RESUMO
Several X-linked genes are involved in neuronal differentiation and may contribute to the generation of sex dimorphisms in the brain. Previous results showed that XX hypothalamic neurons grow faster, have longer axons, and exhibit higher expression of the neuritogenic gene neurogenin 3 (Ngn3) than XY before perinatal masculinization. Here we evaluated the participation of candidate X-linked genes in the development of these sex differences, focusing mainly on Kdm6a, a gene encoding for an H3K27 demethylase with functions controlling gene expression genome-wide. We established hypothalamic neuronal cultures from wild-type or transgenic Four Core Genotypes mice, a model that allows evaluating the effect of sex chromosomes independently of gonadal type. X-linked genes Kdm6a, Eif2s3x and Ddx3x showed higher expression in XX compared to XY neurons, regardless of gonadal sex. Moreover, Kdm6a expression pattern with higher mRNA levels in XX than XY did not change with age at E14, P0, and P60 in hypothalamus or under 17ß-estradiol treatment in culture. Kdm6a pharmacological blockade by GSK-J4 reduced axonal length only in female neurons and decreased the expression of neuritogenic genes Neurod1, Neurod2 and Cdk5r1 in both sexes equally, while a sex-specific effect was observed in Ngn3. Finally, Kdm6a downregulation using siRNA reduced axonal length and Ngn3 expression only in female neurons, abolishing the sex differences observed in control conditions. Altogether, these results point to Kdm6a as a key mediator of the higher axogenesis and Ngn3 expression observed in XX neurons before the critical period of brain masculinization.
Assuntos
Genes Ligados ao Cromossomo X/genética , Histona Desmetilases/genética , Histonas/genética , Hipotálamo/fisiologia , Neurônios/fisiologia , Diferenciação Sexual/genética , Animais , Axônios/fisiologia , Feminino , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Caracteres SexuaisRESUMO
A high-fat diet induces hypothalamic inflammation in rodents which, in turn, contributes to the development of obesity by eliciting both insulin and leptin resistance. However, the mechanism by which long-chain saturated fatty acids trigger inflammation is still contentious. To elucidate this mechanism, the effect of fatty acids on the expression of the pro-inflammatory cytokines IL-6 and TNFα was investigated in the mHypoE-N42 hypothalamic cell line (N42). N42 cells were treated with lauric acid (LA) and palmitic acid (PA). PA challenge was carried out in the presence of either a TLR4 inhibitor, a ceramide synthesis inhibitor (L-cycloserine), oleic acid (OA) or eicosapentaenoic acid (EPA). Intracellular ceramide accumulation was quantified using LC-ESI-MS/MS. PA but not LA upregulated IL-6 and TNFα. L-cycloserine, OA and EPA all counteracted PA-induced intracellular ceramide accumulation leading to a downregulation of IL-6 and TNFα. However, a TLR4 inhibitor failed to inhibit PA-induced upregulation of pro-inflammatory cytokines.In conclusion, PA induced the expression of IL-6 and TNFα in N42 neuronal cells independently of TLR4 but, partially, via ceramide synthesis with OA and EPA being anti-inflammatory by decreasing PA-induced intracellular ceramide build-up. Thus, ceramide accumulation represents one on the mechanisms by which PA induces inflammation in neurons.
Assuntos
Ceramidas/biossíntese , Encefalite/metabolismo , Hipotálamo/metabolismo , Ácido Palmítico/administração & dosagem , Ácido Palmítico/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Encefalite/induzido quimicamente , Hipotálamo/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Sprague-DawleyRESUMO
Aims: To investigate the interactions among narcolepsy-associated genes and reveal the pathways these genes involved through bioinformatics analyses. Methods: The study was performed with the following steps: 1) Selected the previously discovered narcolepsy risk genes through literature review, 2) pathway enrichment analysis, and construction of gene-gene and protein-protein interaction (PPI) networks for narcolepsy. Results: 1) GO analysis revealed the positive regulation of interferon-gamma production as the most enriched terms in biological process, and C-C chemokine receptor activity as the most enriched term in molecular function, 2) KEGG pathway enrichment analysis revealed selective enrichment of genes in cytokine-cytokine receptor interaction signaling pathways, and 3) five hub genes were identified (IFNAR1, IL10RB, DNMT1, TNFSF4 and NFATC2). Conclusion: The bioinformatics results provide new insights into the molecular pathogenesis of narcolepsy and the identification of potential therapeutic targets for narcolepsy treatment.
Assuntos
Redes Reguladoras de Genes/fisiologia , Narcolepsia/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Ontologia Genética , Redes Reguladoras de Genes/genética , Humanos , Mapas de Interação de Proteínas/genética , Mapas de Interação de Proteínas/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Gangliosides are essential components of cell membranes and are involved in a variety of physiological processes, including cell growth, differentiation, and receptor-mediated signal transduction. They regulate functions of proteins in membrane microdomains, notably receptor tyrosine kinases such as insulin receptor (InsR) and epidermal growth factor receptor (EGFR), through lateral association. Studies during the past two decades using knockout (KO) or pharmacologically inhibited cells, or KO mouse models for glucosylceramide synthase (GCS; Ugcg), GM3 synthase (GM3S; St3gal5), and GD3 synthase (GD3S; St8sia1) have revealed essential roles of gangliosides in hypothalamic control of energy balance. The a-series gangliosides GM1 and GD1a interact with leptin receptor (LepR) and promote LepR signaling through activation of the JAK2/STAT3 pathway. Studies of GM3S KO cells have shown that the extracellular signal-regulated kinase (ERK) pathway, downstream of the LepR signaling pathway, is also modulated by gangliosides. Recent studies have revealed crosstalk between the LepR signaling pathway and other receptor signaling pathways (e.g., InsR and EGFR pathways). Gangliosides thus have the ability to modulate the effects of leptin by regulating functions of such receptors, and by direct interaction with LepR to control signaling.
Assuntos
Metabolismo Energético/fisiologia , Gangliosídeos/metabolismo , Hipotálamo/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gangliosídeos/genética , Humanos , Camundongos , Camundongos Knockout , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Sialiltransferases/metabolismoRESUMO
Leptin resistance in the hypothalamus has an essential role in obesity. Saturated fatty acids such as palmitate bind to Toll-like receptor 4 (TLR4) and lead to endoplasmic reticulum (ER) stress and leptin resistance. In this study, we evaluated whether extracts of Ecklonia cava would attenuate the ER stress induced by palmitate and reduce leptin resistance in hypothalamic neurons and microglia. We added palmitate to these cells to mimic the environment induced by high-fat diet in the hypothalamus and evaluated which of the E. cava phlorotannins-dieckol (DK), 2,7-phloroglucinol-6,6-bieckol (PHB), pyrogallol-phloroglucinol-6,6-bieckol (PPB), or phlorofucofuroeckol-A (PFFA)-had the most potent effect on attenuating leptin resistance. TLR4 and NF-κB expression induced by palmitate was attenuated most effectively by PPB in both hypothalamic neurons and microglia. ER stress markers were increased by palmitate and were attenuated by PPB in both hypothalamic neurons and microglia. Leptin resistance, which was evaluated as an increase in SOCS3 and a decrease in STAT3 with leptin receptor expression, was increased by palmitate and was decreased by PPB in hypothalamic neurons. The culture medium from palmitate-treated microglia increased leptin resistance in hypothalamic neurons and this resistance was attenuated by PPB. In conclusion, PPB attenuated leptin resistance by decreasing ER stress in both hypothalamic neurons and microglia.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Leptina/metabolismo , Neurônios/efeitos dos fármacos , Palmitatos/farmacologia , Phaeophyceae/química , Taninos/farmacologia , Animais , Linhagem Celular , Dieta Hiperlipídica/efeitos adversos , Humanos , Hipotálamo/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , NF-kappa B/metabolismo , Neurônios/metabolismo , Obesidade/metabolismo , Floroglucinol/farmacologia , Proibitinas , Receptores para Leptina/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
Developmental actions of estradiol in the hypothalamus are well characterized. This hormone generates sex differences in the development of hypothalamic neuronal circuits controlling neuroendocrine events, feeding, growth, reproduction and behavior. In vitro, estradiol promotes sexually dimorphic effects on hypothalamic neuritogenesis. Previous studies have shown that developmental actions of the phytoestrogen genistein result in permanent sexually dimorphic effects in some behaviors and neural circuits in vivo. In the present study, we have explored if genistein, like estradiol, affects neuritogenesis in primary hypothalamic neurons and investigated the estrogen receptors implicated in this action. Hypothalamic neuronal cultures, obtained from male or female embryonic day 14 (E14) CD1 mice, were treated with genistein (0.1 µM, 0.5 µM or 1 µM) or vehicle. Under basal conditions, female neurons had longer primary neurites, higher number of secondary neurites and higher neuritic arborization compared to male neurons. The treatment with genistein increased neuritic arborization and the number of primary neurites and decreased the number of secondary neurites in female neurons, but not in male neurons. In contrast, genistein resulted in a significant increase in primary neuritic length in male neurons, but not in female neurons. The use of selective estrogen receptor antagonists suggests that estrogen receptor α, estrogen receptor ß and G-protein-coupled estrogen receptors are involved in the neuritogenic action of genistein. In summary, these findings indicate that genistein exerts sexually dimorphic actions on the development of hypothalamic neurons, altering the normal pattern of sex differences in neuritogenesis.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Genisteína/farmacologia , Fitoestrógenos/farmacologia , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Caracteres Sexuais , Animais , Biomarcadores , Feminino , Masculino , Camundongos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurogênese/efeitos dos fármacos , Células Piramidais/metabolismo , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismoRESUMO
Attesting to its intimate peripheral connections, hypothalamic neurons integrate nutritional and hormonal cues to effectively manage energy homeostasis according to the overall status of the system. Extensive progress in the identification of essential transcriptional and post-translational mechanisms regulating the controlled expression and actions of hypothalamic neuropeptides has been identified through the use of animal and cell models. This review will introduce the basic techniques of hypothalamic investigation both in vivo and in vitro and will briefly highlight the key advantages and challenges of their use. Further emphasis will be place on the use of immortalized models of hypothalamic neurons for in vitro study of feeding regulation, with a particular focus on cell lines proving themselves most fruitful in deciphering fundamental basics of NPY/AgRP, Proglucagon, and POMC neuropeptide function.
Assuntos
Metabolismo Energético/fisiologia , Homeostase/fisiologia , Hipotálamo/fisiologia , Neurônios/fisiologia , Animais , Linhagem Celular , Hipotálamo/citologia , Neurônios/citologia , Neuropeptídeos/fisiologiaRESUMO
Neuroinflammation is a feedback mechanism against infection, with recent studies suggesting a neuromodulatory role. The chemokine, (C-C motif) ligand 2 (CCL2), and its receptor, (C-C motif) receptor type 2 (CCR2), affect neuromodulation and migration in response to damage. Although CCL2 co-localizes with neuropeptides in the hypothalamus that control voluntary behavior, the function of CCL2/CCR2 is unknown. This led us to consider the possibility that CCL2 acting through CCR2, under natural conditions, may affect the migration and peptide levels of hypothalamic neurons that control voluntary behavior. This study used primary embryonic hypothalamic neurons to examine the effect of CCL2 on migratory behavior and on levels of the peptides, enkephalin (ENK) and galanin. Treatment with CCL2 led to a significant, dose-dependent increase in the number of migrated neurons and an increase in the velocity and distance traveled. CCL2 also significantly increased the number of ENK-expressing and CCR2/ENK co-expressing neurons and the percentage of neurons that contain higher levels of ENK. Lastly, CCL2 produced a dose-dependent increase in expression of ENK and galanin. These results provide evidence for a stimulatory effect of CCL2 on embryonic hypothalamic neurons involving changes in migratory behavior, expression, and synthesis of neuropeptides that function in controlling behavior. Our results demonstrate that the chemokine, CCL2, functions through its receptor, CCR2, to stimulate the migration and expression of the orexigenic peptides, enkephalin (ENK) and galanin (GAL), in developing embryonic hypothalamic neurons that are important for controlling ingestive behavior. This evidence reveals broad effects of CCL2 in the developing hypothalamus, showing this chemokine system to be tightly linked to the hypothalamic peptide neurons.
Assuntos
Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/farmacologia , Hipotálamo/citologia , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Receptores CCR2/metabolismo , Análise de Variância , Animais , Movimento Celular/fisiologia , Células Cultivadas , Quimiocina CCL2/metabolismo , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Encefalinas/metabolismo , Feminino , Galanina/metabolismo , Neurônios/efeitos dos fármacos , Fosfopiruvato Hidratase/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Prenatal high-fat diet exposure increases hypothalamic neurogenesis events in embryos and programs offspring to be obesity-prone. The molecular mechanism involved in these dietary effects of neurogenesis is unknown. This study investigated the effects of oleic and palmitic acids, which are abundant in a high-fat diet, on the hypothalamic neuronal transcriptome and how these changes impact neurogenesis events. The results show the differential effects of low and high concentrations of oleic or palmitic acid treatment on differential gene transcription. Gene ontology analysis uncovered significant gene enrichment in several cellular pathways involved in gene regulation and protein production, particularly with proliferation, migration, and cell survival. The enriched signaling pathways include Wnt, integrin, PDGF, and apoptosis, in addition to endocrine function signaling pathways CCKR and GnRH. Further examination of proliferation and migration shows low concentrations of oleic acid to stimulate proliferation and high concentrations of both oleic and palmitic acid to stimulate apoptosis. Oleic acid also reduces hypothalamic neuronal migration, with little effect from palmitic acid. The results show the two most abundant fatty acids in a high-fat diet impact hypothalamic neuronal proliferation and migration. The results also uncovered potential signaling pathways affected by oleic and palmitic acid and suggest one mechanism of prenatal high-fat diet-induced neurogenesis events may be through these two abundant fatty acids.
Assuntos
Ácido Palmítico , Ácidos Palmíticos , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/farmacologia , Ácido Oleico/farmacologia , Ácidos Oleicos , Ácido Palmítico/farmacologia , Ácidos Palmíticos/farmacologia , TranscriptomaRESUMO
Olfactory marker protein (OMP) is a cytosolic protein expressed in mature olfactory receptor neurons (ORNs). OMP modulates cAMP signalling and regulates olfactory sensation and axonal targeting. OMP is a small soluble protein, and passive diffusion between nucleus and cytoplasm is expected. However, OMP is mostly situated in the cytosol and is only sparsely detected in the nuclei of a subset of ORNs, hypothalamic neurons and heterologously OMP-expressing cultured cells. OMP can enter the nucleus in association with transcription factors. However, how OMP is retained in the cytosol at rest is unclear. Because OMP is proposed to affect cell differentiation, it is important to understand how OMP is distributed between cytoplasm and nucleus. To elucidate the structural profile of OMP, we applied several bioinformatics methods to a multiple sequence alignment (MSA) of OMP protein sequences and ranked the evolutionarily conserved residues. In addition to the previously reported cAMP-binding domain, we identified a leucine-rich domain in the Ω-loop of OMP. We introduced mutations into the leucine-rich region and heterologously expressed the mutant OMP in HEK293T cells. Mutations into alanine increased the nuclear distribution of OMP quantified by immunocytochemistry and western blotting. Therefore, we concluded that OMP contains a leucine-rich domain important for nuclear transport.
Assuntos
Neurônios Receptores Olfatórios , Humanos , Proteína de Marcador Olfatório , Transporte Ativo do Núcleo Celular , Leucina , Células HEK293 , Fatores de TranscriçãoRESUMO
An endogenous circadian clock system enables organisms to adapt to time-of-day dependent environmental changes. In consequence, most physiological processes exhibit daily rhythms of, e.g., energy metabolism, immune function, sleep, or hormone production. Hypothalamic circadian clocks have been identified to play a particular role in coordinating many of these processes. Primary neuronal cultures are widely used as a physiologically relevant model to study molecular events within neurons. However, as circadian rhythms include dynamic molecular changes over longer timescales that vary between individual cells, longitudinal measurement methods are essential to investigate the regulation of circadian clocks of hypothalamic neurons. Here we provide a protocol for generating primary hypothalamic neuronal cultures expressing a circadian luciferase reporter. Such reporter cells can be used to longitudinally monitor cellular circadian rhythms at high temporal resolution by performing bioluminescence measurements.
RESUMO
Phytoestrogens can control high-fat diet-induced hypothalamic inflammation that is associated with severe consequences, including obesity, type 2 diabetes, cardiovascular and neurodegenerative diseases. However, the phytoestrogen anti-neuroinflammatory action is poorly understood. In this study, we explored the neuroprotection mediated by daidzein in hypothalamic neurons by using a membrane-based model of obesity-related neuroinflammation. To test the daidzein therapeutic potential a biohybrid membrane system, consisting of hfHypo GnRH-neurons in culture on PLGA membranes, was set up. It served as reliable in vitro tool capable to recapitulate the in vivo structure and function of GnRH hypothalamic tissue. Our findings highlighted the neuroprotective role of daidzein, being able to counteract the palmitate induced neuroinflammation. Daidzein protected hfHypo GnRH cells by downregulating cell death, proinflammatory processes, oxidative stress, and apoptosis. It also restored the proper cell morphology and functionality through a mechanism which probably involves the activation of ERß and GPR30 receptors along with the expression of GnRH peptide and KISS1R.
Assuntos
Anti-Inflamatórios/uso terapêutico , Encefalite/tratamento farmacológico , Hipotálamo , Isoflavonas/uso terapêutico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Fitoestrógenos/uso terapêutico , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Células Cultivadas , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Hipotálamo/citologia , Membranas Artificiais , Modelos Biológicos , Neurônios/citologia , Neurônios/metabolismo , Palmitatos/toxicidade , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
17ß-estradiol (E2) induces axonal growth through extracellular signal-regulated kinase 1 and 2 (ERK1/2)-MAPK cascade in hypothalamic neurons of male rat embryos in vitro, but the mechanism that initiates these events is poorly understood. This study reports the intracellular Ca2+ increase that participates in the activation of ERK1/2 and axogenesis induced by E2. Hypothalamic neuron cultures were established from 16-day-old male rat embryos and fed with astroglia-conditioned media for 48 h. E2-induced ERK phosphorylation was completely abolished by a ryanodine receptor (RyR) inhibitor (ryanodine) and partially attenuated by an L-type voltage-gated Ca2+ channel (L-VGCC) blocker (nifedipine), an inositol-1,4,5-trisphosphate receptor (IP3R) inhibitor (2-APB), and a phospholipase C (PLC) inhibitor (U-73122). We also conducted Ca2+ imaging recording using primary cultured neurons. The results show that E2 rapidly induces an increase in cytosolic Ca2+, which often occurs in repetitive Ca2+ oscillations. This response was not observed in the absence of extracellular Ca2+ or with inhibitory ryanodine and was markedly reduced by nifedipine. E2-induced axonal growth was completely inhibited by ryanodine. In summary, the results suggest that Ca2+ mobilization from extracellular space as well as from the endoplasmic reticulum is necessary for E2-induced ERK1/2 activation and axogenesis. Understanding the mechanisms of brain estrogenic actions might contribute to develop novel estrogen-based therapies for neurodegenerative diseases.
RESUMO
Ivabradine (IVA), a heart-rate reducing agent, is recognized as an inhibitor of hyperpolarization-activated cation current (Ih) and also reported to ameliorate inflammatory or neuropathic pain. However, to what extent this agent can perturb another types of membrane ion currents in neurons or endocrine cells remains to be largely unknown. Therefore, the Ih or other types of ionic currents in pituitary tumor (GH3) cells and in hippocampal mHippoE-14 neurons was studied with or without the presence of IVA or other related compounds. The IVA addition caused a time- and concentration-dependent reduction in the amplitude of Ih with an IC50 value of 0.64 µM and a KD value of 0.68 µM. IVA (0.3 µM) shifted the Ih activation curve to a more negative potential by approximately 8 mV, despite no concomitant change in the gating charge. Additionally, IVA was found to increase M-type K+ current (IK(M)) together with a rightward shift in the activation curve. In cell-attached current recordings, IVA (3 µM) applied to the bath increased the open probability of M-type K+ channels; however, it did not modify single-channel conductance of the channel. In current-clamp voltage recordings, IVA suppressed the firing of spontaneous action potentials in GH3 cells; and, further addition of linopirdine attenuated its suppression of firing. In hippocampal mHippoE-14 neurons, IVA also effectively increased IK(M) amplitude. In summary, both inhibition of Ih and activation of IK(M) caused by IVA can synergistically combine to influence electrical behaviors in different types of electrically excitable cells occurring in vivo.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/efeitos dos fármacos , Ivabradina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Células Endócrinas/metabolismo , Hipocampo/metabolismo , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ivabradina/metabolismo , Camundongos , Neurônios/fisiologia , Neoplasias Hipofisárias/fisiopatologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismoRESUMO
The hypothalamus contains neurons that integrate hunger and satiety endocrine signals from the periphery and are implicated in the pathophysiology of obesity. The limited availability of human hypothalamic neurons hampers our understanding of obesity disease mechanisms. To address this, we generated human induced pluripotent stem cells (hiPSCs) from multiple normal body mass index (BMI; BMI ≤ 25) subjects and super-obese (OBS) donors (BMI ≥ 50) with polygenic coding variants in obesity-associated genes. We developed a method to reliably differentiate hiPSCs into hypothalamic-like neurons (iHTNs) capable of secreting orexigenic and anorexigenic neuropeptides. Transcriptomic profiling revealed that, although iHTNs maintain a fetal identity, they respond appropriately to metabolic hormones ghrelin and leptin. Notably, OBS iHTNs retained disease signatures and phenotypes of high BMI, exhibiting dysregulated respiratory function, ghrelin-leptin signaling, axonal guidance, glutamate receptors, and endoplasmic reticulum (ER) stress pathways. Thus, human iHTNs provide a powerful platform to study obesity and gene-environment interactions.
Assuntos
Grelina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Leptina/metabolismo , Neurônios/metabolismo , Obesidade Mórbida/metabolismo , Obesidade Mórbida/patologia , Índice de Massa Corporal , Encéfalo/citologia , Encéfalo/metabolismo , Diferenciação Celular , Feminino , Humanos , Masculino , Obesidade Mórbida/genética , Transdução de Sinais/genéticaRESUMO
The present study was undertaken because no previous developmental studies exist on MCH neurons in any avian species. After validating a commercially-available antibody for use in chickens, immunohistochemical examinations first detected MCH neurons around embryonic day (E) 8 in the posterior hypothalamus. This population increased thereafter, reaching a numerical maximum by E20. MCH-positive cell bodies were found only in the posterior hypothalamus at all ages examined, restricted to a region showing very little overlap with the locations of hypocretin/orexin (H/O) neurons. Chickens had fewer MCH than H/O neurons, and MCH neurons also first appeared later in development than H/O neurons (the opposite of what has been found in rodents). MCH neurons appeared to originate from territories within the hypothalamic periventricular organ that partially overlap with the source of diencephalic serotonergic neurons. Chicken MCH fibers developed exuberantly during the second half of embryonic development, and they became abundant in the same brain areas as in rodents, including the hypothalamus (by E12), locus coeruleus (by E12), dorsal raphe nucleus (by E20) and septum (by E20). These observations suggest that MCH cells may play different roles during development in chickens and rodents; but once they have developed, MCH neurons exhibit similar phenotypes in birds and rodents.
Assuntos
Proteínas Aviárias/metabolismo , Encéfalo/citologia , Encéfalo/embriologia , Hormônios Hipotalâmicos/metabolismo , Melaninas/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Hormônios Hipofisários/metabolismo , Animais , Encéfalo/metabolismo , Embrião de GalinhaRESUMO
We recently developed a technique for generating hypothalamic neurons from human pluripotent stem cells. Here, as proof of principle, we examine the use of these cells in modeling of a monogenic form of severe obesity: PCSK1 deficiency. The cognate enzyme, PC1/3, processes many prohormones in neuroendocrine and other tissues. We generated PCSK1 (PC1/3)-deficient human embryonic stem cell (hESC) lines using both short hairpin RNA and CRISPR-Cas9, and investigated pro-opiomelanocortin (POMC) processing using hESC-differentiated hypothalamic neurons. The increased levels of unprocessed POMC and the decreased ratios (relative to POMC) of processed POMC-derived peptides in both PCSK1 knockdown and knockout hESC-derived neurons phenocopied POMC processing reported in PC1/3-null mice and PC1/3-deficient patients. PC1/3 deficiency was associated with increased expression of melanocortin receptors and PRCP (prolylcarboxypeptidase, a catabolic enzyme for α-melanocyte stimulating hormone (αMSH)), and reduced adrenocorticotropic hormone secretion. We conclude that the obesity accompanying PCSK1 deficiency may not be primarily due to αMSH deficiency.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Neurônios/citologia , Neurônios/metabolismo , Pró-Opiomelanocortina/metabolismo , Pró-Proteína Convertase 1/deficiência , Hormônio Adrenocorticotrópico/metabolismo , Animais , Apoptose , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Células Cultivadas , Estresse do Retículo Endoplasmático , Expressão Gênica , Técnicas de Silenciamento de Genes , Marcação de Genes , Humanos , Imuno-Histoquímica , Camundongos , Mutação , Pró-Proteína Convertase 1/genética , Proteólise , Células Piramidais/citologia , Células Piramidais/metabolismo , alfa-MSH/metabolismoRESUMO
The hypothalamus comprises neuronal clusters that are essential for body weight regulation and other physiological functions. Insights into the complex cellular physiology of this region of the brain are critical to understanding the pathogenesis of obesity, but human hypothalamic cells are largely inaccessible for direct study. Here we describe a technique for generation of arcuate-like hypothalamic neurons from human pluripotent stem (hPS) cells. Early activation of SHH signaling and inhibition of BMP and TGFß signaling, followed by timed inhibition of NOTCH, can efficiently differentiate hPS cells into NKX2.1+ hypothalamic progenitors. Subsequent incubation with BDNF induces the differentiation and maturation of pro-opiomelanocortin and neuropeptide Y neurons, which are major cell types in the arcuate hypothalamus. These neurons have molecular and cellular characteristics consistent with arcuate neurons. © 2016 by John Wiley & Sons, Inc.
Assuntos
Núcleo Arqueado do Hipotálamo/citologia , Diferenciação Celular , Células-Tronco Neurais/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Humanos , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Pró-Opiomelanocortina/metabolismo , Transdução de SinaisRESUMO
Hypothalamic neurons expressing neuropeptide Y (NPY) and agouti related-protein (AgRP) are critical regulators of feeding behavior and body weight, and transduce the action of many peripheral signals including leptin and insulin. However, intracellular signaling molecules involved in regulating NPY/AgRP neuronal activity are incompletely understood. Since phosphodiesterase-3B (PDE3B) mediates the hypothalamic action of leptin and insulin on feeding, and is expressed in NPY/AgRP neurons, PDE3B could play a significant role in regulating NPY/AgRP neuronal activity. To investigate the direct regulation of NPY/AgRP neuronal activity by PDE3B, we examined the effects of gain-of-function or reduced function of PDE3B on NPY/AgRP gene expression in a clonal hypothalamic neuronal cell line, mHypoE-46, which endogenously express NPY, AgRP and PDE3B. Overexpression of PDE3B in mHypoE-46 cells with transfection of pcDNA-3.1-PDE3B expression plasmid significantly decreased NPY and AgRP mRNA levels and p-CREB levels as compared to the control plasmid. For the PDE3B knockdown study, mHypoE-46 cells transfected with lentiviral PDE3BshRNAmir plasmid or non-silencing lentiviral shRNAmir control plasmid were selected with puromycin, and stably transfected cells were grown in culture for 48h. Results showed that PDE3BshRNAmir mediated knockdown of PDE3B mRNA and protein levels (â¼60-70%) caused an increase in both NPY and AgRP gene expression and in p-CREB levels. Together, these results demonstrate a reciprocal change in NPY and AgRP gene expression following overexpression and knockdown of PDE3B, and suggest a significant role for PDE3B in the regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Animais , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Expressão Gênica , Hipotálamo/citologia , CamundongosRESUMO
Taurine is an essential amino-sulfonic acid having a fundamental function in the brain, participating in both cell volume regulation and neurotransmission. Using a whole cell voltage patch clamp technique, the taurine-activated neurotransmitter receptors in the preoptic hypothalamic area (PHA) neurons were investigated. In the first set of experiments, different concentrations of taurine were applied on PHA neurons. Taurine-induced responses were concentration-dependent. Taurine-induced currents were action potential-independent and sensitive to strychnine, suggesting the involvement of glycine receptors. In addition, taurine activated not only α-homomeric, but also αß-heteromeric glycine receptors in PHA neurons. Interestingly, a low concentration of taurine (0.5mM) activated glycine receptors, whereas a higher concentration (3mM) activated both glycine and gamma-aminobutyric acid A (GABAA) receptors in PHA neurons. These results suggest that PHA neurons are influenced by taurine and respond via glycine and GABAA receptors.