RESUMO
The Na+/Ca2+ exchanger 1 (NCX1) participates in the maintenance of neuronal Na+ and Ca2+ homeostasis, and it is highly expressed at synapse level of some brain areas involved in learning and memory processes, including the hippocampus, cortex, and amygdala. Furthermore, NCX1 increases Akt1 phosphorylation and enhances glutamate-mediated Ca2+ influx during depolarization in hippocampal and cortical neurons, two processes involved in learning and memory mechanisms. We investigated whether the modulation of NCX1 expression/activity might influence learning and memory processes. To this aim, we used a knock-in mouse overexpressing NCX1 in hippocampal, cortical, and amygdala neurons (ncx1.4over) and a newly synthesized selective NCX1 stimulating compound, named CN-PYB2. Both ncx1.4over and CN-PYB2-treated mice showed an amelioration in spatial learning performance in Barnes maze task, and in context-dependent memory consolidation after trace fear conditioning. On the other hand, these mice showed no improvement in novel object recognition task which is mainly dependent on non-spatial memory and displayed an increase in the active phosphorylated CaMKIIα levels in the hippocampus. Interestingly, both of these mice showed an increased level of context-dependent anxiety.Altogether, these results demonstrate that neuronal NCX1 participates in spatial-dependent hippocampal learning and memory processes.
Assuntos
Hipocampo/fisiologia , Trocador de Sódio e Cálcio/biossíntese , Aprendizagem Espacial/fisiologia , Memória Espacial/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Cricetinae , Técnicas de Introdução de Genes , Células HEK293 , Hipocampo/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Proteínas Recombinantes/metabolismo , Sódio/metabolismo , Trocador de Sódio e Cálcio/agonistas , Trocador de Sódio e Cálcio/genética , Aprendizagem Espacial/efeitos dos fármacos , Memória Espacial/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
The histone deacetylases (HDACs)-dependent mechanisms regulating gene transcription of the Na+/Ca+ exchanger isoform 3 (ncx3) after stroke are still unknown. Overexpression or knocking-down of HDAC4/HDAC5 down-regulates or increases, respectively, NCX3 mRNA and protein. Likewise, MC1568 (class IIa HDACs inhibitor), but not MS-275 (class I HDACs inhibitor) increased NCX3 promoter activity, gene and protein expression. Furthermore, HDAC4 and HDAC5 physically interacted with the transcription factor downstream regulatory element antagonist modulator (DREAM). As MC1568, DREAM knocking-down prevented HDAC4 and HDAC5 recruitment to the ncx3 promoter. Importantly, DREAM, HDAC4, and HDAC5 recruitment to the ncx3 gene was increased in the temporoparietal cortex of rats subjected to transient middle cerebral artery occlusion (tMCAO), with a consequent histone-deacetylation of ncx3 promoter. Conversely, the tMCAO-induced NCX3 reduction was prevented by intracerebroventricular injection of siDREAM, siHDAC4, and siHDAC5. Notably, MC1568 prevented oxygen glucose deprivation plus reoxygenation and tMCAO-induced neuronal damage, whereas its neuroprotective effect was abolished by ncx3 knockdown. Collectively, we found that: (1) DREAM/HDAC4/HDAC5 complex epigenetically down-regulates ncx3 gene transcription after stroke, and (2) pharmacological inhibition of class IIa HDACs reduces stroke-induced neurodetrimental effects.
Assuntos
Epigênese Genética/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Neurônios/patologia , Proteínas Repressoras/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/patologia , Animais , Córtex Cerebral/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Histona Desacetilases/genética , Humanos , Hipóxia Encefálica/prevenção & controle , Infarto da Artéria Cerebral Média/patologia , Proteínas Interatuantes com Canais de Kv/antagonistas & inibidores , Proteínas Interatuantes com Canais de Kv/genética , Masculino , Fármacos Neuroprotetores , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Trocador de Sódio e Cálcio/genética , Acidente Vascular Cerebral/genéticaRESUMO
[This corrects the article DOI: 10.1038/s41420-017-0018-1.].
RESUMO
In humans, mutation of glycine 93 to alanine of Cu++/Zn++ superoxide dismutase type-1 (SOD1-G93 A) has been associated to some familial cases of Amyotrophic Lateral Sclerosis (ALS). Several evidence proposed the involvement of environmental pollutants that like mercury could accelerate ALS symptoms. SH-SY5Y cells stably transfected with SOD1 and G93 A mutant of SOD1 constructs were exposed to non-toxic concentrations (0.01 µM) of ethylmercury thiosalicylate (thimerosal) for 24 h. Interestingly, we found that thimerosal, in SOD1-G93 A cells, but not in SOD1 cells, reduced cell survival. Furthermore, thimerosal-induced cell death occurred in a concentration dependent-manner and was prevented by the Sirtuin 1 (SIRT1) activator Resveratrol (RSV). Moreover, thimerosal decreased the protein expression of transcription factor Downstream Regulatory Element Antagonist Modulator (DREAM), but not DREAM gene. Interestingly, DREAM reduction was blocked by co-treatment with RSV, suggesting the participation of SIRT1 in determining this effect. Immunoprecipitation experiments in SOD1-G93 A cells exposed to thimerosal demonstrated that RSV increased DREAM deacetylation and reduced its polyubiquitination. In addition, RSV counteracted thimerosal-enhanced prodynorphin (PDYN) mRNA, a DREAM target gene. Furthermore, cortical neurons transiently transfected with SOD1-G93 A construct and exposed to thimerosal (0.5 µM/24 h) showed a reduction of DREAM and an up-regulation of the prodynorphin gene. Importantly, both the treatment with RSV or the transfection of siRNA against prodynorphin signiï¬cantly reduced thimerosal-induced neurotoxicity, while DREAM knocking-down potentiated thimerosal-reduced cell survival. These results demonstrate the particular vulnerability of SOD1-G93 A neuronal cells to thimerosal and that RSV via SIRT1 counteracts the neurodetrimental effect of this toxicant by preventing DREAM reduction and prodynorphin up-regulation.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Resveratrol/administração & dosagem , Transdução de Sinais , Superóxido Dismutase/metabolismo , Timerosal/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Encefalinas/metabolismo , Humanos , Proteínas Interatuantes com Canais de Kv/metabolismo , Precursores de Proteínas/metabolismo , Ratos Wistar , Proteínas Repressoras/metabolismo , Sirtuína 1/metabolismo , Superóxido Dismutase-1/metabolismoRESUMO
Na+-Ca2+ exchanger (NCX) isoforms constitute the major cellular Ca2+ extruding system in neurons and microglia. We herein investigated the role of NCX isoforms in the pathophysiology of Parkinson's disease (PD). Their expression and activity were evaluated in neurons and glia of mice expressing the human A53T variant of α-synuclein (A53T mice), an animal model mimicking a familial form of PD. Western blotting revealed that NCX3 expression in the midbrain of 12-month old A53T mice was lower than that of wild type (WT). Conversely, NCX1 expression increased in the striatum. Immunohistochemical studies showed that glial fibrillary acidic protein (GFAP)-positive astroglial cells significantly increased in the substantia nigra pars compacta (SNc) and in the striatum. However, the number and the density of tyrosine hydroxylase (TH)-positive neurons decreased in both brain regions. Interestingly, ionized calcium binding adaptor molecule 1 (IBA-1)-positive microglial cells increased only in the striatum of A53T mice compared to WT. Double immunostaining studies showed that in A53T mice, NCX1 was exclusively co-expressed in IBA-1-positive microglial cells in the striatum, whereas NCX3 was solely co-expressed in TH-positive neurons in SNc. Beam walking and pole tests revealed a reduction in motor performance for A53T mice compared to WT. In vitro experiments in midbrain neurons from A53T and WT mice demonstrated a reduction in NCX3 expression, which was accompanied by mitochondrial overload of Ca2+ ions, monitored with confocal microscopy by X-Rhod-1 fluorescent dye. Collectively, in vivo and in vitro findings suggest that the reduction in NCX3 expression and activity in A53T neurons from midbrain may cause mitochondrial dysfunction and neuronal death in this brain area, whereas NCX1 overexpression in microglial cells may promote their proliferation in the striatum.
Assuntos
Inflamação/metabolismo , Degeneração Neural/metabolismo , Doença de Parkinson/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Citosol/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/complicações , Inflamação/patologia , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos , Microglia/metabolismo , Mitocôndrias/metabolismo , Atividade Motora , Neostriado/metabolismo , Neostriado/patologia , Degeneração Neural/complicações , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Neurônios/metabolismo , Doença de Parkinson/complicações , Doença de Parkinson/fisiopatologia , Isoformas de Proteínas/metabolismo , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Triticum vulgare has been extensively used in traditional medicine thanks to its properties of accelerating tissue repair. The specific extract of Triticum vulgare manufactured by Farmaceutici Damor (TVE-DAMOR) is already present in some pharmaceutical formulations used in the treatment of decubitus ulcers, skin lesions and burns. It has been recently suggested that this Triticum vulgare extract may possess potential anti-inflammatory properties. In the light of these premises the aim of the present paper was to verify the anti-inflammatory role of TVE, using the LPS-stimulated microglia model of inflammation. In particular the effect of different concentrations of TVE on the release of several mediators of inflammation such as nitric oxide, IL-6, PGE2 and TNF alpha was evaluated. More important, the anti-inflammatory effect of TVE was confirmed also in primary rat microglia cultures. The results of the present study show that TVE exerts anti-inflammatory properties since it reduces the release of all the evaluated markers of inflammation, such as NO, IL6, TNF alpha and PGE2 in LPS-activated BV2 microglial cells. Intriguingly, TVE reduced microglia activation and NO release also in primary microglia. Indeed, to verify the pathway of modulation of the inflammatory markers reported above, we found that TVE restores the cytoplasmic expression of p65 protein, kwown as specific marker associated with activation of inflammatory response. The evidence for an inhibitory activity on inflammation of this specific extract of Triticum vulgare may open the way to the possibility of a therapeutical use of the Triticum vulgare extract as an anti-inflammatory compound in certain pathological states such as burns, decubitus ulcers, folliculitis and inflammation of peripheral nerve.
Assuntos
Inflamação/tratamento farmacológico , Microglia/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Triticum/química , Animais , Dinoprostona/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Interleucina-6/genética , Lipopolissacarídeos/toxicidade , Camundongos , Microglia/patologia , Óxido Nítrico/genética , Extratos Vegetais/química , Ratos , Fator de Necrose Tumoral alfa/genética , eIF-2 Quinase/genéticaRESUMO
Hypoxic-ischemic encephalopathy (HI) accounts for the majority of developmental, motor and cognitive deficits in children, leading to life-long neurological impairments. Since the plasmamembrane sodium/calcium exchanger (NCX) plays a fundamental role in maintaining ionic homeostasis during adult brain ischemia, in the present work we aimed to demonstrate (1)the involvement of NCX in the pathophysiology of neonatal HI and (2)a possible NCX-based pharmacological intervention. HI was induced in neonatal mice at postnatal day 7(P7) by unilateral cut of the right common carotid artery, followed by 60â¯min exposure to 8%O2. Expression profiles of NCX isoforms from embryos stage to adulthood was evaluated in the hippocampus of hypoxic-ischemic and control mice. To assess the effect of NCX pharmacological stimulation, brain infarct volume was evaluated in brain sections, obtained at several time intervals after systemic administration of the newly synthesized NCX activator neurounina. Moreover, the long term effect of NCX activation was evaluated in adult mice (P60) subjected to neonatal HI and daily treated with neurounina for three weeks. Hypoxic-ischemic insult induced a reduction of NCX1 and NCX3 expression starting from day 7 until day 60. Notably, 8 weeks after HI induction in P7 mice, NCX pharmacological stimulation not only reduced infarct volume but improved also motor behaviour, spatial and visual memory. The present study highlights the significant role of NCX in the evolution of neonatal brain injury and in the learning and memory processes that are impaired in mice injured in the neonatal period.
Assuntos
Benzodiazepinonas/farmacologia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/prevenção & controle , Pirrolidinas/farmacologia , Trocador de Sódio e Cálcio/metabolismo , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Benzodiazepinonas/uso terapêutico , Encéfalo/patologia , Feminino , Hipocampo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Masculino , Camundongos , Isoformas de Proteínas , Pirrolidinas/uso terapêutico , Trocador de Sódio e Cálcio/biossíntese , Fatores de TempoRESUMO
Nuclear envelope (NE) is a Ca2+-storing organelle controlling neuronal differentiation through nuclear Ca2+ concentrations ([Ca2+]n). However, how [Ca2+]n regulates this important function remains unknown. Here, we investigated the role of the nuclear form of the Na+/Ca2+ exchanger 1(nuNCX1) during the different stages of neuronal differentiation and the involvement of PTEN/PI3'K/Akt pathway. In neuronal cells, nuNCX1 was detected on the inner membrane of the NE where protein expression and activity of the exchanger increased during NGF-induced differentiation. nuNCX1 activation by Na+-free perfusion induced a time-dependent activation of nuclear-resident PI3K/Akt pathway in isolated nuclei. To discriminate the contribution of nuNCX1 from those of plasma membrane NCX, we generated a chimeric protein composed of the fluorophore EYFP, the exchanger inhibitory peptide, and the nuclear localization signal, named XIP-NLS. Fura-2 measurements on single nuclei and patch-clamp experiments in whole-cell configuration showed that XIP-NLS selectively inhibited nuNCX1. Once it reached the nuclear compartment, XIP-NLS increased the nucleoplasmic Ca2+ peak elicited by ATP and reduced Akt phosphorylation, GAP-43 and MAP-2 expression through nuclear-resident PTEN induction. Furthermore, in accordance with the prevention of the neuronal phenotype, XIP-NLS significantly reduced TTX-sensitive Na+ currents and membrane potential during neuronal differentiation. The selective inhibition of nuNCX1 by XIP-NLS increased the percentage of ß III tubulin-positive immature neurons in mature cultures of MAP-2-positive cortical neurons, thus unraveling a new function for nuNCX1 in regulating neuronal differentiation through [Ca2+]n-dependent PTEN/PI3K/Akt pathway.
RESUMO
Methylmercury (MeHg) causes neuronal death through different pathways. Particularly, we found that in cortical neurons it increased the expression of Repressor Element-1 Silencing Transcription Factor (REST), histone deacetylase (HDAC)4, Specificity Protein (Sp)1, Sp4, and reduced the levels of brain-derived neurotrophic factor (BDNF). Herein, in rat cortical neurons we investigated whether microRNA (miR)206 can modulate MeHg-induced cell death by regulating REST/HDAC4/Sp1/Sp4/BDNF axis. MeHg (1 µM) reduced miR206 expression after both 12 and 24 h and miR206 transfection prevented MeHg-induced neuronal death. Furthermore, miR206 reverted MeHg-induced REST and Sp4 increase and BDNF reduction at gene and protein level, and reverted HDAC4 protein increase, but not HDAC4 mRNA upregulation. Moreover, since no miR206 seed sequences were identified in the 3'-untranslated regions (3'-UTRs) of REST and SP4, we investigated the role of JunD, that presents a consensus motif on REST, Sp4, and BDNF promoters. Indeed, MeHg increased JunD mRNA and protein levels, and JunD knockdown counteracted MeHg-induced REST, Sp4 increase, but not BDNF reduction. Furthermore, we identified a miR206 binding site in the 3'-UTR of JunD mRNA (miR206/JunD) and mutagenesis of miR206/JunD site reverted JunD luciferase activity reduction induced by miR206. Finally, miR206 prevented MeHg-increased JunD binding to REST and Sp4 promoters, and MeHg-reduced BDNF expression was determined by the increase of HDAC4 binding on BDNF promoter IV. Collectively, these results suggest that miR206 downregulation induced by MeHg exposure determines an upregulation of HDAC4, that in turn downregulated BDNF, and the activation of JunD that, by binding REST and Sp4 gene promoters, increased their expression.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , MicroRNAs/metabolismo , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Humanos , MicroRNAs/genética , Neurônios/metabolismo , Neurônios/patologia , Síndromes Neurotóxicas/patologia , Proteínas Proto-Oncogênicas c-jun/genética , RNA Interferente Pequeno/genética , Ratos , TransfecçãoRESUMO
Preconditioning (PC) is a phenomenon wherein a mild insult induces resistance to a later, severe injury. Although PC has been extensively studied in several neurological disorders, no studies have been performed in amyotrophic lateral sclerosis (ALS). Here we hypothesize that a sub-toxic acute exposure to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA) is able to delay ALS progression in SOD1 G93A mice and that NCX3, a membrane transporter able to handle the deregulation of ionic homeostasis occurring during ALS, takes part to this neuroprotective effect. Preconditioning effect was examined on disease onset and duration, motor functions, and motor neurons in terms of functional declines and severity of histological damage in male and female mice. Our findings demonstrate that a sub-toxic dose of L-BMAA works as preconditioning stimulus and is able to delay ALS onset and to prolong ALS mice survival. Interestingly, preconditioning prevented NCX3 downregulation in SOD1 G93A mice spinal cord, leading to an increased number of motor neurons associated to a reduced astrogliosis, and reduced the denervation of neuromuscular junctions observed in SOD1 G93A mice. These protective effects were mitigated in ncx3+/- mice. This study established for the first time an animal model of preconditioning in ALS and candidates NCX3 as a new therapeutic target.
Assuntos
Diamino Aminoácidos/farmacologia , Esclerose Lateral Amiotrófica/metabolismo , Regulação para Baixo/efeitos dos fármacos , Neurotoxinas/farmacologia , Trocador de Sódio e Cálcio/biossíntese , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/terapia , Animais , Toxinas de Cianobactérias , Camundongos , Camundongos Transgênicos , Trocador de Sódio e Cálcio/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Our previous study showed that the environmental neurotoxicant non-dioxin-like polychlorinated biphenyl (PCB)-95 increases RE1-silencing transcription factor (REST) expression, which is related to necrosis, but not apoptosis, of neurons. Meanwhile, necroptosis is a type of a programmed necrosis that is positively regulated by receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) and negatively regulated by caspase-8. Here we evaluated whether necroptosis contributes to PCB-95-induced neuronal death through REST up-regulation. Our results demonstrated that in cortical neurons PCB-95 increased RIPK1, RIPK3, and MLKL expression and decreased caspase-8 at the gene and protein level. Furthermore, the RIPK1 inhibitor necrostatin-1 or siRNA-mediated RIPK1, RIPK3 and MLKL expression knockdown significantly reduced PCB-95-induced neuronal death. Intriguingly, PCB-95-induced increases in RIPK1, RIPK3, MLKL expression and decreases in caspase-8 expression were reversed by knockdown of REST expression with a REST-specific siRNA (siREST). Notably, in silico analysis of the rat genome identified a REST consensus sequence in the caspase-8 gene promoter (Casp8-RE1), but not the RIPK1, RIPK3 and MLKL promoters. Interestingly, in PCB-95-treated neurons, REST binding to the Casp8-RE1 sequence increased in parallel with a reduction in its promoter activity, whereas under the same experimental conditions, transfection of siREST or mutation of the Casp8-RE1 sequence blocked PCB-95-induced caspase-8 reduction. Since RIPK1, RIPK3 and MLKL rat genes showed no putative REST binding site, we assessed whether the transcription factor cAMP Responsive Element Binding Protein (CREB), which has a consensus sequence in all three genes, affected neuronal death. In neurons treated with PCB-95, CREB protein expression decreased in parallel with a reduction in binding to the RIPK1, RIPK3 and MLKL gene promoter sequence. Furthermore, CREB overexpression was associated with reduced promoter activity of the RIPK1, RIPK3 and MLKL genes. Collectively, these results indicate that PCB-95 was associated with REST-induced necroptotic cell death by increasing RIPK1, RIPK3 and MLKL expression and reducing caspase-8 levels. In addition, since REST is involved in several neurological disorders, therapies that block REST-induced necroptosis could be a new strategy to revert the neurodetrimental effects associated to its overexpression.
Assuntos
Caspase 8/metabolismo , Morte Celular/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Neurônios/efeitos dos fármacos , Bifenilos Policlorados/toxicidade , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Repressoras/metabolismo , Animais , Caspase 8/genética , Linhagem Celular Tumoral , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Regulação para Baixo , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Necrose , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Proteínas Quinases/genética , Ratos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas Repressoras/antagonistas & inibidores , Transfecção , Regulação para CimaAssuntos
Antibacterianos/administração & dosagem , Cefalosporinas/administração & dosagem , Fraturas Ósseas/complicações , Osteomielite/tratamento farmacológico , Ácido Penicilânico/análogos & derivados , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Inibidores de beta-Lactamases/administração & dosagem , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Fraturas Ósseas/cirurgia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Ácido Penicilânico/administração & dosagem , Pseudomonas aeruginosa/isolamento & purificação , Tazobactam , Resultado do TratamentoRESUMO
Astrocyte dysfunction emerges early in Alzheimer's disease (AD) and may contribute to its pathology and progression. Recently, the voltage gated potassium channel KV3.4 subunit, which underlies the fast-inactivating K+ currents, has been recognized to be relevant for AD pathogenesis and is emerging as a new target candidate for AD. In the present study, we investigated both in in vitro and in vivo models of AD the expression and functional activity of KV3.4 potassium channel subunits in astrocytes. In primary astrocytes our biochemical, immunohistochemical, and electrophysiological studies demonstrated a time-dependent upregulation of KV3.4 expression and functional activity after exposure to amyloid-ß (Aß) oligomers. Consistently, astrocytic KV3.4 expression was upregulated in the cerebral cortex, hippocampus, and cerebellum of 6-month-old Tg2576 mice. Further, confocal triple labeling studies revealed that in 6-month-old Tg2576 mice, KV3.4 was intensely coexpressed with Aß in nonplaque associated astrocytes. Interestingly, in the cortical and hippocampal regions of 12-month-old Tg2576 mice, plaque-associated astrocytes much more intensely expressed KV3.4 subunits, but not Aß. More important, we evidenced that the selective knockdown of KV3.4 expression significantly downregulated both glial fibrillary acidic protein levels and Aß trimers in the brain of 6-month-old Tg2576 mice. Collectively, our results demonstrate that the expression and function of KV3.4 channel subunits are precociously upregulated in cultured astrocytes exposed to Aß oligomers and in reactive astrocytes of AD Tg2576 mice.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/efeitos adversos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Expressão Gênica , Canais de Potássio Shaw/genética , Canais de Potássio Shaw/metabolismo , Regulação para Cima , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/metabolismo , Ratos Wistar , Canais de Potássio Shaw/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
The molecular pathways involved in methylmercury (MeHg)-induced neurotoxicity are not fully understood. Since pan-Histone deacetylases (HDACs) inhibition has been found to revert the neurodetrimental effect of MeHg, it appeared of interest to investigate whether the pattern of HDACs isoform protein expression is modified during MeHg-induced neurotoxicity and the transcriptional/transductional mechanisms involved. SH-SY5Y neuroblastoma cells treated with MeHg 1 µM for 12 and 24 h showed a significant increase of HDAC4 protein and gene expression, whereas the HDACs isoforms 1-3, 5, and 6 were unmodified. Furthermore, MeHg-induced HDAC4 increase was reverted when cells were transfected with siRNAs against specificity protein 1 (Sp1) and Sp4, that were both increased during MeHg exposure. Next we studied the role of extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) in MeHg-induced increase of Sp1, Sp4, and HDAC4 expression. As shown by Western Blot analysis MeHg exposure increased the phosphorylation of p38, but not of ERK and JNK. Notably, when p38 was pharmacologically blocked, MeHg-induced Sp1, Sp4 protein expression, and HDAC4 protein and gene expression was reverted. In addition, MeHg exposure increased the binding of HDAC4 to the promoter IV of the Brain-derived neurotrophic factor (BDNF) gene, determining its mRNA reduction, that was significantly counteracted by HDAC4 knocking down. Furthermore, rat cortical neurons exposed to MeHg (1 µM/24 h) showed an increased phosphorylation of p38, in parallel with an up-regulation of Sp1, Sp4, and HDAC4 and a down-regulation of BDNF proteins. Importantly, transfection of siRNAs against p38, Sp1, Sp4, and HDAC4 or transfection of vector overexpressing BDNF significantly blocked MeHg-induced cell death in cortical neurons. All these results suggest that p38/Sp1-Sp4/HDAC4/BDNF may represent a new pathway involved in MeHg-induced neurotoxicity.
RESUMO
Amyotrophic lateral sclerosis (ALS) is a severe human adult-onset neurodegenerative disease affecting lower and upper motor neurons. In >20% of cases, the familial form of ALS is caused by mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1). Interestingly, administration of wild-type SOD1 to SOD1G93A transgenic rats ameliorates motor symptoms through an unknown mechanism. Here we investigated whether the neuroprotective effects of SOD1 are due to the Ca2+-dependent activation of such prosurvival signaling pathway and not to its catalytic activity. To this aim, we also examined the mechanism of neuroprotective action of ApoSOD1, the metal-depleted state of SOD1 that lacks dismutase activity, in differentiated motor neuron-like NSC-34 cells and in primary motor neurons exposed to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA). Preincubation of ApoSOD1 and SOD1, but not of human recombinant SOD1G93A, prevented cell death in motor neurons exposed to L-BMAA. Moreover, ApoSOD1 elicited ERK1/2 and Akt phosphorylation in motor neurons through an early increase of intracellular Ca2+ concentration ([Ca2+]i). Accordingly, inhibition of ERK1/2 by siMEK1 and PD98059 counteracted ApoSOD1- and SOD1-induced neuroprotection. Similarly, transfection of the dominant-negative form of Akt in NSC-34 motor neurons and treatment with the selective PI3K inhibitor LY294002 prevented ApoSOD1- and SOD1-mediated neuroprotective effects in L-BMAA-treated motor neurons. Furthermore, ApoSOD1 and SOD1 prevented the expression of the two markers of L-BMAA-induced ER stress GRP78 and caspase-12. Collectively, our data indicate that ApoSOD1, which is devoid of any catalytic dismutase activity, exerts a neuroprotective effect through an early activation of Ca2+/Akt/ERK1/2 pro-survival pathway that, in turn, prevents ER stress in a neurotoxic model of ALS.
Assuntos
Diamino Aminoácidos/toxicidade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Superóxido Dismutase-1/metabolismo , Animais , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Toxinas de Cianobactérias , Chaperona BiP do Retículo Endoplasmático , Flavonoides/farmacologia , Proteínas de Choque Térmico/metabolismo , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Fármacos Neuroprotetores/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genética , Tretinoína/farmacologiaRESUMO
Ethylmercury thiosalicylate (thimerosal) is an organic mercury-based compound commonly used as an antimicrobial preservative that has been found to be neurotoxic. In contrast, histone deacetylases (HDACs) inhibition has been found to be neuroprotective against several environmental contaminants, such as polychlorinated biphenyls, di-2-ethylhexyl phthalate, and methylmercury. The aim of this study was to investigate the effect of HDAC inhibition on thimerosal-induced neurotoxicity in neuroblastoma cells and cortical neurons. Interestingly, we found that thimerosal, at 0.5 µM in SH-SY5Y cells and at 1 µM in neurons, caused cell death by activation of apoptosis, which was prevented by the HDAC class IIA inhibitor MC1568 but not the class I inhibitor MS275. Furthermore, thimerosal specifically increased HDAC4 protein expression but not that of HDACs 5, 6, 7, and 9. Western blot analysis revealed that MC1568 prevented thimerosal-induced HDAC4 increase. In addition, both HDAC4 knocking-down and MC1568 inhibited thimerosal-induced cell death in SH-SY5Y cells and cortical neurons. Importantly, intramuscular injection of 12 µg/kg thimerosal on postnatal days 7, 9, 11, and 15 increased HDAC4 levels in the prefrontal cortex (PFC), which decreased histone H4 acetylation in infant male rats, in parallel increased motor activity changes. In addition, coadministration of 40 mg/kg MC1568 (intraperitoneal injection) moderated the HDAC4 increase which reduced histone H4 deacetylation and caspase-3 cleavage in the PFC. Finally, open-field testing showed that thimerosal-induced motor activity changes are reduced by MC1568. These findings indicate that HDAC4 regulates thimerosal-induced cell death in neurons and that treatment with MC1568 prevents thimerosal-induced activation of caspase-3 in the rat PFC.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Pirróis/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Timerosal/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Citoproteção , Relação Dose-Resposta a Droga , Histona Desacetilases/genética , Humanos , Masculino , Atividade Motora/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Córtex Pré-Frontal/enzimologia , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/fisiopatologia , Interferência de RNA , Ratos Wistar , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Regulação para CimaRESUMO
BACKGROUND AND PURPOSE: The small ubiquitin-like modifier (SUMO), a ubiquitin-like protein involved in posttranslational protein modifications, is activated by several conditions, such as heat stress, hypoxia, and hibernation and confers neuroprotection. Sumoylation enzymes and substrates are expressed also at the plasma membrane level. Among the numerous plasma membrane proteins controlling ionic homeostasis during cerebral ischemia, 1 of the 3 brain sodium/calcium exchangers (NCX3), exerts a protective role during ischemic preconditioning. In this study, we evaluated whether NCX3 is a target for sumoylation and whether this posttranslational modification participates in ischemic preconditioning-induced neuroprotection. To test these hypotheses, we analyzed (1) SUMO1 conjugation pattern after ischemic preconditioning; (2) the effect of SUMO1 knockdown on the ischemic damage after transient middle cerebral artery occlusion and ischemic preconditioning, (3) the possible interaction between SUMO1 and NCX3 and (4) the molecular determinants of NCX3 sequence responsible for sumoylation. METHODS: Focal brain ischemia and ischemic preconditioning were induced in rats by middle cerebral artery occlusion. SUMOylation was evaluated by western blot and immunohistochemistry. SUMO1 and NCX3 interaction was analyzed by site-directed mutagenesis and immunoprecipitation assay. RESULTS: We found that (1) SUMO1 knockdown worsened ischemic damage and reduced the protective effect of preconditioning; (2) SUMO1 bound to NCX3 at lysine residue 590, and its silencing increased NCX3 degradation; and (3) NCX3 sumoylation participates in SUMO1 protective role during ischemic preconditioning. Thus, our results demonstrate that NCX3 sumoylation confers additional neuroprotection in ischemic preconditioning. CONCLUSIONS: Finally, this study suggests that NCX3 sumoylation might be a new target to enhance ischemic preconditioning-induced neuroprotection.
Assuntos
Encéfalo/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Precondicionamento Isquêmico , Neuroproteção/fisiologia , Proteína SUMO-1/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Encéfalo/patologia , Infarto da Artéria Cerebral Média/patologia , Masculino , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , SumoilaçãoRESUMO
Three different Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are expressed in brain where they play a relevant role in maintaining Na+ and Ca2+ homeostasis. Although the neuroprotective roles of NCX2 and NCX3 in stroke have been elucidated, the relevance of NCX1 is still unknown because of embryonic lethality of its knocking-out, heart dysfunctions when it is overexpressed, and the lack of selectivity in currently available drugs. To overcome these limitations we generated two conditional genetically modified mice that upon tamoxifen administration showed a selective decrease or increase of NCX1 in cortical and hippocampal neurons. Interestingly, in cortex and hippocampus NCX1 overexpression increased, where NCX1 knock-out reduced, both exchanger activity and Akt1 phosphorylation, a neuronal survival signaling. More important, mice overexpressing NCX1 showed a reduced ischemic volume and an amelioration of focal and general deficits when subjected to transient middle cerebral artery occlusion. Conversely, NCX1-knock-out mice displayed a worsening of brain damage, focal and neurological deficits with a decrease in Akt phosphorylation. These results support the idea that NCX1 overexpression/activation may represent a feasible therapeutic opportunity in stroke intervention.
Assuntos
Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Córtex Cerebral/patologia , Modelos Animais de Doenças , Regulação para Baixo , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Hipocampo/patologia , Masculino , Camundongos Knockout , Neurônios/patologia , Fosforilação , Trocador de Sódio e Cálcio/genética , Acidente Vascular Cerebral/genética , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia , Regulação para CimaRESUMO
Methylmercury (MeHg) is a highly neurotoxic compound that, in adequate doses, can cause damage to the brain, including developmental defects and in severe cases cell death. The RE-1-silencing transcription factor (REST) has been found to be involved in the neurotoxic effects of environmental pollutants such as polychlorinated biphenyls (PCBs). In this study, we investigated the effects of MeHg treatment on REST expression and its role in MeHg-induced neurotoxicity in neuroblastoma SH-SY5Y cells. We found that MeHg exposure caused a dose- and time- dependent apoptotic cell death, as evidenced by the appearance of apoptotic hallmarks including caspase-3 processing and annexin V uptake. Moreover, MeHg increased REST gene and gene product expression. MeHg-induced apoptotic cell death was completely abolished by REST knockdown. Interestingly, MeHg (1µM/24h) increased the expression of REST Corepressor (Co-REST) and its binding with REST whereas the other REST cofactor mammalian SIN3 homolog A transcription regulator (mSin3A) was not modified. In addition, we demonstrated that the acetylation of histone protein H4 was reduced after MeHg treatment and was critical for MeHg-induced apoptosis. Accordingly, the pan-histone deacetylase inhibitor trichostatin-A (TSA) prevented MeHg-induced histone protein H4 deacetylation, thereby reverting MeHg-induced neurotoxic effect. Male mice subcutaneously injected with 10mg/kg of MeHg for 10 days showed an increase in REST expression in the granule cell layer of the cerebellum together with a decrease in histone H4 acetylation. Collectively, we demonstrated that methylmercury exposure can cause neurotoxicity by activating REST gene expression and H4 deacetylation.