RESUMO
UNLABELLED: Spinal reflex circuit development requires the precise regulation of axon trajectories, synaptic specificity, and synapse formation. Of these three crucial steps, the molecular mechanisms underlying synapse formation between group Ia proprioceptive sensory neurons and motor neurons is the least understood. Here, we show that the Rho GTPase Cdc42 controls synapse formation in monosynaptic sensory-motor connections in presynaptic, but not postsynaptic, neurons. In mice lacking Cdc42 in presynaptic sensory neurons, proprioceptive sensory axons appropriately reach the ventral spinal cord, but significantly fewer synapses are formed with motor neurons compared with wild-type mice. Concordantly, electrophysiological analyses show diminished EPSP amplitudes in monosynaptic sensory-motor circuits in these mutants. Temporally targeted deletion of Cdc42 in sensory neurons after sensory-motor circuit establishment reveals that Cdc42 does not affect synaptic transmission. Furthermore, addition of the synaptic organizers, neuroligins, induces presynaptic differentiation of wild-type, but not Cdc42-deficient, proprioceptive sensory neurons in vitro Together, our findings demonstrate that Cdc42 in presynaptic neurons is required for synapse formation in monosynaptic sensory-motor circuits. SIGNIFICANCE STATEMENT: Group Ia proprioceptive sensory neurons form direct synapses with motor neurons, but the molecular mechanisms underlying synapse formation in these monosynaptic sensory-motor connections are unknown. We show that deleting Cdc42 in sensory neurons does not affect proprioceptive sensory axon targeting because axons reach the ventral spinal cord appropriately, but these neurons form significantly fewer presynaptic terminals on motor neurons. Electrophysiological analysis further shows that EPSPs are decreased in these mice. Finally, we demonstrate that Cdc42 is involved in neuroligin-dependent presynaptic differentiation of proprioceptive sensory neurons in vitro These data suggest that Cdc42 in presynaptic sensory neurons is essential for proper synapse formation in the development of monosynaptic sensory-motor circuits.
Assuntos
Orientação de Axônios/fisiologia , Neurônios Motores/fisiologia , Neurogênese/fisiologia , Terminações Pré-Sinápticas/fisiologia , Células Receptoras Sensoriais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Knockout , Neurônios Motores/citologia , Terminações Pré-Sinápticas/ultraestrutura , Células Receptoras Sensoriais/citologia , Medula Espinal/citologia , Medula Espinal/fisiologiaRESUMO
In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.
RESUMO
Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias Encefálicas/enzimologia , Proliferação de Células , Metabolismo Energético , Glioblastoma/enzimologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Animais , Antineoplásicos/farmacologia , Apoptose , Autofagia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Fator de Transcrição de Proteínas de Ligação GA/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Carga Tumoral , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In the version of this Article originally published, in ref. 34 the first author's name was spelled incorrectly. The correct reference is: Rodón, L. et al. Active CREB1 promotes a malignant TGFß2 autocrine loop in glioblastoma. Cancer Discov. 10, 1230-1241 (2014). This has now been amended in all online versions of the Article.
RESUMO
The RASopathy neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant genetic disorders. In NF1 patients, neurological issues may result from damaged myelin, and mice with a neurofibromin gene (Nf1) mutation show white matter (WM) defects including myelin decompaction. Using mouse genetics, we find that altered Nf1 gene-dose in mature oligodendrocytes results in progressive myelin defects and behavioral abnormalities mediated by aberrant Notch activation. Blocking Notch, upstream mitogen-activated protein kinase (MAPK), or nitric oxide signaling rescues myelin defects in hemizygous Nf1 mutants, and pharmacological gamma secretase inhibition rescues aberrant behavior with no effects in wild-type (WT) mice. Concomitant pathway inhibition rescues myelin abnormalities in homozygous mutants. Notch activation is also observed in Nf1+/- mouse brains, and cells containing active Notch are increased in NF1 patient WM. We thus identify Notch as an Nf1 effector regulating myelin structure and behavior in a RASopathy and suggest that inhibition of Notch signaling may be a therapeutic strategy for NF1.
Assuntos
Bainha de Mielina/metabolismo , Neurofibromina 1/metabolismo , Receptores Notch/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Comportamento Animal , Contagem de Células , Claudinas/metabolismo , Dosagem de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação/genética , Neuroglia/metabolismo , Óxido Nítrico/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Transdução de Sinais , Proteínas ras/metabolismoRESUMO
Magmas, is a 13-kDa mitochondrial protein which is ubiquitously expressed in eukaryotic cells. It was identified as a granulocyte-macrophage-colony stimulating factor (GM-CSF) inducible gene in hematopoietic cells and has a key role in the transport of mitochondrial proteins in yeast. Because GM-CSF receptor levels are elevated in prostate cancer, Magmas expression was examined in normal and neoplastic tissue. Magmas protein levels were barely detectable in non-neoplastic prostate glands. Increased amounts were observed in some samples of intraepithelial neoplasia. Approximately one half of the adenocarcinoma samples examined had weak Magmas expression, while the remainder had intermediate to high levels. The increased Magmas observed in malignant tissue was a result of higher protein expression and not from changes in mitochondrial content. Interestingly, in some patients, the normal prostate tissue had more Magmas message than the malignant portion. The results indicated that Magmas expression in prostate cancer is heterogeneous and independent of clinical stage and Gleason score. Further studies are needed to determine if Magmas expression has prognostic significance in prostate cancer.
Assuntos
Proteínas Mitocondriais/metabolismo , Neoplasias da Próstata/metabolismo , Humanos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/genética , Estadiamento de Neoplasias , Prognóstico , Próstata/química , Próstata/metabolismo , Neoplasias da Próstata/química , Neoplasias da Próstata/diagnóstico , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
Transmission electron microscopy (TEM) provides ultra-structural details of cells at the sub-organelle level. However, details of the cellular ultrastructure, and the cellular organization and content of various organelles in rare populations, particularly in the suspension, like hematopoietic stem cells (HSCs) remained elusive. This is mainly due to the requirement of millions of cells for TEM studies. Thus, there is a vital requirement of a method that will allow TEM studies with low cell numbers of such rare populations. We describe an alternative and novel approach for TEM studies for rare cell populations. Here we performed a TEM study from 10,000 HSC cells with relative ease. In particular, tiny cell pellets were identified by Evans blue staining after PFA-GA fixation. The cell pellet was pre-embedded in agarose in a small microcentrifuge tube and processed for dehydration, infiltration and embedding. Semi-thin and ultra-thin sections identified clusters of numerous cells per sections with well preserved morphology and ultrastructural details of golgi complex and mitochondria. Together, this method provides an efficient, easy and reproducible process to perform qualitative and quantitative TEM analysis from limited biological samples including cells in suspension.
Assuntos
Células-Tronco Hematopoéticas/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Animais , Complexo de Golgi/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microtomia , Mitocôndrias/ultraestrutura , Reprodutibilidade dos Testes , Sefarose , Inclusão do Tecido , Fixação de TecidosRESUMO
A prerequisite to myelination of peripheral axons by Schwann cells (SCs) is SC differentiation, and recent evidence indicates that reprogramming from a glycolytic to oxidative metabolism occurs during cellular differentiation. Whether this reprogramming is essential for SC differentiation, and the genes that regulate this critical metabolic transition are unknown. Here we show that the tumour suppressor Lkb1 is essential for this metabolic transition and myelination of peripheral axons. Hypomyelination in the Lkb1-mutant nerves and muscle atrophy lead to hindlimb dysfunction and peripheral neuropathy. Lkb1-null SCs failed to optimally activate mitochondrial oxidative metabolism during differentiation. This deficit was caused by Lkb1-regulated diminished production of the mitochondrial Krebs cycle substrate citrate, a precursor to cellular lipids. Consequently, myelin lipids were reduced in Lkb1-mutant mice. Restoring citrate partially rescued Lkb1-mutant SC defects. Thus, Lkb1-mediated metabolic shift during SC differentiation increases mitochondrial metabolism and lipogenesis, necessary for normal myelination.
Assuntos
Mitocôndrias/metabolismo , Bainha de Mielina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Diferenciação Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Proteínas Serina-Treonina Quinases/genética , Células de Schwann/citologia , Células de Schwann/enzimologia , Células de Schwann/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Patients with neurofibromatosis type 1 (NF1) and Costello syndrome Rasopathy have behavioral deficits. In NF1 patients, these may correlate with white matter enlargement and aberrant myelin. To model these features, we induced Nf1 loss or HRas hyperactivation in mouse oligodendrocytes. Enlarged brain white matter tracts correlated with myelin decompaction, downregulation of claudin-11, and mislocalization of connexin-32. Surprisingly, non-cell-autonomous defects in perivascular astrocytes and the blood-brain barrier (BBB) developed, implicating a soluble mediator. Nitric oxide (NO) can disrupt tight junctions and gap junctions, and NO and NO synthases (NOS1-NOS3) were upregulated in mutant white matter. Treating mice with the NOS inhibitor NG-nitro-L-arginine methyl ester or the antioxidant N-acetyl cysteine corrected cellular phenotypes. CNP-HRasG12V mice also displayed locomotor hyperactivity, which could be rescued by antioxidant treatment. We conclude that Nf1/Ras regulates oligodendrocyte NOS and that dysregulated NO signaling in oligodendrocytes can alter the surrounding vasculature. The data suggest that antioxidants may improve some behavioral deficits in Rasopathy patients.
Assuntos
Bainha de Mielina/metabolismo , Neurofibromina 1/deficiência , Óxido Nítrico Sintase/metabolismo , Oligodendroglia/metabolismo , Proteínas ras/metabolismo , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/enzimologia , Vasos Sanguíneos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Óxido Nítrico/metabolismo , Oligodendroglia/citologia , Oligodendroglia/enzimologia , Proteínas ras/genéticaRESUMO
Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the NF1 tumor suppressor gene in the Schwann cell lineage. To understand whether neurofibroma formation is possible after birth, we induced Nf1 loss of function with an inducible proteolipid protein Cre allele. Perinatal loss of Nf1 resulted in the development of small plexiform neurofibromas late in life, whereas loss in adulthood caused large plexiform neurofibromas and morbidity beginning 4 months after onset of Nf1 loss. A conditional EGFP reporter allele identified cells showing recombination, including peripheral ganglia satellite cells, peripheral nerve S100ß+ myelinating Schwann cells, and peripheral nerve p75+ cells. Neurofibromas contained cells with Remak bundle disruption but no recombination within GFAP+ nonmyelinating Schwann cells. Extramedullary lympho-hematopoietic expansion was also observed in PlpCre;Nf1fl/fl mice. These tumors contained EGFP+/Sca-1+ stromal cells among EGFP-negative lympho-hematopoietic cells indicating a noncell autonomous effect and unveiling a role of Nf1-deleted microenvironment on lympho-hematopoietic proliferation in vivo. Together these findings define a tumor suppressor role for Nf1 in the adult and narrow the range of potential neurofibroma-initiating cell populations.
Assuntos
Inativação Gênica , Genes da Neurofibromatose 1 , Integrases/biossíntese , Proteína Proteolipídica de Mielina/biossíntese , Neurofibromatose 1/genética , Tamoxifeno/farmacologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Integrases/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteína Proteolipídica de Mielina/genéticaRESUMO
In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g., prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin alpha6, and thus represent a putative anal stem cell population.
Assuntos
Canal Anal/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Reto/citologia , Coloração e Rotulagem , Canal Anal/ultraestrutura , Animais , Biomarcadores/metabolismo , Bromodesoxiuridina/metabolismo , Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Células Epiteliais/ultraestrutura , Epitélio/metabolismo , Epitélio/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Camundongos , Reto/ultraestrutura , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Histoplasma capsulatum is a fungal pathogen that requires the induction of cell-mediated immunity (CMI) for host survival. We have demonstrated that human dendritic cells (DC) phagocytose H. capsulatum yeasts and, unlike human macrophages (Mø) that are permissive for intracellular growth, DC killed and degraded the fungus. In the present study, we sought to determine whether the mechanism(s) by which DC kill Histoplasma is via lysosomal hydrolases, via the production of toxic oxygen metabolites, or both. Phagosome-lysosome fusion (PL-fusion) was quantified by using fluorescein isothiocyanate-dextran and phase and fluorescence microscopy and by electron microscopy with horseradish peroxidase colloidal gold to label lysosomes. Unlike Mphi, Histoplasma-infected DC exhibited marked PL-fusion. The addition of suramin to Histoplasma-infected DC inhibited PL-fusion and DC fungicidal activity. Incubation of Histoplasma-infected DC at 18 degrees C also concomitantly reduced PL-fusion and decreased the capacity of DC to kill and degrade H. capsulatum yeasts. Further, culture of Histoplasma-infected DC in the presence of bafilomycin, an inhibitor of the vacuolar ATPase, did not block DC anti-Histoplasma activity, indicating that phagosome acidification was not required for lysosome enzyme activity. In contrast, culture of Histoplasma-infected DC in the presence of inhibitors of the respiratory burst or inhibitors of NO synthase had little to no effect on DC fungicidal activity. These data suggest that the major mechanism by which human DC mediate anti-Histoplasma activity is through the exposure of yeasts to DC lysosomal hydrolases. Thus, DC can override one of the strategies used by H. capsulatum yeasts to survive intracellularly within Mø.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Histoplasma/crescimento & desenvolvimento , Fagocitose/imunologia , Fagossomos/microbiologia , Catecolaminas/farmacologia , Células Dendríticas/metabolismo , Inibidores Enzimáticos/farmacologia , Histoplasma/patogenicidade , Humanos , Imidazolinas/farmacologia , Imunidade Celular/imunologia , Óxido Nítrico/metabolismo , Fagossomos/efeitos dos fármacos , Fagossomos/ultraestrutura , Explosão Respiratória/efeitos dos fármacos , Suramina/farmacologia , ômega-N-Metilarginina/farmacologiaRESUMO
Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.