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1.
Nat Commun ; 11(1): 640, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005806

RESUMO

Reduced food intake is common to many pathological conditions, such as infection and toxin exposure. However, cortical circuits that mediate feeding responses to these threats are less investigated. The anterior insular cortex (aIC) is a core region that integrates interoceptive states and emotional awareness and consequently guides behavioral responses. Here, we demonstrate that the right-side aIC CamKII+ (aICCamKII) neurons in mice are activated by aversive visceral signals. Hyperactivation of the right-side aICCamKII neurons attenuates food consumption, while inhibition of these neurons increases feeding and reverses aversive stimuli-induced anorexia and weight loss. Similar manipulation at the left-side aIC does not cause significant behavioral changes. Furthermore, virus tracing reveals that aICCamKII neurons project directly to the vGluT2+ neurons in the lateral hypothalamus (LH), and the right-side aICCamKII-to-LH pathway mediates feeding suppression. Our studies uncover a circuit from the cortex to the hypothalamus that senses aversive visceral signals and controls feeding behavior.


Assuntos
Agentes Aversivos/toxicidade , Córtex Cerebral/fisiologia , Comportamento Alimentar/efeitos dos fármacos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/efeitos dos fármacos , Feminino , Região Hipotalâmica Lateral/metabolismo , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vias Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo
2.
Int J Mol Sci ; 20(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614708

RESUMO

RNA binding motif 20 (RBM20) is a key regulator of pre-mRNA splicing of titin and other genes that are associated with cardiac diseases. Hormones, like insulin, triiodothyronine (T3), and angiotensin II (Ang II), can regulate gene-splicing through RBM20, but the detailed mechanism remains unclear. This study was aimed at investigating the signaling mechanism by which hormones regulate pre-mRNA splicing through RBM20. We first examined the role of RBM20 in Z-, I-, and M-band titin splicing at different ages in wild type (WT) and RBM20 knockout (KO) rats using RT-PCR; we found that RBM20 is the predominant regulator of I-band titin splicing at all ages. Then we treated rats with propylthiouracil (PTU), T3, streptozotocin (STZ), and Ang II and evaluated the impact of these hormones on the splicing of titin, LIM domain binding 3 (Ldb3), calcium/calmodulin-dependent protein kinase II gamma (Camk2g), and triadin (Trdn). We determined the activation of mitogen-activated protein kinase (MAPK) signaling in primary cardiomyocytes treated with insulin, T3, and Ang II using western blotting; MAPK signaling was activated and RBM20 expression increased after treatment. Two downstream transcriptional factors c-jun and ETS Transcription Factor (ELK1) can bind the promoter of RBM20. A dual-luciferase activity assay revealed that Ang II, but not insulin and T3, can trigger ELK1 and thus promote transcription of RBM20. This study revealed that Ang II can trigger ELK1 through activation of MAPK signaling by enhancing RBM20 expression which regulates pre-mRNA splicing. Our study provides a potential therapeutic target for the treatment of cardiac diseases in RBM20-mediated pre-mRNA splicing.


Assuntos
Angiotensina II/farmacologia , Sistema de Sinalização das MAP Quinases , Processamento de RNA , Proteínas de Ligação a RNA/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Proteínas Elk-1 do Domínio ets/metabolismo
3.
Artif Cells Nanomed Biotechnol ; 47(1): 2948-2956, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31317779

RESUMO

Neurotoxicity of local anesthetics is often reported in the clinic, more and more people pay attention to them. CaMKIIß, a subtype of CaMKII, is detected in the central nervous system. Previous study found that CaMKIIß mRNA are up-regulated in DRG neurons treated with ropivacaine hydrochloride, as well as inhibition of Cav3.2 and Cav3.3 expression can improve the local anesthetics neurotoxicity. In this study, we observed the effect of CaMKIIß on neurotoxicity injury induced by ropivacaine hydrochloride with DRG cell in vitro. We first constructed the pAd-shRNA-CaMKIIß-DRG to inhibit CaMKIIß mRNA expression and detected the cell viability, cell apoptosis rate, CaMKIIß, Cav3.2 and Cav3.3 expression. The results showed that ropivacaine hydrochloride caused the DRG cell injury with cell viability decreased and cell apoptosis rate increased, CaMKIIß, Cav3.2 and Cav3.3 expression up-regulated. Interestingly, inhibition of CaMKIIß expression protected the DRG cell from the neurotoxicity injury induced by ropivacaine hydrochloride, increased the cell viability and decreased the apoptosis rate, as well as inhibition of CaMKIIß expression down-regulated Cav3.2 and Cav3.3 expression. In other words, CaMKIIß is involved with the DRG injury induced by ropivacaine hydrochloride. Inhibition CaMKIIß expression improved DRG injury, increased the cell viability and decreased cell apoptosis rate.


Assuntos
Anestésicos Locais/toxicidade , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Gânglios Espinais/citologia , Neurotoxinas/toxicidade , Ropivacaina/toxicidade , Animais , Apoptose/efeitos dos fármacos , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Sobrevivência Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley
4.
Int J Mol Sci ; 20(15)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31344789

RESUMO

All-trans retinoic acid (AtRA), an active metabolite of vitamin A, is recognized for its classical action as an endocrine hormone that triggers genomic effects mediated through nuclear receptors RA receptors (RARs). New evidence shows that atRA-mediated cellular responses are biphasic with rapid and delayed responses. Most of these rapid atRA responses are the outcome of its binding to cellular retinoic acid binding protein 1 (CRABP1) that is predominantly localized in cytoplasm and binds to atRA with a high affinity. This review summarizes the most recent studies of such non-genomic outcomes of atRA and the role of CRABP1 in mediating such rapid effects in different cell types. In embryonic stem cells (ESCs), atRA-CRABP1 dampens growth factor sensitivity and stemness. In a hippocampal neural stem cell (NSC) population, atRA-CRABP1 negatively modulates NSC proliferation and affects learning and memory. In cardiomyocytes, atRA-CRABP1 prevents over-activation of calcium-calmodulin-dependent protein kinase II (CaMKII), protecting heart function. These are supported by the fact that CRABP1 gene knockout (KO) mice exhibit multiple phenotypes including hippocampal NSC expansion and spontaneous cardiac hypertrophy. This indicates that more potential processes/signaling pathways involving atRA-CRABP1 may exist, which remain to be identified.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células-Tronco Neurais/metabolismo , Receptores do Ácido Retinoico/genética , Tretinoína/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Proliferação de Células/genética , Humanos , Aprendizagem/fisiologia , Memória/fisiologia , Camundongos , Miócitos Cardíacos
5.
Mol Cell ; 75(1): 13-25.e5, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31151856

RESUMO

Arc is a synaptic protein essential for memory consolidation. Recent studies indicate that Arc originates in evolution from a Ty3-Gypsy retrotransposon GAG domain. The N-lobe of Arc GAG domain acquired a hydrophobic binding pocket in higher vertebrates that is essential for Arc's canonical function to weaken excitatory synapses. Here, we report that Arc GAG also acquired phosphorylation sites that can acutely regulate its synaptic function. CaMKII phosphorylates the N-lobe of the Arc GAG domain and disrupts an interaction surface essential for high-order oligomerization. In Purkinje neurons, CaMKII phosphorylation acutely reverses Arc's synaptic action. Mutant Arc that cannot be phosphorylated by CaMKII enhances metabotropic receptor-dependent depression in the hippocampus but does not alter baseline synaptic transmission or long-term potentiation. Behavioral studies indicate that hippocampus- and amygdala-dependent learning requires Arc GAG domain phosphorylation. These studies provide an atomic model for dynamic and local control of Arc function underlying synaptic plasticity and memory.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células de Purkinje/metabolismo , Sequência de Aminoácidos , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/metabolismo , Animais , Sítios de Ligação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Técnicas de Introdução de Genes , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Células de Purkinje/citologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sinapses/fisiologia , Transmissão Sináptica
6.
Int J Mol Sci ; 20(12)2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31226788

RESUMO

This study reports the identification of splice variants for the calcium/calmodulin-dependent protein kinase II (CaMKII) gene from Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera. CaMKII is a multifunctional serine/threonine protein kinase that transduces Ca2+ signals in cells to control a range of cellular processes in the nervous system and muscular tissue. Sequence analysis showed that CaMKII was 99.0% identical at the amino acid level among three rice planthoppers, with the exception of a variable region located in the association domain. Four kinds of 20-81 amino acid "inserts" were found in the variable region. The phylogenetic tree of the deduced amino acid sequences showed that the NlCaMKII isoforms were more closely related to the LsCaMKII isoforms and were slightly distinct from SfCaMKII. CaMKII-E was the dominant type among the five main isoforms. CaMKII genes were constitutively expressed in various nymphal and adult stages and in tested tissues with the predominant transcription occurring in the head. There was no major tissue specificity of isoform expression, but the expression pattern and relative abundance of isoforms varied when compared with the RT-PCR between tissues. In addition, RNAi in N. lugens with dsRNA at a concentration of 200 ng nymph-1 induced a mortality of 77.7% on the 10th day and a reduction in the mRNA expression level of 67.2%. Unlike the holometabolous insect Helicoverpa armigera, the knockdown of NlCaMKII did not suppress the expression of 20E response genes, such as ECR, USP1, and HR3, in N. lugens. These results indicate that the role of CaMKII in hemimetabolous insects may be different from that in holometabolous insects.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Hemípteros/genética , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Genes de Insetos , Hemípteros/química , Proteínas de Insetos/química , Oryza/parasitologia , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Alinhamento de Sequência
7.
BMC Neurosci ; 20(1): 26, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182018

RESUMO

BACKGROUND: Animal responses to thermal stimuli involve intricate contributions of genetics, neurobiology and physiology, with temperature variation providing a pervasive environmental factor for natural selection. Thermal behavior thus exemplifies a dynamic trait that requires non-trivial phenotypic summaries to appropriately capture the trait in response to a changing environment. To characterize the deterministic and plastic components of thermal responses, we developed a novel micro-droplet assay of nematode behavior that permits information-dense summaries of dynamic behavioral phenotypes as reaction norms in response to increasing temperature (thermal tolerance curves, TTC). RESULTS: We found that C. elegans TTCs shift predictably with rearing conditions and developmental stage, with significant differences between distinct wildtype genetic backgrounds. Moreover, after screening TTCs for 58 C. elegans genetic mutant strains, we determined that genes affecting thermosensation, including cmk-1 and tax-4, potentially play important roles in the behavioral control of locomotion at high temperature, implicating neural decision-making in TTC shape rather than just generalized physiological limits. However, expression of the transient receptor potential ion channel TRPA-1 in the nervous system is not sufficient to rescue rearing-dependent plasticity in TTCs conferred by normal expression of this gene, indicating instead a role for intestinal signaling involving TRPA-1 in the adaptive plasticity of thermal performance. CONCLUSIONS: These results implicate nervous system and non-nervous system contributions to behavior, in addition to basic cellular physiology, as key mediators of evolutionary responses to selection from temperature variation in nature.


Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Canais Iônicos/fisiologia , Locomoção/fisiologia , Canal de Cátion TRPA1/fisiologia , Sensação Térmica/fisiologia , Adaptação Fisiológica/genética , Animais , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Temperatura Alta , Canais Iônicos/genética , Estágios do Ciclo de Vida/fisiologia , Mutação , Sistema Nervoso/metabolismo , Canal de Cátion TRPA1/biossíntese
8.
Mol Med Rep ; 20(2): 863-870, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173191

RESUMO

CaMKII is a calcium­activated kinase, proved to be modulated by oxidation. Currently, the oxidative activation of CaMKII exists in several models of asthma, chronic rhinosinusitis with nasal polyps, cardiovascular disease, diabetes mellitus, acute ischemic stroke and cancer. Oxidized CaMKII (ox­CaMKII) may be important in several of these diseases. The present review examines the mechanism underlying the oxidative activation of CaMKII and summarizes the current findings associated with the function of ox­CaMKII in inflammatory diseases. Taken together, the findings of this review aim to improve current understanding of the function of ox­CaMKII and provide novel insights for future research.


Assuntos
Asma/enzimologia , Isquemia Encefálica/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Doenças Cardiovasculares/enzimologia , Diabetes Mellitus/enzimologia , Pólipos Nasais/enzimologia , Neoplasias/enzimologia , Sinusite/enzimologia , Animais , Asma/tratamento farmacológico , Asma/genética , Asma/patologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Ativação Enzimática , Expressão Gênica , Humanos , Pólipos Nasais/tratamento farmacológico , Pólipos Nasais/genética , Pólipos Nasais/patologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Oxirredução , Estresse Oxidativo , Inibidores de Proteínas Quinases/uso terapêutico , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Sinusite/tratamento farmacológico , Sinusite/genética , Sinusite/patologia
9.
Nat Commun ; 10(1): 2784, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239443

RESUMO

CaMKIIα plays an essential role in decoding Ca2+ signaling in spines by acting as a leaky Ca2+ integrator with the time constant of several seconds. However, the mechanism by which CaMKIIα integrates Ca2+ signals remains elusive. Here, we imaged CaMKIIα-CaM association in single dendritic spines using a new FRET sensor and two-photon fluorescence lifetime imaging. In response to a glutamate uncaging pulse, CaMKIIα-CaM association increases in ~0.1 s and decays over ~3 s. During repetitive glutamate uncaging, which induces spine structural plasticity, CaMKIIα-CaM association did not show further increase but sustained at a constant level. Since CaMKIIα activity integrates Ca2+ signals over ~10 s under this condition, the integration of Ca2+ signal by CaMKIIα during spine structural plasticity is largely due to Ca2+/CaM-independent, autonomous activity. Based on these results, we propose a simple kinetic model of CaMKIIα activation in dendritic spines.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Espinhas Dendríticas/enzimologia , Animais , Cálcio/química , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Calmodulina/química , Calmodulina/metabolismo , Espinhas Dendríticas/genética , Ativação Enzimática , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos C57BL
10.
Biomed Pharmacother ; 117: 109134, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31247466

RESUMO

Proto-oncogene Myc, a key transcription factor, is frequently deregulated in human leukemia with aggressive and poor clinical outcome, but the development of MYC inhibitors remains challenging due to MYC helix-loop-helix topology lacking druggable domains. Here we describe a novel oral active small molecule analog of berbamine, tosyl chloride-berbamine (TCB), that efficiently eliminates MYC-positive leukemia in vitro and in vivo. Mechanistically, TCB potently reduced MYC protein by inhibiting CaMKIIγ, a critical enzyme that stabilizes MYC protein, and induces apoptosis of MYC-positive leukemia cells. In vivo, oral administration of TCB markedly eliminated lethal MYC-positive acute lymphoblastic leukemia (ALL) with well tolerability in orthotopic mouse model. Our studies identify CaMKIIγ/Myc axis as a valid target for developing small molecule-based new therapies for treating MYC-mediated leukemia and demonstrate that TCB is an orally active analog of berbamine that kills MYC-positive leukemia cells.


Assuntos
Benzilisoquinolinas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Leucemia/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Compostos de Tosil/farmacologia , Administração Oral , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzilisoquinolinas/administração & dosagem , Benzilisoquinolinas/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Leucemia/enzimologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Compostos de Tosil/administração & dosagem , Compostos de Tosil/química , Transcrição Genética/efeitos dos fármacos
11.
J Exp Clin Cancer Res ; 38(1): 225, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138329

RESUMO

BACKGROUND: Triple-negative breast cancer (TNBC) is often aggressive and associated with a poor prognosis. Due to the lack of available targeted therapies and to problems of resistance with conventional chemotherapeutic agents, finding new treatments for TNBC remains a challenge and a better therapeutic strategy is urgently required. METHODS: TNBC cells and xenograft mice were treated with a combination of chloroquine (CQ) and isorhamnetin (IH). Mitochondrial fission, apoptosis, and related signaling pathways were determined by flow cytometry, immunofluorescence, and related molecular biological techniques. RESULTS: The inhibition of autophagy/mitophagy by CQ selectively enhances IH-induced mitochondrial fission and apoptosis in TNBC cells but not in estrogen-dependent breast cancer cells. These events were accompanied by mitochondrial translocation of Bax and the release of cytochrome c. Mechanistically, these effects were associated with oxidative stress-mediated phosphorylation of CaMKII (Thr286) and Drp1 (S616), and subsequent mitochondrial translocation of CaMKII and Drp1. The interruption of the CaMKII pathway by genetic approaches (e.g. CaMKII mutant or siRNA) attenuated combination-mediated mitochondrial fission and apoptosis. The combination of CQ/IH was a marked inhibitor tumor growth, inducing apoptosis in the TNBC xenograft mouse model in association with the activation of CaMKII and Drp1 (S616). CONCLUSIONS: Our study highlights the critical role of ROS-mediating CaMKII/Drp1 signaling in the regulation of mitochondrial fission and apoptosis induced by combination of CQ/IH. These findings also suggest that IH could potentially be further developed as a novel chemotherapeutic agent. Furthermore, a combination of IH with classic autophagy/mitophagy inhibitor could represent a novel therapeutic strategy for the treatment of TNBC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cloroquina/administração & dosagem , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Quercetina/análogos & derivados , Espécies Reativas de Oxigênio/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Feminino , Humanos , Camundongos , Dinâmica Mitocondrial/efeitos dos fármacos , Quercetina/administração & dosagem , Quercetina/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Neurochem Res ; 44(7): 1613-1620, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30919283

RESUMO

Ischemic stroke is a leading cause of mortality and morbidity worldwide, and oxidative stress plays a significant role in the ischemia stage and reperfusion stage. Previous studies have indicated that both calcium/calmodulin-dependent protein kinase II (CaMKII) and glucose 6-phosphate dehydrogenase (G6PD) are involved in the oxidative stress. Thus, the aim of this study was to investigate the roles of CaMKIIα, an important isoform of CaMKII, and G6PD in a rat model of middle cerebral artery occlusion (MCAO). Intracerebroventricular injection of small interfering ribonucleic acid (siRNA) for CaMKIIα was performed at 48 h pre-MCAO surgery. Immunofluorescence Staining and western blot were performed to detect the expression of p-CaMKIIα and G6PD in the cortices. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was performed to investigate the infarct volume. In addition, neurological deficit, reactive oxygen species (ROS), ratio of reduced-to-oxidized glutathione (GSH/GSSG) and ratio of reduced-to-oxidized oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) were assessed. The results indicated that both p-CaMKIIα and G6PD were widely located in the neurons and astrocytes, and their expression was gradually increased in the cortices after MCAO, which was accompanied by increased level of ROS and decreased levels of GSH/GSSG and NADPH/NADP+. However, after treatment with siRNA for CaMKIIα, p-CaMKIIα expression was decreased and G6PD expression was increased. Moreover, inhibition of CaMKIIα improved the neurological deficit, reduced the infarct volume, decreased the level of ROS and increased the levels of GSH/GSSG and NADPH/NADP+. The results suggested that CaMKIIα inhibition exerted neuroprotective effects through regulating G6PD expression, which provides a new target for prevention and treatment of stroke.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Estresse Oxidativo/fisiologia , Animais , Astrócitos/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Inativação Gênica , Dissulfeto de Glutationa/metabolismo , Masculino , NADP/metabolismo , Neurônios/metabolismo , RNA Interferente Pequeno/genética , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
13.
Mol Cell Neurosci ; 95: 79-85, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30763690

RESUMO

Trisomy 21, also known as Down syndrome (DS), is the most frequent genetic cause of intellectual impairment. In mouse models of DS, deficits in hippocampal synaptic plasticity have been observed, in conjunction with alterations to local dendritic translation that are likely to influence plasticity, learning and memory. Here we show that expression of a local translational regulator, the Cytoplasmic Polyadenylation Element Binding Protein 1 (CPEB1), is enhanced in hippocampal neurons from the Ts1Cje DS mouse model. Interestingly, this protein, which is also involved in dendritic mRNA transport, is overexpressed in dendrites of neurons derived from DS human induced pluripotent stem cells (hIPSCs). Moreover, there is an increase in the mRNA levels of α-Calmodulin Kinase II (α-CaMKII) and Microtubule-associated protein 1B (MAP1B), two dendritic mRNAs, in Ts1Cje synaptoneurosomes. Taking into account the fundamental role of CPEB1 protein and its target mRNAs in synaptic plasticity, these data could be relevant to the intellectual impairment in the context of DS.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Síndrome de Down/metabolismo , Hipocampo/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dendritos/metabolismo , Síndrome de Down/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/citologia
14.
Gene ; 697: 131-137, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-30802539

RESUMO

Misuse of anabolic androgenic steroids (AAS) increases prevalence of cardiovascular abnormalities in athletes, and the underlying molecular mechanism involved in those abnormalities continues to be investigated. The aim of this study was to investigate the effect of chronic nandrolone exposure on alpha and beta-myosin heavy chain (MHC) isoforms gene expression transition, blood pressure related parameters, calcium/calmodulin-dependent protein kinaseIIδ (CaMKIIδ), and monoamine oxidase (MAO) activities in rats' hearts. It was also planned to evaluate the effect of strenuous exercise on cardiac abnormalities induced by nandrolone. Thirty-two male wistar rats were assigned into four groups, namely control, nandrolone, nandrolone with strenuous exercise, and strenuous exercise groups. Nandrolone consumption significantly increased systolic, diastolic, pulse and dicrotic pressure, mean arterial pressure, as well as the amplitude of first peak (H1). Moreover, exercise combined with nandrolone completely masked this effect. The mRNA expression of ß-MHC and the ratio of ß -MHC/α -MHC showed a significant increase in the nandrolone and nandrolone with strenuous exercise groups compared to those in the control group. The values of heart tissue calcium/calmoldulin-dependent protein kinase IIδ (CaMKIIδ), and monoamine oxidase (MAO) in the nandrolone, nandrolone with strenuous exercise and exercise groups were significantly higher than those values in the control group. These findings indicate that nandrolone-induced heart and hemodynamic abnormalities may in part be associated with MHC isoform changes and Ca2+ homeostasis changes mediated by increased CaMKIIδ and MAO activities and that these effects can be provoked via strenuous exercise.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/efeitos dos fármacos , Monoaminoxidase/efeitos dos fármacos , Nandrolona/farmacologia , Animais , Pressão Arterial , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Frequência Cardíaca , Hipertensão/tratamento farmacológico , Masculino , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/efeitos dos fármacos , Nandrolona/administração & dosagem , Condicionamento Físico Animal/fisiologia , Isoformas de Proteínas , Ratos , Ratos Wistar
15.
Anesth Analg ; 129(2): 608-617, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30303867

RESUMO

BACKGROUND: Propofol can cause degeneration of developing brain cells and subsequent long-term learning or memory impairment. However, at the early stage of embryonic development, the molecular mechanism of propofol-induced inhibition in neural stem cells (NSCs) neurogenesis is still unclear. The aim of this study was to determine the role of propofol in NSCs neurogenesis and, more importantly, to explore the underlying mechanism. METHODS: First, a single intraperitoneal injection of propofol was performed in pregnant mice, and 6 hours after administration of propofol, the hippocampus RNA and the protein of the embryos' brains was extracted to analyze the expression of neuron-specific markers. Second, the primary NSCs were isolated from the hippocampus of mouse embryonic brain and then treated with propofol for cell viability, immunostaining, and transwell assays; more importantly, we performed RNA sequencing (RNA-seq) and q-reverse transcription polymerase chain reaction assays to identify genes regulated by propofol; the Western blot, small interfering RNA (SiRNA), and luciferase reporter assays were used to study the effects of propofol on calmodulin-dependent protein kinase (CaMk) II/5' adenosine monophosphate-activated protein kinase (AMPK)/activating transcription factor 5 (ATF5) signaling pathway. RESULTS: Our results indicated that propofol treatment could inhibit the proliferation, migration, and differentiation of NSCs. The results of RNA-seq assays showed that propofol treatment resulted in downregulation of a group of Ca-dependent genes. The following mechanism studies showed that propofol regulates the proliferation, differentiation, and migration of NSCs through the CaMkII/phosphorylation of serine at amino acid position 485 (pS485)/AMPK/ATF5 signaling pathway. CONCLUSIONS: The results from study demonstrated that propofol inhibits the proliferation, differentiation, and migration of NSCs, and these effects are partially mediated by CaMkII/pS485/AMPK/ATF5 signaling pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fatores Ativadores da Transcrição/metabolismo , Anestésicos Intravenosos/toxicidade , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proliferação de Células/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Propofol/toxicidade , Proteínas Quinases Ativadas por AMP/genética , Fatores Ativadores da Transcrição/genética , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica , Hipocampo/enzimologia , Hipocampo/patologia , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Transdução de Sinais
16.
Psychiatr Genet ; 29(1): 18-25, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30260900

RESUMO

OBJECTIVES: Susceptibility to heroin dependence is strongly influenced by genetic factors with heritability estimates as high as 0.7. A number of genes, as well as environmental factors, are likely to contribute to its etiology. Not all individuals who have ever tried heroin at some stage during their lifetime become dependent on heroin. It has been suggested that genetic factors might be more important in the transition stage to heroin dependence rather than in environmental exposures and experimenting with heroin. As the features of substance dependence and memory formation have been found to be strikingly similar, we have focused on a key enzyme involved in long-term potentiation and synaptic plasticity, namely the calcium-dependent/calmodulin-dependent protein kinase IIα (CAMKIIa). We hypothesized, that CamK2A genetic variation may play a role in the transition from occasional to regular heroin use. MATERIALS AND METHODS: Using quantitative trait association analysis, we addressed this hypothesis by correlating the self-reported time interval between occasional and regular heroin use with the frequency of 12 single nucleotide polymorphisms located within the genomic region of the CamK2A gene. A sample of 570 Caucasian patients was available for analysis. RESULTS: Single marker association analysis (rs10066581, P=0.007), as well as haplotype analysis (global P=0.005), suggested an association with the quantitative trait 'time interval from occasional to regular heroin use.' CONCLUSION: Our results propose that genetic variants located in the genomic region of the CamK2A gene may be involved in transition time from occasional to regular heroin use.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Dependência de Heroína/genética , Adolescente , Adulto , Austrália , Estudos de Casos e Controles , Feminino , Frequência do Gene/genética , Estudos de Associação Genética/métodos , Predisposição Genética para Doença/genética , Variação Genética/genética , Genótipo , Haplótipos/genética , Heroína/efeitos adversos , Humanos , Desequilíbrio de Ligação/genética , Masculino , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Adulto Jovem
17.
Biochim Biophys Acta Mol Basis Dis ; 1865(1): 218-229, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30463690

RESUMO

Pressure overload-induced cardiac hypertrophy occurs in response to chronic blood pressure increase, and dysfunction of CaV1.2 calcium channel involves in cardiac hypertrophic processes by perturbing intracellular calcium concentration ([Ca2+]i) and calcium-dependent signaling. As a carbohydrate-binding protein, galectin-1 (Gal-1) is found to bind with CaV1.2 channel, which regulates vascular CaV1.2 channel functions and blood pressure. However, the potential roles of Gal-1 in cardiac CaV1.2 channel (CaV1.2CM) and cardiomyocyte hypertrophy remain elusive. By whole-cell patch clamp, we find Gal-1 decreases the ICa,L with or without isoproterenol (ISO) application by reducing the channel membrane expression in neonatal rat ventricular myocytes (NRVMs). Moreover, Gal-1 could inhibit the current densities of CaV1.2CM by an alternative exon 9*-dependent manner in heterologously expressed HEK293 cells. Of significance, overexpression of Gal-1 diminishes ISO or KCl-induced [Ca2+]i elevation and attenuates ISO-induced hypertrophy in NRVMs. Mechanistically, Gal-1 decreases the ISO or Bay K8644-induced phosphorylation of intracellular calcium-dependent signaling proteins δCaMKII and HDAC4, and inhibits ISO-triggered translocation of HDAC4 in NRVMs. Pathologically, we observe that the expressions of Gal-1 and CaV1.2E9* channels are synchronously increased in rat hypertrophic cardiomyocytes and hearts. Taken together, our study indicates that Gal-1 reduces the channel membrane expression to inhibit the currents of CaV1.2CM in a splice-variant specific manner, which diminishes [Ca2+]i elevation, and attenuates cardiomyocyte hypertrophy by inhibiting the phosphorylation of δCaMKII and HDAC4. Furthermore, our work suggests that dysregulated Gal-1 and CaV1.2 alternative exon 9* might be attributed to the pathological processes of cardiac hypertrophy, and provides a potential anti-hypertrophic target in the heart.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Galectina 1/antagonistas & inibidores , Galectina 1/metabolismo , Miócitos Cardíacos/metabolismo , Processamento de RNA , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Pressão Sanguínea , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Modelos Animais de Doenças , Células HEK293 , Histona Desacetilases/metabolismo , Humanos , Isoproterenol/farmacologia , Proteínas de Membrana , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fosforilação , Ratos
18.
Behav Brain Res ; 359: 903-909, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29935919

RESUMO

Autistic spectrum disorders (ASDs) are neurodevelopmental disorders for which genetic components have been well defined. However, specific gene deregulations related to synapse function in the autistic brain have not been as extensively described. Based on a candidate genes approach, we present in this study the expression data of 4 transcripts of interest (BDNF, CAMK2a, NR-CAM and RIMS1) located at the synapse in two regions of interest in the context of the ASDs; the lobule VI of cerebellum and the Brodmann area 46. We have also genotyped in our cohort the coding single nucleotide polymorphism rs6265, located in the BDNF gene. After correction for age and sex, whereas no change was observed in the lobule VI between controls and autistic patients, we found a significant increase of BDNF expression level in the BA46 from autistic patients. No significant interaction between the rs6265 genotype and autism was observed for the BDNF expression. However, "A" allele carriers are more likely to have increased BDNF levels. Finally, we found a significant positive correlation between BDNF and RIMS1 expression levels. Our data suggest that these two molecules which are involved in cell signalling at the synapse, might have coordinated expressions and, that BDNF regulation in the brain has to be investigated further in the context of ASDs.


Assuntos
Transtorno Autístico/patologia , Fator Neurotrófico Derivado do Encéfalo/genética , Lobo Frontal/metabolismo , Regulação da Expressão Gênica/fisiologia , RNA Mensageiro/metabolismo , Adolescente , Adulto , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Criança , Pré-Escolar , Diagnóstico , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Genótipo , Humanos , Microdissecção e Captura a Laser , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Adulto Jovem
19.
Glia ; 67(2): 246-262, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30565755

RESUMO

Astrocytes express a complex repertoire of intracellular Ca2+ transients (events) that represent a major form of signaling within individual cells and in astrocytic syncytium. These events have different spatiotemporal profiles, which are modulated by neuronal activity. Spontaneous Ca2+ events appear more frequently in distal astrocytic processes and independently from each other. However, little is known about the mechanisms underlying such subcellular distribution of the Ca2+ events. Here, we identify the initiation points of the Ca2+ events within the territory of single astrocytes expressing genetically encoded Ca2+ indicator GCaMP2 in culture or in hippocampal slices. We found that most of the Ca2+ events start in an optimal range of thin distal processes. Our mathematical model demonstrated that a high surface-to-volume of the thin processes leads to increased amplitude of baseline Ca2+ fluctuations caused by a stochastic opening of Ca2+ channels in the plasma membrane. Suprathreshold fluctuations trigger Ca2+ -induced Ca2+ release from the Ca2+ stores by activating inositol 1,4,5-trisphosphate (IP3 ) receptors. In agreement with the model prediction, the spontaneous Ca2+ events frequency depended on the extracellular Ca2+ concentration. Astrocytic depolarization by high extracellular K+ increased the frequency of the Ca2+ events through activation of voltage-gated Ca2+ channels in cultured astrocytes. Our results suggest that the morphological profile of the astrocytic processes is responsible for tuning of the Ca2+ events frequency. Therefore, structural plasticity of astrocytic processes can be directly translated into changes in astrocytic Ca2+ signaling. This may be important for both physiological and pathological astrocyte remodeling.


Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Benzilaminas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Hipocampo/citologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ácidos Fosfínicos/farmacologia , Ratos , Ratos Wistar , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Transfecção
20.
Biochem Biophys Res Commun ; 508(4): 1227-1232, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30554655

RESUMO

Hepatocellular carcinoma (HCC) is the most prevalent malignancy in liver and a leading cause of cancer-related deaths. Despite the pressing need for treatment options, patients with HCC develop significant resistance and adverse side effects to current approved drugs that becomes a major barrier to effective treatment. A natural product Tetrandrine (TET) is a potential alternative treatment option for HCC, with demonstrated effectiveness and low toxicity. However, the mechanisms by which Tetrandrine inhibits HCC are unclear. In the current study, we identify Ca2+/calmodulin-dependent protein kinase II δ (CaMKIIδ) as a potential TET drug target through structural modeling. Screening of a panel of HCC cell lines reveal differential sensitivities toward TET treatment. Interestingly, IC50 of TET inhibition of HCC cell proliferation is positively correlated with CaMKIIδ expression level in these distinct HCC cells. Furthermore, TET treatment resulted in a marked reduction of CaMKIIδ phosphorylation level, and knockdown of CaMKIIδ reduced the sensitivity of HCC cells to TET. Most importantly, CaMKIIδ protein levels in high-grade human HCC samples were significantly elevated as compared to normal liver tissues. Taken together, our studies demonstrate that the natural compound TET targets CaMKIIδ in HCC cells, and that CaMKIIδ level is a potential biomarker to identify HCC patient populations sensitive to Tetrandrine treatment.


Assuntos
Benzilisoquinolinas/uso terapêutico , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Terapia de Alvo Molecular , Benzilisoquinolinas/química , Benzilisoquinolinas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Modelos Moleculares , Estadiamento de Neoplasias , Fosforilação/efeitos dos fármacos
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