RESUMO
It has been recently established that GPR158, a class C orphan G protein-coupled receptor, serves as a metabotropic glycine receptor. GPR158 is highly expressed in the nucleus accumbens (NAc), a major input structure of the basal ganglia that integrates information from cortical and subcortical structures to mediate goal-directed behaviors. However, whether glycine modulates neuronal activity in the NAc through GPR158 activation has not been investigated yet. Using whole-cell patch-clamp recordings, we found that glycine-dependent activation of GPR158 increased the firing rate of NAc medium spiny neurons (MSNs) while it failed to significantly affect the excitability of cholinergic interneurons (CIN). In MSNs GPR158 activation reduced the latency to fire, increased the action potential half-width, and reduced action potential afterhyperpolarization, effects that are all consistent with negative modulation of potassium M-currents, that in the central nervous system are mainly carried out by Kv7/KCNQ-channels. Indeed, we found that the GPR158-induced increase in MSN excitability was associated with decreased M-current amplitude, and selective pharmacological inhibition of the M-current mimicked and occluded the effects of GPR158 activation. In addition, when the protein kinase A (PKA) or extracellular signal-regulated kinase (ERK) signaling was pharmacologically blocked, modulation of MSN excitability by GPR158 activation was suppressed. Moreover, GPR158 activation increased the phosphorylation of ERK and Kv7.2 serine residues. Collectively, our findings suggest that GPR158/PKA/ERK signaling controls MSN excitability via Kv7.2 modulation. Glycine-dependent activation of GPR158 may significantly affect MSN firing in vivo, thus potentially mediating specific aspects of goal-induced behaviors.
Assuntos
Potenciais de Ação , Glicina , Neurônios , Núcleo Accumbens , Receptores Acoplados a Proteínas G , Animais , Glicina/farmacologia , Glicina/metabolismo , Núcleo Accumbens/metabolismo , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/citologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Masculino , Potenciais de Ação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Glicina/metabolismo , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Neurônios Espinhosos MédiosRESUMO
Extensive research provides evidence that neuroinflammation underlies numerous brain disorders. However, the molecular mechanisms by which inflammatory mediators determine synaptic and cognitive dysfunction occurring in neurodegenerative diseases (e.g., Alzheimer's disease) are far from being fully understood. Here we investigated the role of interleukin 1ß (IL-1ß), and the molecular cascade downstream the activation of its receptor, to the synaptic dysfunction occurring in the mouse model of multiple Herpes simplex virus type-1 (HSV-1) reactivations within the brain. These mice are characterized by neuroinflammation and memory deficits associated with a progressive accumulation of neurodegenerative hallmarks (e.g., amyloid-ß protein and tau hyperphosphorylation). Here we show that mice undergone two HSV-1 reactivations in the brain exhibited increased levels of IL-1ß along with significant alterations of: (1) cognitive performances; (2) hippocampal long-term potentiation; (3) expression synaptic-related genes and pre- and post-synaptic proteins; (4) dendritic spine density and morphology. These effects correlated with activation of the epigenetic repressor MeCP2 that, in association with HDAC4, affected the expression of synaptic plasticity-related genes. Specifically, in response to HSV-1 infection, HDAC4 accumulated in the nucleus and promoted MeCP2 SUMOylation that is a post-translational modification critically affecting the repressive activity of MeCP2. The blockade of IL-1 receptors by the specific antagonist Anakinra prevented the MeCP2 increase and the consequent downregulation of gene expression along with rescuing structural and functional indices of neurodegeneration. Collectively, our findings provide novel mechanistic evidence on the role played by HSV-1-activated IL-1ß signaling pathways in synaptic deficits leading to cognitive impairment.
Assuntos
Doença de Alzheimer , Herpes Simples , Herpesvirus Humano 1 , Camundongos , Animais , Herpesvirus Humano 1/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Doenças Neuroinflamatórias , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Herpes Simples/complicações , Transtornos da Memória/genética , Plasticidade Neuronal/fisiologia , Epigênese Genética , Hipocampo/metabolismo , Modelos Animais de Doenças , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismoRESUMO
AIMS: Early dysfunction in Alzheimer's disease (AD) is characterised by alterations of synapse structure and function leading to dysmorphic neurites, decreased spine density, impaired synaptic plasticity and cognitive deficits. The class II member HDAC4, which recently emerged as a crucial factor in shaping synaptic plasticity and memory, was found to be altered in AD. We investigated how the modulation of HDAC4 may contribute to counteracting AD pathogenesis. METHODS: Using a cytoplasmic HDAC4 mutant (HDAC4SD ), we studied the recovery of synaptic function in hippocampal tissue and primary neurons from the triple-transgenic mouse model of AD (3×Tg-AD). RESULTS: Here, we report that in wild-type mice, HDAC4 is localised at synapses and interacts with postsynaptic proteins, whereas in the 3×Tg-AD, it undergoes nuclear import, reducing its interaction with synaptic proteins. Of note, HDAC4 delocalisation was induced by both amyloid-ß and tau accumulation. Overexpression of the HDAC4SD mutant in CA1 pyramidal neurons of organotypic hippocampal slices obtained from 3×Tg-AD mice increased dendritic length and promoted the enrichment of N-cadherin, GluA1, PSD95 and CaMKII proteins at the synaptic level compared with AD neurons transfected with the empty vector. Moreover, HDAC4 overexpression recovered the level of SUMO2/3ylation of PSD95 in AD hippocampal tissue, and in AD organotypic hippocampal slices, the HDAC4SD rescued spine density and synaptic transmission. CONCLUSIONS: These results highlight a new role of cytoplasmic HDAC4 in providing a structural and enzymatic regulation of postsynaptic proteins. Our findings suggest that controlling HDAC4 localisation may represent a promising strategy to rescue synaptic function in AD, potentially leading to memory improvement.
Assuntos
Doença de Alzheimer , Animais , Camundongos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Modelos Animais de Doenças , Hipocampo/patologia , Camundongos Transgênicos , Sinapses/patologia , Transmissão Sináptica/fisiologia , Citoplasma/metabolismoRESUMO
Although major depressive disorder (MDD) is highly prevalent, its pathophysiology is poorly understood. Recent evidence suggests that glycogen-synthase kinase 3ß (GSK3ß) plays a key role in memory formation, yet its role in mood regulation remains controversial. Here, we investigated whether GSK3ß activity in the nucleus accumbens (NAc) is associated with depression-like behaviors and synaptic plasticity. We performed whole-cell patch-clamp recordings of medium spiny neurons (MSNs) in the NAc and determined the role of GSK3ß in spike timing-dependent long-term potentiation (tLTP) in the chronic unpredictable mild stress (CUMS) mouse model of depression. To assess the specific role of GSK3ß in tLTP, we used in vivo genetic silencing by an adeno-associated viral vector (AAV2) short hairpin RNA against GSK3ß. In addition, we examined the role of the voltage-gated potassium Kv4.2 subunit, a molecular determinant of A-type K+ currents, as a potential downstream target of GSK3ß. We found increased levels of active GSK3ß and augmented tLTP in CUMS mice, a phenotype that was prevented by selective GSK3ß knockdown. Furthermore, knockdown of GSK3ß in the NAc ameliorated depressive-like behavior in CUMS mice. Electrophysiological, immunohistochemical, biochemical, and pharmacological experiments revealed that inhibition of the Kv4.2 channel through direct phosphorylation at Ser-616 mediated the GSK3ß-dependent tLTP changes in CUMS mice. Our results identify GSK3ß regulation of Kv4.2 channels as a molecular mechanism of MSN maladaptive plasticity underlying depression-like behaviors and suggest that the GSK3ß-Kv4.2 axis may be an attractive therapeutic target for MDD.
Assuntos
Transtorno Depressivo Maior/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Plasticidade Neuronal , Núcleo Accumbens/patologia , Canais de Potássio Shal/metabolismo , Potenciais de Ação , Animais , Comportamento Animal , Transtorno Depressivo Maior/etiologia , Transtorno Depressivo Maior/psicologia , Modelos Animais de Doenças , Masculino , Camundongos , Neurônios/patologia , Núcleo Accumbens/citologia , Técnicas de Patch-Clamp , Estresse Psicológico/complicações , Estresse Psicológico/psicologia , Fatores de TempoRESUMO
Histaminergic neurons are exclusively located in the hypothalamic tuberomammillary nucleus, from where they project to many brain areas including the nucleus accumbens (NAc), a brain area that integrates diverse monoaminergic inputs to coordinate motivated behaviours. While the NAc expresses various histamine receptor subtypes, the mechanisms by which histamine modulates NAc activity are still poorly understood. Using whole-cell patch-clamp recordings, we found that pharmacological activation of histamine 2 (H2) receptors elevates the excitability of NAc medium spiny neurons (MSNs), while activation of H1 receptors failed to significantly affect MSN excitability. The evoked firing of MSNs increased after seconds of local H2 agonist administration and remained elevated for minutes. H2 receptor (H2R) activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential afterhyperpolarization and increased the action potential half-width. The increased excitability was protein kinase A-dependent and associated with decreased A-type K+ currents. In addition, selective pharmacological inhibition of the Kv4.2 channel, the main molecular determinant of A-type K+ currents in MSNs, mimicked and occluded the increased excitability induced by H2R activation. Our results indicate that histaminergic transmission in the NAc increases MSN intrinsic excitability through H2R-dependent modulation of Kv4.2 channels. Activation of H2R will significantly alter spike firing in MSNs in vivo, and this effect could be an important mechanism by which these receptors mediate certain aspects of goal-induced behaviours. KEY POINTS: Histamine is synthesized and released by hypothalamic neurons of the tuberomammillary nucleus and serves as a general modulator for whole-brain activity including the nucleus accumbens. Histamine receptors type 2 (HR2), which are expressed in the nucleus accumbens, couple to Gαs/off proteins which elevate cyclic adenosine monophosphate levels and activate protein kinase A. Whole-cell patch-clamp recordings revealed that H2R activation increased the evoked firing in medium spiny neurons of the nucleus accumbens via protein kinase A-dependent mechanisms. HR2 activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential medium after-hyperpolarization and increased the action potential half-width. HR2 activation also reduced A-type potassium current. Selective pharmacological inhibition of the Kv4.2 channel mimicked and occluded the increased excitability induced by H2R activation.
Assuntos
Histamina , Núcleo Accumbens , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Histamina/farmacologia , Neurônios/fisiologia , Núcleo Accumbens/fisiologia , Receptores Histamínicos H2RESUMO
Histamine, a monoamine implicated in stress-related arousal states, is synthesized in neurons exclusively located in the hypothalamic tuberomammillary nucleus (TMN) from where they diffusely innervate striatal and mesolimbic networks including the nucleus accumbens (NAc), a vital node in the limbic loop. Since histamine-containing TMN neuron output increases during stress, we hypothesized that exposure of mice to acute restrain stress (ARS) recruits endogenous histamine type 2 receptor (H2R) signaling in the NAc, whose activation increases medium spiny neurons (MSNs) intrinsic excitability via downregulation of A-type K+ currents. We employed an ARS paradigm in which mice were restrained for 120 min, followed by a 20-min recovery period, after which brain slices were prepared for ex vivo electrophysiology. Using whole-cell patch-clamp recordings, we found that pharmacological activation of H2R failed to affect MSN excitability and A-type K+ currents in mice that underwent ARS. Interestingly, in mice treated with H2R-antagonist prior to ARS paradigm, H2R activation increased evoked firing and decreased A-type K+ currents similarly to what observed in control mice. Furthermore, H2R-antagonist treatment ameliorated anxiety-like behavior in ARS mice. Together, our findings indicate that ARS paradigm recruits endogenous H2R signaling in MSNs and suggest the involvement of H2R signaling in stress-related motivational states.
Assuntos
Histamina , Núcleo Accumbens , Camundongos , Animais , Potenciais de Ação/fisiologia , Neurônios Espinhosos Médios , Técnicas de Patch-ClampRESUMO
Nucleoporins (Nups) are components of the nuclear pore complex that, besides regulating nucleus-cytoplasmic transport, emerged as a hub for chromatin interaction and gene expression modulation. Specifically, Nups act in a dynamic manner both at specific gene level and in the topological organization of chromatin domains. As such, they play a fundamental role during development and determination of stemness/differentiation balance in stem cells. An increasing number of reports indicate the implication of Nups in many central nervous system functions with great impact on neurogenesis, neurophysiology, and neurological disorders. Nevertheless, the role of Nup-mediated epigenetic regulation in embryonic and adult neural stem cells (NSCs) is a field largely unexplored and the comprehension of their mechanisms of action is only beginning to be unveiled. After a brief overview of epigenetic mechanisms, we will present and discuss the emerging role of Nups as new effectors of neuroepigenetics and as dynamic platform for chromatin function with specific reference to the biology of NSCs.
Assuntos
Células-Tronco Neurais , Complexo de Proteínas Formadoras de Poros Nucleares , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Células-Tronco Neurais/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
Consistent body of evidence shows that transcranial direct-current stimulation (tDCS) over the primary motor cortex (M1) facilitates motor learning and promotes recovery after stroke. However, the knowledge of molecular mechanisms behind tDCS effects needs to be deepened for a more rational use of this technique in clinical settings. Here we characterized the effects of anodal tDCS of M1, focusing on its impact on glutamatergic synaptic transmission and plasticity. Mice subjected to tDCS displayed increased long-term potentiation (LTP) and enhanced basal synaptic transmission at layer II/III horizontal connections. They performed better than sham-stimulated mice in the single-pellet reaching task and exhibited increased forelimb strength. Dendritic spine density of layer II/III pyramidal neurons was also increased by tDCS. At molecular level, tDCS enhanced: 1) BDNF expression, 2) phosphorylation of CREB, CaMKII, and GluA1, and 3) S-nitrosylation of GluA1 and HDAC2. Blockade of nitric oxide synthesis by L-NAME prevented the tDCS-induced enhancement of GluA1 phosphorylation at Ser831 and BDNF levels, as well as of miniature excitatory postsynaptic current (mEPSC) frequency, LTP and reaching performance. Collectively, these findings demonstrate that anodal tDCS engages plasticity mechanisms in the M1 and highlight a role for nitric oxide (NO) as a novel mediator of tDCS effects.
Assuntos
Córtex Motor/fisiologia , Plasticidade Neuronal/fisiologia , Óxido Nítrico/fisiologia , Transdução de Sinais/fisiologia , Estimulação Transcraniana por Corrente Contínua/métodos , Animais , Eletrodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Motor/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Técnicas de Cultura de Órgãos , Transdução de Sinais/efeitos dos fármacosRESUMO
Spike timing-dependent plasticity (STDP) is a form of activity-dependent remodeling of synaptic strength that underlies memory formation. Despite its key role in dictating learning rules in the brain circuits, the molecular mechanisms mediating STDP are still poorly understood. Here, we show that spike timing-dependent long-term depression (tLTD) and A-type K+ currents are modulated by pharmacological agents affecting the levels of active glycogen-synthase kinase 3 (GSK3) and by GSK3ß knockdown in layer 2/3 of the mouse somatosensory cortex. Moreover, the blockade of A-type K+ currents mimics the effects of GSK3 up-regulation on tLTD and occludes further changes in synaptic strength. Pharmacological, immunohistochemical and biochemical experiments revealed that GSK3ß influence over tLTD induction is mediated by direct phosphorylation at Ser-616 of the Kv4.2 subunit, a molecular determinant of A-type K+ currents. Collectively, these results identify the functional interaction between GSK3ß and Kv4.2 channel as a novel mechanism for tLTD modulation providing exciting insight into the understanding of GSK3ß role in synaptic plasticity.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Neurônios/fisiologia , Canais de Potássio Shal/metabolismo , Córtex Somatossensorial/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Fosforilação , Córtex Somatossensorial/metabolismoRESUMO
OBJECTIVES: The purpose of this study was to determine the effects of the Nutrition and Culinary in the Kitchen (NCK) Program to evaluate the improvement of cooking skills and healthy eating of university students. METHODS: A randomized controlled trial with six months follow-up was used incorporating an intervention group (IG) and a control group (CG). The IG participated in the NCK program over a six-week period, three hours weekly, based on five hands-on cooking classes and one food market visit, while CG continued their usual activities. Outcome measures included changes in relation to a) accessibility and availability of fruits and vegetables (AAFV); b) cooking attitudes (CA); c) cooking behaviors at home (CBH); d) cooking behaviors away from home (CBAH) e) produce consumption self-efficacy (SEPC); f) self-efficacy for using cooking techniques (SECT); g) self-efficacy for using fruits, vegetables, and seasonings (while cooking) (SEFVS); and h) knowledge of cooking terms and techniques (CTT). An online self-completed validated survey was answered, at three time points: baseline (T1), after intervention (T2) and six months after intervention (T3). Statistical analyses were conducted to evaluate changes in outcomes within and between groups over time. RESULTS: 76 students completed the online questionnaire at the 3 time points. Findings revealed a statistically significant increase (pâ¯<â¯0.05) in all outcomes evaluated in the IG, except for CBH and CBAH. This effect was sustained at T3 (pâ¯<â¯0.001). Results were similar to the adjusted model (pâ¯<â¯0.001), thus, indicating that IG changes, when compared to CG, were due to the intervention effect. CONCLUSION: NCK culinary intervention program demonstrated efficacy for the increased on AAFV, CA, SEPC, SECT, SEFVS and CTT with university students.
Assuntos
Culinária , Dieta Saudável , Autoeficácia , Brasil , Feminino , Seguimentos , Frutas , Humanos , Aprendizagem , Masculino , Estudantes , Inquéritos e Questionários , Verduras , Adulto JovemRESUMO
BACKGROUND: Community-based intervention studies that aim at developing cooking skills have increased in the scientific literature and are related to healthier food practices. However, methodological limitations are observed and only a few studies have university students as the target. The university entrance period has been related to negative changes in eating habits among young people and it represents an important period for developing interventions for health promotion. This study describes the study protocol and the evaluation framework for the Nutrition and Culinary in the Kitchen program. This program aims to develop cooking skills in university students, and is based on the Cooking with a Chef program in the United States. METHODS: This ongoing, randomized controlled intervention was designed with a six month follow-up study. The intervention consisted of three-hour weekly classes during a six week period with printed materials provided. Five of the classes were hands-on cooking and one was a tour to a popular food market. There were eight primary outcome measures: changes in relation to i) accessibility and availability of fruits and vegetables; ii) cooking attitudes; iii) cooking behaviors at home; iv) cooking behaviors away from home; v) produce consumption self-efficacy; vi) self-efficacy for using basic cooking techniques; vii) self-efficacy for using fruits, vegetables, and seasonings (while cooking); and viii) knowledge of cooking terms and techniques. Secondary outcomes included changes in body mass index and in personal characteristics related to cooking. Repeated measures were collected through the application of an online self-completed survey, at baseline, after intervention and six months after intervention. A sample of 80 university students (40: intervention group; 40: control group) was estimated to detect a mean change of 1.5 points in cooking knowledge, with study power of 80%, and 95% level of confidence, plus 20% for random losses and 10% for confounding factors. The control group participants have continued with their usual activities. Data analyses will evaluate the intervention effect on changes in outcomes within and between groups, as well as explore relations with personal characteristics. DISCUSSION: This method provides new evidence about whether or not a culinary intervention targeting university students has an impact on the improvement of cooking skills and healthy eating practices. TRIAL REGISTRATION: Brazilian Clinical Trials Registry - RBR-8nwxh5 ( http://www.ensaiosclinicos.gov.br/rg/RBR-8nwxh5/ ).
Assuntos
Culinária/métodos , Dieta Saudável , Educação em Saúde , Estudantes , Universidades , Brasil , Dietética/educação , Comportamento Alimentar , Alimentos , Frutas , Conhecimentos, Atitudes e Prática em Saúde , Promoção da Saúde , Humanos , Fenômenos Fisiológicos da Nutrição , Ciências da Nutrição/educação , VerdurasRESUMO
Communication between cardiomyocytes depends upon gap junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylase (HAT) and deacetylase (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5 Hz for 24 h significantly reduced connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation. In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43.
Assuntos
Comunicação Celular/genética , Conexina 43/biossíntese , Junções Comunicantes/genética , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Acetilação/efeitos dos fármacos , Ácidos Anacárdicos/administração & dosagem , Animais , Conexina 43/genética , Cães , Estimulação Elétrica , Junções Comunicantes/patologia , Ventrículos do Coração/patologia , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/patologia , RNA Mensageiro/biossínteseRESUMO
In a mouse model of skin repair we found that the class I-IIa histone deacetylase inhibitor trichostatin A accelerated tissue regeneration. Unexpectedly, this effect was suppressed by Sirtinol, a class III histone deacetylase (HDAC) (sirtuin)-selective inhibitor. The role of sirtuins (SIRTs) was then investigated by using resveratrol and a novel SIRT1-2-3 activator, the MC2562 compound we synthesized recently. Both resveratrol and MC2562 were effective in accelerating wound repair. The local administration of natural or synthetic SIRT activators, in fact, significantly accelerated skin regeneration by increasing keratinocyte proliferation. In vitro experiments revealed that the activation of SIRTs stimulated keratinocyte proliferation via endothelial NO synthase phosphorylation and NO production. In this condition, the class I member HDAC2 was found S-nitrosylated on cysteine, a post-transduction modification associated with loss of activity and DNA binding capacity. After deacetylase inhibitor or SIRT activator treatment, ChIP showed, in fact, a significant HDAC2 detachment from the promoter region of insulin growth factor I (IGF-I), fibroblast growth factor 10 (FGF-10), and Epithelial Growth Factor (EGF), which may be the final recipients and effectors of the SIRT-NO-HDAC signaling cascade. Consistently, the effect of SIRT activators was reduced in the presence of NG-nitro-L-arginine methyl ester (L-NAME), a general inhibitor of NO synthesis. In conclusion, the NO-dependent cross-talk among class III and I histone deacetylases suggests an unprecedented signaling pathway important for skin repair.
Assuntos
Histona Desacetilases do Grupo III/metabolismo , Histona Desacetilase 2/metabolismo , Óxido Nítrico/metabolismo , Pele/enzimologia , Pele/lesões , Cicatrização/fisiologia , Animais , Linhagem Celular Transformada , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Fator 10 de Crescimento de Fibroblastos/metabolismo , Histona Desacetilases do Grupo III/antagonistas & inibidores , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Cicatrização/efeitos dos fármacosRESUMO
Histone deacetylase inhibitors (DIs) are promising drugs for the treatment of several pathologies including ischemic and failing heart where they demonstrated efficacy. However, adverse side effects and cardiotoxicity have also been reported. Remarkably, no information is available about the effect of DIs during tissue regeneration following acute peripheral ischemia. In this study, mice made ischemic by femoral artery excision were injected with the DIs MS275 and MC1568, selective for class I and IIa histone deacetylases (HDACs), respectively. In untreated mice, soon after damage, class IIa HDAC phosphorylation and nuclear export occurred, paralleled by dystrophin and neuronal nitric-oxide synthase (nNOS) down-regulation and decreased protein phosphatase 2A activity. Between 14 and 21 days after ischemia, dystrophin and nNOS levels recovered, and class IIa HDACs relocalized to the nucleus. In this condition, the MC1568 compound increased the number of newly formed muscle fibers but delayed their terminal differentiation, whereas MS275 abolished the early onset of the regeneration process determining atrophy and fibrosis. The selective DIs had differential effects on the vascular compartment: MC1568 increased arteriogenesis whereas MS275 inhibited it. Capillarogenesis did not change. Chromatin immunoprecipitations revealed that class IIa HDAC complexes bind promoters of proliferation-associated genes and of class I HDAC1 and 2, highlighting a hierarchical control between class II and I HDACs during tissue regeneration. Our findings indicate that class-selective DIs interfere with normal mouse ischemic hindlimb regeneration and suggest that their use could be limited by alteration of the regeneration process in peripheral ischemic tissues.
Assuntos
Benzamidas/efeitos adversos , Membro Posterior/irrigação sanguínea , Inibidores de Histona Desacetilases/efeitos adversos , Ácidos Hidroxâmicos/efeitos adversos , Isquemia , Músculo Esquelético , Piridinas/efeitos adversos , Pirróis/efeitos adversos , Regeneração/efeitos dos fármacos , Animais , Benzamidas/farmacologia , Distrofina/metabolismo , Membro Posterior/metabolismo , Membro Posterior/patologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Óxido Nítrico Sintase Tipo I/metabolismo , Proteína Fosfatase 2/metabolismo , Piridinas/farmacologia , Pirróis/farmacologia , Fatores de TempoRESUMO
Wanting to explore the epigenetic basis of Duchenne cardiomyopathy, we found that global histone acetylase activity was abnormally elevated and the acetylase P300/CBP-associated factor (PCAF) coimmunoprecipitated with connexin 43 (Cx43), which was N(ε)-lysine acetylated and lateralized in mdx heart. This observation was paralleled by Cx43 dissociation from N-cadherin and zonula occludens 1, whereas pp60-c-Src association was unaltered. In vivo treatment of mdx with the pan-histone acetylase inhibitor anacardic acid significantly reduced Cx43 N(ε)-lysine acetylation and restored its association to GAP junctions (GJs) at intercalated discs. Noteworthy, in normal as well as mdx mice, the class IIa histone deacetylases 4 and 5 constitutively colocalized with Cx43 either at GJs or in the lateralized compartments. The class I histone deacetylase 3 was also part of the complex. Treatment of normal controls with the histone deacetylase pan-inhibitor suberoylanilide hydroxamic acid (MC1568) or the class IIa-selective inhibitor 3-{4-[3-(3-fluorophenyl)-3-oxo-1-propen-1-yl]-1-methyl-1H-pyrrol-2-yl}-N-hydroxy-2-propenamide (MC1568) determined Cx43 hyperacetylation, dissociation from GJs, and distribution along the long axis of ventricular cardiomyocytes. Consistently, the histone acetylase activator pentadecylidenemalonate 1b (SPV106) hyperacetylated cardiac proteins, including Cx43, which assumed a lateralized position that partly reproduced the dystrophic phenotype. In the presence of suberoylanilide hydroxamic acid, cell to cell permeability was significantly diminished, which is in agreement with a Cx43 close conformation in the consequence of hyperacetylation. Additional experiments, performed with Cx43 acetylation mutants, revealed, for the acetylated form of the molecule, a significant reduction in plasma membrane localization and a tendency to nuclear accumulation. These results suggest that Cx43 N(ε)-lysine acetylation may have physiopathological consequences for cell to cell coupling and cardiac function.
Assuntos
Cardiomiopatias/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Lisina/metabolismo , Distrofia Muscular de Duchenne/complicações , Miócitos Cardíacos/metabolismo , Acetilação/efeitos dos fármacos , Ácidos Anacárdicos/farmacologia , Animais , Cardiomiopatias/etiologia , Histona Acetiltransferases/metabolismo , Ácidos Hidroxâmicos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos mdx , Microscopia de Fluorescência , Vorinostat , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
OBJECTIVE: To address the role of epigenetic enzymes in the process of arterial vasorelaxation and nitrate tolerance, in vitro and in vivo experiments were performed in the presence or absence of glyceryl trinitrate (GTN) or histone deacetylases/histone acetylases modulators. METHODS AND RESULTS: In vitro single GTN administration rapidly increased cGMP synthesis and protein N(ε)-lysine acetylation in rat smooth muscle cells, including myosin light chain and smooth muscle actin. This phenomenon determined a decrease in myosin light chain phosphorylation and actomyosin formation. These effects were abolished by prolonged exposure to GTN and rescued by treatment with trichostatin A. In vivo, adult male rats were treated for 72 hours with subcutaneous injections of GTN alone or in combination with the histone deacetylases inhibitors trichostatin A, suberoylanilide hydroxamic acid, MS-27-275, or valproic acid. Ex vivo experiments performed on aortic rings showed that the effect of tolerance was reversed by all proacetylation drugs, including the p300/CREB binding protein-associated factor activator pentadecylidenemalonate 1b (SPV106). Any response to GTN was abolished by anacardic acid, a potent histone acetylases inhibitor. CONCLUSIONS: This study establishes the following points: (1) GTN treatment increases histone acetylases activity; (2) GTN-activated p300/CREB binding protein-associated factor increases protein N(ε)-lysine acetylation; (3) N(ε)-lysine acetylation of contractile proteins influences GTN-dependent vascular response. Hence, combination of epigenetic drugs and nitroglycerin may be envisaged as a novel treatment strategy for coronary artery disease symptoms and other cardiovascular accidents of ischemic origin.
Assuntos
Actinas/metabolismo , Aorta/fisiologia , Lisina/metabolismo , Cadeias Leves de Miosina/metabolismo , Nitroglicerina/metabolismo , Vasodilatação/fisiologia , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Aorta/efeitos dos fármacos , Benzamidas/farmacologia , GMP Cíclico/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Injeções Subcutâneas , Masculino , Modelos Animais , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Nitroglicerina/administração & dosagem , Nitroglicerina/farmacologia , Piridinas/farmacologia , Ratos , Ácido Valproico/farmacologia , Fatores de Transcrição de p300-CBP/efeitos dos fármacosRESUMO
The purpose of this exploratory, descriptive, and cross-sectional study was to investigate the relationship between the price and availability of food products with and without trans fatty acids in food stores near elementary schools located in low- and medium-income neighborhoods of a Brazilian city. The supply of products containing trans fatty acids was higher in both regions, and these products were also cheaper. It is noteworthy that this availability may influence food choices and, consequently, the health status of children and adolescents, since this population is more likely to buy less-healthy foods when these are more available, accessible, and financially attractive.
Assuntos
Comércio , Dieta/economia , Abastecimento de Alimentos/economia , Renda , Características de Residência , Instituições Acadêmicas , Ácidos Graxos trans/economia , Dieta/normas , Abastecimento de Alimentos/normas , Humanos , PobrezaRESUMO
OBJECTIVE: Smad-interacting protein-1 (Sip1/ZEB2) is a transcriptional repressor of the telomerase reverse transcriptase catalytic subunit (Tert) and has recently been identified as a key regulator of embryonic cell fate with a phenotypic effect similar, in our opinion, to that reported for nitric oxide (NO). Remarkably, SIP1/ZEB2 is a known target of the microRNA 200 (miR-200) family. In this light, we postulated that Sip1/ZEB2 and the miR-200 family could play a role during the NO-dependent differentiation of mES. METHODS AND RESULTS: The results of the present study show that Sip1/ZEB2 expression is downregulated during the NO-dependent expression of mesendoderm and early cardiovascular precursor markers, including Flk1 and CXCR4 in mES. Coincidently, members of the miR-200 family, namely miR-429, -200a, -200b, and -200c, were transcriptionally induced in parallel to mouse Tert. This regulation occurred at the level of chromatin. Remarkably, miR-429/miR-200a overexpression or Sip1/ZEB2 knockdown by short hairpin RNA interference elicited a gene expression pattern similar to that of NO regardless of the presence of leukemia inhibitory factor. CONCLUSIONS: These results are the first demonstrating that the miR-200 family and Sip1/ZEB2 transcription factor are regulated by NO, indicating an unprecedented molecular circuitry important for telomerase regulation and early differentiation of mES.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Óxido Nítrico/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Montagem e Desmontagem da Cromatina , Células-Tronco Embrionárias/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fator Inibidor de Leucemia/metabolismo , Camundongos , Doadores de Óxido Nítrico/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Telomerase/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.