RESUMO
The evolutionarily conserved minor spliceosome (MiS) is required for protein expression of â¼714 minor intron-containing genes (MIGs) crucial for cell-cycle regulation, DNA repair, and MAP-kinase signaling. We explored the role of MIGs and MiS in cancer, taking prostate cancer (PCa) as an exemplar. Both androgen receptor signaling and elevated levels of U6atac, a MiS small nuclear RNA, regulate MiS activity, which is highest in advanced metastatic PCa. siU6atac-mediated MiS inhibition in PCa in vitro model systems resulted in aberrant minor intron splicing leading to cell-cycle G1 arrest. Small interfering RNA knocking down U6atac was â¼50% more efficient in lowering tumor burden in models of advanced therapy-resistant PCa compared with standard antiandrogen therapy. In lethal PCa, siU6atac disrupted the splicing of a crucial lineage dependency factor, the RE1-silencing factor (REST). Taken together, we have nominated MiS as a vulnerability for lethal PCa and potentially other cancers.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Íntrons/genética , Neoplasias da Próstata/metabolismo , Splicing de RNA/genética , Spliceossomos/metabolismo , Transdução de Sinais , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Neoplasias de Próstata Resistentes à Castração/genéticaRESUMO
Advanced prostate cancer initially responds to hormonal treatment, but ultimately becomes resistant and requires more potent therapies. One mechanism of resistance observed in around 10-20% of these patients is lineage plasticity, which manifests in a partial or complete small cell or neuroendocrine prostate cancer (NEPC) phenotype. Here, we investigate the role of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex in NEPC. Using large patient datasets, patient-derived organoids and cancer cell lines, we identify mSWI/SNF subunits that are deregulated in NEPC and demonstrate that SMARCA4 (BRG1) overexpression is associated with aggressive disease. We also show that SWI/SNF complexes interact with different lineage-specific factors in NEPC compared to prostate adenocarcinoma. These data point to a role for mSWI/SNF complexes in therapy-related lineage plasticity, which may also be relevant for other solid tumors.
Assuntos
Linhagem da Célula , Plasticidade Celular , Proteínas Cromossômicas não Histona/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fatores de Transcrição/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Estudos de Coortes , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Modelos Biológicos , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Subunidades Proteicas/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
Olfaction declines with aging and appears to be a prodromal sign of cognitive decline in progressive neurodegenerative diseases. Nevertheless, very little is known about the pathophysiological changes underlying smell loss that may reflect early network dysfunction. A cross-sectional histoanatomical study was conducted on postmortem olfactory nerves of patients with increasing severity of dementia from mild cognitive impairment (MCI) to moderate and severe Alzheimer's disease. The olfactory bulbs and tracts show a prominent and progressive tauopathy in contrast to a weaker amyloid pathology localized to the glomerular region. Topological analysis of Notch signaling components reveals a transient increase in Jagged1 expression in mitral cells of the olfactory bulb of patients with MCI and a gradual decline onwards. Analysis of the olfactory tract reveals an abundance of corpora amylacea, which declines starting from the MCI stage. With the increasing severity of dementia, corpora amylacea are characterized by a gradual shift in cytoskeletal proteins, tau, MAP2 and glial fibrillary acid protein, as well as by a decrease in their Reelin and Jagged1 content. Our research indicates that the olfactory nerve undergoes early and sequential morphological and signaling alterations that correlate with the development of dementia suggesting that this structure may capture and propagate neuronal network imbalances to connected higher brain centers of the entorhinal cortex and hippocampus.
Assuntos
Doença de Alzheimer/patologia , Bulbo Olfatório/patologia , Transdução de Sinais/fisiologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Estudos Transversais , Progressão da Doença , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Humanos , Proteína Jagged-1/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Bulbo Olfatório/metabolismo , Bulbo Olfatório/fisiopatologia , Proteína Reelina , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Notch signaling plays an instrumental role in hippocampus-dependent memory formation and recent evidence indicates a displacement of Notch1 and a reduction its activity in hippocampal and cortical neurons from Alzheimer's disease (AD) patients. As Notch activation depends on ligand availability, we investigated whether Jagged1 expression was altered in brain specimen of AD patients. We found that Jagged1 expression was reduced in the CA fields and that there was a gradual reduction of Jagged1 in the cerebrospinal fluid (CSF) with the progression of dementia. Given the role of Notch signaling in memory encoding, we investigated whether targeted loss of Jagged1 in neurons may be responsible for the memory loss seen in AD patients. Using a transgenic mouse model, we show that the targeted loss of Jagged1 expression during adulthood is sufficient to cause spatial memory loss and a reduction in exploration-dependent Notch activation. We also show that Jagged1 is selectively enriched at the presynaptic terminals in mice. Overall, the present data emphasizes the role of the Notch ligand, Jagged1, in memory formation and the potential deficit of the signaling ligand in AD patients.
RESUMO
The small intestine is a dynamic and complex organ that is characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota. Lymphatic capillaries of the small intestine, called lacteals, play key roles in dietary fat absorption and the gut immune response; however, little is known about the molecular regulation of lacteal function. Here, we performed a high-resolution analysis of the small intestinal stroma and determined that lacteals reside in a permanent regenerative, proliferative state that is distinct from embryonic lymphangiogenesis or quiescent lymphatic vessels observed in other tissues. We further demonstrated that this continuous regeneration process is mediated by Notch signaling and that the expression of the Notch ligand delta-like 4 (DLL4) in lacteals requires activation of VEGFR3 and VEGFR2. Moreover, genetic inactivation of Dll4 in lymphatic endothelial cells led to lacteal regression and impaired dietary fat uptake. We propose that such a slow lymphatic regeneration mode is necessary to match a unique need of intestinal lymphatic vessels for both continuous maintenance, due to the constant exposure to dietary fat and mechanical strain, and efficient uptake of fat and immune cells. Our work reveals how lymphatic vessel responses are shaped by tissue specialization and uncover a role for continuous DLL4 signaling in the function of adult lymphatic vasculature.
Assuntos
Gorduras na Dieta/metabolismo , Intestino Delgado/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfangiogênese , Vasos Linfáticos/fisiologia , Proteínas de Membrana/metabolismo , Regeneração , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Gorduras na Dieta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Receptores Notch/genética , Receptores Notch/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.
Assuntos
Células Endoteliais/citologia , Fatores de Transcrição Forkhead/fisiologia , Sistema Linfático/crescimento & desenvolvimento , Vasos Linfáticos/citologia , Reologia , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Apoptose , Ciclo Celular , Divisão Celular , Células Cultivadas , Citoesqueleto/ultraestrutura , Células Endoteliais/patologia , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/deficiência , Humanos , Junções Intercelulares/ultraestrutura , Vasos Linfáticos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/fisiologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Fibras de Estresse/ultraestrutura , Estresse Mecânico , Fatores de Transcrição/fisiologia , Transcrição Gênica , Transfecção , Proteínas de Sinalização YAPRESUMO
Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.
Assuntos
Linfangiogênese , Vasos Linfáticos/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Animais Geneticamente Modificados , Células Cultivadas , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Junções Intercelulares/metabolismo , Linfonodos/crescimento & desenvolvimento , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fenótipo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/antagonistas & inibidores , Canais de Cátion TRPP/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.
Assuntos
Células Endoteliais/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Transcrição Gênica/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Células Cultivadas , Chlorocebus aethiops , Fatores de Transcrição Forkhead/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Immunoblotting , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Prolina/genética , Prolina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismoRESUMO
Lymphatic valves are essential for efficient lymphatic transport, but the mechanisms of early lymphatic-valve morphogenesis and the role of biomechanical forces are not well understood. We found that the transcription factors PROX1 and FOXC2, highly expressed from the onset of valve formation, mediate segregation of lymphatic-valve-forming cells and cell mechanosensory responses to shear stress in vitro. Mechanistically, PROX1, FOXC2, and flow coordinately control expression of the gap junction protein connexin37 and activation of calcineurin/NFAT signaling. Connexin37 and calcineurin are required for the assembly and delimitation of lymphatic valve territory during development and for its postnatal maintenance. We propose a model in which regionally increased levels/activation states of transcription factors cooperate with mechanotransduction to induce a discrete cell-signaling pattern and morphogenetic event, such as formation of lymphatic valves. Our results also provide molecular insights into the role of endothelial cell identity in the regulation of vascular mechanotransduction.
Assuntos
Calcineurina/metabolismo , Conexinas/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Proteínas de Homeodomínio/fisiologia , Linfangiogênese/fisiologia , Vasos Linfáticos/citologia , Mecanotransdução Celular/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Western Blotting , Calcineurina/genética , Proliferação de Células , Conexinas/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Proteína alfa-4 de Junções ComunicantesRESUMO
This work is believed to be the first report on the physiological and biochemical characterization of alpha-l-rhamnosidases in lactic acid bacteria. A total of 216 strains representing 37 species and eight genera of food-grade bacteria were screened for alpha-l-rhamnosidase activity. The majority of positive bacteria (25 out of 35) were Lactobacillus plantarum strains, and activity of the L. plantarum strain NCC245 was examined in more detail. The analysis of alpha-l-rhamnosidase activity under different growth conditions revealed dual regulation of the enzyme activity, involving carbon catabolite repression and induction: the enzyme activity was downregulated by glucose and upregulated by l-rhamnose. The expression of the two alpha-l-rhamnosidase genes rhaB1 and rhaB2 and two predicted permease genes rhaP1 and rhaP2, identified in a probable operon rhaP2B2P1B1, was repressed by glucose and induced by l-rhamnose, showing regulation at the transcriptional level. The two alpha-l-rhamnosidase genes were overexpressed and purified from Escherichia coli. RhaB1 activity was maximal at 50 degrees C and at neutral pH and RhaB2 maximal activity was detected at 60 degrees C and at pH 5, with high residual activity at 70 degrees C. Both enzymes showed a preference for the alpha-1,6 linkage of l-rhamnose to beta-d-glucose, hesperidin and rutin being their best substrates, but, surprisingly, no activity was detected towards the alpha-1,2 linkage in naringin under the tested conditions. In conclusion, we identified and characterized the strain L. plantarum NCC245 and its two alpha-l-rhamnosidase enzymes, which might be applied for improvement of bioavailability of health-beneficial polyphenols, such as hesperidin, in humans.
Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Lactobacillus plantarum/enzimologia , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/análise , DNA Bacteriano/genética , Glucose/metabolismo , Glucosídeos/metabolismo , Glicosídeo Hidrolases/genética , Hesperidina/metabolismo , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
The mechanisms of blood vessel maturation into distinct parts of the blood vasculature such as arteries, veins, and capillaries have been the subject of intense investigation over recent years. In contrast, our knowledge of lymphatic vessel maturation is still fragmentary. In this study, we provide a molecular and morphological characterization of the major steps in the maturation of the primary lymphatic capillary plexus into collecting lymphatic vessels during development and show that forkhead transcription factor Foxc2 controls this process. We further identify transcription factor NFATc1 as a novel regulator of lymphatic development and describe a previously unsuspected link between NFATc1 and Foxc2 in the regulation of lymphatic maturation. We also provide a genome-wide map of FOXC2-binding sites in lymphatic endothelial cells, identify a novel consensus FOXC2 sequence, and show that NFATc1 physically interacts with FOXC2-binding enhancers. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.
Assuntos
Fatores de Transcrição Forkhead/fisiologia , Vasos Linfáticos/fisiologia , Fatores de Transcrição NFATC/fisiologia , Animais , Membrana Basal/fisiologia , Vasos Sanguíneos/fisiologia , Capilares/fisiologia , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Valvas Cardíacas/fisiologia , Vasos Linfáticos/patologia , Camundongos , Camundongos Knockout , Fenômenos Fisiológicos da PeleRESUMO
The impact of a moderate consumption of an instant coffee on the general composition of the human intestinal bacterial population was assessed in this study. Sixteen (16) healthy adult volunteers consumed a daily dose of 3 cups of coffee during 3 weeks. Faecal samples were collected before and after the consumption of coffee, and the impact of the ingestion of the product on the intestinal bacteria as well as the quantification of specific bacterial groups was assessed using nucleic acid-based methods. Although faecal profiles of the dominant microbiota were not significantly affected after the consumption of the coffee (Dice's similarity index=92%, n=16), the population of Bifidobacterium spp. increased after the 3-week test period (P=0.02). Moreover, in some subjects, there was a specific increase in the metabolic activity of Bifidobacterium spp. Our results show that the consumption of the coffee preparation resulting from water co-extraction of green and roasted coffee beans produce an increase in the metabolic activity and/or numbers of the Bifidobacterium spp. population, a bacterial group of reputed beneficial effects, without major impact on the dominant microbiota.
Assuntos
Bactérias/classificação , Café/química , Intestinos/microbiologia , Eletroforese em Gel Bidimensional , Fezes/microbiologia , Humanos , RNA Bacteriano/classificação , RNA Ribossômico 16S/classificaçãoRESUMO
Recent data suggest that the gut microbiota plays a significant role in fat accumulation. However, it is not clear whether gut microbiota is involved in the pathophysiology of type 2 diabetes. To assess this issue, we modulated gut microbiota via antibiotics administration in two different mouse models with insulin resistance. Results from dose-determination studies showed that a combination of norfloxacin and ampicillin, at a dose of 1 g/L, maximally suppressed the numbers of cecal aerobic and anaerobic bacteria in ob/ob mice. After a 2-wk intervention with the antibiotic combination, both ob/ob and diet-induced obese and insulin-resistant mice showed a significant improvement in fasting glycemia and oral glucose tolerance. The improved glycemic control was independent of food intake or adiposity because pair-fed ob/ob mice were as glucose intolerant as the control ob/ob mice. Reduced liver triglycerides and increased liver glycogen correlated with improved glucose tolerance in the treated mice. Concomitant reduction of plasma lipopolysaccharides and increase of adiponectin further supported the antidiabetic effects of the antibiotic treatment in ob/ob mice. In summary, modulation of gut microbiota ameliorated glucose tolerance of mice by altering the expression of hepatic and intestinal genes involved in inflammation and metabolism, and by changing the hormonal, inflammatory, and metabolic status of the host.
Assuntos
Ampicilina/farmacologia , Bacteroides/efeitos dos fármacos , Bifidobacterium/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Lactobacillus/efeitos dos fármacos , Norfloxacino/farmacologia , Animais , Antibacterianos/farmacologia , Bacteroides/fisiologia , Bifidobacterium/fisiologia , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Enterobacteriaceae/fisiologia , Lactobacillus/fisiologia , Camundongos , Camundongos Obesos , Testes de Sensibilidade Microbiana , Obesidade/microbiologia , Obesidade/fisiopatologiaAssuntos
Terapia Genética/métodos , Vetores Genéticos , Lentivirus/genética , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/terapia , Epitélio Pigmentado Ocular/metabolismo , Regiões Promotoras Genéticas , Animais , Proteínas de Transporte/genética , Eletrorretinografia/métodos , Proteínas do Olho/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lentivirus/metabolismo , Camundongos , Camundongos Knockout , Retina/metabolismo , cis-trans-IsomerasesRESUMO
Excitotoxicity may be a critical factor in the formation of brain lesions associated with cerebral palsy. When injected into the murine neopallium at postnatal day 5, the glutamatergic analog N-methyl-D-aspartate (NMDA) produces transcortical neuronal death and periventricular white matter cysts, which mimic brain damage observed in human term and preterm neonates at risk for developing cerebral palsy. We previously showed that intracerebral injection of brain-derived neurotrophic factor (BDNF) was neuroprotective in this model. Because BDNF does not easily cross the blood-brain barrier, alternative strategies to avoid repeated intracerebral injections of BDNF should be tested, particularly when the goal of such translational research is ultimately to achieve clinical application. The goal of the present study was to assess the protective role of lentiviral-mediated gene transfer of BDNF against excitotoxic lesions induced by NMDA in newborn mice. We first assessed the biological activity of BDNF gene transfer in vitro and determined the efficiency of gene transfer in our in vivo model. We next administered the BDNF-expressing vector by intracerebral injection in neonatal mice, 3 days before inducing NMDA lesions. When compared with a control green fluorescent protein-expressing lentiviral vector, administration of BDNF-expressing vector induced a significant protection of the periventricular white matter and cortical plate against the NMDA-mediated insult. Intraventricular delivery of the BDNF-expressing lentiviral vector was more efficient in terms of neuroprotection than the intraparenchymal route. Altogether, the present study shows that viral-mediated gene transfer of BDNF to newborn mouse brain is feasible and affords significant neuroprotection against an excitotoxic insult.
Assuntos
Animais Recém-Nascidos/fisiologia , Fator Neurotrófico Derivado do Encéfalo/genética , Técnicas de Transferência de Genes , Lentivirus/genética , Fármacos Neuroprotetores , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/toxicidade , Animais , Astrócitos/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática , Agonistas de Aminoácidos Excitatórios/toxicidade , Feminino , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , HIV-1/genética , Células HeLa , Humanos , Injeções Intraventriculares , Masculino , Camundongos , N-Metilaspartato/toxicidade , Ratos , Proteínas Recombinantes/farmacologiaRESUMO
The polycomb transcriptional repressor Bmi1 promotes cell cycle progression, controls cell senescence, and is implicated in brain development. Loss of Bmi1 leads to a decreased brain size and causes progressive ataxia and epilepsy. Recently, Bmi1 was shown to control neural stem cell (NSC) renewal. However, the effect of Bmi1 loss on neural cell fate in vivo and the question whether the action of Bmi1 was intrinsic to the NSCs remained to be investigated. Here, we show that Bmi1 is expressed in the germinal zone in vivo and in NSCs as well as in progenitors proliferating in vitro, but not in differentiated cells. Loss of Bmi1 led to a decrease in proliferation in zones known to contain progenitors: the newborn cortex and the newborn and adult subventricular zone. This decrease was accentuated in vitro, where we observed a drastic reduction in NSC proliferation and renewal because of NSC-intrinsic effects of Bmi1 as shown by the means of RNA interference. Bmi1(-/-) mice also presented more astrocytes at birth, and a generalized gliosis at postnatal day 30. At both stages, colocalization of bromodeoxyuridine and GFAP demonstrated that Bmi1 loss did not prevent astrocyte precursor proliferation. Supporting these observations, Bmi1(-/-) neurospheres generate preferentially astrocytes probably attributable to a different responsiveness to environmental factors. Bmi1 is therefore necessary for NSC renewal in a cell-intrinsic mode, whereas the altered cell pattern of the Bmi1(-/-) brain shows that in vivo astrocyte precursors can proliferate in the absence of Bmi1.
Assuntos
Astrócitos/citologia , Neurônios/fisiologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Sequência de Bases , Núcleo Caudado/fisiologia , Diferenciação Celular , Divisão Celular , Córtex Cerebral/fisiologia , Primers do DNA , Regulação da Expressão Gênica no Desenvolvimento , Triagem de Portadores Genéticos , Gliose/genética , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Neurônios/citologia , Complexo Repressor Polycomb 1 , Putamen , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Type 1 diabetes is characterized by the infiltration of activated leukocytes within the pancreatic islets, leading to beta-cell dysfunction and destruction. The exact role played by interferon-gamma, tumor necrosis factor (TNF)-alpha, and interleukin-1beta in this pathogenic process is still only partially understood. To study cytokine action at the cellular level, we are working with the highly differentiated insulin-secreting cell line, betaTc-Tet. We previously reported that it was susceptible to apoptosis induced by TNF-alpha, in combination with interleukin-1beta and interferon-gamma. Here, we report that cytokine-induced apoptosis was correlated with the activation of caspase-8. We show that in betaTc-Tet cells, overexpression of cFLIP, the cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein, completely abolished cytokine-dependent activation of caspase-8 and protected the cells against apoptosis. Furthermore, cFLIP overexpression increased the basal and interleukin-1beta-mediated transcriptional activity of nuclear factor (NF)-kappaB, whereas it did not change cytokine-induced inducible nitric oxide synthase gene transcription and nitric oxide secretion. The presence of cFLIP prevented the weak TNF-alpha-induced reduction in cellular insulin content and secretion; however, it did not prevent the decrease in glucose-stimulated insulin secretion induced by the combined cytokines, in agreement with our previous data demonstrating that interferon-gamma alone could induce these beta-cell dysfunctions. Together, our data demonstrate that overexpression of cFLIP protects mouse beta-cells against TNF-alpha-induced caspase-8 activation and apoptosis and is correlated with enhanced NF-kappaB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor-triggered responses by directing the intracellular signals from beta-cell death to beta-cell survival.