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1.
Epilepsy Res ; 88(2-3): 196-207, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20015616

RESUMO

EFHC1, a gene mutated in juvenile myoclonic epilepsy, encodes EFHC1, a protein with three DM10 domains and one EF-hand motif. We recently demonstrated that this molecule is a microtubule-associated protein (MAP) implicated in neuronal migration. Because some controversies persist about the precise localization in the CNS, we studied the neuroanatomical distribution of EFHC1 in mature and developing mouse brain. In the adult, low mRNA expression was detected in several brain structures such as cortex, striatum, hippocampus and cerebellum. At E16, EFHC1 mRNA was shown to be expressed in cortex and not only in cells lining ventricles. Using a purified polyclonal antibody, EFHC1 staining was observed in all cortical layers, in piriform cortex, in hippocampus and in Purkinje cells of cerebellum. In the cortex, the majority of EFHC1 positive cells correspond to neurons, however some glial cells were also stained. In agreement with a previous study, we demonstrated strong EFHC1 expression in cilia of ependymal cells lining cerebral ventricles. Moreover, at E16, the protein was observed at the borders of brain ventricles, in choroid plexus, but also, although to a lesser extent, in piriform and neocortex. In these latter structures, the pattern of expression seems to correspond to the extensions of the radial glia fibers as demonstrated by BLBP immunostaining. Finally, we confirmed that EFHC1 was also expressed and co-localized with the mitotic spindle of neural stem cells. These results confirm that EFHC1 is a protein with a likely low expression level in mouse brain but detectable both in adult and embryonic brain supporting our previous data and hypothesis that EFHC1 could play an important role during brain development. As discussed, this opens the door to a new conceptual approach viewing some idiopathic generalized epilepsies as developmental diseases instead of classical channelopathies.


Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Ciclo Celular/genética , Movimento Celular/genética , Células Cultivadas , Imuno-Histoquímica , Camundongos , Mitose/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
2.
BMC Neurosci ; 8: 104, 2007 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-18053121

RESUMO

BACKGROUND: Bone marrow stromal cells and radial glia are two stem cell types with neural phenotypic plasticity. Bone marrow mesenchymal stem cells can differentiate into osteocytes, chondrocytes and adipocytes, but can also differentiate into non-mesenchymal cell, i.e. neural cells in appropriate in vivo and in vitro experimental conditions. Likewise, radial glial cells are the progenitors of many neurons in the developing cortex, but can also generate astrocytes. Both cell types express nestin, an intermediate filament protein which is the hallmark of neural precursors. RESULTS: In this study, we demonstrate that thrombin, a multifunctional serine protease, stimulates the growth of radial glial cells (RG) and mesenchymal stem cells (MSCs) in a dose-dependent manner. In RG, the mitogenic effect of thrombin is correlated with increased expression of nestin but in MSCs, this mitogenic effect is associated with nestin down-regulation. Both cell types express the PAR-1 type receptor for Thrombin and the effect of Thrombin on both cell types can be mimicked by its analogue TRAP-6 activating specifically this receptor subtype or by serum which contains various amount of thrombin. Moreover, we also demonstrate that serum deprivation-induced expression of nestin in MSCs is inhibited by high cell density (> 50,000 cells/cm2). CONCLUSION: This work shows that thrombin stimulates the growth of both RG and MSCs and that nestin expression by MSCs and RG is regulated in opposite manner by thrombin in vitro. Thrombin effect is thus associated in both cell types with a proliferating, undifferentiated state but in RG this involves the induction of nestin expression, a marker of immaturity for neural progenitors. In MSCs however, nestin expression, as it corresponds to a progression from the mesenchymal "undifferentiated", proliferating phenotype toward acquisition of a neural fate, is inhibited by the mitogenic signal.


Assuntos
Proteínas de Filamentos Intermediários/genética , Células-Tronco Mesenquimais/citologia , Mitógenos/farmacologia , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Trombina/farmacologia , Animais , Proteínas Sanguíneas/farmacologia , Células da Medula Óssea/citologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Proteínas de Filamentos Intermediários/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Fenótipo , Ratos
3.
Exp Cell Res ; 312(15): 2872-9, 2006 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-16824517

RESUMO

A novel gene, EFHC1, mutated in juvenile myoclonic epilepsy (JME) encodes a protein with three DM10 domains of unknown function and one putative EF-hand motif. To study the properties of EFHC1, we expressed EGFP-tagged protein in various cell lines. In interphase cells, the fusion protein was present in the cytoplasm and in the nucleus with specific accumulation at the centrosome. During mitosis EGFP-EFHC1 colocalized with the mitotic spindle, especially at spindle poles and with the midbody during cytokinesis. Using a specific antibody, we demonstrated the same distribution of the endogenous protein. Deletion analyses revealed that the N-terminal region of EFHC1 is crucial for the association with the mitotic spindle and the midbody. Our results suggest that EFHC1 could play an important role during cell division.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Divisão Celular , Fuso Acromático/metabolismo , Animais , Células COS , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Imunofluorescência , Células HeLa , Humanos , Camundongos , Mutação , Epilepsia Mioclônica Juvenil/genética , Células NIH 3T3 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais Cultivadas
4.
FEBS Lett ; 527(1-3): 205-10, 2002 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-12220661

RESUMO

Melanin-concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated MCH function in human and mouse immune cells. RT-PCR analysis revealed a low expression of prepro-MCH and MCH receptor 1 (MCHR1) but not of MCHR2 transcript in tissular and peripheral blood immune cells. FACS and in vitro assay studies demonstrated that MCHR1 receptor expression on most cell types can trigger, in the presence of MCH, cAMP synthesis and calcium mobilization in peripheral blood mononuclear cells (PBMCs). Moreover, MCH treatment decreases the CD3-stimulated PBMC proliferation in vitro. Accordingly, our data indicate for the first time that MCH and MCHR1 may exert immunomodulatory functions.


Assuntos
Hormônios Hipotalâmicos/genética , Linfócitos/imunologia , Precursores de Proteínas/genética , Receptores do Hormônio Hipofisário/metabolismo , Animais , Complexo CD3/farmacologia , Células CHO , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Criança , Pré-Escolar , Colforsina/farmacologia , Cricetinae , AMP Cíclico/metabolismo , Humanos , Hormônios Hipotalâmicos/metabolismo , Hormônios Hipotalâmicos/farmacologia , Imunidade Celular/fisiologia , Lactente , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Melaninas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Hormônios Hipofisários/farmacologia , Precursores de Proteínas/metabolismo , RNA Mensageiro/análise , Receptores do Hormônio Hipofisário/efeitos dos fármacos , Receptores do Hormônio Hipofisário/genética , Baço/citologia , Baço/imunologia , Baço/metabolismo , Timo/imunologia , Timo/metabolismo
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