RESUMO
Purpose: This study investigates alterations in intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells (DACs) in lid suture myopia (LSM) rats. Methods: LSM was induced in rats by suturing the right eyes for 4 weeks. Double immunofluorescence staining of ipRGCs and DACs in whole-mount retinas was performed to analyze changes in the density and morphology of control, LSM, and fellow eyes. Real-time quantitative PCR and Western blotting were used to detect related genes and protein expression levels. Results: Significant myopia was induced in the lid-sutured eye, but the fellow eye was not different to control. Decreased ipRGC density with paradoxically increased overall melanopsin expression and enlarged dendritic beads was observed in both the LSM and fellow eyes of the LSM rat retinas. In contrast, DAC changes occurred only in the LSM eyes, with reduced DAC density and tyrosine hydroxylase (TH) expression, sparser dendritic processes, and fewer varicosities. Interestingly, contacts between ipRGCs and DACs in the inner plexiform layer (IPL) and the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and vesicular monoamine transporter protein 2 (VMAT2) mRNA were decreased in the LSM eyes. Conclusions: The ipRGCs and DACs in LSM rat retinas undergo multiple alterations in density, morphology, and related molecule expressions. However, the ipRGC changes alone appear not to be required for the development of myopia, given that myopia is only induced in the lid-sutured eye, and they are unlikely alone to drive the DAC changes. Reduced contacts between ipRGCs and DACs in the LSM eyes may be the structural foundation for the impaired signaling between them. PACAP and VMAT2, strongly associated with ipRGCs and DACs, may play important roles in LSM through complex mechanisms.
Assuntos
Células Amácrinas , Western Blotting , Modelos Animais de Doenças , Miopia , Células Ganglionares da Retina , Opsinas de Bastonetes , Animais , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/metabolismo , Ratos , Miopia/metabolismo , Células Amácrinas/metabolismo , Células Amácrinas/patologia , Opsinas de Bastonetes/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/genética , Masculino , Ratos Sprague-Dawley , Pálpebras/patologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Contagem de Células , Proteína Vesicular 2 de Transporte de GlutamatoRESUMO
The importance of vesicular monoamine transporter 2 (VMAT2) in dopamine regulation, which is considered crucial for neuropsychiatric disorders, is currently being studied. Moreover, the development of disease treatments using histone deacetylase (HDAC) inhibitors (HDACi) is actively progressing in various fields. Recently, research on the possibility of regulating neuropsychiatric disorders has been conducted. In this study, we evaluated whether VMAT2 expression increased by an HDACi can fine-tune neuropsychotic behavior, such as attention deficit hyperactivity disorder (ADHD) and protect against the cell toxicity through oxidized dopamine. First, approximately 300 candidate HDACi compounds were added to the SH-SY5Y dopaminergic cell line to identify the possible changes in the VMAT2 expression levels, which were measured using quantitative polymerase chain reaction. The results demonstrated, that treatment with pimelic diphenylamide 106 (TC-H 106), a class I HDACi, increased VMAT2 expression in both the SH-SY5Y cells and mouse brain. The increased VMAT2 expression induced by TC-H 106 alleviated the cytotoxicity attributed to 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium (MPP+ ) and free dopamine treatment. Moreover, dopamine concentrations, both intracellularly and in the synaptosomes, were significantly elevated by increased VMAT2 expression. These results suggest that dopamine concentration regulation by VMAT2 expression induced by TC-H 106 could alter several related behavioral aspects that was confirmed by attenuation of hyperactivity and impulsivity, which were major characteristics of animal model showing ADHD-like behaviors. These results indicate that HDACi-increased VMAT2 expression offers sufficient protections against dopaminergic cell death induced by oxidative stress. Thus, the epigenetic approach could be considered as therapeutic candidate for neuropsychiatric disease regulation.
Assuntos
Inibidores de Histona Desacetilases , Neuroblastoma , Humanos , Animais , Camundongos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Proteínas Vesiculares de Transporte de Monoamina/genética , Citoproteção , Dopamina , OxidopaminaRESUMO
Paragangliomas and pheochromocytomas are rare neuroendocrine tumors, carrying a germ-line mutation in 40% patients. Sclerosis is a rare histological feature in these tumors. We investigated the possible correlations between histological findings, first sclerosis, immunoreactivity for vesicular catecholamine transporters (VMAT1/VMAT2) and patients' genotype in a consecutive series of 57 tumors (30 paragangliomas and 27 pheochromocytomas) from 55 patients. The M-GAPP grading system, sclerosis (0-3 scale) and VMAT1/VMAT2 (0-6 scale) immunoreactivity scores were assessed. Germ-line mutations of Succinate Dehydrogenase genes, RET proto-oncogene and Von Hippel Lindau tumor suppressor gene were searched. A germ-line mutation was found in 25/55 (45.5%) patients, mainly with paraganglioma (N = 14/30, 46,66%). Significant (score ≥ 2) tumor sclerosis was found in 9 (16.1%) tumors, i.e., 7 paragangliomas and 2 pheochromocytomas, most of them (8/9) from patients with a germ-line mutation. M-GAPP score was higher in the mutation status (in 76% of patients involving the SDHx genes, in 12% the RET gene and in the remaining 12% the VHL gene) and in tumors with sclerosis (p < 0.05). Spearman's rank correlation showed a strong correlation of germ-line mutations with M-GAPP (p < 0.0001) and sclerosis (p = 0.0027) scores; a significant correlation was also found between sclerosis and M-GAPP scores (p = 0.029). VMAT1 expression was higher in paragangliomas than in pheochromocytomas (p = 0.0006), the highest scores being more frequent in mutation-bearing patients' tumors (p < 0.01). VMAT2 was highly expressed in all but two negative tumors. Sclerosis and VMAT1 expression were higher in paragangliomas than in pheochromocytomas; tumor sclerosis, M-GAPP and VMAT1 scores were associated to germ-line mutations. Sclerosis might represent a histological marker of tumor susceptibility, prompting to genetic investigations in paragangliomas.
Assuntos
Proteínas Vesiculares de Transporte de Monoamina , Humanos , Proteínas Vesiculares de Transporte de Monoamina/genética , EscleroseRESUMO
Heightened activity of glycogen synthase kinase-3ß (GSK-3ß) is linked to the degeneration of dopaminergic neurons in Parkinson's disease (PD). Phytic acid (PA), a naturally occurring compound with potent antioxidant property, has been shown to confer neuroprotection on dopaminergic neurons in PD. However, the underlying mechanism remains unclear. In the present study, MPTP and MPP+ treatments were used to model PD in mice and SH-SY5Y cells, respectively. We observed reduced tissue dopamine, disrupted synaptic vesicle recycling, and defective neurotransmitter exocytosis. Furthermore, expression of GSK-3ß was upregulated while that of ß-catenin was downregulated, concentration of cytosolic calcium was increased, and expressions of two dopamine carriers, dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) were decreased. PA treatment attenuated the MPTP-induced upregulation of GSK-3ß, increase in cytosolic calcium concentration, decreases in the levels of DAT, VMAT2, tissue dopamine, and synaptic vesicle recycling. Importantly, disturbances in synaptic vesicle recycling are thought to be early events in PD pathology. These findings suggest that PA is a promising therapeutic agent to treat early events in PD.
Assuntos
Antiparkinsonianos/uso terapêutico , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Intoxicação por MPTP/tratamento farmacológico , Ácido Fítico/uso terapêutico , Vesículas Sinápticas/efeitos dos fármacos , Animais , Antiparkinsonianos/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/biossíntese , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/biossíntese , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Intoxicação por MPTP/metabolismo , Camundongos Endogâmicos C57BL , Neuroblastoma/patologia , Ácido Fítico/farmacologia , Teste de Desempenho do Rota-Rod , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/biossíntese , Proteínas Vesiculares de Transporte de Monoamina/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Aim: To determine if selected serotonergic and noradrenergic gene variants are associated with heroin addiction. Subjects & methods: A total of 126 variants in 19 genes in subjects with Dutch European ancestry from The Netherlands. Subjects included 281 opioid-dependent volunteers in methadone maintenance or heroin-assisted treatment, 163 opioid-exposed but not opioid-dependent volunteers who have been using illicit opioids but never became opioid-dependent and 153 healthy controls. Results: Nominal associations were indicated for 20 variants in six genes including an experiment-wise significant association from the combined effect of three SLC18A2 SNPs (rs363332, rs363334 and rs363338) with heroin dependence (pfinal = 0.047). Conclusion: Further studies are warranted to confirm and elucidate the role of these variants in the vulnerability to opioid addiction.
Assuntos
Transtornos Relacionados ao Uso de Opioides/genética , Polimorfismo de Nucleotídeo Único , Proteínas Vesiculares de Transporte de Monoamina/genética , Adulto , Analgésicos Opioides/administração & dosagem , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Dependência de Heroína/tratamento farmacológico , Dependência de Heroína/genética , Humanos , Masculino , Metadona/uso terapêutico , Pessoa de Meia-Idade , Países Baixos , Tratamento de Substituição de Opiáceos , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Testes Farmacogenômicos , Fatores de Risco , População Branca/genéticaRESUMO
CD8+ T regulatory/suppressor cells (Treg) affect peripheral tolerance and may be involved in autoimmune diseases as well as in cancer. In view of our previous data showing the ability of DA to affect adaptive immune responses, we investigated the dopaminergic phenotype of human CD8+ Treg as well as the ability of DA to affect their generation and activity. Results show that CD8+ T cells express both D1-like and D2-like dopaminergic receptors (DR), tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA, and vesicular monoamine transporter (VMAT) 2 and contain high levels of intracellular DA. Preferential upregulation of DR mRNA levels in the CD8+CD28- T cell compartment occurs during generation of CD8+ Treg, which is reduced by DA and by the D1-like DR agonist SKF-38393. DA and SKF-38393 also reduce the suppressive activity of CD8+ Treg on human peripheral blood mononuclear cells. Treg are crucial for tumor escape from the host immune system, thus the ability of DA to inhibits Treg function supports dopaminergic pathways as a druggable targets to develop original and innovative antitumor strategies.
Assuntos
Dopamina/farmacologia , Receptores de Dopamina D1/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Citometria de Fluxo , Humanos , Neuroimunomodulação/fisiologia , RNA Mensageiro/biossíntese , Receptores de Dopamina D1/análise , Receptores de Dopamina D1/biossíntese , Receptores de Dopamina D1/genética , Linfócitos T Reguladores/metabolismo , Tirosina 3-Mono-Oxigenase/biossíntese , Tirosina 3-Mono-Oxigenase/genética , Proteínas Vesiculares de Transporte de Monoamina/biossíntese , Proteínas Vesiculares de Transporte de Monoamina/genéticaRESUMO
Epithelial-neuronal signaling is essential for sensory encoding in touch, itch, and nociception; however, little is known about the release mechanisms and neurotransmitter receptors through which skin cells govern neuronal excitability. Merkel cells are mechanosensory epidermal cells that have long been proposed to activate neuronal afferents through chemical synaptic transmission. We employed a set of classical criteria for chemical neurotransmission as a framework to test this hypothesis. RNA sequencing of adult mouse Merkel cells demonstrated that they express presynaptic molecules and biosynthetic machinery for adrenergic transmission. Moreover, live-cell imaging directly demonstrated that Merkel cells mediate activity- and VMAT-dependent release of fluorescent catecholamine neurotransmitter analogs. Touch-evoked firing in Merkel-cell afferents was inhibited either by pre-synaptic silencing of SNARE-mediated vesicle release from Merkel cells or by neuronal deletion of ß2-adrenergic receptors. Together, these results identify both pre- and postsynaptic mechanisms through which Merkel cells excite mechanosensory afferents to encode gentle touch. VIDEO ABSTRACT.
Assuntos
Adrenérgicos/metabolismo , Vias Aferentes/fisiologia , Células de Merkel/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Animais , Cápsulas Bacterianas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Feminino , Gânglios Espinais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Adrenérgicos beta 2 , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores 5-HT3 de Serotonina/genética , Receptores 5-HT3 de Serotonina/metabolismo , Pele/citologia , Pele/inervação , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/genética , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
BACKGROUND: One aspect of smoking and lung cancer that has not been closely examined, is that regarding genes that may predispose to tobacco dependence. Smoking and mental illness are tightly linked, apparently the result of smokers using cigarettes to self-medicate for mental problems. The gene for solute carrier family 18 member A1 (vesicular monoamine transporter; SLC18A1) is of particular interest in this regard because of its association with schizophrenia, autism and bipolar illness as well as with cancer. In the current study, the relationship of SLC18A1 expression with smoking and lung cancer was analyzed. MATERIALS AND METHODS: The association between smoking, SLC18A1 expression and overall survival in the lung cancer dataset in The Cancer Genome Atlas was evaluated using the Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov), as well as CbioPortal for Cancer Genomics (http://www.cbioportal.org) and the University of California Santa Cruz Xena browser (https://xenabrowser.net). RESULTS: Increased expression of SLC18A1 was found to be associated with a significantly increased survival in patients with adenocarcinoma (p=0.0058), but not those with squamous carcinoma (p=0.96). Lifelong never-smokers had the highest SLC18A1 expression. In the Pan Cancer Atlas, increased expression of SLC18A1 places such a tumor in group C5, among immunologically-quiet tumors. CONCLUSION: Most never-smokers with lung cancer do not respond to immune checkpoint inhibitors (ICIs). But for unknown reasons, a small proportion do show clinical benefit from the ICI pembrolizumab. Because of the good response of this group, it may be worthwhile assessing their SLC18A1 expression pre-treatment as a marker for potential clinical benefit. If SLC18A1 expression is low, a never-smoker may respond well to ICIs. High levels of expression would indicate a C5 tumor less likely to respond to ICIs. SLC18A1 might complement other biomarkers currently under study in relation to programmed cell death protein 1/programmed cell death protein ligand 1 inhibition.
Assuntos
Adenocarcinoma/genética , Antígeno B7-H1/genética , Neoplasias Pulmonares/genética , Receptor de Morte Celular Programada 1/genética , Proteínas Vesiculares de Transporte de Monoamina/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/etiologia , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adulto , Idoso , Anticorpos Monoclonais Humanizados/uso terapêutico , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Fumar/efeitos adversosRESUMO
Many studies have shown that the pollutant simazine (6-chloro-N,N'-diethyl-1,3,5-triazine-2,4-diamine), which has been overused, inhibits the proliferation of mammalian dopaminergic cells, and affects the developmental differentiation of mammalian dopaminergic neurons. However, few studies have shown the effects of simazine on dopaminergic metabolism in these cells. Therefore, we aim to examine the metabolic effects of simazine exposure in mouse dopaminergic progenitor neurons (MN9D) at different exposure times. The cells were treated with simazine at 0, 150, 300 and 600 µM for 12, 24 and 48 h, respectively. The content of dopamine in these cells was then examined using the enzyme-linked immunosorbent assay (ELISA) kit. Real-time quantitative polymerase chain reaction (PCR) and western blotting were performed to analyze the mRNA and protein expression of aromatic amino acid decarboxylase (AADC), tyrosine hydroxylase (DYT5b), dopamine transporter (DAT), monoamine vesicular transporter 2 (VMAT2), monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT). The results showed that simazine influenced the metabolism of dopamine and led to a decrease in dopamine level in these cells which may eventually lead to neurological disorders of the dopaminergic system.
Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Herbicidas/toxicidade , Células-Tronco Neurais/efeitos dos fármacos , Simazina/toxicidade , Animais , Descarboxilases de Aminoácido-L-Aromático/genética , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Catecol O-Metiltransferase/genética , Catecol O-Metiltransferase/metabolismo , Linhagem Celular Tumoral , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Camundongos , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Células-Tronco Neurais/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/genética , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Copy number variations (CNVs) contribute to the development of colorectal cancer (CRC). We conducted a two-stage association study to identify CNV risk loci for CRC. We performed a gene-based rare CNV study on 694 sporadic CRC and 1641 controls using Illumina Human-OmniExpress-12v1.0 BeadChips, and further replicated in 934 CRC cases and 2680 controls for risk CNVs by using TaqMan Copy Number Assay. Tumor buddings, cancer cells in the center of primary tumor and normal intestinal epithelial cells were captured using laser capture microdissection (LCM) and were assayed using AffymetrixGeneChip® Human Genome U133 Plus 2.0 Array. In addition, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus data were assessed for the effects of risk CNVs. We found that germline deletions affecting the last six exons of SLC18A1 significantly associated with CRC with a combined P value of 6.4 × 10-5 by a two-stage analysis. Both in TCGA CRC RNA seq dataset and GDS4382, SLC18A1 was significantly down regulated in CRC tissues than in paired normal tissues (N = 32 and 17 pairs, P = 0.004 and 0.009, respectively). In LCM samples, similar observations were obtained that the expression levels of SLC18A1 in the tumor buddings, cancer cells in the center of primary tumor, and stroma of both tumor budding and cancer cells were lower than normal intestinal epithelial and stromal cells (fold change = 0.17-0.62, 0.12-0.57 and 0.37-0.68, respectively). In summary, the germline deletions at SLC18A1 contributed to the development of CRC. The role of SLC18A1 required further exploration.
Assuntos
Neoplasias Colorretais/genética , Deleção de Sequência/genética , Proteínas Vesiculares de Transporte de Monoamina/genética , Adulto , Estudos de Casos e Controles , China , Variações do Número de Cópias de DNA/genética , Regulação para Baixo/genética , Células Epiteliais/metabolismo , Éxons/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Células Estromais/metabolismoRESUMO
The type 2 vesicular monoamine transporter (VMAT2), by regulating the storage of monoamines transmitters into synaptic vesicles, has a protective role against their cytoplasmic toxicity. Increasing evidence suggests that impairment of VMAT2 neuroprotection contributes to the pathogenesis of Parkinson's disease (PD). Several transgenic VMAT2 mice models have been developed, however these models lack specificity regarding the monoaminergic system targeting. To circumvent this limitation, we created VMAT2-KO mice specific to the dopamine (DA) nigrostriatal pathway to analyze VMAT2's involvement in DA depletion-induced motor features associated to PD and examine the relevance of DA toxicity in the pathogenesis of neurodegeneration. Adult VMAT2 floxed mice were injected in the substancia nigra (SN) with an adeno-associated virus (AAV) expressing the Cre-recombinase allowing VMAT2 removal in DA neurons of the nigrostriatal pathway solely. VMAT2 deletion in the SN induced both DA depletion exclusively in the dorsal striatum and motor dysfunction. At 16 weeks post-injection, motor symptoms were accompanied with a decreased in food and water consumption and weight loss. However, despite an accelerating death, degeneration of nigrostriatal neurons was not observed in this model during this time frame. This study highlights a non-cytotoxic role of DA in our genetic model of VMAT2 deletion exclusively in nigrostriatal neurons.
Assuntos
Dopamina/deficiência , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson Secundária/genética , Substância Negra/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/genética , Animais , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Dependovirus/genética , Dependovirus/metabolismo , Neurônios Dopaminérgicos/patologia , Ingestão de Líquidos , Ingestão de Alimentos , Deleção de Genes , Expressão Gênica , Injeções Intraventriculares , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Doença de Parkinson Secundária/metabolismo , Doença de Parkinson Secundária/patologia , Doença de Parkinson Secundária/fisiopatologia , Substância Negra/patologia , Vesículas Sinápticas/patologia , Proteínas Vesiculares de Transporte de Monoamina/deficiência , Redução de PesoRESUMO
Norepinephrine transporter (NET) transfection leads to significant uptake of iodine-131-labeled metaiodobenzylguanidine (131I-MIBG) in non-neuroendocrine tumors. However, the use of 131I-MIBG is limited by its short retention time in target cells. To prolong the retention of 131I-MIBG in target cells, we infected hepatocarcinoma (HepG2) cells with Lentivirus-encoding human NET and vesicular monoamine transporter 2 (VMAT2) genes to obtain NET-expressing, NET-VMAT2-coexpressing, and negative-control cell lines. We evaluated the uptake and efflux of 131I-MIBG both in vitro and in vivo in mice bearing transfected tumors. NET-expressing and NET-VMAT2-coexpressing cells respectively showed 2.24 and 2.22 times higher 131I-MIBG uptake than controls. Two hours after removal of 131I-MIBG-containing medium, 25.4% efflux was observed in NET-VMAT2-coexpressing cells and 38.6% in NET-expressing cells. In vivo experiments were performed in nude mice bearing transfected tumors; results revealed that NET-VMAT2-coexpressing tumors had longer 131I-MIBG retention time than NET-expressing tumors. Meanwhile, NET-VMAT2-coexpressing and NET-expressing tumors displayed 0.54% and 0.19%, respectively, of the injected dose per gram of tissue 24 h after 131I-MIBG administration. Cotransfection of HepG2 cells with NET and VMAT2 resulted in increased 131I-MIBG uptake and retention. However, the degree of increase was insufficient to be therapeutically effective in target cells.
Assuntos
Carcinoma Hepatocelular/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , 3-Iodobenzilguanidina , Animais , Modelos Animais de Doenças , Terapia Genética , Células Hep G2 , Humanos , Radioisótopos do Iodo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Cintilografia , Compostos Radiofarmacêuticos , Transfecção , Proteínas Vesiculares de Transporte de Monoamina/genéticaRESUMO
STUDY QUESTION: Are genetic variations at the human solute carrier family 18 member A2 (SLC18A2) locus associated with the etiology of polycystic ovary syndrome (PCOS) and/or with follicle stimulating hormone (FSH) levels and insulin secretion in PCOS? SUMMARY ANSWER: We found two common genetic variants in the 3'-untranslated region of SLC18A2 (rs363282 and rs363238) that are associated with serum FSH concentration in the PCOS group. WHAT IS KNOWN ALREADY: SLC18A2 is a vesicular monoamine transporter that is essential in dopamine regulation. Dopamine can negatively regulate FSH and insulin secretion through the D2 receptor. STUDY DESIGN, SIZE, DURATION: This study was a cross-sectional examination in women with PCOS (n = 319) and controls (n = 220) from China. PARTICIPANTS/MATERIALS, SETTING, METHODS: The PCOS patients were diagnosed based on the criteria of the Androgen Excess Society, including clinical and/or biochemical signs of hyperandrogenemia plus oligoamenorrhea or polycystic ovaries. Controls had regular menstrual cycles and no hyperandrogenism or other endocrine disorders related to PCOS. Tag single nucleotide polymorphisms (SNPs) were selected based on resequencing data in 48 PCOS patients and linkage disequilibrium analysis. Allele frequencies for variants (rs363282 and rs363238) were examined in PCOS cases and controls along with their relationship to quantitative traits. The samples were further divided into two subgroups for association analysis: AA + AG group and GG group (rs363282), CC + AC group and AA group (rs363238). The functional effects of SLC18A2 variants were measured by luciferase assay. The gene expression of SLC18A2 was compared with the NCBI's Gene Expression Omnibus datasets. MAIN RESULTS AND THE ROLE OF CHANCE: Two common genetic variants in the 3'-untranslated region (rs363282 and rs363238) are associated with serum FSH in the PCOS group (P= 0.005 and P= 0.001, respectively), while no associations were found in controls. Functional studies showed that minor alleles of the two variants (rs363282-G and rs363238-A) had significantly lower luciferase activities than rs363282-A (P= 0.009) and rs363238-C (P = 0.009). LIMITATIONS, REASONS FOR CAUTION: Results were not validated in another independent cohort, though we provided functional evidence of the two SNPs. Because of limited condition, more specific parameters, including ovarian follicle count and anti-Müllerian hormone were not included and relationship between SLC18A2 and these parameters cannot be evaluated. WIDER IMPLICATIONS OF THE FINDINGS: We found a novel association between two genetic variants in SLC18A2 and FSH levels in PCOS patients. These findings might indicate a novel regulatory mechanism in follicular development and maturation in PCOS. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (grant numbers 81571501 and 81270747), National Basic Research Program of China (grant number 2015CB943300). No competing interests declared.
Assuntos
Regiões 3' não Traduzidas , Hormônio Foliculoestimulante/sangue , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/genética , Polimorfismo de Nucleotídeo Único , Proteínas Vesiculares de Transporte de Monoamina/genética , Adulto , Alelos , Estudos Transversais , Feminino , Regulação da Expressão Gênica , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Adulto JovemRESUMO
Transplantation of dopaminergic (DA) neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson's disease (PD), thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs) derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX), bone morphogenetic proteins 7 (BMP-7), and growth factors, including acidic fibroblast factor (aFGF), forskolin, and phorbol-12-myristae-13-acetate (TPA), to induce differentiation of forebrain-derived hNSCs toward DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2), and tyrosine hydroxylase (TH) were significantly increased after 24 h of differentiation by RT-PCR analysis (p < 0.01). Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24 h, about 5% of cells became VMAT2 (vascular monoamine transporter 2)-positive neurons, and less than 5% of cells became DAT (dopamine transporter)-positive neurons at 72 h following differentiation in cultures. Importantly, these TH-, VMAT2-, and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P < 0.01), as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons.
Assuntos
Benzotiazóis/farmacologia , Proteína Morfogenética Óssea 7/farmacologia , Diferenciação Celular/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Análise de Variância , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Combinação de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Pramipexol , Prosencéfalo/citologia , RNA Mensageiro/metabolismo , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/genética , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Vesicular monoamine transporter 2 (VMAT2) is responsible for sequestering cytosolically toxic dopamine into intracellular secretory vesicles. Animal genetic studies have suggested that reduced VMAT2 activity contributes to the genetic etiology of Parkinson's disease (PD), but this role has not been established in humans. Based on human genetic association and meta-analysis, we first confirm the human VMAT2 (hVMAT2 or SLC18A2) promoter as a risk factor for PD in both family and unrelated US white people: marker rs363324 at -11.5 kb in the hVMAT2 promoter is reproducibly associated with PD in a cohort of nuclear families (p = 0.04506 in early-onset PD) and 3 unrelated US white people (meta-analysis p = 0.01879). In SH-SY5Y cells, low activity-associated hVMAT2 promoter confers high methylpiperidinopyrazole iodide cytotoxicity, which is likely attributed to functional polymorphisms bound by nuclear proteins. Interestingly, treatments with the dopamine neuron-protecting agent puerarin upregulates the promoter activity in a haplotype- and cell line-dependent manner. These pharmacogenetic findings suggest that hVMAT2 could be a risk factor and imply it as a target of genetic medications for PD.
Assuntos
Doença de Parkinson/genética , Medicina de Precisão , Proteínas Vesiculares de Transporte de Monoamina/genética , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Epigênese Genética , Feminino , Estudos de Associação Genética , Haplótipos , Humanos , Isoflavonas/administração & dosagem , Masculino , Pessoa de Meia-Idade , Fármacos Neuroprotetores/administração & dosagem , Doença de Parkinson/metabolismo , Doença de Parkinson/prevenção & controle , Parte Compacta da Substância Negra/efeitos dos fármacos , Parte Compacta da Substância Negra/metabolismo , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Fatores de Risco , Estados Unidos , Regulação para Cima , População BrancaRESUMO
The aim of this study was to evaluate the played by oxidative stress in the apoptotic response in different brain areas of rats chronically treated with supra-physiological doses of nandrolone decanoate (ND). Immunohistochemical study and Western blot analysis were performed to evaluate cells' apoptosis and to measure the effects of expression of specific mediators, such as NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), Bcl-2 (B-cell lymphoma 2), SMAC/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low PI) and VMAT2 (vesicular monoamine transporter 2) on apoptosis. The results of the present study indicate that a long-term administration of ND promotes oxidative injury in rat brain specific areas. A link between oxidative stress and NF-κB signalling pathways is supported by our results. In addition to high levels of oxidative stress, we consistently observed a strong immunopositivity to NF-κB. It has been argued that one of the pathways leading to the activation of NF-κB could be under reactive oxygen species (ROS)-mediated control. In fact, growing evidence suggests that although in limited doses, endogenous ROS may play an activating role in NF-κB signalling, while above a certain threshold, they may negatively impact upon this signalling. However, a mutual crosstalk between ROS and NF-κB exists and recent studies have shown that ROS activity is subject to negative feedback regulation by NF-κB, and that this negative regulation of ROS is the means through which NF-κB counters programmed cells.
Assuntos
Encéfalo/efeitos dos fármacos , NF-kappa B/genética , Nandrolona/análogos & derivados , Espécies Reativas de Oxigênio/metabolismo , Congêneres da Testosterona/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Nandrolona/efeitos adversos , Decanoato de Nandrolona , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/agonistas , Transdução de Sinais , Proteínas Vesiculares de Transporte de Monoamina/genética , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Limitations of current diagnostic and prognostic tools for prostate cancer (PC) have led to over-diagnosis and over-treatment. Here, we investigate the biomarker potential of the SLC18A2 (VMAT2) gene for PC at three molecular levels. Thus, SLC18A2 promoter methylation was analyzed in 767 malignant and 78 benign radical prostatectomy (RP) samples using methylation-specific qPCR and Illumina 450K methylation microarray data. SLC18A2 transcript levels were assessed in 412 malignant and 45 benign RP samples using RNAseq data. SLC18A2 protein was evaluated by immunohistochemistry in 502 malignant and 305 benign RP samples. Cancer-specificity of molecular changes was tested using Mann-Whitney U tests and/or receiver operating characteristic (ROC) analyses. Log rank, uni- and multivariate Cox regression tests were used for survival analyses. We found that SLC18A2 promoter hypermethylation was highly cancer-specific (area under the curve (AUC): 0.923-0.976) and associated with biochemical recurrence (BCR) after RP in univariate analyses. SLC18A2 transcript levels were reduced in PC and had independent prognostic value for BCR after RP (multivariate HR 0.13, P < 0.05). Likewise, SLC18A2 protein was down-regulated in PC (AUC 0.898) and had independent prognostic value for BCR (multivariate HR 0.51, P < 0.05). Reduced SLC18A2 protein expression was also associated with poor overall survival in univariate analysis (HR 0.29, P < 0.05). Our results highlight SLC18A2 as a new promising methylation marker candidate for PC diagnosis. Furthermore, SLC18A2 expression (RNA and protein) showed promising prognostic potential beyond routine clinicopathological variables. Thus, novel SLC18A2-based molecular tests could have useful future applications for PC detection and identification of high-risk patients.
Assuntos
Regulação Neoplásica da Expressão Gênica , Próstata/patologia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Proteínas Vesiculares de Transporte de Monoamina/genética , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Metilação de DNA , Humanos , Masculino , Prognóstico , Regiões Promotoras Genéticas , Próstata/metabolismo , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/cirurgia , RNA Mensageiro/análise , RNA Mensageiro/genética , Análise de Sobrevida , Proteínas Vesiculares de Transporte de Monoamina/análiseRESUMO
The identification of an effective disease-modifying treatment for the neurodegenerative progression in Parkinson's disease (PD) remains a major challenge. Epidemiological studies have reported that intake of statins, cholesterol lowering drugs, could be associated to a reduced risk of developing PD. In-vivo studies suggest that statins may reduce the severity of dopaminergic neurodegeneration. The trophic potential of statins and their impact on the expression of dopaminergic synaptic markers and dopamine (DA) transport function in SH-SY5Y cells has been investigated. The findings showed that statin treatment induces neurite outgrowth involving a specific effect on the complexity of the neurite branching pattern. Statins increased the levels of presynaptic dopaminergic biomarkers such as vesicular monoamine transporter 2 (VMAT2), synaptic vesicle glycoproteins 2A and 2C (SV2C), and synaptogyrin-3 (SYNGR3). Gene expression analysis confirmed a rapid statin-induced up-regulation of VMAT2-, SV2C-, and SYNGR3-mRNA levels. Assessment of [(3) H]DA transport in statin-treated cells showed a reduction in DA uptake concomitant to a modification of VMAT2 pharmacological properties. It was also observed that a nuclear translocation of the sterol regulatory element-binding protein 1 (SREBP-1). The results suggested that statins induced phenotypic changes in dopaminergic cells characterized by an increase of growth, complexity of structural synaptic elements, and expression of key presynaptic proteins with functional impact on the DA transport capacity. Statin-induced changes are likely the result of a downstream modulation of SREBP-1 pathway. Overall, these mechanisms may contribute to the neuroprotective or neurorestorative effects observed in the dopaminergic system and strengthen the therapeutic potential of statins for PD.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Inibidores da Captação Adrenérgica/farmacologia , Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Relação Dose-Resposta a Droga , Humanos , Lovastatina/farmacologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , RNA Mensageiro/metabolismo , Reserpina/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Sinaptogirinas/metabolismo , Tetrabenazina/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/genética , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
AIMS: To address questions regarding onset and progression of types 1 and 2 diabetes (T1D/T2D), surrogate imaging biomarkers for beta cell function and mass are needed. Here, we assess the potential of GPR44 as a surrogate marker for beta cells, in a direct comparison with clinically used biomarker VMAT2. METHODS: GPR44 surface availability was assessed by flow cytometry of human beta cells. RNA transcription levels in different pancreas compartments were evaluated. The density of GPR44 receptor in endocrine and exocrine tissues was assessed by the radiolabeled GPR44 ligand [(3)H]AZD 3825. A direct comparison with the established beta cell marker VMAT2 was performed by radiolabeled [(3)H]DTBZ. RESULTS: GPR44 was available on the cell surface, and pancreatic RNA levels were restricted to the islets of Langerhans. [(3)H]AZD 3825 had nanomolar affinity for GPR44 in human islets and EndoC-ßH1 beta cells, and the specific binding to human beta cells was close to 50 times higher than in exocrine preparations. The endocrine-to-exocrine binding ratio was approximately 10 times higher for [(3)H]AZD 3825 than for [(3)H]DTBZ. CONCLUSION: GPR44 is a highly beta cell-specific target, which potentially offers improved imaging contrast between the human beta cell and the exocrine pancreas.
Assuntos
Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Especificidade de Órgãos , Ratos , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Proteínas Vesiculares de Transporte de Monoamina/genética , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
AIMS: To examine the role of genetic and environmental factors in the pathogenesis of alcohol dependence in a Spanish cohort of women and men. METHODS: We analyzed the relationship between 56 genetic variants in 7 genes associated with the dopaminergic reward pathway and excessive alcohol consumption. The study sample (N = 1533, of which 746 were women) consisted of 653 heavy consumers and 880 very low consumers from the Spanish subcohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Single nucleotide polymorphisms (SNPs) were genotyped using a customized array. Lifestyle variables were also examined to assess associations between genetic and environmental factors. RESULTS: No statistically significant differences were found between cases and controls for the allele frequencies in five genes: TH, SLC18A2, DRD1, DRD3 and COMT. Conversely, some alleles of the 12 SNPs from the DRD2 locus and the 5 from the MAOA locus showed significant associations with excessive alcohol consumption. Namely, rs10891556 (DRD2) proved to be the only SNP positively correlated with excessive alcohol consumption in both sexes. DRD2 rs1800497 and rs877138 were significantly associated in men, whereas DRD2 rs17601612 and rs4936271 and MAOA rs5906898 were associated with excessive alcohol consumption in women. A correspondence analysis provided an overall lifestyle profile of excessive drinkers, who were predominantly men who smoked, had large intakes of meat, small intakes of fruit and vegetables, whose jobs did not require high education levels and who engaged in little physical activity. CONCLUSIONS: It has shown the influence of dopaminergic pathway in the genetics of alcohol dependence with differences between men and women and providing a lifestyle profile of excessive drinkers.