoxidation of peroxiredoxins by oxidative insults. These NMDA receptor-dependent effects are mediated in part by a coordinated program of gene expression changes centered on the thioredoxin-peroxiredoxin system, a thiol-based enzymatic system which is an important reducer of oxidative stressors such as hydroperoxides. We show here that while too little glutamatergic activity can render neurons vulnerable to peroxiredoxin hyperoxidation, so can too much. Exposure of neurons to toxic concentrations of glutamate, activating both synaptic and extrasynaptic NMDA receptors, acutely induces peroxiredoxin hyperoxidation. Thus, the effect of NMDA receptor activity on the activity of neuronal peroxiredoxins follows the classical U-shaped dose response curve.
Assuntos
Neurotoxinas/toxicidade , Peroxirredoxinas/metabolismo , Animais , Células Cultivadas , Ácido Glutâmico/toxicidade , Estresse Oxidativo , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Tiorredoxinas/metabolismoRESUMO
In the last few years, it has been evidenced that serine proteases play key roles in the mammalian brain, both in physiological and pathological conditions. It has been well established that among these serine proteases, the tissue-type plasminogen activator (t-PA) is critically involved in development, plasticity, and pathology of the nervous system. However, its mechanism of action remains to be further investigated. By using pharmacological and immunological approaches, we have evidenced in the present work that t-PA should be considered as a neuromodulator. Indeed, we have observed that: (i). neuronal depolarization induces a release of t-PA; (ii). this release of t-PA is sensitive to exocytosis inhibition and calcium chelation; (iii). released t-PA modulates NMDA receptor signaling and (iv). astrocytes are able to recapture extracellular t-PA through a low-density lipoprotein (LDL) receptor-related protein (LRP)-dependent mechanism.