RESUMO
Prefrontal control of cognitive functions critically depends upon glutamatergic transmission and N-methyl D-aspartate (NMDA) receptors, the activity of which is regulated by dopamine. Yet whether the NMDA receptor coagonist d-serine is implicated in the dopamine-glutamate dialogue in the prefrontal cortex (PFC) and other brain areas remains unexplored. Here, using electrophysiological recordings, we show that d-serine is required for the fine-tuning of glutamatergic neurotransmission, neuronal excitability, and synaptic plasticity in the PFC through the actions of dopamine at D1 and D3 receptors. Using in vivo microdialysis, we show that D1 and D3 receptors exert a respective facilitatory and inhibitory influence on extracellular levels and activity of d-serine in the PFC, with actions expressed primarily via the cAMP/protein kinase A (PKA) signaling cascade. Further, using functional magnetic resonance imaging (fMRI) and behavioral assessment, we show that d-serine is required for the potentiation of cognition by D3R blockade as revealed in a test of novel object recognition memory. Collectively, these results unveil a key role for d-serine in the dopaminergic neuromodulation of glutamatergic transmission and PFC activity, findings with clear relevance to the pathogenesis and treatment of diverse brain disorders involving alterations in dopamine-glutamate cross-talk.
Assuntos
Dopamina/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo , Animais , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Knockout , Racemases e Epimerases/deficiência , Racemases e Epimerases/genética , Receptores Dopaminérgicos/metabolismo , Esquizofrenia , Transmissão Sináptica/efeitos dos fármacosRESUMO
Approximately 80% of breast cancer (BC) cases express the estrogen receptor (ER), and 30-40% of these cases acquire resistance to endocrine therapies over time. Hyperactivation of Akt is one of the mechanisms by which endocrine resistance is acquired. Apigenin (Api), a flavone found in several plant foods, has shown beneficial effects in cancer and chronic diseases. Here, we studied the therapeutic potential of Api in the treatment of ER-positive, endocrine therapy-resistant BC. To achieve this objective, we stably overexpressed the constitutively active form of the Akt protein in MCF-7 cells (named the MCF-7/Akt clone). The proliferation of MCF-7/Akt cells is partially independent of estradiol (E2) and exhibits an incomplete response to the anti-estrogen agent 4-hydroxytamoxifen, demonstrating the resistance of these cells to hormone therapy. Api exerts an antiproliferative effect on the MCF-7/Akt clone. Api inhibits the proliferative effect of E2 by inducing G2/M phase cell cycle arrest and apoptosis. Importantly, Api inhibits the Akt/FOXM1 signaling pathway by decreasing the expression of FOXM1, a key transcription factor involved in the cell cycle. Api also alters the expression of genes regulated by FOXM1, including cell cycle-related genes, particularly in the MCF-7/Akt clone. Together, our results strengthen the therapeutic potential of Api for the treatment of endocrine-resistant BC.
Assuntos
Apigenina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteína Forkhead Box M1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/antagonistas & inibidores , Linhagem Celular Tumoral , Células Endócrinas/efeitos dos fármacos , Células Endócrinas/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Células MCF-7 , Transdução de Sinais/efeitos dos fármacosRESUMO
At sub-anaesthetic doses, ketamine, a non competitive N-methyl-d-aspartate (NMDA) receptor antagonist, has demonstrated remarkable and rapid antidepressant (AD) efficacy in patients with treatment-resistant depression (TRD). However, its mechanism of action of ketamine is not fully understood. Since comorbid depression and anxiety disorders often occur, GABAergic/inhibitory and glutamatergic/excitatory drug treatments may be co-administered in these patients. Information regarding this combination is critical to establish efficacy or treatment restrictions to maximize translation from animal models to TRD patients, effectiveness and safety. To assess the specific role of excitatory/inhibitory neurotransmission in the medial prefrontal cortex-raphe nuclei (mPFC-DRN) circuit in the sustained antidepressant-like activity (AD) of ketamine (at t24h post dose), AMPA-R antagonist (intra-DRN) and GABAA-R agonist (intra-mPFC) were co-administered with ketamine (intra-mPFC). Twenty-four hours later, responses in the forced swim test (FST) and neurochemical consequences on extracellular mPFC glutamate, GABA and 5-HT levels were measured in BALB/cJ mice. Intra-DRN NBQX prevented the sustained AD-like activity of ketamine evidenced by decreases in FST swimming duration and blunted cortical 5-HText and Gluext. Intra-mPFC muscimol blocked ketamine AD-like activity and its effects on cortical 5-HText. Moreover, a selective glutamate transporter GLT-1 inhibitor, dihydrokainic acid (DHK) locally perfused into the mPFC produced an AD-like activity at t24h associated with robust increases in mPFC 5-HText, Gluext and GABAext. Thus, the sustained AD-like activity of ketamine is triggered by AMPA-R activation in the DRN and 5-HT - glutamate release in the mPFC, but limited by GABAA-R activation - GABA release in the mPFC. The local blockade of GLT-1 in the mPFC also mimics the rapid responses of ketamine, thus highlighting the role of neuronal-glial adaptation in these effects. These results also suggests the need to test for the concomitant prescription of ketamine and BZD to see whether its sustained antidepressant activity is maintained in TRD patients.
Assuntos
Antidepressivos/administração & dosagem , Depressão/tratamento farmacológico , Núcleo Dorsal da Rafe/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Ketamina/administração & dosagem , Neuroglia/metabolismo , Córtex Pré-Frontal/metabolismo , Receptores de AMPA/metabolismo , Receptores de GABA-A/metabolismo , Animais , Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Benzodiazepinas/administração & dosagem , Benzodiazepinas/farmacologia , Núcleo Dorsal da Rafe/efeitos dos fármacos , Transportador 2 de Aminoácido Excitatório/antagonistas & inibidores , Agonistas de Receptores de GABA-A/administração & dosagem , Agonistas de Receptores de GABA-A/farmacologia , Ácido Caínico/administração & dosagem , Ácido Caínico/análogos & derivados , Ácido Caínico/farmacologia , Ketamina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microdiálise , Neuroglia/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Quinoxalinas/administração & dosagem , Quinoxalinas/farmacologia , Receptores de AMPA/antagonistas & inibidores , Resultado do TratamentoRESUMO
Recent studies strongly support the use of the aryl hydrocarbon receptor (AhR) as a therapeutic target in breast cancer. Glyceollins, a group of soybean phytoalexins, are known to exert therapeutic effects in chronic human diseases and also in cancer. To investigate the interaction between glyceollin I (GI), glyceollin II (GII) and AhR, a computational docking analysis, luciferase assays, immunofluorescence and transcriptome analyses were performed with different cancer cell lines. The docking experiments predicted that GI and GII can enter into the AhR binding pocket, but their interactions with the amino acids of the binding site differ, in part, from those interacting with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Both GI and GII were able to weakly and partially activate AhR, with GII being more potent. The results from the transcriptome assays showed that approximately 10% of the genes regulated by TCDD were also modified by both GI and GII, which could have either antagonistic or synergistic effects upon TCDD activation. In addition, we report here, on the basis of phenotype, that GI and GII inhibit the migration of triple-negative (ER-, PgR-, HER2NEU-) MDA-MB-231 breast cancer cells, and that they inhibit the expression of genes which code for important regulators of cell migration and invasion in cancer tissues. In conclusion, GI and GII are AhR ligands that should be further investigated to determine their usefulness in cancer treatments.
Assuntos
Movimento Celular/efeitos dos fármacos , Pterocarpanos/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Pterocarpanos/química , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Ketamine, a non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptor, displays a fast antidepressant activity in treatment-resistant depression and in rodent models of anxiety/depression. A large body of evidence concerning the cellular and molecular mechanisms underlying its fast antidepressant-like activity comes from animal studies. Although structural remodeling of frontocortical/hippocampal neurons has been proposed as critical, the role of excitatory/inhibitory neurotransmitters in this behavioral effect is unclear. Neurochemical and behavioral changes are maintained 24h after ketamine administration, well beyond its plasma elimination half-life. Thus, ketamine is believed to initiate a cascade of cellular mechanisms supporting its fast antidepressant-like activity. To date, the underlying mechanism involves glutamate release, then downstream activation of AMPA receptors, which trigger mammalian target of rapamycin (mTOR)-dependent structural plasticity via brain-derived neurotrophic factor (BDNF) and protein neo-synthesis in the medial prefrontal cortex (mPFC), a brain region strongly involved in ketamine therapeutic effects. However, these mPFC effects are not restricted to glutamatergic pyramidal cells, but extend to other neurotransmitters (GABA, serotonin), glial cells, and brain circuits (mPFC/dorsal raphe nucleus-DRN). It could be also mediated by one or several ketamine metabolites (e.g., (2R,6R)-HNK). The present review focuses on evidence for mPFC neurotransmission abnormalities in major depressive disorder (MDD) and their potential impact on neural circuits (mPFC/DRN). We will integrate these considerations with results from recent preclinical studies showing that ketamine, at antidepressant-relevant doses, induces neuronal adaptations that involve the glutamate-excitatory/GABA-inhibitory balance. Our analyses will help direct future studies to further elucidate the mechanism of action of fast-acting antidepressant drugs, and to inform development of novel, more efficacious therapeutics.
Assuntos
Antidepressivos/administração & dosagem , Encéfalo/efeitos dos fármacos , Ketamina/administração & dosagem , Animais , Antidepressivos/farmacocinética , Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Ketamina/farmacocinética , Serotonina/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
Glyphosate is the most widely used herbicide in the world. Several studies have investigated the effects of glyphosate and glyphosate-based herbicides (GBHs) on male reproduction, but there is still little and conflicting evidence for its toxicity. In this study, we analyzed the effects of glyphosate, alone or in formula, on the male reproductive system. Pregnant mice were treated from E10.5 to 20 days postpartum by adding glyphosate or a GBH (Roundup 3 Plus) to their drinking water at 0.5 (the acceptable daily intake, ADI dose), 5 and 50 mg/kg/day. Male offspring derived from treated mice were sacrificed at 5, 20, and 35 days old (d.o.) and 8 months old (m.o.) for analysis. Our result showed that exposure to glyphosate, but not GBH, affects testis morphology in 20 d.o. and decrease serum testosterone concentrations in 35 d.o. males. We identified that the spermatozoa number decreased by 89% and 84% in 0.5 and 5 mg/kg/day of GBH and glyphosate groups, respectively. Moreover, the undifferentiated spermatogonia numbers were decreased by 60% in 5 mg/kg/day glyphosate group, which could be due to the alterations in the expression of genes involved in germ cell differentiation such as Sall4 and Nano3 and apoptosis as Bax and Bcl2. In 8 m.o. animals, a decreased testosterone level was observed in GBH groups. Our data demonstrate that glyphosate and GBHs could cause endocrine-disrupting effects on male reproduction at low doses. As glyphosate has effects at the ADI level, our data suggest that the current ADI for glyphosate could be overestimated.
Assuntos
Disruptores Endócrinos/toxicidade , Glicina/análogos & derivados , Herbicidas/toxicidade , Infertilidade Masculina/induzido quimicamente , Espermatogênese/efeitos dos fármacos , Espermatogônias/efeitos dos fármacos , Testículo/efeitos dos fármacos , Fatores Etários , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Glicina/toxicidade , Infertilidade Masculina/sangue , Infertilidade Masculina/patologia , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos , Nível de Efeito Adverso não Observado , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Medição de Risco , Contagem de Espermatozoides , Espermatogônias/metabolismo , Espermatogônias/patologia , Testículo/metabolismo , Testículo/patologia , Testículo/fisiopatologia , Testosterona/sangue , GlifosatoRESUMO
The flavone apigenin and the mycotoxin zearalenone are two major compounds found in the human diet which bind estrogen receptors (ERs), and therefore influence ER activity. However, the underlying mechanisms are not well known. To unravel the molecular mechanisms that could explain the differential effect of zearalenone and apigenin on ER-positive breast cancer cell proliferation, gene-reporter assays, chromatin immunoprecipitation (ChIP) experiments, proliferation assays and transcriptomic analysis were performed. We found that zearalenone and apigenin transactivated ERs and promoted the expression of estradiol (E2)-responsive genes. However, zearalenone clearly enhanced cellular proliferation, while apigenin appeared to be antiestrogenic in the presence of E2 in both ER-positive breast cancer cell lines, MCF-7 and T47D. The transcriptomic analysis showed that both compounds regulate gene expression in the same way, but with differences in intensity. Two major sets of genes were identified; one set was linked to the cell cycle and the other set was linked to stress response and growth arrest. Our results show that the transcription dynamics in gene regulation induced by apigenin were somehow different with zearalenone and E2 and may explain the differential effect of these compounds on the phenotype of the breast cancer cell. Together, our results confirmed the potential health benefit effect of apigenin, while zearalenone appeared to be a true endocrine-disrupting compound.
Assuntos
Apigenina/toxicidade , Neoplasias da Mama/metabolismo , Receptores de Estrogênio/metabolismo , Zearalenona/toxicidade , Linhagem Celular Tumoral , Estrogênios não Esteroides/toxicidade , Feminino , Humanos , Fitoestrógenos , Receptores de Estrogênio/genéticaRESUMO
Biologically active plant-based compounds, commonly referred to as phytochemicals, can influence the expression and function of various receptors and transcription factors or signaling pathways that play vital roles in cellular functions and are then involved in human health and diseases. Thus, phytochemicals may have a great potential to prevent and treat chronic diseases. Glyceollins, a group of phytoalexins that are isolated from soybeans, have attracted attention because they exert numerous effects on human functions and diseases, notably anticancer effects. In this review, we have presented an update on the effects of glyceollins in relation to their potential beneficial roles in human health. Despite a growing number of studies suggesting that this new family of phytochemicals can be involved in critical cellular pathways, such as estrogen receptor, protein kinase, and lipid kinase signaling pathways, future investigations will be needed to better understand their molecular mechanisms and their specific significance in biomedical applications.
Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Pterocarpanos/química , Pterocarpanos/farmacologia , Humanos , Compostos Fitoquímicos , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Glycine max/química , FitoalexinasRESUMO
In vivo measurement of multiple neurotransmitters is highly interesting but remains challenging in the field of neuroscience. GABA and l-glutamic acid are the major inhibitory and excitatory neurotransmitters, respectively, in the central nervous system, and their changes are related to a variety of diseases such as anxiety and major depressive disorder. This study described a simple method allowing the simultaneous LC-MS/MS quantification of l-glutamic acid, glutamine and GABA. Analytes were acquired from samples of the prefrontal cortex by microdialysis technique in freely moving mice. The chromatographic separation was performed by hydrophilic interaction liquid chromatography (HILIC) with a core-shell ammonium-sulfonic acid modified silica column using a gradient elution with mobile phases consisting of a 25â¯mM pH 3.5 ammonium formate buffer and acetonitrile. The detection of l-glutamic acid, glutamine and GABA, as well as the internal standards [d6]-GABA and [d5]-glutamate was performed on a triple quadrupole mass spectrometer in positive electrospray ionization and multiple reaction monitoring mode. The limit of quantification was 0.63â¯ng/ml for GABA, 1.25â¯ng/ml for l-glutamic acid and 3.15â¯ng/ml for glutamine, and the intra-day and inter-day accuracy and precision have been assessed for the three analytes. Therefore, the physiological relevance of the method was successfully applied for the determination of basal extracellular levels and potassium-evoked release of these neuroactive substances in the prefrontal cortex in adult awake C57BL/6 mice.
Assuntos
Lobo Frontal/química , Ácido Glutâmico/química , Glutamina/química , Ácido gama-Aminobutírico/química , Acetonitrilas/química , Compostos de Amônio/química , Animais , Cromatografia Líquida de Alta Pressão/métodos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microdiálise/métodos , Neurotransmissores/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Ácidos Sulfônicos/química , Espectrometria de Massas em Tandem/métodosAssuntos
Antidepressivos/uso terapêutico , Depressão/tratamento farmacológico , Ketamina/uso terapêutico , Neurotransmissores/metabolismo , Espermidina/análogos & derivados , Animais , Modelos Animais de Doenças , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microdiálise , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/fisiologia , Espermidina/uso terapêutico , Natação/psicologia , Fatores de TempoRESUMO
The dentate gyrus (DG) has distinct roles along its dorso-ventral axis. In the mouse, we recently demonstrated that dorsal DG (dDG) stimulation enhances exploratory behavior (Kheirbek et al., 2013). Dopamine (DA) release in the Nucleus Accumbens (NAcc), which belongs to the reward system, could be a key target of dDG mediating this motivation-related behavior. Here, an optogenetic stimulation of either ventral (vDG) or dDG granule cells was coupled with NAcc DA release monitoring using in vivo microdialysis. Only dDG stimulation enhanced NAcc DA release, indicating differential interconnections between dDG and vDG to the reward system.
Assuntos
Giro Denteado/citologia , Dopamina/metabolismo , Vias Neurais/fisiologia , Núcleo Accumbens/metabolismo , Optogenética , Transmissão Sináptica/fisiologia , Análise de Variância , Animais , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Estimulação Elétrica , Camundongos , Camundongos Transgênicos , Microdiálise , Núcleo Accumbens/citologia , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Transmissão Sináptica/genética , Fatores de TempoRESUMO
Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.
Assuntos
Agrobacterium tumefaciens/fisiologia , Aspergillus oryzae/genética , Genes Fúngicos , Genes Reporter , Vetores Genéticos/genética , Regiões Promotoras Genéticas , Engenharia de Proteínas , Proteínas Recombinantes/metabolismo , Transformação GenéticaRESUMO
BACKGROUND: Stress exposure is one of the greatest risk factors for psychiatric illnesses like major depressive disorder and posttraumatic stress disorder. However, not all individuals exposed to stress develop affective disorders. Stress resilience, the ability to experience stress without developing persistent psychopathology, varies from individual to individual. Enhancing stress resilience in at-risk populations could potentially protect against stress-induced psychiatric disorders. Despite this fact, no resilience-enhancing pharmaceuticals have been identified. METHODS: Using a chronic social defeat (SD) stress model, learned helplessness (LH), and a chronic corticosterone (CORT) model in mice, we tested if ketamine could protect against depressive-like behavior. Mice were administered a single dose of saline or ketamine and then 1 week later were subjected to 2 weeks of SD, LH training, or 3 weeks of CORT. RESULTS: SD robustly and reliably induced depressive-like behavior in control mice. Mice treated with prophylactic ketamine were protected against the deleterious effects of SD in the forced swim test and in the dominant interaction test. We confirmed these effects in LH and the CORT model. In the LH model, latency to escape was increased following training, and this effect was prevented by ketamine. In the CORT model, a single dose of ketamine blocked stress-induced behavior in the forced swim test, novelty suppressed feeding paradigm, and the sucrose splash test. CONCLUSIONS: These data show that ketamine can induce persistent stress resilience and, therefore, may be useful in protecting against stress-induced disorders.
Assuntos
Antidepressivos/administração & dosagem , Depressão/prevenção & controle , Ketamina/administração & dosagem , Resiliência Psicológica/efeitos dos fármacos , Estresse Psicológico/complicações , Animais , Ansiedade , Corticosterona/administração & dosagem , Depressão/etiologia , Medo/efeitos dos fármacos , Desamparo Aprendido , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Comportamento SocialRESUMO
The use of H(2), He and O(2) during batch fermentation of Saccharomyces cerevisiae BRAS291 increased the final intracellular glycogen contents of the cells from 2-fold to 10-fold compared with a gas-free condition, and this depended on the gas applied. Differently, the intracellular trehalose contents increased from 2-fold to 10-fold in reducing conditions compared with more oxidizing conditions. During storage at 4 degrees C, the viability of cells cultivated with gas was twice that of cells cultivated without gas. These results could be explained by the intracellular carbohydrate contents as well as yeast ultrastructural modifications observed previously.