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1.
Cell Death Discov ; 2: 16062, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27777788

RESUMO

We have discovered that the accumulation of an anti-calcitonin receptor (anti-CTR) antibody conjugated to a fluorophore (mAb2C4:AF568) provides a robust signal for cells undergoing apoptotic programmed cell death (PCD). PCD is an absolute requirement for normal development of metazoan organisms. PCD is a hallmark of common diseases such as cardiovascular disease and tissue rejection in graft versus host pathologies, and chemotherapeutics work by increasing PCD. This robust signal or high fluorescent events were verified by confocal microscopy and flow cytometry in several cell lines and a primary culture in which PCD had been induced. In Jurkat cells, GBM-L2 and MG63 cells, the percentage undergoing PCD that were positive for both mAb2C4:AF568 and annexin V ranged between 70 and >90%. In MG63 cells induced for the preapoptotic cell stress response (PACSR), the normal expression of α-tubulin, a key structural component of the cytoskeleton, and accumulation of mAb2C4:AF568 were mutually exclusive. Our data support a model in which CTR is upregulated during PACSR and recycles to the plasma membrane with apoptosis. In cells committed to apoptosis (α-tubulin negative), there is accumulation of the CTR-ligand mAb2C4:AF568 generating a high fluorescent event. The reagent mAb2C4:AF568 effectively identifies a novel event linked to apoptosis.

2.
Neuroscience ; 247: 75-83, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-23707981

RESUMO

Adult neurogenesis occurs throughout life; however the majority of new neurons do not survive. Enhancing the survival of these new neurons will increase the likelihood that these neurons could return function following injury. Inhibition of Rho kinase is known to increase neurite outgrowth and regeneration. Previous work in our lab has demonstrated a role for Rho kinase inhibition and survival of new born neurons from the sub-ventricular zone. In this study we examined the role of Rho kinase inhibition on hippocampal neurogenesis. Two concentrations of Rho kinase inhibitor Y27632 (20 and 100 µM) and the proliferative marker EdU were infused in the lateral ventricle for 7 days. Quantification of doublecortin+/EdU+ cells on the 7th day showed that cell numbers were not significantly different, suggesting no effect on neuroblast generation. Following infusion of 100µM Y27632, the number of newborn NeuN+/EdU+ neurons at 35 days in the granular cell layer of the dentate gyrus of the ipsilateral side of the infusion did not display a significant difference; however there was an increase on the contralateral side, suggesting a dose effect. Infusion of a lower dose (20 µM) of Y27632 resulted in an increase in NeuN+/EdU+ cells in the granular cell layer of the ipsilateral side at 35 days. These mice also demonstrated enhanced spatial memory as tested by the Y maze with no significant changes in anxiety or novel object recognition. Rho kinase inhibition enhanced the survival of new born neurons in the dentate gyrus with a specific dosage effect. These results suggest that inhibition of Rho kinase following injury could be beneficial for increasing the survival of new neurons that may aid recovery.


Assuntos
Sobrevivência Celular/fisiologia , Hipocampo/enzimologia , Neurogênese/fisiologia , Neurônios/enzimologia , Inibidores de Proteínas Quinases/administração & dosagem , Quinases Associadas a rho/metabolismo , Amidas/administração & dosagem , Animais , Sobrevivência Celular/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Infusões Intraventriculares , Camundongos , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Piridinas/administração & dosagem , Quinases Associadas a rho/antagonistas & inibidores
3.
Growth Horm IGF Res ; 19(3): 212-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18976947

RESUMO

OBJECTIVES: Growth hormone (GH) and its receptor (GHR) are widely expressed in the CNS. During development, GH signaling regulates both proliferation of neural progenitor cells as well as their differentiation into neurons and glia. Here we have examined the effect of GH signaling on adult subventricular zone derived neural progenitor cells cultured as neurospheres. DESIGN: GH was added to adult wild-type (WT) neurosphere cultures and neurosphere growth measured using the MTT cell proliferation assay. To examine the influence of endogenous GH production on neural progenitors, neurospheres derived from GH receptor knockout (GHRKO) mice were examined by measuring neurosphere sizes and Ki67 and TUNEL immunoreactivity. In addition, neurosphere growth curves were compared following long term culture. Finally, the differentiation of WT vs. GHRKO neurospheres was compared using immunocytochemistry for betaIII-tubulin and GFAP. RESULTS: While GH alone was insufficient to support neurosphere formation, it enhanced neurosphere growth by 20% in the presence of epidermal growth factor and fibroblast growth factor-2. Compared to wildtype neurospheres, GHRKO neurospheres were smaller, contained fewer proliferating cells and exhibited reduced self-renewal in long term culture. Addition of GH increased STAT5 phosphorylation levels in neurosphere cells. Upon differentiation, GHRKO neurospheres showed accelerated neurogenesis, although over time similar numbers of betaIII-tubulin positive neurons were generated by cells of both genotypes. CONCLUSIONS: GH functions as an autocrine mitogen in adult neurosphere cultures and promotes proliferation of neural progenitor cells as well as self-renewal of neurosphere cultures. In addition, signaling through the GHR appeared to delay neuronal differentiation in adult neurospheres.


Assuntos
Proliferação de Células/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Neurogênese , Neurônios/citologia , Neurônios/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Células-Tronco/fisiologia , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Técnicas Imunoenzimáticas , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Fosforilação , Fator de Transcrição STAT5 , Esferoides Celulares/metabolismo , Tubulina (Proteína)/metabolismo
4.
J Neuroimmunol ; 206(1-2): 32-8, 2009 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-19027965

RESUMO

Interferons are produced following neural damage as part of the inflammatory response and may thus affect neural stem cell function. We compared the effects of interferon-gamma and interferon-beta on the proliferation and differentiation of adult murine neural progenitors. Both interferons inhibited neurosphere proliferation due to cell cycle arrest in G1 but only interferon-gamma induced neuronal differentiation. Both interferons induced differential phosphorylation of STAT proteins and a modest and late upregulation of the cell cycle regulator p27 but not several other likely cell cycle regulators. Thus in neural progenitor cells, anti-proliferative effects of interferons are not necessarily linked to differentiation.


Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Interferon beta/farmacologia , Interferon gama/farmacologia , Neurônios/fisiologia , Análise de Variância , Animais , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Citometria de Fluxo/métodos , Imunoprecipitação , Ventrículos Laterais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Neuritos/efeitos dos fármacos , Neurônios/citologia , Fosforilação/efeitos dos fármacos , Fatores de Transcrição STAT/metabolismo , Fatores de Tempo , Tubulina (Proteína)/metabolismo
5.
Neuroscience ; 136(2): 405-15, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16226389

RESUMO

PURPOSE: Proliferation of neural precursors adjacent to the granule cell layer of the dentate gyrus has been identified in previous epilepsy models. Convincingly demonstrating that seizure activity is the stimulant for neurogenesis, rather than neuronal death or other insults inherent to seizure models, is difficult. To address this we derived a rapid electrical amygdala kindling model in mice known to be resistant to seizure-induced neuronal death as an experimental model of focal seizures and to analyze subsequent neurogenesis. METHODS: Mice were implanted with bipolar electrodes in the left amygdala and given electrical stimulation (3 s, 100 Hz, 1 ms monophasic square wave pulses every 5 min, 40 in total) while being observed and graded for the development of seizures. Neurogenesis in the hippocampus was assessed by counting bromodeoxyuridine-immunoreactive cells co-labeled for astrocyte (glial fibrillary acidic protein) and neuronal nuclear markers. RESULTS: Bromodeoxyuridine-reactive cell numbers were three-fold higher in stimulated mice compared with controls at 1 week in the subgranular region and at three weeks extensive co-labeling with neuronal nuclear was noted in cells which had migrated into the body of the granule cell layer, while mice receiving stimulation but failing to kindle did not differ significantly from controls. No increase in neuronal death was detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling, Fluorojade or fluorescent examination of hematoxylin and eosin-stained sections in any inter-group comparison. CONCLUSIONS: We propose that this kindling paradigm, not previously applied to mice, demonstrates more convincingly than previously the surge in neurogenesis in response to seizures, and the effects of seizures alone in regard to neuronal injury and regeneration.


Assuntos
Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Excitação Neurológica/fisiologia , Neurônios/patologia , Convulsões/patologia , Animais , Antimetabólitos , Comportamento Animal/fisiologia , Bromodesoxiuridina , Morte Celular , Proliferação de Células , Grânulos Citoplasmáticos/fisiologia , Giro Denteado/patologia , Eletroencefalografia , Genótipo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Mitose/fisiologia , Degeneração Neural/patologia
6.
Neuroscience ; 132(3): 673-87, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15837129

RESUMO

Developing an understanding of factors that regulate development of the nervous system is important if we hope to be able to repair the nervous system after injury or disease. Suppressor of cytokine signaling-2 (SOCS2) is an intracellular regulator of cytokine signaling that blocks the inhibitory effects of growth hormone on neuronal differentiation and promotes neurogenesis. Here we examine the effect of SOCS2 over-expression on brain development by assessing density and soma size of different neuronal populations in the somatosensory cortex and striatum of SOCS2 transgenic mice compared with wildtype C57BL/6 mice. There were no significant differences in brain weight, cortical thickness or striatal area between mice of either genotype. Analysis of NeuN positive neuronal cell density showed a modest but significant 9% increase across layers 2-6 of SOCS2 transgenic cortex, while cortical interneuron subpopulations were variably affected. In the cortex, parvalbumin and somatostatin expressing neuron densities were unaffected, while calretinin and calbindin positive neuronal densities increased by 48% and 45% respectively. There was no apparent difference in glial fibrillary acidic protein positive astrocyte numbers in layers 1 or 6b of cortex. Furthermore, soma sizes of calretinin and calbindin positive cortical neurons were significantly smaller than wildtype, although there was no difference in size of Cresyl Violet-stained layer 5 projection neurons nor of parvalbumin or somatostatin positive cortical neurons. Additionally, synaptic density and dendritic branching were found to be increased in SOCS2 transgenic cortex. These effects on calretinin and calbindin positive cortical neurons and cortical neuronal circuitry were not observed in the striatum of SOCS2-Tg brains. However, striatal cholinergic interneurons were significantly smaller in SOCS2-Tg brains. At embryonic day 14.5, proliferation and apoptosis in the developing telencephalon were similar in each genotype. Therefore, over-expression of SOCS2 variably affects different cortical regions and neuronal populations, with the predominant effect appearing to be on interneurons and neuronal connectivity in the cortex.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Animais , Apoptose/fisiologia , Western Blotting/métodos , Calbindina 2 , Calbindinas , Contagem de Células/métodos , Proliferação de Células , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Indóis , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/classificação , Parvalbuminas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Repressoras/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Somatostatina/metabolismo , Coloração e Rotulagem/métodos , Proteínas Supressoras da Sinalização de Citocina , Sinaptofisina/metabolismo , Transativadores/genética , Ácido gama-Aminobutírico/metabolismo
7.
J Peripher Nerv Syst ; 7(3): 181-9, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12365566

RESUMO

The pathogenesis of tomacula in mice with a null mutation of the myelin-associated glycoprotein (MAG) gene is not well understood. This study, using a novel teased nerve fiber technique, demonstrates that tomacula in MAG-deficient mice are formed by redundant myelin infoldings and outfoldings in the paranodal regions as early as 4 weeks after birth and increase in size and frequency with age. Although tomacula show degenerative changes with increasing age, there was no significant evidence of demyelination/remyelination. Longitudinal sections of normal teased nerve fibers show early redundant myelin foldings in externally normal paranodal regions. These data and the absence of internodal tomacula support a role for MAG in the maintenance of myelin at the paranodal regions.


Assuntos
Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Glicoproteína Associada a Mielina/deficiência , Animais , Neuropatia Hereditária Motora e Sensorial/genética , Neuropatia Hereditária Motora e Sensorial/metabolismo , Neuropatia Hereditária Motora e Sensorial/patologia , Camundongos , Camundongos Knockout , Bainha de Mielina/genética , Glicoproteína Associada a Mielina/genética , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia
8.
Neuroreport ; 12(16): 3443-5, 2001 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-11733687

RESUMO

Differentiation and survival of sensory neurons is regulated by factors such as NGF and LIF. Regulation of signal transduction pathways downstream of such factor signalling by suppressor of cytokine signalling (SOCS) proteins, which negatively regulate the JAK/STAT pathway, may modulate biological outcome. In this study, SOCS1 regulation of growth factor mediated sensory neuron survival was examined. SOCS1 expression by sensory neurons was up-regulated by IFNgamma. Survival of sensory neurons from SOCS1 null mice in NGF or LIF was similar to wildtype mice. IFNgamma partially supported survival of wildtype neurons but supported survival of SOCS1 null neurons as effectively as NGF or LIF. Thus it appears that SOCS1 is a major regulator of sensory neuron responses to the inflammatory cytokine, IFNgamma.


Assuntos
Proteínas de Transporte/fisiologia , Interferon gama/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Proteínas Repressoras/fisiologia , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Camundongos , Camundongos Mutantes , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina
9.
J Neurosci ; 21(15): 5587-96, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11466430

RESUMO

The differentiation of precursor cells into neurons has been shown to be influenced by both the Notch signaling pathway and growth factor stimulation. In this study, the regulation of neuronal differentiation by these mechanisms was examined in the embryonic day 10 neuroepithelial precursor (NEP) population. By downregulating Notch1 expression and by the addition of a Delta1 fusion protein (Delta Fc), it was shown that signaling via the Notch pathway inhibited neuron differentiation in the NEP cells, in vitro. The expression of two of the Notch receptor homologs, Notch1 and Notch3, and the ligand Delta1 in these NEP cells was found to be influenced by a number of different growth factors, indicating a potential interaction between growth factors and Notch signaling. Interestingly, none of the growth factors examined promoted neuron differentiation; however, the fibroblast growth factors (FGFs) 1 and 2 potently inhibited differentiation. FGF1 and FGF2 upregulated the expression of Notch and decreased expression of Delta1 in the NEP cells. In addition, the inhibitory response of the cells to the FGFs could be overcome by downregulating Notch1 expression and by disrupting Notch cleavage and signaling by the ablation of the Presenilin1 gene. These results indicate that FGF1 and FGF2 act via the Notch pathway, either directly or indirectly, to inhibit differentiation. Thus, signaling through the Notch receptor may be a common regulator of neuronal differentiation within the developing forebrain.


Assuntos
Diferenciação Celular/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Fatores de Transcrição , Animais , Proteínas Sanguíneas/farmacologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Expressão Gênica/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Mutantes , Neurônios/citologia , Oligonucleotídeos Antissenso/farmacologia , Presenilina-1 , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch1 , Receptor Notch2 , Receptor Notch3 , Receptor Notch4 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Notch , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
10.
J Comp Neurol ; 423(2): 348-58, 2000 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-10867663

RESUMO

Growth factor and cytokine signalling in the developing nervous system has multiple effects, ranging from cell differentiation and cell survival to modulation of cell phenotype. Molecules that can regulate growth factor signalling pathways will therefore be of importance in determining the cellular response to factor stimulation. Members of a recently described gene family, the suppressor of cytokine signalling (SOCS) family, can regulate signalling events downstream of predominantly cytokine stimulation and may have important roles in the nervous system. We have examined the temporal and spatial expression of SOCS-1, SOCS-2, and SOCS-3 in the developing and adult nervous system by use of Northern analysis and in situ hybridisation. All three genes were expressed in the brain, with maximal expression from embryonic day 14 to postnatal day 8 and declining thereafter, with SOCS-2 being the most highly expressed. In situ hybridisation analysis showed that SOCS-1 and SOCS-3 had a low and widespread pattern of expression, whereas SOCS-2 expression was higher and tightly regulated. Its expression pattern indicated that SOCS-2 was expressed exclusively in neurons and that it was switched on developmentally at the time of neuronal differentiation.


Assuntos
Proteínas de Transporte/genética , Citocinas/genética , Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Supressores/genética , Camundongos Endogâmicos C57BL/genética , Sistema Nervoso/crescimento & desenvolvimento , Proteínas Repressoras , Transdução de Sinais/genética , Transativadores , Fatores de Transcrição , Fatores Etários , Animais , Mapeamento Encefálico , Citocinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL/anatomia & histologia , Camundongos Endogâmicos C57BL/metabolismo , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/crescimento & desenvolvimento , Sistema Nervoso Periférico/metabolismo , Prosencéfalo/citologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Proteínas/genética , Organismos Livres de Patógenos Específicos , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina
11.
J Neurochem ; 74(3): 889-99, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10693919

RESUMO

Cytokines that signal through the leukemia inhibitory factor (LIF) receptor, such as LIF and ciliary neuronotrophic factor, have a wide range of roles within both the developing and mature nervous system. They play a vital role in the differentiation of neural precursor cells into astrocytes and can prevent or promote neuronal differentiation. One of the conundrums regarding signalling through the LIF receptor is how it can have multiple, often conflicting roles in different cell types, such as enhancing the differentiation of astrocytes while inhibiting the differentiation of some neuronal cells. Factors that can modulate signal transduction downstream of cytokine signalling, such as "suppressor of cytokine signalling" proteins, which inhibit the JAK/STAT but not the mitogen-activated protein kinase pathway, may therefore play an important role in determining how a given cell will respond to cytokine signalling. This review discusses the general effects of cytokine signalling within the nervous system. Special emphasis is placed on differentiation of neural precursor cells and the role that regulation of cytokine signalling may play in how a given precursor cell responds to cytokine stimulation.


Assuntos
Citocinas/fisiologia , Inibidores do Crescimento , Interleucina-6 , Linfocinas , Fenômenos Fisiológicos do Sistema Nervoso , Receptores de Citocinas/fisiologia , Transdução de Sinais/fisiologia , Animais , Astrócitos/fisiologia , Proteínas de Transporte/fisiologia , Senescência Celular/fisiologia , Citocinas/antagonistas & inibidores , Fator Inibidor de Leucemia , Modelos Neurológicos , Isoformas de Proteínas/fisiologia , Receptores de OSM-LIF
12.
Clin Exp Pharmacol Physiol ; 26(9): 746-8, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10499166

RESUMO

1. The development of ventral cell types in the spinal cord, including motor neurons, requires the growth factor Sonic hedgehog (Shh). However, it is still unknown whether Shh acts directly on precursors to induce these cell types and whether additional factors are required for induction. 2. To further investigate the precise role of Shh in spinal motoneuron development, we used low-density cultures of murine spinal cord precursor cells. 3. Although purified Shh stimulated neuronal differentiation, it did not increase the proportion of neurons expressing the motoneuron marker Islet-1 (Isl-1). In contrast, purified Shh induced Isl-1 expression in neural tube explants, suggesting that additional neural tube-derived factors are required to induce motoneuron differentiation. 4. A factor implicated in motoneuron development, neurotrophin 3 (NT3), had no effect on Isl-1 expression; however, in combination with Shh, it induced Isl-1 expression in the majority of neurons in the low-density cultures. Furthermore, in explant cultures, Shh-induced Isl-1 expression was blocked by an anti-NT3 antibody. 5. In addition, previous studies have shown expression of NT3 in the region of motoneuron differentiation and the loss of spinal fusimotor neurons in NT3-knockout animals. 6. Taken together, these findings suggest that Shh can act directly on spinal cord precursors to promote neuronal differentiation, but induction of Isl-1 expression and motor differentiation requires additional factors, including NT3.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Neurônios Motores/citologia , Neurotrofina 3/fisiologia , Proteínas Nucleares/metabolismo , Proteínas/fisiologia , Medula Espinal/embriologia , Transativadores/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Hedgehog , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurotrofina 3/farmacologia , Proteínas Nucleares/efeitos dos fármacos , Proteínas/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologia , Transativadores/efeitos dos fármacos
13.
Int J Dev Neurosci ; 17(2): 109-19, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10221670

RESUMO

Myelin-associated glycoprotein (MAG) is a molecule expressed by myelinating cells at the myelin/axon interface, which binds to an as yet unidentified molecule on neurons. We have used a MAG-immunoglobulin Fc fusion protein to examine the expression and regulation of the MAG binding molecule on sensory neurons in culture. Binding of the MAG-Fc reached a maximum at 24-48 h and was higher on neurons which expressed high levels of neurofilament. Nerve growth factor (NGF) upregulated expression of the MAG binding molecule in a dose dependent manner. Schwann cells co-cultured with sensory neurons in serum-free medium stimulated maximal expression of the MAG binding molecule, which was decreased by addition of anti-NGF to the co-cultures. This indicated that Schwann cells can modulate expression of the MAG binding molecule via production of NGF and may represent a physiological mechanism for regulation of MAG-MAG binding molecule interactions during myelination and remyelination.


Assuntos
Interleucina-6 , Glicoproteína Associada a Mielina/metabolismo , Fatores de Crescimento Neural/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Células de Schwann/metabolismo , Animais , Axônios/metabolismo , Sítios de Ligação , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura Livres de Soro/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/crescimento & desenvolvimento , Glicoconjugados/metabolismo , Inibidores do Crescimento/farmacologia , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Cinética , Fator Inibidor de Leucemia , Lipossomos , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos CBA , Bainha de Mielina/fisiologia , Glicoproteína Associada a Mielina/genética , Ácido N-Acetilneuramínico/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas de Neurofilamentos/biossíntese , Neurônios Aferentes/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo
14.
J Neurosci ; 19(7): 2601-8, 1999 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-10087073

RESUMO

Sonic hedgehog (Shh) is strongly implicated in the development of ventral structures in the nervous system. Addition of Sonic hedgehog protein to chick spinal cord explants induces floor plate and motoneuron development. Whether Shh acts directly to induce these cell types or whether their induction is mediated by additional factors is unknown. To further investigate the role of Shh in spinal neuron development, we have used low-density cultures of murine spinal cord precursor cells. Shh stimulated neuronal differentiation; however, it did not increase the proportion of neurons expressing the first postmitotic motoneuron marker Islet-1. Moreover, Shh did induce Islet-1 expression in neural tube explants, suggesting that it acts in combination with neural tube factors to induce motoneurons. Another factor implicated in motoneuron development is neurotrophin 3 (NT3), and when assayed in isolated precursor cultures, it had no effect on Islet-1 expression. However, the combination of N-terminal Shh and NT3 induced Islet-1 expression in the majority of neurons in low-density cultures of caudal intermediate neural plate. Furthermore, in explant cultures, Shh-mediated Islet-1 expression was blocked by an anti-NT3 antibody. Previous studies have shown expression of NT3 in the region of motoneuron differentiation and that spinal fusimotor neurons are lost in NT3 knock-out animals. Taken together, these findings suggest that Shh can act directly on spinal cord precursors to promote neuronal differentiation, but induction of Islet-1 expression is regulated by factors additional to Shh, including NT3.


Assuntos
Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Proteínas de Homeodomínio/análise , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Endogâmicos CBA , Proteínas do Tecido Nervoso/análise , Sistema Nervoso/citologia , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/embriologia , Neurônios/citologia , Neurotrofina 3 , Medula Espinal/citologia , Células-Tronco/efeitos dos fármacos , Fatores de Transcrição
15.
J Neurochem ; 72(4): 1707-16, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10098881

RESUMO

The mammalian AMP-activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (alpha, beta, and gamma) and is activated under conditions of metabolic stress. It is widely expressed in many tissues, including the brain, although its expression pattern throughout the CNS is unknown. We show that brain mRNA levels for the alpha2 and beta2 subunits were increased between embryonic days 10 and 14, whereas expression of alpha1, beta1, and gamma1 subunits was consistent at all ages examined. Immunostaining revealed a mainly neuronal distribution of all isoforms. The alpha2 catalytic subunit was highly expressed in neurons and activated astrocytes, whereas the alpha1 catalytic subunit showed low expression in neuropil. The gamma1 noncatalytic subunit was highly expressed by neurons, but not by astrocytes. Expression of the beta1 and beta2 noncatalytic subunits varied, but some neurons, such as granule cells of olfactory bulb, did not express detectable levels of either beta isoform. Preferential nuclear localization of the alpha2, beta1, and gamma1 subunits suggests new functions of the AMP-activated protein kinase, and the different expression patterns and cellular localization between the two catalytic subunits alpha1 and alpha2 point to different physiological roles.


Assuntos
Adenilato Quinase/genética , Sistema Nervoso Central/enzimologia , Sistema Nervoso Central/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Adenilato Quinase/análise , Adenilato Quinase/imunologia , Fatores Etários , Animais , Anticorpos , Astrócitos/enzimologia , Núcleo Celular/enzimologia , Sistema Nervoso Central/citologia , Camundongos , Camundongos Endogâmicos CBA , Neurônios/enzimologia , RNA Mensageiro/análise
16.
Immunol Cell Biol ; 76(5): 414-8, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9797460

RESUMO

In the developing forebrain, mounting evidence suggests that neural stem cell proliferation and differentiation is regulated by growth factors. In vitro in the presence of serum, stem cell proliferation is predominantly mediated by fibroblast growth factor-2 (FGF-2) whereas neuronal differentiation can be triggered by FGF-1 in association with a specific heparan sulphate proteoglycan. On the other hand, astrocyte differentiation in vivo and in vitro appears to be dependent on signalling through the leukaemia inhibitory factor receptor (LIFR). The evidence suggests that in the absence of LIFR signalling, the stem cell population is present at approximately the same frequency and can generate neurons but is blocked from producing astrocytes that express glial fibrillary acidic protein (GFAP) or have trophic functions. The block can be overcome by other growth factors such as BMP-2/4 or interferon-gamma, providing further evidence that the inhibition to astrocyte development does not result from loss of a precursor population. Signalling through the LIFR, in addition to stimulating astrocyte differentiation, may also inhibit neuronal differentiation, which may explain why this receptor is expressed at the earliest stages of neurogenesis. Another signalling system which also exerts its influence on neurogenesis through active inhibition is Delta-Notch. We show in vitro that at high cell densities which impede neuronal production by FGF-1, lowering the levels of expression of the receptor Notch by antisense oligonucleotide results in a significant increase in neuronal production. Thus, stem cell differentiation appears to be dependent on the outcome of interactions between a number of signalling pathways, some which promote specific lineages and some which inhibit.


Assuntos
Diferenciação Celular , Inibidores do Crescimento , Interleucina-6 , Linfocinas , Neurônios/citologia , Prosencéfalo/citologia , Prosencéfalo/embriologia , Células-Tronco/fisiologia , Fator de Crescimento Transformador beta , Animais , Astrócitos/citologia , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Proteína Glial Fibrilar Ácida/fisiologia , Interferon gama/fisiologia , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Camundongos , Oligonucleotídeos Antissenso/farmacologia , Proteoglicanas/fisiologia , Receptores de Citocinas/fisiologia , Receptores de OSM-LIF , Receptores Opioides delta/antagonistas & inibidores
17.
Neuroreport ; 9(9): 1987-90, 1998 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-9674579

RESUMO

Myelin-associated glycoprotein (MAG) inhibits neurite outgrowth of postnatal spinal cord neurons, but its effect on embryonic neurons is unknown. The effect on neurite outgrowth of another myelin protein, myelin-oligodendrocyte glycoprotein (MOG) is also unknown. We determined the effect of MAG and MOG on embryonic day 17 spinal cord neurons, which were cultured on MAG, MOG or control transfected CHO cells. Neurite outgrowth was examined and both total neurite length and longest neurite length were significantly enhanced by both MAG and MOG. These findings show that, in contrast to postnatal spinal cord neurons, MAG can enhance neurite outgrowth of embryonic spinal cord neurons. In addition, another myelin protein, MOG, can also modulate neurite outgrowth.


Assuntos
Glicoproteína Associada a Mielina/farmacologia , Neuritos/ultraestrutura , Neurônios/ultraestrutura , Oligodendroglia/ultraestrutura , Medula Espinal/ultraestrutura , Animais , Células CHO , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/ultraestrutura , Cricetinae , Proteína GAP-43/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Proteínas da Mielina , Glicoproteína Mielina-Oligodendrócito , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/crescimento & desenvolvimento , Estimulação Química , Transfecção
18.
Proc Natl Acad Sci U S A ; 95(6): 3178-81, 1998 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-9501236

RESUMO

The differentiation of precursor cells into neurons or astrocytes in the developing brain has been thought to be regulated in part by growth factors. We show here that neural precursors isolated from the developing forebrain of mice that are deficient in the gene for the low-affinity leukemia inhibitory factor receptor (LIFR-/-) fail to generate astrocytes expressing glial fibrillary acidic protein (GFAP) when cultured in vitro. Precursors from mice heterozygous for the null allele show normal levels of GFAP expression. These findings support the in vivo findings that show extremely low levels of GFAP mRNA in brains of embryonic day 19 LIFR-/- mice. In addition, monolayers of neural cells from LIFR-/- mice are far less able to support the neuronal differentiation of normal neural precursors than are monolayers from heterozygous or wild-type animals, indicating that endogenous signaling through the LIFR is required for the expression of both functional and phenotypic markers of astrocyte differentiation. LIFR-/- precursors are not irreversibly blocked from differentiating into astrocytes: they express GFAP after long-term passaging or stimulation with bone morphogenetic protein-2. These findings strongly implicate the LIF family of cytokines in the regulation of astrocyte differentiation and indeed the LIF-deficient animals show a significant reduction in the number of GFAP cells in the hippocampus. However, because this reduction is only partial it suggests that LIF may not be the predominant endogenous ligand signaling through the LIFR.


Assuntos
Astrócitos/citologia , Inibidores do Crescimento/deficiência , Interleucina-6 , Linfocinas/deficiência , Prosencéfalo/citologia , Receptores de Citocinas/deficiência , Células-Tronco/citologia , Fator de Crescimento Transformador beta , Animais , Astrócitos/metabolismo , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular , Células Clonais , Células Epiteliais/citologia , Proteína Glial Fibrilar Ácida/biossíntese , Inibidores do Crescimento/genética , Heterozigoto , Hipocampo/citologia , Hipocampo/metabolismo , Homozigoto , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Linfocinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Mutantes , Neurônios/citologia , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , RNA Mensageiro/análise , Receptores de Citocinas/genética , Receptores de OSM-LIF , Transdução de Sinais , Células-Tronco/metabolismo
19.
Development ; 122(12): 3911-9, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9012511

RESUMO

Melanocytes in the skin are derived from the embryonic neural crest. Recently, mutations in endothelin 3 and the endothelin receptor B genes have been shown to result in gross pigment defects, indicating that this signalling pathway is required for melanocyte development. We have examined the effects of endothelins on melanocyte progenitors in cultures of mouse neural crest. Firstly, they stimulate an increase in progenitor number and act synergistically with another factor, Steel factor, in the survival and proliferation of the progenitors. These findings are consistent with findings from mice with natural mutations in the endothelin receptor B gene, which show an early loss of melanocyte progenitors. Secondly, endothelins induce differentiation of the progenitors into fully mature pigmented melanocytes. This finding is consistent with the expression of endothelins in the skin of mice at the initiation of pigmentation. The melanocytes generated in endothelin-treated cultures also become responsive to alpha melanocyte-stimulating hormone, which then acts to regulate the activity of the pigmentation pathway. These findings indicate two key roles for endothelin in melanocyte development: regulation of expansion of the progenitor pool and differentiation of progenitors into mature melanocytes.


Assuntos
Endotelinas/farmacologia , Melanócitos/efeitos dos fármacos , Crista Neural/citologia , Pele/embriologia , Células-Tronco/efeitos dos fármacos , Contagem de Células , Diferenciação Celular , Técnicas de Cultura , Interações Medicamentosas , Endotelina-1/metabolismo , Endotelina-1/farmacologia , Endotelina-3/metabolismo , Endotelina-3/farmacologia , Endotelinas/metabolismo , Cor de Cabelo/genética , Melanócitos/citologia , Pigmentação/efeitos dos fármacos , Ligação Proteica , Receptores de Endotelina/isolamento & purificação , Fator de Células-Tronco/farmacologia , Células-Tronco/citologia , Distribuição Tecidual , alfa-MSH/farmacologia
20.
Microsc Res Tech ; 32(4): 286-94, 1995 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-8573778

RESUMO

Why is it that oligodendrocytes do not normally express major histocompatibility complex (MHC) molecules? To examine the effect of aberrant MHC expression in oligodendrocytes, transgenic mice have been produced which expressed the class I MHC gene, H-2Kb, under direction of the MBP promoter [Turnley et al. (1991b) Nature, 353:566-569; Yoshioka et al. (1991) Mol. Cell. Biol., 11:5479-5486]. A proportion of these mice exhibited a shivering phenotype, with tonic seizures and early death. Oligodendrocyte function and viability was shown to be affected, resulting in severe dysmyelination of the CNS. Is this phenomenon of cell damage due to aberrant expression of MHC molecules restricted to oligodendrocytes, and could other, non-MHC molecules, when aberrantly expressed, result in similar cell damage? This paper discusses these questions and examines possible mechanisms for the oligodendrocyte damage and hypomyelination observed in these transgenic mice. Finally, the implications of aberrant MHC expression in oligodendrocytes for demyelinating diseases such as multiple sclerosis are discussed.


Assuntos
Doenças Desmielinizantes/genética , Regulação da Expressão Gênica/fisiologia , Genes MHC Classe I , Oligodendroglia/metabolismo , Animais , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Retículo Endoplasmático/metabolismo , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Oligodendroglia/patologia , Fenótipo
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