RESUMEN
Microtubules are subject to a variety of posttranslational modifications that potentially regulate cytoskeletal functions. Two modifications, glutamylation and glycylation, are highly enriched in the axonemes of most eukaryotes, and might therefore play particularly important roles in cilia and flagella. Here we systematically analyze the dynamics of glutamylation and glycylation in developing mouse ependymal cilia and the expression of the corresponding enzymes in the brain. By systematically screening enzymes of the TTLL family for specific functions in ependymal cilia, we demonstrate that the glycylating enzymes TTLL3 and TTLL8 were required for stability and maintenance of ependymal cilia, whereas the polyglutamylase TTLL6 was necessary for coordinated beating behavior. Our work provides evidence for a functional separation of glutamylating and glycylating enzymes in mammalian ependymal cilia. It further advances the elucidation of the functions of tubulin posttranslational modifications in motile cilia of the mammalian brain and their potential importance in brain development and disease.
Asunto(s)
Cilios/enzimología , Epéndimo/citología , Epéndimo/enzimología , Péptido Sintasas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Cilios/metabolismo , Epéndimo/metabolismo , Ratones , Péptido Sintasas/genéticaRESUMEN
We studied activity of 3ß-hydroxysteroid dehydrogenase in rat brain ependymocytes. Enzyme activity was found in the cytoplasm of cells lining the villi in the vascular plexuses in the lateral ventricles and cells lining the ventricles. These data suggest that ependymocyte can synthesize neurosteroids.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Epéndimo/enzimología , Células Ependimogliales/enzimología , Animales , Epéndimo/metabolismo , Células Ependimogliales/metabolismo , Femenino , Ventrículos Laterales/enzimología , Ventrículos Laterales/metabolismo , Masculino , RatasRESUMEN
Using the histochemical method, the activity of 3beta-hydroxysteroid dehydrogenase (HSDH) was studied in the brain of laboratory male albino rats of different age groups: 5-6 days (n = 6), 45-50 days (n = 12), and 6 months (n = 15). The quantitative assessment of reaction intensity was performed with the cytospectrophotometer. The results obtained indicate that the ependimocytes lining the brain lateral ventricles and covering the villi of their vascular plexuses are characterized by the presence of HSDH activity typical to that of steroid-producing cells. In this regard ependimocytes may be attributed to the cells that can produce neurosteroids. It was established that HSDH activity in ependimocytes was minimal in the early postnatal period and considerably increased by the prepuberty period, remaining at this level in adult animals.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Plexo Coroideo , Epéndimo , Ventrículos Laterales , Factores de Edad , Animales , Plexo Coroideo/citología , Plexo Coroideo/enzimología , Epéndimo/citología , Epéndimo/enzimología , Epéndimo/metabolismo , Ventrículos Laterales/citología , Ventrículos Laterales/enzimología , Masculino , Neurotransmisores/metabolismo , RatasRESUMEN
Intraperitoneal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration induces apoptosis of subventricular zone (SVZ) doublecortin (Dcx)-positive neural progenitor cells (migrating neuroblasts, A cells). Actually, a metabolite of MPTP, 1-methy-4-phenylpiridinium (MPP(+)), is responsible for neural progenitor cell toxicity. In the present study, to examine whether the MPTP-induced SVZ cell apoptosis is caused directly by MPP(+) metabolized through monoamine oxidase B (MAO-B), MPTP or MPP(+) was intracerebroventricularly (icv) injected into C57BL/6 mice. At Day 1 postinjection, many terminal deoxynucleotidyl transferase-mediated dUTP endlabeling (TUNEL)-positive cells were observed in the SVZ of both low (36 µg) and high (162 µg) dose MPTP- and MPP(+)-injected mice. The number of Dcx-positive A cells showed a significant decrease following high dose of MPTP- or MPP(+)-injection on Days 1 and 3, respectively, whereas that of EGFR-positive C cells showed no change in mice with any treatment. In addition, prior icv injection of a MAO-B inhibitor, R(-)-deprenyl (deprenyl), inhibited MPTP-induced apoptosis, but not MPP(+)-induced apoptosis. MAO-B- and GFAP-double positive cells were detected in the ependyma and SVZ in all mice. It is revealed from these results that icv injection of MPTP induces apoptosis of neural progenitor cells (A cells) in the SVZ via MPP(+) toxicity. In addition, it is suggested that the conversion from MPTP to MPP(+) is caused mainly by MAO-B located in ependymal cells and GFAP-positive cells in the SVZ.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , 1-Metil-4-fenilpiridinio/metabolismo , Apoptosis/efectos de los fármacos , Ventrículos Cerebrales/efectos de los fármacos , Monoaminooxidasa/metabolismo , Células-Madre Neurales/efectos de los fármacos , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , 1-Metil-4-fenilpiridinio/farmacología , Animales , Ventrículos Cerebrales/enzimología , Ventrículos Cerebrales/patología , Relación Dosis-Respuesta a Droga , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Epéndimo/efectos de los fármacos , Epéndimo/enzimología , Epéndimo/patología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Células-Madre Neurales/enzimología , Células-Madre Neurales/patología , Neuropéptidos/metabolismo , Selegilina/farmacologíaRESUMEN
The mitochondrial enzyme, pyruvate carboxylase (PC; EC 6.4.1.1) is considered to play a significant role in the intermediary metabolism of neural tissue. PC-catalyzed carboxylation of pyruvate to oxaloacetate is a major anaplerotic reaction in brain. Anaplerosis is essential for homeostasis of the members of the tricarboxylic acid (TCA) cycle. Several biochemical pathways rely on withdrawing TCA cycle members. Prominent among these are biosynthesis of fatty acids and of non-essential amino acids such as aspartate, asparagine, glutamate and glutamine, gluconeogenesis, glycogen synthesis, and regeneration of NADPH. The expression of PC in brain has already been described and assigned to astrocytes. Since pyruvate carboxylase deficiency is associated with malformations of the brain, e.g., inadequate development of the corpus callosum and the lack of myelination, one can hypothesize that PC may be expressed also in glial cells other than astrocytes. Therefore, the expression of PC was investigated in cultured oligodendroglial, microglial, and ependymal cells. As assessed by RT-PCR, all these cultures contain PC mRNA. This mRNA is generated in a transcription process that is regulated by the "distal class" of promoters of the PC gene. The expression of PC among cultured glial cells was studied with a rabbit antiserum by immunoblotting and immunocytochemistry. The results indicate that PC is not only expressed in cultured astroglial cells but also in cultured oligodendrocytes, microglial cells, and ependymocytes. It appears that the intermediary metabolism of these cells includes the anaplerotic action of PC as well as possibly also functions of the enzyme in biosynthetic pathways and the provision of NADPH for defense against reactive oxygen species.
Asunto(s)
Epéndimo/enzimología , Microglía/enzimología , Oligodendroglía/enzimología , Piruvato Carboxilasa/biosíntesis , Animales , Animales Recién Nacidos , Células Cultivadas , Epéndimo/citología , Inmunohistoquímica , ARN Mensajero/biosíntesis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
To evaluate the ability of ependymal, microglial and oligodendroglial cells to degrade leucine, the presence of 3-methylcrotonyl-CoA carboxylase (MCC) was investigated in cultures of these cells. MCC is a biotin-containing heterodimeric enzyme that is specific for the irreversible part of the leucine catabolic pathway. It has been reported previously that in cell culture MCC is expressed in astrocytes and a subpopulation of neurones. In the present study ependymal, microglial and oligodendroglial cell cultures, derived from the brains of newborn rats, were examined for the expression of MCC by RT-PCR, western blotting and immunocytochemistry. The results of RT-PCR and western blotting showed the presence of mRNA as well as protein of both subunits of MCC in ependymal, microglial and oligodendroglial cell cultures. Immunocytochemical investigation of the cellular and subcellular distribution of MCC demonstrated a mitochondrial location of MCC in all neuroglial cell types investigated. The ubiquitous expression of MCC in glial cells demonstrates the ability of the cells to engage in the catabolism of leucine transported into the brain, mainly for the generation of energy.
Asunto(s)
Encéfalo/citología , Encéfalo/enzimología , Ligasas de Carbono-Carbono/metabolismo , Epéndimo/enzimología , Microglía/enzimología , Oligodendroglía/enzimología , Animales , Animales Recién Nacidos , Encéfalo/crecimiento & desarrollo , Ligasas de Carbono-Carbono/química , Ligasas de Carbono-Carbono/genética , Células Cultivadas , Metabolismo Energético/fisiología , Epéndimo/citología , Inmunohistoquímica , Leucina/metabolismo , Microglía/citología , Mitocondrias/enzimología , Oligodendroglía/citología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Brain edema and severe alterations of the glial and endothelial cells have recently been demonstrated in the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy, and an increase in microvessel density in patients affected by Duchenne muscular dystrophy has also been shown. In order to further elucidate the mechanisms underlying the angiogenetic processes occurring in Duchenne muscular dystrophy, in this study we analyzed matrix-metalloproteinase-2 and -9 expression in the brain of 20-month-old mdx and control mice by means of immunohistochemistry, in situ hybridization, immunoblotting and gelatin zymography. Moreover, we studied vascular endothelial growth factor expression by means of Western blot and immunohistochemistry, and by dual immunofluorescence using anti-vascular endothelial growth factor and anti matrix-metalloproteinase-2 and-9 antibodies. Ultrastructural features of the brain choroidal plexuses were evaluated by electron microscopy. Spatial relationships between endothelium and astrocyte processes were studied by confocal laser microscopy, using an anti-CD31 antibody as a marker of endothelial cells, and anti-glial fibrillary acidic protein (GFAP) as a marker of glial cells. The results demonstrate that high expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 protein content occurs in mdx brain and in choroidal plexuses where, by in situ hybridization, matrix-metalloproteinase-2 and matrix-metalloproteinase-9 mRNA was localized in the epithelial cells. Moreover, matrix-metalloproteinase-2 mRNA was found in both mdx perivascular astrocytes and blood vessels, while matrix-metalloproteinase-9 mRNA was localized in mdx vessels. Through zymography, increased expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in mdx brain compared with the controls. These enhanced matrix-metalloproteinase levels in mdx mice were found to be associated with increased vascular endothelial growth factor expression, as determined by immunoblotting and immunocytochemistry and with ultrastructural alterations of the mdx choroidal epithelial cells and brain vessels, as previously reported [Nico B, Frigeri A, Nicchia GP, Corsi P, Ribatti D, Quondamatteo F, Herken R, Girolamo F, Marzullo A, Svelto M, Roncali L (2003) Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice. Glia 42:235-251]. Indeed, in the mdx epithelial cells of the plexuses, the apical microvilli were located on the lateral membranes, whereas in the controls they were uniformly distributed over the free ventricular surface. Moreover, by dual immunofluorescence, a colocalization of vascular endothelial growth factor and matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in the ependymal and epithelial cells of plexuses in mdx mice and, under confocal laser microscopy, mdx CD-31 positive vessels were enveloped by less GFAP-positive astrocyte processes than the controls. Overall, these data point to a specific pathogenetic role of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 in neurological dysfunctions associated with Duchenne muscular dystrophy.
Asunto(s)
Barrera Hematoencefálica/enzimología , Encéfalo/enzimología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Microcirculación/enzimología , Distrofia Muscular de Duchenne/enzimología , Animales , Astrocitos/enzimología , Astrocitos/patología , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/fisiopatología , Encéfalo/patología , Encéfalo/fisiopatología , Plexo Coroideo/enzimología , Plexo Coroideo/patología , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Células Endoteliales/patología , Epéndimo/enzimología , Epéndimo/patología , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Microcirculación/patología , Microcirculación/fisiopatología , Microscopía Electrónica de Transmisión , Microvellosidades/enzimología , Microvellosidades/patología , Distrofia Muscular de Duchenne/fisiopatología , Neovascularización Patológica/enzimología , Neovascularización Patológica/patología , Neovascularización Patológica/fisiopatología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN Mensajero/metabolismo , Regulación hacia Arriba/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Neuronal nitric oxide synthase (nNOS) regulates neurogenesis in normal developing brain, but the role of nNOS in neurogenesis in the ischemic brain remains unclear. To investigate the temporal and spatial relationship between cell proliferation of the ependymal/subventricular zone (SVZ), a principal neuroproliferative region in the adult brain, and nNOS expression, the male Sprague-Dawley rats weighing 250-350 g were used. The focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). 10 microl of 0.2% fluorescence dye DiI was injected into the right lateral ventricle to prelabel ependymal/subventricular zone cells before ischemia. The rats were killed immediately after ischemia and days 1, 3, 7, 11, 14, 21 and 28 after ischemia. DiI-labeled cell counting was employed to assess cell proliferation. Immunohistochemistry and grayscale analysis were performed to determine nNOS localization and its quantity in the specific regions. Compared with control, the density of DiI-labeled cells in the ipsilateral ependyma/SVZ was significantly higher at days 1, 3, 7 and 11 after ischemia, whereas the quantity of nNOS expression in the ependyma/SVZ adjacent regions was significantly lower at the above time points. Additionally, nNOS positive cells were largely excluded from SVZ, and their long processes did not enter the ependyma/SVZ. Our results indicate that after focal cerebral ischemia, decreased nNOS expression in the ipsilateral ependymal/SVZ adjacent regions might be related to cell proliferation in the ependymal/SVZ.
Asunto(s)
Isquemia Encefálica/patología , Proliferación Celular , Ventrículos Cerebrales/patología , Epéndimo/enzimología , Epéndimo/patología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Isquemia Encefálica/enzimología , Carbocianinas , Recuento de Células/métodos , Ventrículos Cerebrales/metabolismo , Modelos Animales de Enfermedad , Expresión Génica/fisiología , Inmunohistoquímica/métodos , Masculino , Óxido Nítrico Sintasa de Tipo I/genética , Ratas , Factores de TiempoRESUMEN
Atrial natriuretic peptide-(1-28) (ANP), brain natriuretic peptide-(1-32) (BNP) and C-Type natriuretic polypeptide (CNP) occur in the brain, are concentrated in the anteroventral area of the third cerebral ventricle and participate in the regulation of body fluid homeostasis. The ventricles of the mammalian brain are lined by a continuous monolayered epithelium of polyciliated ependymal cells. In the adult rat, the ependymocytes continue to express the intermediate filament vimentin, but do not contain glial fibrillary acidic protein. Ependymal functions are poorly understood, but may extend to osmoregulation and volume sensing. Ependymal cells possess receptors for the natriuretic peptides, and in cell culture respond to them with an increase in their cyclic GMP content. In this study, a cyclic GMP-specific antibody was employed together with an ex vivo brain slice system to assess the ependymal response to ANP, BNP and CNP under close to life-like conditions. While ANP in concentrations of 0.1 nM and 1 nM had no effect, at concentrations of 10nM and 100 nM it increased ependymal cyclic GMP levels in a concentration-dependent manner. The other natriuretic peptides BNP, and CNP, also increased the cyclic GMP content of ependymocytes, while nitric oxide (NO) donors had no effect. However, in contrast to the natriuretic peptides, the NO donors elevated the level of cyclic GMP in the brain parenchyma below the ependymal layer.
Asunto(s)
Encéfalo/citología , GMP Cíclico/metabolismo , Epéndimo/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Péptidos Natriuréticos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Epéndimo/enzimología , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica/métodos , Técnicas In Vitro , Péptido Natriurético Encefálico/farmacología , Péptido Natriurético Tipo-C/farmacología , Péptidos Natriuréticos/clasificación , Donantes de Óxido Nítrico/farmacología , RatasRESUMEN
Inducible nitric oxide synthase (iNOS) is an enzyme that produces nitric oxide (NO) and is thought to contribute to the pathogenesis of multiple sclerosis (MS). The extent of iNOS expression was examined using laser scanning confocal microscopy of 13 chronic active plaques from seven MS patients displaying both acute demyelination and active inflammation. iNOS expression in these plaques was substantial and diverse in cellular distribution. Expression of iNOS was observed in ependymal cells located in periventricular lesions, inflammatory cells, and occasionally in astrocytes. iNOS was found in microglial/macrophage cells that expressed CD64, the high affinity Fc gamma receptor associated with cells that have phagocytic function and participate in antibody-dependent cellular cytotoxicity (ADCC). Scavenger microglial/macrophage cells that expressed the marker CD14 were also present and may express iNOS. The markers for myelin damage, nitrotyrosine (an index of iNOS mediated damage via peroxynitrite formation), along with MBP fragments, were also observed associated with iNOS in MS plaques. Together, these findings support a central role for iNOS in the pathogenesis of multiple sclerosis.
Asunto(s)
Enfermedades Desmielinizantes/patología , Inflamación/inmunología , Esclerosis Múltiple/enzimología , Esclerosis Múltiple/patología , Óxido Nítrico Sintasa/metabolismo , Astrocitos/enzimología , Enfermedades Desmielinizantes/inmunología , Epéndimo/enzimología , Técnica del Anticuerpo Fluorescente , Humanos , Receptores de Lipopolisacáridos/metabolismo , Microglía/enzimología , Microscopía Confocal , Óxido Nítrico Sintasa de Tipo IIRESUMEN
We examined the expression profile of catechol O-methyltransferase (COMT) mRNA and its protein in the neonatal rat hypothalamus by in situ hybridization and immunohistochemistry to clarify the sites of dopamine degradation. Strong COMT mRNA expression was observed in the suprachiasmatic nucleus (SCN) throughout its rostrocaudal extent at postnatal day 1 (P1) and P2, and the mRNA levels decreased gradually until P16. COMT mRNA was predominantly localized to the ventral and medial parts of the SCN. Intense COMT immunoreactivity was demonstrated in the ventral SCN and was detected in neuronal perikarya and processes at P1. Ependymal and microglial cells also exhibited strong COMT immunoreactivity. These results indicate that COMT may directly be involved in dopaminergic signaling in the neonatal SCN.
Asunto(s)
Catecol O-Metiltransferasa/metabolismo , Ritmo Circadiano/genética , Dopamina/metabolismo , Neuronas/enzimología , ARN Mensajero/metabolismo , Núcleo Supraquiasmático/enzimología , Animales , Animales Recién Nacidos , Catecol O-Metiltransferasa/genética , Diferenciación Celular/genética , Epéndimo/citología , Epéndimo/enzimología , Epéndimo/crecimiento & desarrollo , Femenino , Inmunohistoquímica , Masculino , Microglía/citología , Microglía/enzimología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/crecimiento & desarrollo , Regulación hacia Arriba/genéticaRESUMEN
The minibrain kinase (Mnb/Dyrk1A) gene is localized in the Down syndrome (DS) critical region of chromosome 21. This gene encodes a proline-directed serine/threonine protein kinase (minibrain kinase-Mnb/Dyrk1A), which is required for the proliferation of distinct neuronal cell types during postembryonic neurogenesis. To study the distribution of Mnb/Dyrk1A during human brain development and aging, we raised Mnb/Dyrk1A-specific antibody (mAb 7F3) and examined 22 brains of normal subjects from 8 months to 90 years of age. We found that neurons were the only cells showing the presence of 7F3-positive product in both cell nucleus and cytoplasm. Nuclear localization supports the concept that Mnb/Dyrk1A may be involved in control of gene expression. Synaptic localization of Mnb/Dyrk1A also supports our previous studies suggesting that Mnb/Dyrk1A is a regulator of assembly of endocytic apparatus and appears to be involved in synaptic vesicle recycling and synaptic signal transmission. Accumulation of numerous 7F3-positive corpora amylacea in the memory and motor system subdivisions in subjects older than 33 years of age indicates that Mnb/Dyrk1A is colocalized with markers of astrocyte and neuron degeneration. Differences in the topography and the amount of Mnb/Dyrk1A in neurons, astrocytes, and ependymal and endothelial cells appear to reflect cell type- and brain structure-specific patterns in trafficking and utilization of Mnb/Dyrk1A.
Asunto(s)
Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Neuronas/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Anticuerpos , Astrocitos/citología , Astrocitos/enzimología , Biomarcadores , Encéfalo/citología , Núcleo Celular/enzimología , Citoplasma/enzimología , Endocitosis/fisiología , Células Endoteliales/enzimología , Epéndimo/enzimología , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/fisiopatología , Neuronas/citología , Terminales Presinápticos/enzimología , Proteínas Tirosina Quinasas , Transmisión Sináptica/fisiología , Quinasas DyrKRESUMEN
The molecular mechanisms responsible for seasonal time measurement have yet to be fully described. Recently, we used differential analysis to identify that the type 2 iodothyronine deiodinase (Dio2) gene is responsible for the photoperiodic response of gonads in Japanese quail. It was found that expression of Dio2 in the mediobasal hypothalamus is induced by light and that T(3) content in the mediobasal hypothalamus increased under long day conditions. In addition, we showed that intracerebroventricular infusion of T(3) mimics photoperiodically induced testicular growth. Because it is well known that thyroid hormone is also essential for the maintenance of the seasonal reproductive changes in a number of mammals, we examined expression of Dio2 in Djungarian hamsters and found expression in the ependymal cell layer lining the infralateral walls of the third ventricle and the cell-clear zone overlying the tuberoinfundibular sulcus. Signal intensity was high under long days and weak under short days. Although light pulse did not affect Dio2 expression, melatonin injections decreased Dio2 expression under long days. These results indicate that Dio2 may be involved in the regulation of seasonal reproduction in mammals in the same way as observed in birds.
Asunto(s)
Yoduro Peroxidasa/metabolismo , Fotoperiodo , Animales , Núcleo Arqueado del Hipotálamo/irrigación sanguínea , Aves/metabolismo , Vasos Sanguíneos/enzimología , Cricetinae , Epéndimo/citología , Epéndimo/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Hipotálamo/enzimología , Inyecciones Intraperitoneales , Yoduro Peroxidasa/antagonistas & inhibidores , Yoduro Peroxidasa/genética , Masculino , Melatonina/administración & dosificación , Tamaño de los Órganos , Phodopus/metabolismo , Reproducción/genética , Homología de Secuencia , Testículo/anatomía & histología , Tercer Ventrículo/citología , Tercer Ventrículo/enzimología , Distribución Tisular , Yodotironina Deyodinasa Tipo IIRESUMEN
Prostaglandin F synthase has at least two isozymes, i.e. prostaglandin F synthase I and II. Recently, we demonstrated immunocytochemically that prostaglandin F synthase I was localized in neuronal dendrites and somata, and in endothelial cells of blood vessels in the whole area of rat spinal cord. In the present study, we immunocytochemically localized prostaglandin F synthase II in ependymal cells and tanycytes surrounding the central canal and in endothelial cells of blood vessels, but not in any neuronal elements at all segmental levels of the rat spinal cord. Immunoelectron microscopy and confocal laser scanning microscopy confirmed these findings and further revealed that strong immunoreactivity was found in the basal processes of the tanycytes. Our present and recent studies using antibodies against the two isozymes of prostaglandin F synthase clearly indicated that they were localized differentially in ependymal (prostaglandin F synthase II) and neuronal elements (prostaglandin F synthase I), but were co-localized in blood vessels in the rat spinal cord. The distinct localization of the two isozymes suggests that prostaglandin F(2) has different transcellular biological actions via different cell groups.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/metabolismo , Médula Espinal/citología , Médula Espinal/enzimología , Animales , Endotelio Vascular/enzimología , Endotelio Vascular/ultraestructura , Epéndimo/enzimología , Epéndimo/ultraestructura , Inmunohistoquímica , Isoenzimas/metabolismo , Masculino , Microscopía Confocal , Microscopía Inmunoelectrónica , Neuronas/enzimología , Neuronas/ultraestructura , Ratas , Ratas Wistar , Médula Espinal/ultraestructuraRESUMEN
The purine nucleotide cycle enzyme AMP deaminase (AMPD) catalyzes the irreversible hydrolytic deamination of AMP. The physiological function of the purine nucleotide cycle in the brain is unknown. In situ hybridization and immunocytochemical studies were performed to identify the regional and cellular expression of AMPD in rat brain with the goal of elucidating the neural function of the purine nucleotide cycle. AMPD messenger RNA was detected in ventricular ependymal cells and cells of the choroid plexus and in neurons of distinct brain areas. Although only low antibody titers were obtained by immunization with the purified sheep brain AMPD, immunization of mice with synthetic lipopeptide vaccines containing oligopeptides derived from a known partial complementary DNA sequence of the enzyme yielded an antiserum suitable for immunocytochemistry. Immunostaining of cells in culture showed that neurons but not astroglial cells express appreciable amounts of the enzyme. Results of immunocytochemical staining performed on rat brain slices were in accord with the localization of AMPD messenger RNA, thus confirming the expression of AMPD in neurons of the brain stem, hippocampus, cerebellar nuclei and mesencephalic nuclei, as well as in ventricular ependymal cells and their cilia.
Asunto(s)
AMP Desaminasa/genética , AMP Desaminasa/metabolismo , Encéfalo/enzimología , AMP Cíclico/metabolismo , Epéndimo/enzimología , Neuronas/enzimología , ARN Mensajero/metabolismo , AMP Desaminasa/aislamiento & purificación , Animales , Animales Recién Nacidos , Especificidad de Anticuerpos , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/citología , Epéndimo/citología , Feto , Inmunohistoquímica , Hibridación in Situ , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Sondas de Oligonucleótidos , Ratas , Ratas Wistar , OvinosRESUMEN
Kin17 and 8-Oxoguanine DNA glycosylase (Ogg1) are proteins, respectively, involved in illegitimate recombination and DNA repair in eukaryotic cells. To characterize the expression of these proteins in cell types of rodent and avian brains, we combined immunocytochemistry for either Kin17 or Ogg1 proteins with glial fibrillary acidic protein (GFAP, an astrocyte marker) immunodetection on the same tissue section. Both Kin17 and Ogg1 proteins were localized in cell nuclei and were extensively distributed in neuronal populations of quail and rodent brains. However, GFAP-immunoreactive cells were never labeled by Kin17 protein. This was observed in nerve fiber tracts, in the cerebral cortex, the hippocampal formation, the hypothalamic region, and the periventricular regions of the brain of both species studied. These results were confirmed by combining in situ hybridization of kin17 mRNA and GFAP immunodetection. On the contrary, GFAP-immunoreactive cells were often labeled by the Ogg1 protein in brain structures such as fiber tracts, the cortical surface, the cerebellum, and the ependymal surface of both quail and mouse brains. Our results suggest that the expression of the Kin17 protein (observed in neurons) and that of the Ogg1 protein (observed in neurons and glial cells) is conserved in brain phylogeny.
Asunto(s)
Sistema Nervioso Central/enzimología , Proteínas de Unión al ADN/metabolismo , N-Glicosil Hidrolasas/metabolismo , Neuroglía/enzimología , Neuronas/enzimología , Proteínas Nucleares , Codorniz/metabolismo , Roedores/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Axones/enzimología , Axones/ultraestructura , Sistema Nervioso Central/citología , Reparación del ADN/fisiología , ADN-Formamidopirimidina Glicosilasa , Epéndimo/citología , Epéndimo/enzimología , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Ratones , Neuroglía/citología , Neuronas/citología , Codorniz/anatomía & histología , Ratas , Ratas Sprague-Dawley , Roedores/anatomía & histologíaRESUMEN
Histamine mediates many types of physiologic signals in multicellular organisms. To clarify the developmental role of histamine, we have examined the developmental expression of L-histidine decarboxylase (HDC) mRNA and the production of histamine during mouse development. The predominant expression of HDC in mouse development was seen in mast cells. The HDC expression was evident from embryonal day 13 (Ed13) until birth, and the mast cells were seen in most peripheral tissues. Several novel sites with a prominent HDC mRNA expression were revealed. In the brain, the choroid plexus showed HDC expression at Ed14 and the raphe neurons at Ed15. Close to the parturition, at Ed19, the neurons in the tuberomammillary (TM) area and the ventricular neuroepithelia also displayed a clear HDC mRNA expression and histamine immunoreactivity (HA-ir). From Ed14 until birth, the olfactory and nasopharyngeal epithelia showed an intense HDC mRNA expression and HA-ir. In the olfactory epithelia, the olfactory receptor neurons (ORN) were shown to have very prominent histamine immunoreactivity. The bipolar nerve cells in the epithelium extended both to the epithelial surface and into the subepithelial layers to be collected into thick nerve bundles extending caudally toward the olfactory bulbs. Also, in the nasopharynx, an extensive subepithelial network of histamine-immunoreactive nerve fibers were seen. Furthermore, in the peripheral tissues, the degenerating mesonephros (Ed14) and the convoluted tubules in the developing kidneys (Ed15) showed HDC expression, as did the prostate gland (Ed15). In adult mouse brain, the HDC expression resembled the neuronal pattern observed in rat brain. The expression was restricted to the TM area in the ventral hypothalamus, with the main expression in the five TM subgroups called E1-E5. A distinct mouse HDC mRNA expression was also seen in the ependymal wall of the third ventricle, which has not been reported in the rat. The tissue- and cell-specific expression patterns of HDC and histamine presented in this work indicate that histamine could have cell guidance or regulatory roles in development.
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Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Histidina Descarboxilasa/genética , Histidina Descarboxilasa/metabolismo , Histidina/metabolismo , Factores de Edad , Animales , Anticuerpos , Epéndimo/embriología , Epéndimo/enzimología , Femenino , Histidina/análisis , Histidina/inmunología , Área Hipotalámica Lateral/embriología , Área Hipotalámica Lateral/enzimología , Inmunohistoquímica , Hibridación in Situ , Riñón/embriología , Riñón/enzimología , Masculino , Ratones , Ratones Endogámicos BALB C , Mucosa Olfatoria/embriología , Mucosa Olfatoria/enzimología , Embarazo , Próstata/embriología , Próstata/enzimología , ARN Mensajero/análisisRESUMEN
Studies in rodents and monkeys suggest that neuronal precursor cells continue to exist and differentiate well into adulthood in these species. These results challenge the long held assumption that neurogenesis does not occur in the postnatal human brain. We examined the rostral subependymal zone (SEZ) of postnatal human brain for expression of cell phenotypic markers that have been associated with neuronal precursors and neuroblasts in rodent brain. We found epidermal growth factor receptor (EGF-R) mRNA and protein to be expressed in infant, teen, young adult, and adult human SEZ. Some SEZ cells expressed the polysialic acid form of neural cell adhesion molecule (PSA-NCAM), characteristic of migrating neuroblasts, as well as class III beta-tubulin and Hu protein, characteristic of neuroblasts and early neurons. These neuroblast-like cells were negative for glial fibrillary acidic protein (GFAP), 2;,3;-cyclic nucleotide 3;-phosphohydrolase (CNPase), and vimentin, suggesting that they were not differentiating as glia. Our results show that neuroblast-like cells exist in the human SEZ and support the theory that SEZ of postnatal human brain has neurogenic potential.
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Epéndimo/química , Receptores ErbB/análisis , Receptores ErbB/genética , Molécula L1 de Adhesión de Célula Nerviosa , Neuronas/química , 2',3'-Nucleótido Cíclico Fosfodiesterasas/análisis , Adolescente , Adulto , Especificidad de Anticuerpos , Biomarcadores , Movimiento Celular , Preescolar , Proteínas ELAV , Epéndimo/enzimología , Receptores ErbB/inmunología , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Etiquetado Corte-Fin in Situ , Lactante , Masculino , Proteínas del Tejido Nervioso/análisis , Moléculas de Adhesión de Célula Nerviosa/análisis , Neuroglía/química , Neuronas/citología , Neuronas/enzimología , ARN Mensajero/análisis , Proteínas de Unión al ARN/análisis , Ácidos Siálicos/análisis , Tubulina (Proteína)/análisisRESUMEN
Type 2 iodothyronine deiodinase, an enzyme involved in the conversion of thyroxin to the biologically active 3,5, 3'-triiodothyronine, is highly concentrated in a group of specialized ependymal cells, tanycytes, lining the wall and floor of the third ventricle. As this distribution is highly reminiscent of the distribution of cells containing the phosphatase inhibitor, DARPP-32, we raised the possibility that these two proteins may coexist in tanycytes and that DARPP-32 may modulate type 2 deiodinase activity by regulating the phosphorylation state of the cAMP regulatory factor, CREB. To address this question, double-labeling histochemical studies were performed for type 2 deiodinase mRNA and DARPP-32 immunoreactivity (IR), or DARPP-32- and CREB-IR in the same tissue sections. Type 2 deiodinase mRNA was found in the cell bodies of all DARPP-32-immunolabeled tanycytes. Both type 2 deiodinase mRNA and DARPP-32-IR also extended into tanycyte processes that ramified in the arcuate nucleus and median eminence, in close association with blood vessels and portal capillaries. In contrast, type 2 deiodinase mRNA was not present in the same cells that contained DARPP-32-IR in the pituitary gland. All tanycytes containing DARPP-32-IR also contained CREB-IR in their nucleus. Since type 2 deiodinase activity can be induced by substances that increase cAMP, we hypothesize that DARPP-32 may regulate the activity of type 2 deiodinase by prolonging the activation of CREB. Selectivity for the colocalization of these factors to tanycytes but not the pituitary gland, may explain the heterogeneous response of type 2 deiodinase activity in these two loci in response to specific stimuli such as fasting.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/análisis , Epéndimo/química , Epéndimo/enzimología , Yoduro Peroxidasa/genética , Fosfoproteínas/análisis , Animales , Fosfoproteína 32 Regulada por Dopamina y AMPc , Activación Enzimática/genética , Epéndimo/citología , Regulación Enzimológica de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Yoduro Peroxidasa/análisis , Masculino , Proteínas del Tejido Nervioso/análisis , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Radioisótopos de Azufre , Yodotironina Deyodinasa Tipo IIRESUMEN
The subependymal zone (SEZ) of the adult mammalian forebrain contains a population of progenitor cells that proliferate in response to brain injury. This study examined the effect of cortical injury on metabolic activity in the SEZ using quantitative histochemistry of cytochrome oxidase. The SEZ showed significantly enhanced cytochrome oxidase activity in rats with electrolytic cortical injuries relative to sham-operated controls, while other brain regions showed no such changes. The results indicate that the SEZ had increased oxidative energy demands, and thus provide metabolic evidence that SEZ cells are activated in response to brain injury.