RESUMO
The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity. OBJECTIVE: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly. METHODS: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors. RESULTS: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation. CONCLUSIONS: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.
Assuntos
Proteínas de Transporte/metabolismo , Obesidade/metabolismo , Receptores para Leptina/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Peso Corporal/fisiologia , Proteínas de Transporte/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Leptina/metabolismo , Leptina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Receptores para Leptina/fisiologia , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.
Assuntos
Aminoácidos Essenciais/administração & dosagem , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Administração Oral , Aminoácidos Essenciais/farmacocinética , Animais , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Ingestão de Alimentos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Transgenes/genética , Resultado do TratamentoRESUMO
Glioblastoma multiform (GBM) are devastating brain tumors containing a fraction of multipotent stem-like cells which are highly tumorigenic. These cells are resistant to treatments and are likely to be responsible for tumor recurrence. One approach to eliminate GBM stem-like cells would be to force their terminal differentiation. During development, neurons formation is controlled by neurogenic transcription factors such as Ngn1/2 and NeuroD1. We found that in comparison with oligodendrogenic genes, the expression of these neurogenic genes is low or absent in GBM tumors and derived cultures. We thus explored the effect of overexpressing these neurogenic genes in three CD133(+) Sox2(+) GBM stem-like cell cultures and the U87 glioma line. Introduction of Ngn2 in CD133(+) cultures induced massive cell death, proliferation arrest and a drastic reduction of neurosphere formation. Similar effects were observed with NeuroD1. Importantly, Ngn2 effects were accompanied by the downregulation of Olig2, Myc, Shh and upregulation of Dcx and NeuroD1 expression. The few surviving cells adopted a typical neuronal morphology and some of them generated action potentials. These cells appeared to be produced at the expense of GFAP(+) cells which were radically reduced after differentiation with Ngn2. In vivo, Ngn2-expressing cells were unable to form orthotopic tumors. In the U87 glioma line, Ngn2 could not induce neuronal differentiation although proliferation in vitro and tumoral growth in vivo were strongly reduced. By inducing cell death, cell cycle arrest or differentiation, this work supports further exploration of neurogenic proteins to oppose GBM stem-like and non-stem-like cell growth.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Neoplasias Encefálicas/patologia , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Fatores de Transcrição/farmacologia , Antígeno AC133 , Antígenos CD/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Morte Celular , Citometria de Fluxo , Proteína Glial Fibrilar Ácida/metabolismo , Glicoproteínas/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Fator de Transcrição 2 de Oligodendrócitos , Proteína Oncogênica p55(v-myc)/metabolismo , Peptídeos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
In a perspective of regenerative medicine, multipotent human neural progenitor cells (hNPCs) offer a therapeutic advantage over pluripotent stem cells in that they are already invariantly "neurally committed" and lack tumorigenicity. However, some of their intrinsic properties, such as slow differentiation and uncontrolled multipotency, remain among the obstacles to their routine use for transplantation. Although rodent NPCs have been genetically modified in vitro to overcome some of these limitations, the translation of this strategy to human cells remains in its early stages. In the present study, we compare the actions of 4 basic helix-loop-helix transcription factors on the proliferation, specification, and terminal differentiation of hNPCs isolated from the fetal dorsal telencephalon. Consistent with their proneural activity, Ngn1, Ngn2, Ngn3, and Mash1 prompted rapid commitment of the cells. The Ngns induced a decrease in proliferation, whereas Mash1 maintained committed progenitors in a proliferative state. As opposed to Ngn1 and Ngn3, which had no effect on glial differentiation, Ngn2 induced an increase in astrocytes in addition to neurons, whereas Mash1 led to both neuronal and oligodendroglial specification. GABAergic, cholinergic, and motor neuron differentiations were considerably increased by overexpression of Ngn2 and, to a lesser extent, of Ngn3 and Mash1. Thus, we provide evidence that hNPCs can be efficiently, rapidly, and safely expanded in vitro as well as rapidly differentiated toward mature neural (typically neuronal) lineages by the overexpression of select proneural genes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Diferenciação Celular/fisiologia , Córtex Cerebral/metabolismo , Neurônios Colinérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Células-Tronco/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Córtex Cerebral/citologia , Neurônios Colinérgicos/citologia , Feto , Neurônios GABAérgicos/citologia , Regulação da Expressão Gênica/fisiologia , Humanos , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/genética , Células-Tronco/citologiaRESUMO
We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.
RESUMO
Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1(-/-)) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT(2A) and 5-HT(2B) receptors toward terminal differentiation. In addition, red blood cells from 5-HT-deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1(-/-) animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT-deficient and normal cells. Our thorough analysis of TPH1(-/-) mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.
Assuntos
Eritrócitos/patologia , Eritropoese , Serotonina/deficiência , Anemia Macrocítica/complicações , Anemia Macrocítica/enzimologia , Anemia Macrocítica/patologia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Suplementos Nutricionais , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patologia , Eritropoese/efeitos dos fármacos , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Serotonina/metabolismo , Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Siderose/complicações , Siderose/patologia , Baço/efeitos dos fármacos , Baço/patologia , Triptofano Hidroxilase/deficiência , Triptofano Hidroxilase/metabolismoRESUMO
The gut contains a large 5-HT pool in enterochromaffin (EC) cells and a smaller 5-HT pool in the enteric nervous system (ENS). During development, enteric neurons are generated asynchronously. We tested hypotheses that serotonergic neurons, which arise early, affect development/survival of later-born dopaminergic, GABAergic, nitrergic, and calcitonin gene-related peptide-expressing neurons and are essential for gastrointestinal motility. 5-HT biosynthesis depends on tryptophan hydroxylase 1 (TPH1) in EC cells and on TPH2 in neurons; therefore, mice lacking TPH1 and/or TPH2 distinguish EC-derived from neuronal 5-HT. Deletion of TPH2, but not TPH1, decreased myenteric neuronal density and proportions of dopaminergic and GABAergic neurons but did not affect the extrinsic sympathetic innervation of the gut; intestinal transit slowed in mice lacking TPH2 mice, but gastric emptying accelerated. Isolated enteric crest-derived cells (ENCDCs) expressed the serotonin reuptake transporter (SERT) and 15 subtypes of 5-HT receptor. Addition of 5-HT to cultures of isolated ENCDCs promoted total and dopaminergic neuronal development. Rings of SERT-immunoreactive terminal axons surrounded myenteric dopaminergic neurons and SERT knock-out increased intestinal levels of dopamine metabolites, implying that enteric dopaminergic neurons receive a serotonergic innervation. Observations suggest that constitutive gastrointestinal motility depends more on neuronal than EC cell serotonin; moreover, serotonergic neurons promote development/survival of some classes of late-born enteric neurons, including dopaminergic neurons, which appear to innervate and activate in the adult ENS.
Assuntos
Dopamina/metabolismo , Sistema Nervoso Entérico/citologia , Motilidade Gastrointestinal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , Serotonina/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 3 , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Esvaziamento Gástrico/efeitos dos fármacos , Esvaziamento Gástrico/genética , Motilidade Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Ácido Homovanílico/análogos & derivados , Ácido Homovanílico/metabolismo , Técnicas In Vitro , Intestino Delgado/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/metabolismo , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Triptofano Hidroxilase/deficiência , Tirosina 3-Mono-Oxigenase/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Mucosal inflammation in the gut is characterized by infiltration of innate and adaptive immune cells and by an alteration in serotonin-producing enterochromaffin cells. We investigated the role of serotonin in the function of dendritic cells (DCs) and sequential T-cell activation in relation to generation of gut inflammation. DCs isolated from tryptophan hydroxylase-1-deficient (TPH1(-/-)) mice, which have reduced serotonin in the gut, and wild-type (TPH1(+/+)) mice with or without dextran sulfate sodium (DSS)-induced colitis were stimulated with lipopolysaccharide to assess interleukin-12 (IL-12) production. Isolated DCs from TPH1(+/+) and TPH1(-/-) mice were also cocultured with CD4(+) T cells of naive TPH1(+/+) mice to assess the role of serotonin in priming T cells. In addition, serotonin-pulsed DCs were transferred to TPH1(-/-) mice to assess the effect on DSS-induced colitis. Consistent with a reduced severity of colitis, DCs from DSS-induced TPH1(-/-) mice produced less IL-12 compared with the TPH1(+/+) mice. In vitro serotonin stimulation restored the cytokine production from TPH1(-/-) DCs and adoptive transfer of serotonin-pulsed DCs into TPH1(-/-) up-regulated colitis. Furthermore, CD4(+) T cells primed by TPH1(-/-) DCs produce reduced the levels of IL-17 and interferon-γ. This study provides novel information on serotonin-mediated immune signaling and promotion of interactions between innate and adaptive immune responses in the context of gut inflammation, which may ultimately lead to improved strategies to combat gut inflammatory disorders.
Assuntos
Células Dendríticas/metabolismo , Sistema Digestório/patologia , Inflamação/patologia , Serotonina/metabolismo , Transferência Adotiva , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Separação Celular , Colite/induzido quimicamente , Colite/patologia , Apresentação Cruzada/efeitos dos fármacos , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Sulfato de Dextrana , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/metabolismo , Regulação para Baixo/efeitos dos fármacos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Serotonina/farmacologiaAssuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Esclerose Lateral Amiotrófica/diagnóstico , Estudos de Casos e Controles , Músculo Deltoide/metabolismo , Humanos , Miosite de Corpos de Inclusão/diagnósticoRESUMO
BACKGROUND: Spinal cord injury (SCI) is a widely spread pathology with currently no effective treatment for any symptom. Regenerative medicine through cell transplantation is a very attractive strategy and may be used in different non-exclusive ways to promote functional recovery. We investigated functional and structural outcomes after grafting human embryonic neural progenitors (hENPs) in spinal cord-lesioned rats. METHODS AND PRINCIPAL FINDINGS: With the objective of translation to clinics we have chosen a paradigm of delayed grafting, i.e., one week after lesion, in a severe model of spinal cord compression in adult rats. hENPs were either naïve or engineered to express Neurogenin 2 (Ngn2). Moreover, we have compared integrating and non-integrating lentiviral vectors, since the latter present reduced risks of insertional mutagenesis. We show that transplantation of hENPs transduced to express Ngn2 fully restore weight support and improve functional motor recovery after severe spinal cord compression at thoracic level. This was correlated with partial restoration of serotonin innervations at lumbar level, and translocation of 5HT1A receptors to the plasma membrane of motoneurons. Since hENPs were not detectable 4 weeks after grafting, transitory expression of Ngn2 appears sufficient to achieve motor recovery and to permit axonal regeneration. Importantly, we also demonstrate that transplantation of naïve hENPs is detrimental to functional recovery. CONCLUSIONS AND SIGNIFICANCE: Transplantation and short-term survival of Ngn2-expressing hENPs restore weight support after SCI and partially restore serotonin fibers density and 5HT1A receptor pattern caudal to the lesion. Moreover, grafting of naïve-hENPs was found to worsen the outcome versus injured only animals, thus pointing to the possible detrimental effect of stem cell-based therapy per se in SCI. This is of major importance given the increasing number of clinical trials involving cell grafting developed for SCI patients.
Assuntos
Axônios/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/terapia , Células-Tronco/metabolismo , Animais , Comportamento Animal , Feminino , Humanos , Neurônios Motores/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1A de Serotonina/metabolismo , Medicina Regenerativa/métodos , Serotonina/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Células-Tronco/citologiaRESUMO
BACKGROUND: Postulating that serotonin (5-HT), released from smoking-activated platelets could be involved in smoking-induced vascular modifications, we studied its catabolism in a series of 115 men distributed as current smokers (S), never smokers (NS) and former smokers (FS) who had stopped smoking for a mean of 13 years. METHODOLOGY/PRINCIPAL FINDINGS: 5-HT, monoamine oxidase (MAO-B) activities and amounts were measured in platelets, and 5-hydroxyindolacetic acid (5-HIAA)--the 5-HT/MAO catabolite--in plasma samples. Both platelet 5-HT and plasma 5-HIAA levels were correlated with the 10-year cardiovascular Framingham relative risk (P<0.01), but these correlations became non-significant after adjustment for smoking status, underlining that the determining risk factor among those taken into account in the Framingham risk calculation was smoking. Surprisingly, the platelet 5-HT content was similar in S and NS but lower in FS with a parallel higher plasma level of 5-HIAA in FS. This was unforeseen since MAO-B activity was inhibited during smoking (P<0.00001). It was, however, consistent with a higher enzyme protein concentration found in S and FS than in NS (P<0.001). It thus appears that MAO inhibition during smoking was compensated by a higher synthesis. To investigate the persistent increase in MAO-B protein concentration, a study of the methylation of its gene promoter was undertaken in a small supplementary cohort of similar subjects. We found that the methylation frequency of the MAOB gene promoter was markedly lower (P<0.0001) for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity. CONCLUSIONS/SIGNIFICANCE: This is one of the first reports that smoking induces an epigenetic modification. A better understanding of the epigenome may help to further elucidate the physiopathology and the development of new therapeutic approaches to tobacco addiction. The results could have a larger impact than cardiovascular damage, considering that MAO-dependent 5-HT catabolism is also involved in addiction, predisposition to cancer, behaviour and mental health.
Assuntos
Epigênese Genética , Monoaminoxidase/genética , Fumar/genética , Adulto , Animais , Plaquetas/metabolismo , Predisposição Genética para Doença , Humanos , Ácido Hidroxi-Indolacético/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Monoaminoxidase/fisiologia , Agregação Plaquetária , Risco , Serotonina/metabolismo , Tabagismo/genéticaRESUMO
BACKGROUND & AIMS: Mucosal changes in inflammatory bowel disease are characterized by ulcerative lesions accompanied by a prominent infiltrate of immune cells as well as alteration in serotonin (5-hydroxytryptamine [5-HT])-producing enterochromaffin cells. We investigated the role of 5-HT in colonic inflammation in mice. METHODS: Colitis was induced with dextran sulfate sodium or dinitrobenzene sulfonic acid in tryptophan hydroxylase 1-deficient (TPH1(-/-)) mice, which have markedly reduced 5-HT in the gastrointestinal tract, and in mice given the 5-HT synthesis inhibitor parachlorophenylalanine. RESULTS: Delayed onset, decreased severity of clinical disease, and significantly lower macroscopic and histologic damage scores were observed in TPH1(-/-) mice, compared with wild-type mice, and in mice given parachlorophenylalanine after induction of colitis by dextran sulfate sodium. This was associated with down-regulation of macrophage infiltration and production of proinflammatory cytokines. 5-HT stimulated production of proinflammatory cytokines from macrophages collected from the peritoneal cavity of wild-type mice; this process was inhibited by a nuclear factor kappaB inhibitor, indicating a critical role for nuclear factor kappaB signaling in 5-HT-mediated activation of immune cells. Restoration of 5-HT levels in TPH1(-/-) mice by the 5-HT precursor 5-hydroxytryptophan increased the severity of DSS-induced colitis. We also observed significant reduction in severity of colitis in TPH1(-/-) mice after induction of dinitrobenzene sulfonic acid-induced colitis. CONCLUSIONS: 5-HT is involved in the pathogenesis of inflammation in experimental colitis. These findings provide insight into the mechanisms of gastrointestinal inflammation and could lead to new therapeutic strategies for inflammatory disorders.
Assuntos
Colite/etiologia , Colite/patologia , Serotonina/fisiologia , Animais , Benzenossulfonatos , Proteína C-Reativa/metabolismo , Colite/metabolismo , Sulfato de Dextrana , Fenclonina , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , Antagonistas da Serotonina , Triptofano Hidroxilase/deficiência , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The identification of the factors that allow better monitoring of stem cell renewal and differentiation is of paramount importance for the implementation of new regenerative therapies, especially with regard to the nervous and hematopoietic systems. In this article, we present new information on the function of zinc finger protein 191 (ZNF/Zfp191), a factor isolated in hematopoietic cell lines, within progenitors of the central nervous system (CNS). ZNF/Zfp191 has been found to be principally expressed in progenitors of the developing CNS of humans and mice. Such an overlap of the expression patterns in addition to the high homology of the protein in mammals suggested that ZNF/Zfp191 exerts a conserved function within such progenitors. Indeed, ZNF191 knockdown in human neural progenitors inhibits proliferation and leads to the exit of the cell cycle. Conversely, ZNF191 misexpression maintains progenitors in cycle and exerts negative control on the Notch pathway, which prevents them from differentiating. The present data, together with the fact that the inactivation of Zfp191 leads to embryonic lethality, confirm ZNF191 as an essential factor acting for the promotion of the cell cycle and thus maintenance in the progenitor stage. On the bases of expression data, such a function can be extended to progenitor cells of other tissues such as the hematopoietic system, which emphasizes the important issue of further understanding the molecular events controlled by ZNF/Zfp191.
Assuntos
Proteínas de Transporte/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco/citologia , Animais , Proteínas de Transporte/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Embrião de Galinha , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Lentivirus/genética , Masculino , Camundongos , Gravidez , Receptores Notch/genética , Receptores Notch/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células-Tronco/metabolismoRESUMO
BACKGROUND: There is increasing interest in developing human cell lines to be used to better understand cell biology, but also for drug screening, toxicology analysis and future cell therapy. In the endocrine pancreatic field, functional human beta cell lines are extremely scarce. On the other hand, rodent insulin producing beta cells have been generated during the past years with great success. Many of such cell lines were produced by using transgenic mice expressing SV40T antigen under the control of the insulin promoter, an approach clearly inadequate in human. Our objective was to develop and validate in rodent an alternative transgenic-like approach, applicable to human tissue, by performing somatic gene transfer into pancreatic progenitors that will develop into beta cells. METHODS AND FINDINGS: In this study, rat embryonic pancreases were transduced with recombinant lentiviral vector expressing the SV40T antigen under the control of the insulin promoter. Transduced tissues were next transplanted under the kidney capsule of immuno-incompetent mice allowing insulinoma development from which beta cell lines were established. Gene expression profile, insulin content and glucose dependent secretion, normalization of glycemia upon transplantation into diabetic mice validated the approach to generate beta cell lines. CONCLUSIONS: Somatic gene transfer into pancreatic progenitors represents an alternative strategy to generate functional beta cell lines in rodent. Moreover, this approach can be generalized to derive cells lines from various tissues and most importantly from tissues of human origin.
Assuntos
Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/transplante , Pâncreas/citologia , Células-Tronco/citologia , Transdução Genética , Animais , Linhagem Celular , Vetores Genéticos , Insulina/genética , Células Secretoras de Insulina/metabolismo , Insulinoma , Lentivirus/genética , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ratos , Transplante de Células-Tronco , Células-Tronco/metabolismoRESUMO
BACKGROUND: The sodium- and potassium-activated adenosine triphosphatase (Na+, K+-ATPase) is a major plasma membrane transporter for sodium and potassium. We recently suggested that bipolar disorders (BD) may be associated with alterations in brain Na+, K+-ATPase. We further conjectured that the differences in Na+, K+-ATPase in BD patients could result partially from genetic variations in Na+, K+-ATPase alpha isoforms. METHODS: To test our hypothesis, we undertook a comprehensive study of 13 tagged single nucleotide polymorphisms (SNPs) across the three genes of the brain alpha isoforms of Na+, K+- ATPase (ATP1A1, ATP1A2, and ATP1A3, which encode the three alpha isoforms, alpha1, alpha2, and alpha3, respectively) identified using HapMap data and the Haploview algorithm. Altogether, 126 subjects diagnosed with BD from 118 families were genotyped (parents and affected siblings). Both individual SNPs and haplotypes were tested for association using family-based association tests as provided in the UNPHASED and PBAT set of programs. RESULTS: Significant nominal association with BD was observed for six single SNPs (alpha1: rs11805078; alpha2: rs2070704, rs1016732, rs2854248, and rs2295623; alpha3: rs919390) in the three genes of Na+, K+-ATPase alpha isoforms. Haplotype analysis of the alpha2 isoform (ATP1A2 gene) showed a significant association with two loci haplotypes with BD (rs2295623: rs2070704; global p value = .0198, following a permutation test). CONCLUSIONS: This study demonstrates for the first time that genetic variations in Na+, K+-ATPase are associated with BD, suggesting a role of this enzyme in the etiology of this disease.
Assuntos
Transtorno Bipolar/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Isoformas de Proteínas/genética , ATPase Trocadora de Sódio-Potássio/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , ATPase Trocadora de Sódio-Potássio/classificaçãoRESUMO
Reactive oxygen species (ROS) modulate intracellular signaling but are also responsible for neuronal damage in pathological states. Microglia, the resident CNS macrophages, are prominent sources of ROS through expression of the phagocyte oxidase which catalytic subunit Nox2 generates superoxide ion (O2(.-)). Here we show that microglia also express Nox1 and other components of nonphagocyte NADPH oxidases, including p22(phox), NOXO1, NOXA1, and Rac1/2. The subcellular distribution and functions of Nox1 were determined by blocking Nox activity with diphenylene iodonium or apocynin, and by silencing the Nox1 gene in microglia purified from wild-type (WT) or Nox2-KO mice. [Nox1-p22(phox)] dimers localized in intracellular compartments are recruited to phagosome membranes during microglial phagocytosis of zymosan, and Nox1 produces O2(.-) in zymosan-loaded phagosomes. In microglia activated with lipopolysaccharide (LPS), Nox1 produces O2(.-), which enhances cell expression of inducible nitric oxide synthase and secretion of interleukin-1beta. Comparisons of microglia purified from WT, Nox2-KO, or Nox1-KO mice indicate that both Nox1 and Nox2 are required to optimize microglial production of nitric oxide. By injecting LPS in the striatum of WT and Nox1-KO mice, we show that Nox1 also enhances microglial production of cytotoxic nitrite species and promotes loss of presynaptic proteins in striatal neurons. These results demonstrate the functional expression of Nox1 in resident CNS phagocytes, which can promote production of neurotoxic compounds during neuroinflammation. Our study also shows that Nox1- and Nox2-dependent oxidases play distinct roles in microglial activation and that Nox1 is a possible target for the treatment of neuroinflammatory states.
Assuntos
Encefalite/enzimologia , Gliose/enzimologia , Microglia/enzimologia , NADH NADPH Oxirredutases/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Corpo Estriado/fisiopatologia , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Encefalite/fisiopatologia , Feminino , Gliose/fisiopatologia , Mediadores da Inflamação/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neurotoxinas/farmacologia , Nitritos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas/genética , Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zimosan/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
BACKGROUND: In both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets. METHODS AND FINDINGS: In this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets. CONCLUSION: The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.
Assuntos
Diferenciação Celular , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/fisiologia , Células-Tronco/fisiologia , Adolescente , Animais , Células Cultivadas , Feminino , Transplante de Tecido Fetal/métodos , Técnicas de Transferência de Genes , Glucagon/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Lentivirus/genética , Masculino , Camundongos , Camundongos SCID , Morfogênese/genética , Transplante HeterólogoRESUMO
Gene transfer may become a powerful clinical tool for the delivery of secreted therapeutic polypeptides, provided that the in situ production of these peptides can be tightly regulated by the administration of a small inducer molecule. Particularly efficient control may be achieved by simultaneously using two regulation systems that interfere with the biosynthesis of the therapeutic factor at two different levels. Therefore, we have developed a set of two lentiviral vectors containing two regulation systems. These systems are induced by nonimmunosuppressive derivatives of rapamycin ("rapalogs") and allow simultaneous control of expression and of exocytosis of secreted therapeutic polypeptides. The set of vectors was used to produce green fluorescent protein (GFP) and glial cell line-derived neurotrophic factor (GDNF); GFP served as a model factor to demonstrate expression and entry into the exocytotic pathway in transduced cells. The constructs allowed robust in vitro expression and secretion of the polypeptides in the presence of rapalog AP21967. Withdrawal of the inducer resulted in efficient downregulation. In vivo, tightly regulated production of GFP and GDNF was observed after injection of the constructs into the striata of mice. The vectors thus fulfill key requirements for application in gene therapy.
Assuntos
Encéfalo/metabolismo , Exocitose , Vetores Genéticos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Lentivirus/genética , Sirolimo/análogos & derivados , Transcrição Gênica , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Linhagem Celular , Exocitose/efeitos dos fármacos , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunossupressores/farmacologia , Camundongos , Peptídeos/metabolismo , Sirolimo/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transdução GenéticaRESUMO
The human zinc finger protein 191 (ZNF191) is a Krüppel-like protein and can specifically interact with the widespread TCAT motif which constitutes the HUMTH01 microsatellite in the tyrosine hydroxylase (TH) gene (encoding the rate-limiting enzyme in the synthesis of catecholamines). Allelic variations of HUMTH01 are known to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. This factor has been isolated from bone marrow and promyelocytic leukemia cell lines indicating that ZNF191 also plays a role in hematopoiesis. Thus, ZNF191 could participate in the regulation of several genes implicated in different functions. Moreover, mice that are deficient in Zfp191, the murine homologue of ZNF191, have been shown to be severely retarded in development and to die approximately at embryonic day 7.5. In order to gain further insight into its biological functions, we have analysed the localisation of Zfp191 throughout mouse development. Expression was detected early during embryogenesis in ectodermal, endodermal, mesodermal and extra-embryonic tissues. In particular, Zfp191 was observed in the developing central nervous system. Interestingly, its expression levels were prominent in areas of proliferation such as the subventricular zone. Zfp191 expression pattern during development can account for the phenotypic features of Zfp191(-/-) embryos.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Animais , Desenvolvimento Embrionário/fisiologia , Feminino , Camundongos , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Fenótipo , Dedos de Zinco/genéticaRESUMO
We generated mice with doxycycline control of a human neurofilament light (NF-L) transgene in the context of the absence (tTA;hNF-L;NF-L(-/-)) or presence (tTA;hNF-L;NF-L(+/-)) of endogenous mouse NF-L proteins. Doxycycline treatment caused the rapid disappearance of human NF-L (hNF-L) mRNA in tTA;hNF-L mice, but the hNF-L proteins remained with a half-life of 3 weeks in the brain. In the sciatic nerve, the disappearance of hNF-L proteins after doxycycline treatment occurred in synchrony along the sciatic nerve, suggesting a proteolysis of NF proteins along the entire axon. The presence of permanent NF network in tTA;hNF-L;NF-L(+/-) mice further stabilized and extended longevity of hNF-L proteins by several months. Surprisingly, after cessation of doxycycline treatment, there was no evidence of leading front of newly synthesized hNF-L proteins migrating into sciatic nerve axons devoid of NF structures. The hNF-L proteins detected at weekly intervals reappeared and accumulated in synchrony at similar rate along nerve segments, a phenomenon consistent with a fast hNF-L transport into axons. We estimated the hNF-L transport rate to be of approximately 10 mm/d in axons devoid of NF structures based on the use of an adenovirus encoding tet-responsive transcriptional activator to transactivate the hNF-L transgene in hypoglossal motor neurons. These results provide in vivo evidence that the stationary NF network in axons is a key determinant of half-life and transport rate of NF proteins.