RESUMEN
Glial fibrillary acidic protein (GFAP) is a promising biomarker for brain and spinal cord disorders. Recent studies have highlighted the differences in the reliability of GFAP measurements in different biological matrices. The reason for these discrepancies is poorly understood as our knowledge of the protein's 3-dimensional conformation, proteoforms, and aggregation remains limited. Here, we investigate the structural properties of GFAP under different conditions. For this, we characterized recombinant GFAP proteins from various suppliers and applied hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a snapshot of the conformational dynamics of GFAP in artificial cerebrospinal fluid (aCSF) compared to the phosphate buffer. Our findings indicate that recombinant GFAP exists in various conformational species. Furthermore, we show that GFAP dimers remained intact under denaturing conditions. HDX-MS experiments show an overall decrease in H-bonding and an increase in solvent accessibility of GFAP in aCSF compared to the phosphate buffer, with clear indications of mixed EX2 and EX1 kinetics. To understand possible structural interface regions and the evolutionary conservation profiles, we combined HDX-MS results with the predicted GFAP-dimer structure by AlphaFold-Multimer. We found that deprotected regions with high structural flexibility in aCSF overlap with predicted conserved dimeric 1B and 2B domain interfaces. Structural property predictions combined with the HDX data show an overall deprotection and signatures of aggregation in aCSF. We anticipate that the outcomes of this research will contribute to a deeper understanding of the structural flexibility of GFAP and ultimately shed light on its behavior in different biological matrices.
Asunto(s)
Medición de Intercambio de Deuterio , Proteína Ácida Fibrilar de la Glía , Fosfatos , Humanos , Medición de Intercambio de Deuterio/métodos , Proteína Ácida Fibrilar de la Glía/química , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/fisiología , Conformación Proteica , Reproducibilidad de los Resultados , Proteínas RecombinantesRESUMEN
In adult mammals, neural stem cells are localized in three neurogenic regions, the subventricular zone of the lateral ventricle (SVZ), the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the hypothalamus. In the SVZ and the SGZ, neural stem/progenitor cells (NSPCs) express the glial fibrillary acidic protein (GFAP) and selective depletion of these NSPCs drastically decreases cell proliferation in vitro and in vivo. In the hypothalamus, GFAP is expressed by α-tanycytes, which are specialized radial glia-like cells in the wall of the third ventricle also recognized as NSPCs. To explore the role of these hypothalamic GFAP-positive tanycytes, we used transgenic mice expressing herpes simplex virus thymidine kinase (HSV-Tk) under the control of the mouse Gfap promoter and a 4-week intracerebroventricular infusion of the antiviral agent ganciclovir (GCV) which kills dividing cells expressing Tk. While GCV significantly reduced the number and growth of hypothalamus-derived neurospheres from adult transgenic mice in vitro, it causes hypogonadotropic hypogonadism in vivo. The selective death of dividing tanycytes expressing GFAP indeed results in a marked decrease in testosterone levels and testicular weight, as well as vacuolization of the seminiferous tubules and loss of spermatogenesis. Additionally, GCV-treated GFAP-Tk mice show impaired sexual behavior, but no alteration in food intake or body weight. Our results also show that the selective depletion of GFAP-expressing tanycytes leads to a sharp decrease in the number of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons and a blunted LH secretion. Overall, our data show that GFAP-expressing tanycytes play a central role in the regulation of male reproductive function.
Asunto(s)
Células Ependimogliales , Proteína Ácida Fibrilar de la Glía , Hipogonadismo , Animales , Células Ependimogliales/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Ácida Fibrilar de la Glía/fisiología , Hipogonadismo/genética , Hipogonadismo/metabolismo , Masculino , Mamíferos/metabolismo , Ratones , Neurogénesis/fisiología , Neuronas/metabolismoRESUMEN
Intermediate filaments (also termed nanofilaments) are involved in many cellular functions and play important roles in cellular responses to stress. The upregulation of glial fibrillary acidic protein (GFAP) and vimentin (Vim), intermediate filament proteins of astrocytes, is the hallmark of astrocyte activation and reactive gliosis in response to injury, ischemia or neurodegeneration. Reactive gliosis is essential for the protective role of astrocytes at acute stages of neurotrauma or ischemic stroke. However, GFAP and Vim were also linked to neural plasticity and regenerative responses in healthy and injured brain. Mice deficient for GFAP and vimentin (GFAP-/-Vim-/-) exhibit increased post-traumatic synaptic plasticity and increased basal and post-traumatic hippocampal neurogenesis. Here we assessed the locomotor and exploratory behavior of GFAP-/-Vim-/- mice, their learning, memory and memory extinction, by using the open field, object recognition and Morris water maze tests, trace fear conditioning, and by recording reversal learning in IntelliCages. While the locomotion, exploratory behavior and learning of GFAP-/-Vim-/- mice, as assessed by object recognition, the Morris water maze, and trace fear conditioning tests, were comparable to wildtype mice, GFAP-/-Vim-/- mice showed more pronounced memory extinction when tested in IntelliCages, a finding compatible with the scenario of an increased rate of reorganization of the hippocampal circuitry.
Asunto(s)
Proteína Ácida Fibrilar de la Glía/fisiología , Aprendizaje/fisiología , Memoria/fisiología , Vimentina/fisiología , Animales , Proteína Ácida Fibrilar de la Glía/genética , Hipocampo/fisiología , Filamentos Intermedios/metabolismo , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Noqueados , Neurogénesis , Vimentina/genéticaRESUMEN
BACKGROUND: Mice with pilocarpine-induced temporal lobe epilepsy (TLE) are characterized by intense hippocampal neuroinflammation, a prominent pathological hallmark of TLE that is known to contribute to neuronal hyperexcitability. Recent studies indicate that Adam10, a member of a disintegrin and metalloproteinase domain-containing protein (Adam) family, has been involved in the neuroinflammation response. However, it remains unclear whether and how Adam10 modulates neuroinflammation responses in the context of an epileptic brain or whether Adam10 affects epileptogenesis via the neuroinflammation pathway. METHODS: Adult male C57BL/6J mice were subjected to intraperitoneal injection of pilocarpine to induce TLE. Adeno-associated viral (AAV) vectors carrying Adam10 (AAV-Adam10) or lentiviral vectors carrying short hairpin RNA, which is specific to the mouse Adam10 mRNA (shRNA-Adam10), were bilaterally injected into the hippocampus to induce overexpression or knockdown of Adam10, respectively. The specific anti-inflammatory agent minocycline was administered following status epilepticus (SE) to block hippocampal neuroinflammation. Continuous video EEG recording was performed to analyze epileptic behavior. Western blot, immunofluorescence staining, and ELISA were performed to determine Adam10 expression as well as hippocampal neuroinflammation. RESULTS: In this study, we demonstrate that overexpression of Adam10 in the hippocampus suppresses neuroinflammation and reduces seizure activity in TLE mice, whereas knockdown of Adam10 exacerbates hippocampal neuroinflammation and increases seizure activity. Furthermore, increased seizure activity in Adam10 knockdown TLE mice is dependent on hippocampal neuroinflammation. CONCLUSION: These results suggest that Adam10 suppresses epilepsy through repression of hippocampal neuroinflammation. Our findings provide new insights into the Adam10 regulation of development of epilepsy via the neuroinflammation pathway and identify a potential therapeutic target for epilepsy.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Encefalitis/etiología , Proteína Ácida Fibrilar de la Glía/fisiología , Hipocampo/metabolismo , Proteínas de la Membrana/metabolismo , Estado Epiléptico , Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Proteínas de Unión al Calcio/metabolismo , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Encefalitis/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/efectos de los fármacos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Agonistas Muscarínicos/toxicidad , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Pilocarpina/toxicidad , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Estado Epiléptico/inducido químicamente , Estado Epiléptico/complicaciones , Estado Epiléptico/patología , Estado Epiléptico/terapiaRESUMEN
Intermediate filament proteins are structural components of the cellular cytoskeleton with cell-type specific expression and function. Glial fibrillary acidic protein (GFAP) is a type III intermediate filament protein and is up-regulated in glia of the nervous system in response to injury and during neurodegenerative diseases. In the retina, GFAP levels are dramatically increased in Müller glia and are thought to play a role in the extensive structural changes resulting in Müller cell hypertrophy and glial scar formation. In spite of similar changes to the morphology of Xenopus Müller cells following injury, we found that Xenopus lack a gfap gene. Other type III intermediate filament proteins were, however, significantly induced following rod photoreceptor ablation and retinal ganglion cell axotomy. The recently available X. tropicalis and X. laevis genomes indicate a small deletion most likely resulted in the loss of the gfap gene during anuran evolution. Lastly, a survey of representative species from all three extant amphibian orders including the Anura (frogs, toads), Caudata (salamanders, newts), and Gymnophiona (caecilians) suggests that deletion of the gfap locus occurred in the ancestor of all Anura after its divergence from the Caudata ancestor around 290 million years ago. Our results demonstrate that extensive changes in Müller cell morphology following retinal injury do not require GFAP in Xenopus, and other type III intermediate filament proteins may be involved in the gliotic response.
Asunto(s)
Células Ependimogliales/patología , Gliosis/fisiopatología , Proteínas de Filamentos Intermediarios/fisiología , Retina/lesiones , Proteínas de Xenopus/fisiología , Xenopus laevis/fisiología , Animales , Animales Modificados Genéticamente , Anuros/genética , Axotomía , Evolución Biológica , Femenino , Eliminación de Gen , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/fisiología , Gliosis/patología , Humanos , Larva , Masculino , Metronidazol/toxicidad , Ratones , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Ganglionares de la Retina/patología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/patología , Especificidad de la Especie , Sintenía , Urodelos/genética , Vimentina/fisiología , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
Spinal cord injury is a devastating pathology with heavy social and personal sequels,. Therapeutic strategies are organized around three research directions: Neuroprotection, axonal regeneration and substitutive therapies. Of particular interest is axonal regeneration. Since it may be applicable to other spinal or brain pathologies. The condition for regeneration in the central nervous system is the control of tissular surrounding after the lesion, and essentially the constitution of a glial scar. A key factor in the glial scar is the massive synthesis of glial fibrous proteins GFAP and Vimentin. We have devised a transgenic mouse model in which the genes coding for these proteins have been inactivated. After a lateral hemisection of the spinal cord, transgenic mice recover within five weeks the function of the paralyzed hind limb and the absence of glial scar formation is correlated with the regeneration of descending serotonergic axons. The same results have been obtained in native mice injected locally with a lentiviral vector carrying a siRNA for GRAP. This tool, which can potentially be used in injured patients, will be tested on a primate model with a non-invasive follow-up with high resolution MRI.
Asunto(s)
Traumatismos de la Médula Espinal/terapia , Animales , Axones/fisiología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/fisiología , Humanos , Regeneración Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Serotonina/fisiología , Vimentina/genética , Vimentina/fisiologíaRESUMEN
The aim of this study was to examine the structural organization of processes of ependymocytes lining the lateral ventricles of the rat brain using vimentin immunocytochemistry and confocal laser microscopy. The study was performed on adult male rats (n = 3). It was found that most typical ependymocytes had basal processes, while 1/3 of these cells had none. Some vimentin-immunopositive tanycyte-like cells with long processes appoaching blood vessels, were found inside the ependymal lining In some typical ependymocytes, cytroskeleton wa s formed by intermediate filaments of mixed type containing both vimentin and glial fibrillary acidic protein.
Asunto(s)
Epéndimo/anatomía & histología , Proteína Ácida Fibrilar de la Glía/fisiología , Ventrículos Laterales/anatomía & histología , Morfogénesis , Animales , Astrocitos/citología , Epéndimo/citología , Inmunohistoquímica , Ventrículos Laterales/fisiología , Masculino , Microscopía Confocal , Neuroglía/citología , RatasRESUMEN
Aberrant sympathetic sprouting is seen in the uninjured trigeminal ganglia of transgenic mice that ectopically express nerve growth factor under the control of the glial fibrillary acidic protein promoter. These sympathetic axons form perineuronal plexuses around a subset of sensory somata in 2- to 3-month-old transgenic mice. Here, we show that aged transgenic mice (i.e., 11-14 and 16-18 months old) have dystrophic sympathetic plexuses (i.e., increased densities of swollen axons), and that satellite glial cells, specifically those in contact with dystrophic plexuses in the aged mice display strong immunostaining for tumor necrosis factor alpha. The colocalization of dystrophic plexuses and reactive satellite glial cells in the aged mice coincides with degenerative features in the enveloped sensory somata. Collectively, these novel results show that, with advancing age, sympathetic plexuses undergo dystrophic changes that heighten satellite glial cell reactivity and that together these cellular events coincide with neuronal degeneration.
Asunto(s)
Envejecimiento/genética , Envejecimiento/patología , Ganglios Simpáticos/patología , Regulación del Desarrollo de la Expresión Génica , Expresión Génica , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Ganglio del Trigémino/patología , Animales , Axones/patología , Proteína Ácida Fibrilar de la Glía/fisiología , Inmunohistoquímica , Ratones Transgénicos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Alexander disease (AxD) is a rare neurodegenerative disorder characterized pathologically by the presence of eosinophilic inclusions known as Rosenthal fibers (RFs) within astrocytes, and is caused by dominant mutations in the coding region of the gene encoding glial fibrillary acidic protein (GFAP). GFAP is the major astrocytic intermediate filament, and in AxD patient brain tissue GFAP is a major component of RFs. TAR DNA binding protein of 43 kDa (TDP-43) is the major pathological protein in almost all cases of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and â¼50% of frontotemporal lobar degeneration (FTLD), designated as FTLD-TDP. In ALS and FTLD-TDP, TDP-43 becomes insoluble, ubiquitinated, and pathologically phosphorylated and accumulates in cytoplasmic inclusions in both neurons and glia of affected brain and spinal cord regions. Previously, TDP-43 was detected in RFs of human pilocytic astrocytomas; however, involvement of TDP-43 in AxD has not been determined. Here we show that TDP-43 is present in RFs in AxD patient brains, and that insoluble phosphorylated full-length and high molecular weight TDP-43 accumulates in white matter of such brains. Phosphorylated TDP-43 also accumulates in the detergent-insoluble fraction from affected brain regions of Gfap(R236H/+) knock-in mice, which harbor a GFAP mutation homologous to one that causes AxD in humans, and TDP-43 colocalizes with astrocytic RF pathology in Gfap(R236H/+) mice and transgenic mice overexpressing human wild-type GFAP. These findings suggest common pathogenic mechanisms in ALS, FTLD, and AxD, and this is the first report of TDP-43 involvement in a neurological disorder primarily affecting astrocytes.
Asunto(s)
Enfermedad de Alzheimer/patología , Astrocitos/patología , Proteinopatías TDP-43/patología , Adolescente , Adulto , Anciano , Envejecimiento/fisiología , Animales , Western Blotting , Niño , Citoplasma/metabolismo , Proteínas de Unión al ADN , Femenino , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/fisiología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Lactante , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Fosforilación , Adulto JovenRESUMEN
One of the plastic base material, widely used in the plastics industry in various countries, is a ester phthalate. These compounds will be oxidizedin the body to 2-methoxyethanol (2-ME). Effect of 2-ME on human health and the environment depends on the number, duration and frequency of exposure. 2-ME and its metabolites in the body can damage cells and tissues. The body can be exposed by 2-ME through the air, water and soil. Western blot results showed that the protein Vimentin was detectable in the control group at GD-11 to 17, meanwhile GFAP protein was detachable in the control group atGD- 12 to GD-18. After administration 2-ME, the expression of Vimentinprotein were changed, and started at GD- 12 up to GD-18. whereas the expression of GFAP protein began at GD-11 up to GD-17. The Changes on timetable protein expression of Vimentin and GFAP affect corticogenesis disorder. The disorder caused by the existence of these proteins as a result of 2-Methoxyethanol. Disorder of corticogenesis process were sub-plate and cortical plate of the cerebral cortex of fetus brains of mice at GD-18. Generally, it can be concluded that changes inprotein expression of Vimentin and GFAP causedby 2-ME. The Vimentin more important during the period of fetal brain development. GFAP and Vimentin is a protein involved in response to damage caused by a teratogenic agent, so that cells in the cerebral cortex, has dedifferentiation.
Uno de los materiales a base de plástico, ampliamente utilizado en la industria en varios países, es un éster de ftalato. Estos compuestos se oxidan en el cuerpo a 2-metoxietanol (2-ME). El efecto del 2-ME en la salud humana y el medio ambiente depende de la cantidad, duración y frecuencia de exposición. El 2-ME y sus metabolitos en el cuerpo puede dañar las células y tejidos. El cuerpo puede ser expuesto al 2-ME a través del aire, agua y suelo. Los resultados de Western blot mostraron que la proteína vimentina fue detectable en el grupo de control en GD-11 a 17, por su parte proteína GFAP fue detectable en el grupo de control en GD-12 a GD-18. Después de la administración de 2-ME, la expresión de la proteína vimentina cambió, y comenzó a detectarse en GD-12 hasta GD-18, mientras que la expresión de la proteína GFAP se inició en GD-11 hasta GD-17. Los cambios en el momento de expresión de las proteínas vimentina y GFAP afectan produciendo trastornos de la corticogénesis. El trastorno causado por la existencia de estas proteínas como resultado de 2-metoxietanol a nivel del proceso corticogénesis fue en la subplaca y la placa cortical de la corteza cerebral del cerebro de fetos de ratones en GD-18. En general, se puede concluir que existen cambios en la expresión de las proteínas vimentina y GFAP causados por el 2-ME. La vimentina es muy importante durante el período de desarrollo del cerebro fetal. GFAP y vimentina son proteínas implicadas en la respuesta a los daños causados por un agente teratogénico, de modo que las células en la corteza cerebral presentan desdiferenciación.
Asunto(s)
Animales , Ratones , Corteza Cerebral , Glicol de Etileno/toxicidad , Proteína Ácida Fibrilar de la Glía , Vimentina , Western Blotting , Corteza Cerebral/crecimiento & desarrollo , Proteína Ácida Fibrilar de la Glía/fisiología , Teratógenos , Vimentina/fisiologíaRESUMEN
Axons and glial cells are the main components of white matter. The corpus callosum (CC) is the largest white matter tract in mammals; in rodents, 99% of the cells correspond to glia after postnatal day 5 (P5). The area of the CC varies through life and regional differences related to the number of axons have been previously described. Whether glial cell density varies accordingly is unknown; thus the aim of this study was to estimate glial cell density for the genu, body and splenium -the three main regions of CC-, of P6 and P30 rats. Here we report that the density of CC glial cells reduced by ~10% from P6 to P30. Even so, the density of astrocytes showed a slight increase (+6%), probably due to differentiation of glioblasts. Interestingly, glial cell density decreased for the genu (-21%) and the body (-13%), while for the splenium a minor increase (+5%) was observed. The astrocyte/glia ratio increased (from P6 to P30) for the genu (+27%), body (+17%) and splenium (+4%). Together, our results showed regional differences in glial cell density of the CC. Whether this pattern is modified in some neuropathologies remains to be explored.
Asunto(s)
Cuerpo Calloso/citología , Proteína Ácida Fibrilar de la Glía/fisiología , Neuroglía/citología , Factores de Edad , Animales , Astrocitos/citología , Recuento de Células , Diferenciación Celular/fisiología , Cuerpo Calloso/crecimiento & desarrollo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Morfogénesis , RatasRESUMEN
Neuromyelitis optica (NMO) is an autoimmune disease targeting aquaporin 4 (AQP4), localized mainly at the astrocytic foot processes. Loss of AQP4 and glial fibrillary acidic protein (GFAP) was reported, but the pathological significance of astrocytopathy is still controversial. Here we show that active lesions in NMO display a wide spectrum of pathology even within a single tissue block of an individual patient. We have distinguished six different lesion types. The first reflects complement deposition at the surface of astrocytes, associated with granulocyte infiltration and astrocyte necrosis and followed by demyelination, global tissue destruction and the formation of cystic, necrotic lesions (lesion type 2). Such destructive lesions lead to Wallerian degeneration in lesion-related tracts (lesion type 3). Around active NMO lesions AQP4 may selectively be lost in the absence of aquaporin 1 (AQP1) loss or other structural damage (lesion type 4). Another pattern is characterized by clasmatodendrosis of astrocytes, defined by cytoplasmic swelling and vacuolation, beading and dissolution of their processes and nuclear alterations resembling apoptosis, which was associated with internalization of AQP4 and AQP1 and astrocyte apoptosis in the absence of complement activation. Such lesions give rise to extensive astrocyte loss, which may occur in part in the absence of any other tissue injury, such as demyelination or axonal degeneration (lesion type 5). Finally, lesions with a variable degree of astrocyte clasmatodendrosis are found, which show plaque-like primary demyelination that is associated with oligodendrocyte apoptosis, but with preservation of axons (lesion type 6). In active multiple sclerosis (MS) lesions astrocytes reveal changes of reactive protoplasmatic or fibrillary gliosis. Only in a subset of lesions, in patients with aggressive disease, loss of AQP4 is observed in the initial stage of their formation, which is associated with retraction of astrocyte processes in the absence of complement deposition, granulocyte infiltration or loss of AQP1 or astrocytes. Our data underline the primary assault of astrocytes in NMO lesions, but also indicate that different mechanisms of tissue injury operate in parallel in the same patient and even within the same lesion.
Asunto(s)
Encéfalo/patología , Neuromielitis Óptica/metabolismo , Neuromielitis Óptica/patología , Médula Espinal/patología , Adulto , Anciano , Anciano de 80 o más Años , Acuaporina 1/fisiología , Acuaporina 4/fisiología , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Estudios de Cohortes , Femenino , Proteína Ácida Fibrilar de la Glía/fisiología , Humanos , Masculino , Persona de Mediana Edad , Neuromielitis Óptica/etiología , Médula Espinal/metabolismo , Adulto JovenRESUMEN
INTRODUCTION: We have recently reported that the signal of pulp injury induces both neuronal and glial cell activation in the contralateral thalamus in rats, although the mechanisms of the glial cell/neuronal interaction remain unclear. This study was undertaken to test our hypothesis that p38 mitogen-activated protein kinase (MAPK) signaling pathways are involved in the pulp injury-induced glial cell/neuronal interaction in the thalamus. METHODS: A local anesthetic (lidocaine with epinephrine) or saline (control) was injected into the tissue surrounding the left mandibular first molar of Wistar rats. The tooth was then pulp-exposed, and the cavity was sealed with flowable composite. After 0 (normal pulp with local anesthetic or saline pretreatment), 24, and 72 hours, the contralateral side of thalamus was retrieved and subjected to immunohistochemistry for phospho-p38 MAPK and glial fibrillary acidic protein and real-time polymerase chain reaction analysis of p38-MAPK family (MAPK 13 and MAPK 14) mRNAs. RESULTS: The area immunopositive to phospho-p38 MAPK increased until 72 hours after pulp exposure in both local anesthetic-pretreated and saline-pretreated animals, but the rate of increase was lower in the local anesthetic-pretreated animals. The density of glial fibrillary acidic protein-expressing astrocytes showed a significant increase only in the saline-pretreated animals. Expression levels of MAPK 13 and MAPK 14 mRNAs increased at 24 hours and still higher at 72 hours in the saline-pretreated animals. Notably, MAPK 13 and MAPK 14 mRNA levels at 24 and 72 hours in the local anesthetic-pretreated animals showed significantly lower levels than those in the saline-pretreated animals. CONCLUSIONS: It was concluded that pulp injury-induced up-regulation of MAPK 13, MAPK 14, and phospho-p38 MAPK in the thalamus was suppressed by the local anesthetic pretreatment, suggesting the involvement of p38 MAPK signaling pathways in the glial cell-neuronal interaction induced by pulpal nociception.
Asunto(s)
Astrocitos/fisiología , Exposición de la Pulpa Dental/enzimología , Sistema de Señalización de MAP Quinasas/fisiología , Neuronas/fisiología , Nocicepción/fisiología , Tálamo/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Anestésicos Locales/farmacología , Animales , Comunicación Celular , Proteína Ácida Fibrilar de la Glía/fisiología , Sistema de Señalización de MAP Quinasas/genética , Masculino , Nocicepción/efectos de los fármacos , Ratas , Ratas Wistar , Tálamo/citología , Regulación hacia ArribaRESUMEN
The present paper reviews astrocyte pathology in major depressive disorder (MDD) and proposes that reductions in astrocytes and related markers are key features in the pathology of MDD. Astrocytes are the most numerous and versatile of all types of glial cells. They are crucial to the neuronal microenvironment by regulating glucose metabolism, neurotransmitter uptake (particularly for glutamate), synaptic development and maturation and the blood brain barrier. Pathology of astrocytes has been consistently noted in MDD as well as in rodent models of depressive-like behavior. This review summarizes evidence from human postmortem tissue showing alterations in the expression of protein and mRNA for astrocyte markers such as glial fibrillary acidic protein (GFAP), gap junction proteins (connexin 40 and 43), the water channel aquaporin-4 (AQP4), a calcium-binding protein S100B and glutamatergic markers including the excitatory amino acid transporters 1 and 2 (EAAT1, EAAT2) and glutamine synthetase. Moreover, preclinical studies are presented that demonstrate the involvement of GFAP and astrocytes in animal models of stress and depressive-like behavior and the influence of different classes of antidepressant medications on astrocytes. In light of the various astrocyte deficits noted in MDD, astrocytes may be novel targets for the action of antidepressant medications. Possible functional consequences of altered expression of astrocytic markers in MDD are also discussed. Finally, the unique pattern of cell pathology in MDD, characterized by prominent reductions in the density of astrocytes and in the expression of their markers without obvious neuronal loss, is contrasted with that found in other neuropsychiatric and neurodegenerative disorders.
Asunto(s)
Astrocitos/patología , Encéfalo/patología , Trastorno Depresivo Mayor/patología , Acuaporina 4/fisiología , Astrocitos/fisiología , Biomarcadores/análisis , Encéfalo/metabolismo , Conexinas/fisiología , Trastorno Depresivo Mayor/etiología , Proteína Ácida Fibrilar de la Glía/fisiología , Humanos , Neurotransmisores/fisiologíaRESUMEN
Axons and glial cells are the main components of white matter. The corpus callosum (CC) is the largest white matter tract in mammals; in rodents, 99% of the cells correspond to glia after postnatal day 5 (P5). The area of the CC varies through life and regional differences related to the number of axons have been previously described. Whether glial cell density varies accordingly is unknown; thus the aim of this study was to estimate glial cell density for the genu, body and splenium -the three main regions of CC-, of P6 and P30 rats. Here we report that the density of CC glial cells reduced by ~10% from P6 to P30. Even so, the density of astrocytes showed a slight increase (+6%), probably due to differentiation of glioblasts. Interestingly, glial cell density decreased for the genu (-21%) and the body (-13%), while for the splenium a minor increase (+5%) was observed. The astrocyte/glia ratio increased (from P6 to P30) for the genu (+27%), body (+17%) and splenium (+4%). Together, our results showed regional differences in glial cell density of the CC. Whether this pattern is modified in some neuropathologies remains to be explored.
Asunto(s)
Animales , Femenino , Ratas , Cuerpo Calloso/citología , Proteína Ácida Fibrilar de la Glía/fisiología , Neuroglía/citología , Factores de Edad , Astrocitos/citología , Recuento de Células , Diferenciación Celular/fisiología , Cuerpo Calloso/crecimiento & desarrollo , Técnica del Anticuerpo Fluorescente Indirecta , MorfogénesisAsunto(s)
Enfermedad de Alexander/patología , Edad de Inicio , Enfermedad de Alexander/genética , Enfermedad de Alexander/terapia , Animales , Astrocitos/patología , Encéfalo/patología , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/fisiología , Proteína Ácida Fibrilar de la Glía/toxicidad , Humanos , Modelos Neurológicos , Vías Nerviosas/patologíaRESUMEN
We previously showed that human intravenous immunoglobulin (IVIG) can lower seizure severity and prolong seizure latency in picrotoxin-kindled rats. The aim of this study was to further characterize the effects of IVIG on seizure activity and investigate its influence on astrocytes in the hippocampus of picrotoxin-kindled rats. A rat kindling model was established by peritoneal injections of picrotoxin for 21 days in Wistar rats. Seventy-five rats were equally divided into five groups: picrotoxin, IVIG pretreatment, IVIG post-treatment, normal saline control, and IVIG control. Seizure severity was evaluated according to a six-stage classification. The number and morphology of glial fibrillary acidic protein (GFAP)-positive astrocytes were studied by immunohistochemistry using the anti-GFAP antibody. The cross-sectional area and grayscale of GFAP-positive astrocytes were also determined. In picrotoxin-kindled rats, pretreatment with IVIG appeared to inhibit full kindling rates, and it significantly reduced the number of GFAP-positive cells in the hippocampus (p < .001). IVIG also significantly (p < .001) attenuated the increase in the cross-sectional area and grayscale of GFAP-positive astrocytes in the hippocampus. Our results suggest that by suppressing the expression of GFAP, IVIGs may reduce seizure activity and inhibit the activation of GFAP-positive astrocytes in picrotoxin-kindled rats.
Asunto(s)
Astrocitos/efectos de los fármacos , Convulsivantes , Proteína Ácida Fibrilar de la Glía/fisiología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Inmunoglobulinas Intravenosas/uso terapéutico , Excitación Neurológica/efectos de los fármacos , Picrotoxina , Convulsiones/tratamiento farmacológico , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/efectos de los fármacos , Región CA2 Hipocampal/citología , Región CA2 Hipocampal/efectos de los fármacos , Giro Dentado/citología , Giro Dentado/efectos de los fármacos , Humanos , Masculino , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Convulsiones/fisiopatologíaRESUMEN
Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in preparing APC candidates from developing mouse cerebellum, characterized them in vitro, and found that BMPs are survival factors for these cells.
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Astrocitos/metabolismo , Diferenciación Celular/fisiología , Cerebelo/crecimiento & desarrollo , Receptores de Hialuranos/biosíntesis , Células-Madre Neurales/metabolismo , Animales , Animales Recién Nacidos , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/metabolismo , Astrocitos/citología , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 4/fisiología , Supervivencia Celular/fisiología , Cerebelo/citología , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/fisiología , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/citología , Cultivo Primario de Células/métodosRESUMEN
The directed generation of pure astrocyte cultures from pluripotent stem cells has proven difficult. Generation of defined pluripotent-stem-cell derived astrocytes would allow new approaches to the investigation of plasticity and heterogeneity of astrocytes. We here describe a two-step differentiation scheme resulting in the generation of murine embryonic stem cell (mESC) derived astrocytes (MEDA), as characterized by the upregulation of 19 astrocyte-associated mRNAs, and positive staining of most cells for GFAP (glial fibrillary acidic protein), aquaporin-4 or glutamine synthetase. The MEDA cultures could be cryopreserved, and they neither contained neuronal, nor microglial cells. They also did not react to the microglial stimulus lipopolysaccharide, while inflammatory activation by a complete cytokine mix (CCM) or its individual components (TNF-α, IL1-ß, IFN-γ) was readily observed. MEDA, stimulated by CCM, became susceptible to CD95 ligand-induced apoptosis and produced NO and IL-6. This was preceded by NF-kB activation, and up-regulation of relevant mRNAs. Also GFAP-negative astrocytes were fully inflammation-competent. Neurotrophic support by MEDA was found to be independent of GFAP expression. In summary, we described here the generation and functional characterization of microglia-free murine astrocytes, displaying phenotypic heterogeneity as is commonly observed in brain astrocytes.
Asunto(s)
Astrocitos/patología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/genética , Células Madre Embrionarias/patología , Proteína Ácida Fibrilar de la Glía/fisiología , Inflamación/patología , Factores de Crecimiento Nervioso/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Línea Celular , Linaje de la Célula/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteína Ácida Fibrilar de la Glía/deficiencia , Inflamación/metabolismo , Ratones , Ratones Endogámicos BALB C , Fenotipo , Cultivo Primario de CélulasRESUMEN
BACKGROUNDS: Glial fibrillary acidic protein (GFAP) is a monomeric intermediate filament protein found in the astroglial cytoskeleton and is not found outside the central nervous system. It is a brain-specific protein that is released after traumatic brain injury (TBI). METHODS: This prospective study enrolled 59 children who had TBI, as verified by computed tomography. Daily GFAP measurement began at admission (<12 hours after trauma) and continued for 6 days. Blood samples were analyzed for GFAP by enzyme-linked immunosorbent assay. Outcome was assessed using the Glasgow Outcome Scale (GOS) at 6 months after injury. RESULTS: The median serum levels of GFAP at admission were 7.47 ng/mL in patients who died, compared with 0.12 ng/mL in patients who survived (p=0.002). GFAP levels were significantly higher in patients who had a poor outcome 6 months after injury than in those who were alive or had good outcome (p<0.001). The area under the receiver operating characteristic curve for GFAP was 0.833 for day 0 and 0.884 for day 2. CONCLUSIONS: These results suggest that determination of serum levels of GFAP may add to the clinical assessment of the primary damage and prediction of outcome after severe TBI.