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1.
PLoS Genet ; 16(8): e1009003, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866139

RESUMO

Sensory systems rely on neuromodulators, such as serotonin, to provide flexibility for information processing as stimuli vary, such as light intensity throughout the day. Serotonergic neurons broadly innervate the optic ganglia of Drosophila melanogaster, a widely used model for studying vision. It remains unclear whether serotonin modulates the physiology of interneurons in the optic ganglia. To address this question, we first mapped the expression patterns of serotonin receptors in the visual system, focusing on a subset of cells with processes in the first optic ganglion, the lamina. Serotonin receptor expression was found in several types of columnar cells in the lamina including 5-HT2B in lamina monopolar cell L2, required for spatiotemporal luminance contrast, and both 5-HT1A and 5-HT1B in T1 cells, whose function is unknown. Subcellular mapping with GFP-tagged 5-HT2B and 5-HT1A constructs indicated that these receptors localize to layer M2 of the medulla, proximal to serotonergic boutons, suggesting that the medulla neuropil is the primary site of serotonergic regulation for these neurons. Exogenous serotonin increased basal intracellular calcium in L2 terminals in layer M2 and modestly decreased the duration of visually induced calcium transients in L2 neurons following repeated dark flashes, but otherwise did not alter the calcium transients. Flies without functional 5-HT2B failed to show an increase in basal calcium in response to serotonin. 5-HT2B mutants also failed to show a change in amplitude in their response to repeated light flashes but other calcium transient parameters were relatively unaffected. While we did not detect serotonin receptor expression in L1 neurons, they, like L2, underwent serotonin-induced changes in basal calcium, presumably via interactions with other cells. These data demonstrate that serotonin modulates the physiology of interneurons involved in early visual processing in Drosophila.


Assuntos
Receptor 5-HT1B de Serotonina/genética , Receptores 5-HT1 de Serotonina/genética , Receptores 5-HT2 de Serotonina/genética , Neurônios Serotoninérgicos/metabolismo , Serotonina/metabolismo , Animais , Ritmo Circadiano/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica/genética , Interneurônios/metabolismo , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurotransmissores/genética , Receptores de Serotonina/genética , Serotonina/genética , Percepção Visual/genética
2.
Pharmacogenomics ; 20(17): 1199-1223, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31686592

RESUMO

Identifying biomarkers which can be used as a diagnostic tool is a major objective of pharmacogenetic studies. Most mental and many neurological disorders have a compiled multifaceted nature, which may be the reason why this endeavor has hitherto not been very successful. This is also true for tardive dyskinesia (TD), an involuntary movement complication of long-term treatment with antipsychotic drugs. The observed associations of specific gene variants with the prevalence and severity of a disorder can also be applied to try to elucidate the pathogenesis of the condition. In this paper, this strategy is used by combining pharmacogenetic knowledge with theories on the possible role of a dysfunction of specific cellular elements of neostriatal parts of the (dorsal) extrapyramidal circuits: various glutamatergic terminals, medium spiny neurons, striatal interneurons and ascending monoaminergic fibers. A peculiar finding is that genetic variants which would be expected to increase the neostriatal dopamine concentration are not associated with the prevalence and severity of TD. Moreover, modifying the sensitivity to glutamatergic long-term potentiation (and excitotoxicity) shows a relationship with levodopa-induced dyskinesia, but not with TD. Contrasting this, TD is associated with genetic variants that modify vulnerability to oxidative stress. Reducing the oxidative stress burden of medium spiny neurons may also be the mechanism behind the protective influence of 5-HT2 receptor antagonists. It is probably worthwhile to discriminate between neostriatal matrix and striosomal compartments when studying the mechanism of TD and between orofacial and limb-truncal components in epidemiological studies.


Assuntos
Discinesia Induzida por Medicamentos/genética , Estresse Oxidativo/efeitos dos fármacos , Esquizofrenia/tratamento farmacológico , Discinesia Tardia/genética , Antipsicóticos/efeitos adversos , Antipsicóticos/uso terapêutico , Dopamina/genética , Dopamina/metabolismo , Discinesia Induzida por Medicamentos/patologia , Fármacos Atuantes sobre Aminoácidos Excitatórios/efeitos adversos , Fármacos Atuantes sobre Aminoácidos Excitatórios/uso terapêutico , Humanos , Neostriado/efeitos dos fármacos , Neostriado/patologia , Farmacogenética , Células Piramidais/efeitos dos fármacos , Células Piramidais/patologia , Receptores 5-HT2 de Serotonina/genética , Esquizofrenia/complicações , Esquizofrenia/genética , Esquizofrenia/patologia , Antagonistas do Receptor 5-HT2 de Serotonina/efeitos adversos , Antagonistas do Receptor 5-HT2 de Serotonina/uso terapêutico , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Discinesia Tardia/induzido quimicamente , Discinesia Tardia/patologia
3.
Cell Rep ; 27(7): 1960-1966.e6, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091436

RESUMO

JC polyomavirus (JCPyV) is a ubiquitous human pathogen that causes progressive multifocal leukoencephalopathy (PML). The entry receptors for JCPyV belong to the 5-hydroxytryptamine 2 receptor (5-HT2R) family, but how individual members of the family function to facilitate infection is not known. We used proximity ligation assay (PLA) to determine that JCPyV interacts with each of the 5-HT2 receptors (5-HT2Rs) in a narrow window of time during entry. We used CRISPR-Cas9 to randomly introduce stop codons in the gene for each receptor and discovered that the second intracellular loop of each was necessary for infection. This loop contains a motif possibly involved in receptor internalization by ß-arrestin. Mutation of this motif and small interfering RNA (siRNA) knockdown of ß-arrestin recapitulated the results of our CRISPR-Cas9 screen, showing that this motif is critical. Our results have implications for the role these receptors play in virus infection and for their normal functioning as receptors for serotonin.


Assuntos
Vírus JC/genética , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Internalização do Vírus , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus JC/patogenicidade , beta-Arrestinas/genética , beta-Arrestinas/metabolismo
4.
Brain Res ; 1715: 21-26, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30880116

RESUMO

Lower urinary tract (LUT) dysfunction is the most common complication of diabetes mellitus (DM). An involvement of the 5-HT2A receptor in spinal micturition control has been demonstrated in urethane anaesthetized DM rats in which i.v. administration of the 5-HT2A/2C receptor agonist 2,5-methoxy-4-iodoamphetamine (DOI), stimulated high frequency oscillations (HFOs) and improved micturition. However, the mechanisms involved in these effects are not completely understood. The present work showed that 5-HT2A and -2C receptors were upregulated in lumbosacral cord motoneurons, and the number of serotonergic paraneurons were downregulated in the urethra in DM group. The importance of the downregulation of urethral paraneurons in DM remains to be elucidated but may be related to the reduced urethral sensation caused by the disease. We suggest that targets of 5-HT receptor agonists for improvement of voiding function may be found both in the LUT and lumbosacral spinal cord.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Receptores 5-HT2 de Serotonina/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Feminino , Região Lombossacral/fisiopatologia , Neurônios Motores/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Receptores 5-HT2 de Serotonina/genética , Neurônios Serotoninérgicos/metabolismo , Serotonina/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Medula Espinal/efeitos dos fármacos , Estreptozocina/farmacologia , Ativação Transcricional/efeitos dos fármacos , Uretra/fisiopatologia , Bexiga Urinária/fisiopatologia
5.
PLoS One ; 14(1): e0203980, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30695038

RESUMO

Despite the conserved function of aggression across taxa in obtaining critical resources such as food and mates, serotonin's (5-HT) modulatory role on aggressive behavior appears to be largely inhibitory for vertebrates but stimulatory for invertebrates. However, critical gaps exist in our knowledge of invertebrates that need to be addressed before definitively stating opposing roles for 5-HT and aggression. Specifically, the role of 5-HT receptor subtypes are largely unknown, as is the potential interactive role of 5-HT with other neurochemical systems known to play a critical role in aggression. Similarly, the influence of these systems in driving sex differences in aggressive behavior of invertebrates is not well understood. Here, we investigated these questions by employing complementary approaches in a novel invertebrate model of aggression, the stalk-eyed fly. A combination of altered social conditions, pharmacological manipulation and 5-HT2 receptor knockdown by siRNA revealed an inhibitory role of this receptor subtype on aggression. Additionally, we provide evidence for 5-HT2's involvement in regulating neuropeptide F activity, a suspected inhibitor of aggression. However, this function appears to be stage-specific, altering only the initiation stage of aggressive conflicts. Alternatively, pharmacologically increasing systemic concentrations of 5-HT significantly elevated the expression of the neuropeptide tachykinin, which did not affect contest initiation but instead promoted escalation via production of high intensity aggressive behaviors. Notably, these effects were limited solely to males, with female aggression and neuropeptide expression remaining unaltered by any manipulation that affected 5-HT. Together, these results demonstrate a more nuanced role for 5-HT in modulating aggression in invertebrates, revealing an important interactive role with neuropeptides that is more reminiscent of vertebrates. The sex-differences described here also provide valuable insight into the evolutionary contexts of this complex behavior.


Assuntos
Agressão/fisiologia , Comportamento Animal/fisiologia , Dípteros/fisiologia , Caracteres Sexuais , 5-Hidroxitriptofano/administração & dosagem , 5-Hidroxitriptofano/farmacologia , Agressão/efeitos dos fármacos , Animais , Técnicas de Observação do Comportamento/métodos , Comportamento Animal/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Masculino , Modelos Animais , Neuropeptídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/metabolismo , Serotonina/metabolismo , Taquicininas/metabolismo
6.
Asian J Psychiatr ; 39: 178-184, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30389402

RESUMO

The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in DRD2 and 5-HT2 receptor genes are associated with schizophrenia in North Indian population. Four hundred forty-three patients who met ICD10-DCR criteria for schizophrenia were enrolled from six participating centers along with 443 genetically related healthy subjects and 150 genetically unrelated healthy participants. A total of 7 gene polymorphisms from DRD2 (rs1800497, rs1079597, rs1800498, rs1801028) and 5-HT2 A (rs6313, rs6311, rs6305) were genotyped for their association with schizophrenia. No significant difference was found in frequency of various genotypes and alleles of the studied markers for DRD2 and 5-HT2 A genes between the cases and their genetic controls. However, significant differences were noted for rs1079597 genotype (Taq1B; p = 0.039) and its allele frequencies (p = 0.029) in persons with schizophrenia and the unrelated healthy controls. The DRD2 (Taq1 A-B-D) and 5-HT2 A (rs6311-rs6313-rs6305) haplotype frequencies differed significantly for A2B1D2 [p = 0.038; OR = 0.685 (95%CI = 0.479-0.981)] and ACC [p = 0.001; OR = 0.621 (95%CI = 0.461-0.838)] for the cases vs genetically related healthy controls. Similarly, significant difference was observed for the frequencies of GCC [p = 0.006; OR = 0.692 (95%CI = 0.532-0.900)] and ACC [p < 0.001; OR = 3.622 (95%CI = 1.73-7.585)] in the cases and unrelated healthy controls. Unlike previous research from India as well as abroad, the predominance of B1 allele of rs1079597 in patients with schizophrenia and absence of Cys311 in all study participants is a salient difference. Concluding, the B2 allele of rs1079597 may increase the risk of schizophrenia while the A2B1D2 haplotype may be protective in North Indian population.


Assuntos
Polimorfismo Genético/genética , Receptores de Dopamina D2/genética , Receptores 5-HT2 de Serotonina/genética , Esquizofrenia/genética , Adolescente , Adulto , Idoso , Feminino , Predisposição Genética para Doença/genética , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
7.
Cell Physiol Biochem ; 48(6): 2409-2428, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30121645

RESUMO

BACKGROUND/AIMS: Previously, we confirmed that liver-synthesized 5-HT rather than non-liver 5-HT, acting on the 5-HT2 receptor (5-HT2R), modulates lipid-induced excessive lipid synthesis (ELS). Here, we further revealed the effects of the hepatocellular 5-HT system in diabetes-related disorders. METHODS: Studies were conducted in male ICR mice, human HepG2 cells, and primary mouse hepatocytes (PMHs) under gene or chemical inhibition of the 5-HT system, key lipid metabolism, and inflammation-related factors. Protein and messenger RNA expression and levels of the factors were determined via western blotting, reverse transcription PCR, and quantitative assay kits, respectively. Hepatic steatosis with inflammation and fibrosis, intracellular lipid droplet accumulation (LDA), and reactive oxygen species (ROS) location were determined via hematoxylin and eosin, Masson's trichrome, Oil red O, and fluorescent-specific staining, respectively. RESULTS: Palmitic acid induced the activation of the 5-HT system: the activation of 5-HT2R, primarily 5-HT2AR, in addition to upregulating monoamine oxidase A (MAO-A) expression and 5-HT synthesis, by activating the G protein/ phospholipase C pathway modulated PKCε activation, resulting in ELS with LDA; the activation of NF-κB, which mediates the generation of pro-inflammatory cytokines, was primarily due to ROS generation in the mitochondria induced by MAO-A-catalyzed 5-HT degradation, and secondarily due to the activation of PKCε. These effects of the 5-HT system were also detected in palmitic acid- or high glucose-treated PMHs and regulated multiple inflammatory signaling pathways. In diabetic mice, co-treatment with antagonists of both 5-HT synthesis and 5-HT2R significantly abolished hepatic steatosis, inflammation, and fibrosis as well as hyperglycemia and dyslipidemia. CONCLUSION: Activation of the hepatocellular 5-HT system plays a crucial role in inducing diabetes-related hepatic dysfunction and is a potential therapeutic target.


Assuntos
Citocinas/metabolismo , Receptores 5-HT2 de Serotonina/metabolismo , Serotonina/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Monoaminoxidase/química , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Ácido Palmítico/farmacologia , Proteína Quinase C-épsilon/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores 5-HT2 de Serotonina/química , Receptores 5-HT2 de Serotonina/genética , Serotonina/farmacologia , Triptofano Hidroxilase/antagonistas & inibidores , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo
8.
J Child Adolesc Psychopharmacol ; 28(10): 711-718, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29975559

RESUMO

Objective: Fluoxetine is an effective and well-tolerated pharmacological treatment for children and adolescents with major depressive disorder (MDD). However, a high percentage of patients do not respond. There is a substantial genetic contribution to this variable clinical outcome. Based on previous genetic results of our group and given the lack of pharmacogenetics studies of antidepressant response with a long follow-up period, we evaluated the influence of single nucleotide polymorphisms (SNPs) in genes related to the serotonergic pathway on remission and recovery in children and adolescents diagnosed with MDD after 12 months of initiating fluoxetine treatment. Methods: The assessment was performed in 46 patients. All of them were visited at least once a month during the 12-month follow-up. Psychiatrists interviewed patients and their parents to explore clinical improvement. A total of 75 genotyped SNPs in 10 candidate genes were included in the genetic association analysis with remission and recovery. Bonferroni correction for multiple testing was applied to avoid false positive results. Results: The HTR2A rs7997012 SNP was significantly associated after Bonferroni correction with clinical improvement. Particularly, the homozygotes for the major allele (GG) showed the highest percentage of remitters and the highest score reductions on the Clinical Global Impressions-Severity (CGI-S) scale. Moreover, although the results were on the border of statistical significance, the GG homozygotes also tended to experience fewer readmissions during the follow-up period Conclusions: These results provide more evidence of the involvement of genetic variants related to the serotonergic pathway in the antidepressant response. Studies with larger cohorts are needed to integrate all relevant variants into clinical predictors of antidepressant response.


Assuntos
Transtorno Depressivo Maior , Fluoxetina , Receptores 5-HT2 de Serotonina/genética , Adolescente , Antidepressivos/farmacocinética , Antidepressivos/uso terapêutico , Criança , Transtorno Depressivo Maior/diagnóstico , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/genética , Feminino , Fluoxetina/farmacocinética , Fluoxetina/uso terapêutico , Variação Genética , Humanos , Masculino , Farmacogenética , Polimorfismo de Nucleotídeo Único , Escalas de Graduação Psiquiátrica , Indução de Remissão/métodos , Resultado do Tratamento
9.
Rev. Hosp. Ital. B. Aires (2004) ; 38(1): 5-10, mar. 2018. tab.
Artigo em Inglês | LILACS | ID: biblio-1023455

RESUMO

The dysfunction in the serotoninergic neurotransmission has been classically associated with major depressive disorder (MDD); however, other pathways and processes seem to have a role in this illness, such as neurogenesis and related molecules: the Brain-Derived Neurotrophic Factor (BDNF) and the Apolipoprotein E (APOE). There are many reports that indicate an association between certain polymorphism in these genes and MDD. The aim of our study was to analyze the possible association between MDD and polymorphisms in HTR2A (5-hydroxytryptamine receptor 2A), BDNF and APOE genes in a sample of the Argentinean population previously studied for 2 polymorphisms in SLC6A4 (Solute Carrier Family 6 Member 4) gene. Five polymorphisms were studied (rs6311 and rs6313 in HTR2A; rs429358 and rs7412 in APOE, and rs6265 in BDNF) in 95 MDD patients and 107 non-related controls. No statistically significant differences were observed between groups when analyzing the association with a single marker using logistic regression; however, when a possible combinatory effect of the polymorphisms (including previously studied polymorphisms in SLC6A4 gene) was analyzed using a dominant model for the risk alleles, the genotypes L/S_10/12_G/A (OR=3.57(95%CI=1.43-8.93); p=0.004, adjusted p-value=0.01) in SLC6A4 and BDNF genes and L/S_10/12_T/C_3/3_G/A in SLC6A4, HTR2A, APOE and BDNF genes (OR=5.99(95%CI=1.66-21.56); p=0.002, adjusted p-value=0.07), were more prevalent in patients than in controls (20%vs.6% and 15%vs.3%, respectively). Even though it is necessary to replicate these findings in a larger population, our results suggest a possible interaction between molecules involved in neurogenesis (BDNF and APOE), serotoninergic neurotransmission (SLC6A4 and HTR2A) and the pathogenesis of MDD. (AU)


La disfunción en la neurotransmisión serotoninérgica ha sido clásicamente asociada con el trastorno depresivo mayor (TDM); sin embargo, otras vías y procesos parecerían tener un rol en esta enfermedad, como la neurogénesis y moléculas asociadas: el factor neurotrófico derivado del cerebro (BDNF) y la apoliproteína E (APOE). Existen reportes en los que se establecen asociaciones entre polimorfismos en estos genes y el TDM. El objetivo de nuestro trabajo fue analizar la posible asociación entre el TDM y polimorfismos en los genes HTR2A (receptor 5-hidroxitriptamina 2A), BDNF y APOE en una muestra de la población argentina previamente estudiada para 2 polimorfismos en el gen SLC6A4 (transportador soluble familia 6 miembro 4). Se estudiaron 5 polimorfismos (rs6311 y rs6313 en HTR2A; rs429358 y rs7412 en APOE; rs6265 en BDNF) en 95 pacientes con TDM y 107 controles no relacionados. No se observaron diferencias significativas entre grupos al analizar la asociación por regresión logística con un único marcador; cuando se analizó el posible efecto combinatorio de polimorfismos (incluyendo los previamente estudiados para el gen SCL6A4) usando un modelo dominante para los alelos de riesgo, los genotipos L/S_10/12_G/A (OR=3,57(95%CI=1,43-8,93); p=0,004, valor-p-ajustado=0,01) en SLC6A4 y BDNF y L/S_10/12_T/C_3/3_G/A en SLC6A4, HTR2A, APOE y BDNF (OR=5,99(95%CI=1,66-21,56); p=0,002, valor-p-ajustado=0,07), fueron más prevalentes en pacientes que controles (20%vs.6% y 15%vs.3% respectivamente). Si bien es necesario replicar estos hallazgos en una población más grande, nuestros resultados sugieren una posible interacción entre moléculas involucradas en la neurogénesis (BDNF y APOE), la neurotransmisión serotoninérgica (SLC6A4 y HTR2A) y la patogenia de la depresión mayor. (AU)


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Adulto Jovem , Apolipoproteínas E/deficiência , Polimorfismo Genético , Fator Neurotrófico Derivado do Encéfalo/deficiência , Receptores 5-HT2 de Serotonina/deficiência , Transtorno Depressivo Maior/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Apolipoproteínas E/genética , Argentina/epidemiologia , Fator Neurotrófico Derivado do Encéfalo/genética , Receptores 5-HT2 de Serotonina/genética , Transtorno Depressivo Maior/patologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética
10.
Protein Pept Lett ; 25(4): 390-397, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29412102

RESUMO

BACKGROUND: The 5HT2A G-Protein Coupled Receptor (GPCR) is an important family of receptors involved in an array of neuromodulatory functions. Their dysregulation has been implicated in a number of psychiatric diseases. In spite of the importance of this GPCR, high resolution structure and mechanistic details of its function is unknown. Cholesterol plays an important role in the function of many receptors and reduced cholesterol levels can lead to disruption of serotonergic pathways. However, the role of cholesterol in the formation of GPCR oligomers has not been previously shown for this receptor. Given that receptor dimers have been shown to be the functional unit of this receptor, it is important to investigate the effect of cholesterol in the oligomeric state of 5HT2A receptor. OBJECTIVES: The main objective of this work is to clone, over-express and purify the 5HT2A receptor and investigate the effect of cholesterol in its oligomer formation. METHODS: The 5HT2A receptor (5HT2AR) DNA construct was subcloned into pFastBac-HT vector and the purified bacmid was used to transfect healthy Sf9 cells. After subsequent passages, a high titer baculovirus was used for over-expression in Sf9 cells. To verify whether the over-expressed receptor was localized in the membrane or cytosolic fraction, cells with and without baculoviral infection were analyzed by immunocytochemistry. Subsequently, the over-expression conditions required to obtain sufficient quantity of the receptor was optimized followed by the optimization of the purification conditions. Finally, the culture was scaled up and the receptor was purified by affinity chromatography. The over-expression of the receptor was checked by Western blotting and purity was analyzed by Coomassie stained SDS PAGE. Cryo-electron microscopy experiments were performed on the purified receptor in presence and absence of cholesterol and at multiple concentrations to rule out any concentration dependent effect on the oligomer formation. RESULTS: Immunocytochemistry experiments showed prominent nuclear staining; however, bright green staining along the cell membrane was observed only for the infected cells, suggesting appropriate trafficking of majority of the over-expressed receptors to the cell membrane. Results of cryoelectron microscopy show that the receptor with cholesterol had particles that were bigger in size (~11 - 12 nm) compared to the dimension of known GPCR homologs. In contrast, the receptor after removal of cholesterol revealed a uniform distribution of smaller particles (~5 - 6 nm) that is approximately half the size of 5HT2AR particles with cholesterol. Comparing the 2D average views of detergent-encapsulated 5HT2AR particles with the overall dimensions of other 5HT receptor analogs, we show that while a 5HT2AR dimer more closely matches the dimensions of particles with CHS, only a monomer can be fit to particles without CHS. Importantly, even at higher receptor concentration and particle density, the size for 5HT2AR particles without CHS remains the same, suggesting that dimerization is unlikely an effect of concentration. CONCLUSION: Our results indicated that 5HT2A receptor primarily forms a dimer in presence of cholesterol whereas it predominantly forms a monomer when cholesterol is removed.


Assuntos
Receptores 5-HT2 de Serotonina/química , Animais , Técnicas de Cultura de Células , Colesterol/química , Nanopartículas/metabolismo , Tamanho da Partícula , Multimerização Proteica , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Células Sf9 , Transdução de Sinais
11.
Sci Rep ; 7(1): 1310, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28465511

RESUMO

Berberine has been reports to have antidepressant-like effects. However, it is seldom known whether berberine produces antidepressant-like effects in ovariectomized mice, which exhibit depressive-like responses. To examine the antidepressant-like effects of berberine in ovariectomized mice, behavioral tests were conducted, including the forced swimming test and the open field test. To elucidate the mechanisms, levels of BDNF, phosphorylated CREB and phosphorylated eEF2 were analyzed by western blotting, and c-Fos induction was examined by immunohistochemistry. In the forced swimming test, berberine decreased the immobility time in a dose-dependent manner, reversing the depressive-like effect observed in ovariectomized mice, and this effect was blocked by the 5-HT2 antagonist ketanserin. In addition, western blotting indicated that BDNF and peEF2 in the hippocampus, but not pCREB/CREB in the frontal cortex, were affected by berberine treatment. Furthermore, immunohistochemistry demonstrated that the reduction in c-Fos induced by ovariectomy were greater after berberine treatment. Ketanserin also antagonized the effect of berberine on the c-Fos expression. Our findings suggest that berberine exerts antidepressant-like effects in ovariectomized mice, and 5-HT2 receptor activation may be partially related to the antidepressant-like effects of the berberine by BDNF-CREB and eEF2 pathways.


Assuntos
Antidepressivos/administração & dosagem , Berberina/administração & dosagem , Depressão/tratamento farmacológico , Receptores 5-HT2 de Serotonina/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Depressão/genética , Depressão/patologia , Quinase do Fator 2 de Elongação/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ketanserina/administração & dosagem , Camundongos , Ovariectomia , Proteínas Proto-Oncogênicas c-fos/genética , Antagonistas do Receptor 5-HT2 de Serotonina/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Natação
12.
J Biol Chem ; 292(15): 6352-6368, 2017 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-28258217

RESUMO

The serotonin receptor subtypes 2 comprise 5-HT2A, 5-HT2B, and 5-HT2C, which are Gαq-coupled receptors and display distinct pharmacological properties. Although co-expressed in some brain regions and involved in various neurological disorders, their functional interactions have not yet been studied. We report that 5-HT2 receptors can form homo- and heterodimers when expressed alone or co-expressed in transfected cells. Co-immunoprecipitation and bioluminescence resonance energy transfer studies confirmed that 5-HT2C receptors interact with either 5-HT2A or 5-HT2B receptors. Although heterodimerization with 5-HT2C receptors does not alter 5-HT2C Gαq-dependent inositol phosphate signaling, 5-HT2A or 5-HT2B receptor-mediated signaling was totally blunted. This feature can be explained by a dominance of 5-HT2C on 5-HT2A and 5-HT2B receptor binding; in 5-HT2C-containing heterodimers, ligands bind and activate the 5-HT2C protomer exclusively. This dominant effect on the associated protomer was also observed in neurons, supporting the physiological relevance of 5-HT2 receptor heterodimerization in vivo Accordingly, exogenous expression of an inactive form of the 5-HT2C receptor in the locus ceruleus is associated with decreased 5-HT2A-dependent noradrenergic transmission. These data demonstrate that 5-HT2 receptors can form functionally asymmetric heterodimers in vitro and in vivo that must be considered when analyzing the physiological or pathophysiological roles of serotonin in tissues where 5-HT2 receptors are co-expressed.


Assuntos
Locus Cerúleo/metabolismo , Receptores 5-HT2 de Serotonina/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Células HEK293 , Humanos , Masculino , Camundongos , Multimerização Proteica , Receptores 5-HT2 de Serotonina/genética
13.
J Biol Chem ; 292(14): 5943-5956, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28213524

RESUMO

G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans, and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway.


Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Quinases de Receptores Acoplados a Proteína G/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Monoaminoxidase/metabolismo , Serotonina/metabolismo , Transdução de Sinais/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Quinases de Receptores Acoplados a Proteína G/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Monoaminoxidase/genética , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/metabolismo , Serotonina/genética
14.
Dialogues Clin Neurosci ; 18(3): 323-337, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27757066

RESUMO

Optimizing antipsychotic pharmacotherapy is often challenging due to significant variability in effectiveness and tolerability. Genetic factors influencing pharmacokinetics and pharmacodynamics may contribute to some of this variability. Research studies have characterized these pharmacogenetic relationships, and some genetic markers are now available as clinical tests. These advances in pharmacogenetics research and test availability have great potential to improve clinical outcomes and quality of life in psychiatric patients. For clinicians considering using pharmacogenetics, it is important to understand the clinical implications and also the limitations of markers included in currently available tests. This review focuses on pharmacokinetic and pharmacodynamic gene variants that are currently available in commercial genetic testing panels. Associations of these variants with clinical efficacy and adverse effects, as well as other clinical implications, in antipsychotic pharmacotherapy are discussed.


Assuntos
Antipsicóticos/farmacologia , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/genética , Testes Farmacogenômicos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Antipsicóticos/metabolismo , Antipsicóticos/farmacocinética , Catecol O-Metiltransferase/genética , Sistema Enzimático do Citocromo P-450/genética , Marcadores Genéticos , Humanos , Transtornos Mentais/metabolismo , Receptores de Dopamina D2/genética , Receptores 5-HT2 de Serotonina/genética
15.
Elife ; 52016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27572262

RESUMO

Research into how protein restriction improves organismal health and lengthens lifespan has largely focused on cell-autonomous processes. In certain instances, however, nutrient effects on lifespan are independent of consumption, leading us to test the hypothesis that central, cell non-autonomous processes are important protein restriction regulators. We characterized a transient feeding preference for dietary protein after modest starvation in the fruit fly, Drosophila melanogaster, and identified tryptophan hydroxylase (Trh), serotonin receptor 2a (5HT2a), and the solute carrier 7-family amino acid transporter, JhI-21, as required for this preference through their role in establishing protein value. Disruption of any one of these genes increased lifespan up to 90% independent of food intake suggesting the perceived value of dietary protein is a critical determinant of its effect on lifespan. Evolutionarily conserved neuromodulatory systems that define neural states of nutrient demand and reward are therefore sufficient to control aging and physiology independent of food consumption.


Assuntos
Envelhecimento , Drosophila melanogaster/fisiologia , Comportamento Alimentar , Transdução de Sinais , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Técnicas de Inativação de Genes , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo
16.
Int J Biol Sci ; 12(2): 219-34, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26884719

RESUMO

Hepatic triglycerides production and adipose lipolysis are pivotal for long-term stress (LTS) or hyperglucocorticoidemia-induced insulin resistance. 5-hydroxytryptamine (5-HT) has been demonstrated to induce hepatic lipid metabolic abnormality by activating mammalian target of rapamycin (mTOR). In present study, we explored whether 5-HT is involved in LTS effects in liver using restraint stress-exposed rats and cultured primary rat hepatocytes and HepG2 cells. LTS with hyperglucocorticoidemia induced hepatic 5-HT synthetic increase with tryptophan hydroxylase 1 (Tph1) up-regulation, and 5-HT2 receptor (5-HT2R, including 5-HT2A, 2B receptor) up-regulation in liver and visceral adipose, as well as hepatic mTOR activation with triglycerides and VLDL overproduction with steatosis, and visceral adipose lipolytic increase with high blood free fatty acids (FFAs) level. 5-HT exposure exhibited LTS-like effects in both tissues, and both LTS and 5-HT effects could be abolished significantly by blocking 5-HT2R. In HepG2 cells dexamethasone or palmitate-induced mTOR activation with triglycerides and VLDL overproduction were accompanied by up-regulations of 5-HT synthesis and 5-HT2R, which were significantly abolished by gene silencing Tph1 or 5-HT2R and were almost fully abolished by co-silencing of both, especially on VLDL overproduction. Chemical inhibition of Tph1 or/and 5-HT2R in both hepatocytes exhibited similar abolishment with genetic inhibition on dexamethason-induced effects. 5-HT-stimulated effects in both hepatocytes were fully abolished by blocking 5-HT2R, while 5-HT itself also up-regulated 5-HT2R. In conclusion, up-regulated hepatic 5-HT synthesis and 5-HT2R induced by both glucocorticoid and FFAs are crucial for LTS-induced hepatic steatosis with VLDL overproduction, while 5-HT by acting on 5-HT2R mediates mTOR activation in liver.


Assuntos
VLDL-Colesterol/biossíntese , Fígado/metabolismo , Receptores 5-HT2 de Serotonina/biossíntese , Serotonina/biossíntese , Estresse Fisiológico , Animais , Células Cultivadas , Ácidos Graxos não Esterificados/sangue , Fígado Gorduroso/metabolismo , Inativação Gênica , Glucocorticoides/sangue , Células Hep G2 , Humanos , Masculino , Modelos Biológicos , Ratos Sprague-Dawley , Receptores 5-HT2 de Serotonina/genética , Transdução de Sinais , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Regulação para Cima
17.
Sleep ; 39(12): 2189-2199, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28057087

RESUMO

STUDY OBJECTIVE: The expression of the immediate early gene early growth response 3 (Egr3) is a functional marker of brain activity including responses to novelty, sustained wakefulness, and sleep. We examined the role of this gene in regulating wakefulness and sleep. METHODS: Electroencephalogram/electromyogram (EEG/EMG) were recorded in Egr3-/- and wild-type (WT) mice during 24 h baseline, 6 h sleep disruption and 6 h recovery. Serotonergic signaling was assessed with 6 h EEG/EMG recordings after injections of nonselective 5-HT2 antagonist (clozapine), selective 5-HT2 antagonists (5-HT2A; MDL100907 and 5-HT2BC; SB206553) and a cocktail of both selective antagonists, administered in a randomized order to each animal. RESULTS: Egr3-/- mice did not exhibit abnormalities in the timing of wakefulness and slow wave sleep (SWS); however, EEG dynamics in SWS (suppressed 1-3 Hz power) and in quiet wakefulness (elevated 3-8 Hz and 15-35 Hz power) differed in comparison to WT-mice. Egr3-/- mice showed an exaggerated response to sleep disruption as measured by active wakefulness, but with a blunted increase in homeostatic sleep drive (elevated 1-4 Hz power) relative to WT-mice. Egr3-/-mice exhibit greatly reduced sedative effects of clozapine at the electroencephalographic level. In addition, clozapine induced a previously undescribed dissociated state (low amplitude, low frequency EEG and a stable, low muscle tone) lasting up to 2 h in WT-mice. Egr3-/- mice did not exhibit this phenomenon. Selective 5-HT2A antagonist, alone or in combination with selective 5-HT2BC antagonist, caused EEG slowing coincident with behavioral quiescence in WT-mice but not in Egr3-/- mice. CONCLUSION: Egr3 has an essential role in regulating cortical arousal, wakefulness, and sleep, presumably by its regulation of 5-HT2 receptors.


Assuntos
Homeostase/genética , Homeostase/fisiologia , Fenótipo , Canais de Potássio/genética , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/fisiologia , Privação do Sono/genética , Privação do Sono/fisiopatologia , Sono/genética , Sono/fisiologia , Vigília/genética , Vigília/fisiologia , Animais , Cruzamentos Genéticos , Eletroencefalografia , Eletromiografia , Feminino , Homeostase/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas da Serotonina/farmacologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
18.
Biol Pharm Bull ; 39(1): 121-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26567725

RESUMO

The involvement of serotonin (5-hydroxytryptamine; 5-HT) and the 5-HT2 receptor subtypes in the induction of DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction mechanisms. Hepatocyte parenchymal cells maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of 5-HT or a selective 5-HT2B receptor agonist, BW723C86, but not in the presence of 5-HT2A, or 5-HT2C receptor agonists (TCB-2 and CP809101, respectively), in a time- and dose-dependent manner. A selective 5-HT2B receptor antagonist, LY272015 (10(-7) M), and a specific phospholipase C (PLC) inhibitor, U-73122 (10(-6) M), as well as specific inhibitors of growth-related signal transducers-including AG1478, LY294002, PD98059, and rapamycin-completely inhibited 5-HT (10(-6) M)- or BW723C86 (10(-6) M)-induced hepatocyte DNA synthesis and proliferation. Both 5-HT and BW723C86 were shown to significantly stimulate the phosphorylation of epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (p175 kDa) and extracellular signal-regulated kinase (ERK) 2 on Western blot analysis. These results suggest that the proliferative mechanism of activating 5-HT is mediated mainly through 5-HT2B receptor-stimulated Gq/PLC and EGF/TGF-α-receptor/phosphatidylinositol 3-kinase (PI3K)/ERK2/mammalian target of rapamycin (mTOR) signaling pathways in primary cultured hepatocytes.


Assuntos
Proliferação de Células/fisiologia , DNA/biossíntese , Hepatócitos/efeitos dos fármacos , Receptores 5-HT2 de Serotonina/metabolismo , Serotonina/farmacologia , Transdução de Sinais/fisiologia , Animais , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hepatócitos/metabolismo , Indóis/farmacologia , Fosforilação , Ratos , Receptores 5-HT2 de Serotonina/classificação , Receptores 5-HT2 de Serotonina/genética , Serotonina/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia
19.
PLoS One ; 10(8): e0134444, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26244344

RESUMO

Evidence from systems biology indicates that promiscuous drugs, i.e. those that act simultaneously at various protein targets, are clinically better in terms of efficacy, than those that act in a more selective fashion. This has generated a new trend in drug development called polypharmacology. However, the rational design of promiscuous compounds is a difficult task, particularly when the drugs are aimed to act at receptors with diverse structure, function and endogenous ligand. In the present work, using docking and molecular dynamics methodologies, we established the most probable binding sites of SB-206553, a drug originally described as a competitive antagonist of serotonin type 2B/2C metabotropic receptors (5-HT2B/2CRs) and more recently as a positive allosteric modulator of the ionotropic α7 nicotinic acetylcholine receptor (nAChR). To this end, we employed the crystal structures of the 5-HT2BR and acetylcholine binding protein as templates to build homology models of the 5-HT2CR and α7 nAChR, respectively. Then, using a statistical algorithm, the similarity between these binding sites was determined. Our analysis showed that the most plausible binding sites for SB-206553 at 5-HT2Rs and α7 nAChR are remarkably similar, both in size and chemical nature of the amino acid residues lining these pockets, thus providing a rationale to explain its affinity towards both receptor types. Finally, using a computational tool for multiple binding site alignment, we determined a consensus binding site, which should be useful for the rational design of novel compounds acting simultaneously at these two types of highly different protein targets.


Assuntos
Indóis/química , Piridinas/química , Receptores 5-HT2 de Serotonina/química , Receptor Nicotínico de Acetilcolina alfa7/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Ligação Competitiva , Humanos , Indóis/metabolismo , Indóis/farmacologia , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Piridinas/metabolismo , Piridinas/farmacologia , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/metabolismo , Homologia de Sequência de Aminoácidos , Antagonistas da Serotonina/química , Antagonistas da Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
20.
Curr Psychiatry Rep ; 17(7): 50, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25980509

RESUMO

The pharmacogenetics of antidepressants has been not only a challenging but also frustrating research field since its birth in the 1990s. Indeed, great expectations followed the first evidence of familiar aggregation of antidepressant response. Despite the progress from candidate gene studies to genome-wide association studies (GWAS), results fell out the expectations and they were often inconsistent. Anyway, the cumulative evidence supports the involvement of some genes and molecular pathways in antidepressant efficacy. The best single genes are SLC6A4, HTR2A, BDNF, GNB3, FKBP5, ABCB1, and cytochrome P450 genes (CYP2D6 and CYP2C19). Molecular pathways involved in inflammation and neuroplasticity show the greatest support. The first studies evaluating benefits of genotype-guided antidepressant treatments provided encouraging results and confirmed the relevance of SLC6A4, HTR2A, ABCB1, and cytochrome P450 genes. Further progress in genotyping and data analysis would allow to move forward and complete the understanding of antidepressant pharmacogenetics and its translation into clinical applications.


Assuntos
Antidepressivos/farmacologia , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/metabolismo , Transdução de Sinais , Subfamília B de Transportador de Cassetes de Ligação de ATP/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Antidepressivos/uso terapêutico , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Transtorno Depressivo Maior/genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Farmacogenética , Receptores 5-HT2 de Serotonina/efeitos dos fármacos , Receptores 5-HT2 de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
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