Glial Intermediate Filament Protein Research Articles

Effect of Prenatal Topiramate Administration on Early Postnatal Expression of GFAP in Radial Glial Cells in Albino Rat Cerebellum and Spinal Cord

Background: Topiramate is known as a modern drug approved for the treatment of patients with partial seizures. Radial glial cells are bipolar-shaped cells in the developing central nervous system of vertebrates. It can differentiate into other neurons and astrocytes. GFAP, Glial fibrillary acidic protein, is a glial intermediate filament protein that is known as the most comprehensive marker for astroglial cells including the radial glia Aim of the work: There are few reports on topiramate administration during pregnancy. Therefore, this drug was chosen to investigate its effects, if any, especially the expression of GFAP in radial glia in the pups’ cerebellum and spinal cord. Material and Methods: Male and female rats were coupled in one cage for mating. On the next morning, mating was approved by the presence of sperms in the female vaginal swab, it was considered as Gestation Day zero:0. Twelve female pregnant rats were divided equally into two groups; control and topiramate treated. Topiramate drug was given to the treated group via orogastric tube from GD zero (0) till the time of delivery in a dose of 100mg/kg the control rats were administered tap water by orogastric tube. At the end of the experiment, the newly born pups (PN1) were weighed, anesthetized with Ketamine (60 mg/kg i.p.). The cerebellum and spinal cord were dissected and weighted. The specimens were fixed in Bouin’s solution then processed for preparation of H& E and GFAP immunostained sections. Results:In H& E-stained sections, the cerebellum in the control group was formed of subsequent layers, purkinje cells were arranged in one row, rounded or fusiform shape. GFAP positive Immunostained radial fibers (Bregman glia) were detected in granular and purkinje cell layers. The treated group showed that cells were disorganized and degenerated with focal areas of neuronal loss. Weak GFAP reaction was observed in Bregman glia in granular, purkinje and white matter cell layers. The lumbosacral segment of the spinal cord ventral horn showed large multipolar motor neurons with vesicular nucleus (“owl-eye” appearance), dendrites and axons were obvious. Glial cells with deeply stained nuclei were noticed. In the treated group, neurons appeared degenerated, vacuolated cytoplasm and pyknotic nuclei with focal areas of cellular loss. GFAP immunostained section in the control group showed an intense reaction in the radial fibers of the glia of the ventral funiculus, in contrast to the treated group which showed a faint reaction in both ventral funiculus and horn. Conclusion: it could be concluded that daily regular administration of topiramate during a childbearing period in females may result in a neurotoxic effect in the cerebellum and spinal cord. So, the administration of this drug should be under strict medical observation.

MeCP2 Deficiency in Neuroglia: New Progress in the Pathogenesis of Rett Syndrome.

Rett syndrome (RTT) is an X-linked neurodevelopmental disease predominantly caused by mutations of the methyl-CpG-binding protein 2 (MeCP2) gene. Generally, RTT has been attributed to neuron-centric dysfunction. However, increasing evidence has shown that glial abnormalities are also involved in the pathogenesis of RTT. Mice that are MeCP2-null specifically in glial cells showed similar behavioral and/or neuronal abnormalities as those found in MeCP2-null mice, a mouse model of RTT. MeCP2 deficiency in astrocytes impacts the expression of glial intermediate filament proteins such as fibrillary acidic protein (GFAP) and S100 and induces neuron toxicity by disturbing glutamate metabolism or enhancing microtubule instability. MeCP2 deficiency in oligodendrocytes (OLs) results in down-regulation of myelin gene expression and impacts myelination. While MeCP2-deficient microglia cells fail in response to environmental stimuli, release excessive glutamate, and aggravate impairment of the neuronal circuit. In this review, we mainly focus on the progress in determining the role of MeCP2 in glial cells involved in RTT, which may provide further insight into a therapeutic intervention for RTT.

Signaling mechanisms and disrupted cytoskeleton in the diphenyl ditelluride neurotoxicity.

Evidence from our group supports that diphenyl ditelluride (PhTe)2 neurotoxicity depends on modulation of signaling pathways initiated at the plasma membrane. The (PhTe)2-evoked signal is transduced downstream of voltage-dependent Ca2+ channels (VDCC), N-methyl-D-aspartate receptors (NMDA), or metabotropic glutamate receptors activation via different kinase pathways (protein kinase A, phospholipase C/protein kinase C, mitogen-activated protein kinases (MAPKs), and Akt signaling pathway). Among the most relevant cues of misregulated signaling mechanisms evoked by (PhTe)2 is the cytoskeleton of neural cells. The in vivo and in vitro exposure to (PhTe)2 induce hyperphosphorylation/hypophosphorylation of neuronal and glial intermediate filament (IF) proteins (neurofilaments and glial fibrillary acidic protein, resp.) in different brain structures of young rats. Phosphorylation of IFs at specific sites modulates their association/disassociation and interferes with important physiological roles, such as axonal transport. Disrupted cytoskeleton is a crucial marker of neurodegeneration and is associated with reactive astrogliosis and apoptotic cell death. This review focuses the current knowledge and important results on the mechanisms of (PhTe)2 neurotoxicity with special emphasis on the cytoskeletal proteins and their differential regulation by kinases/phosphatases and Ca2+-mediated mechanisms in developmental rat brain. We propose that the disrupted cytoskeletal homeostasis could support brain damage provoked by this neurotoxicant.

Thrombospondin‐1 and L‐selectin are associated with silent cerebral infarct in children with sickle cell anaemia

Thrombospondin‐1 and <scp>L</scp>‐selectin are associated with silent cerebral infarct in children with sickle cell anaemia

Ciliary neurotrophic factor induces glial fibrillary acidic protein in retinal Müller cells through the JAK/STAT signal transduction pathway

Purpose. Intravitreal injection of ciliary neurotrophic factor (CNTF) is known to induce glial intermediate filament protein (GFAP) expression in retinal Müller cells. Because CNTF binding can activate multiple signaling kinases, we have examined the involvement of JAK/STAT pathway in GFAP induction in Müller cells. Methods. CNTF was injected intravitreally into mouse eyes. Immunocytochemistry and immunoblotting were used to study GFAP and STAT3-p (phosphorylated STAT3) levels either in mouse eyes, retinal explant cultures or in a Müller cell line, rMC-1. Results. In protein extracts of CNTF-injected eyes, retinal explants and the Müller cells, there was a substantial increase in STAT3-p level. Immunocytochemistry showed that STAT3-p was now present in many cell bodies in the INL and the GCL. To prove that CNTF acted via the JAK-STAT pathway, rMC-1 cells were transfected with a dominant-negative STAT3 mutant prior to treatment with CNTF. In the immunoblots of transfected cells, there was decrease in GFAP level. Conclusions. The results establish that CNTF can induce GFAP expression in retinal Müller cells through the JAK/STAT signaling pathway.

Intermediate filament proteins define different glial subpopulations.

In certain species, specialized glial cells delineate cell domains in the central nervous system and assist in the elongation of axons by providing mechanical and chemical barriers. We showed previously, that the glial intermediate filament proteins vimentin and glial fibrillary acidic protein are extensively coexpressed in radial glia in the developing hindbrain, and that subsequently, the two proteins define distinct rhombomere domains: vimentin is localized in radial glia at the rhombomere boundaries and glial fibrillary acidic protein expression is restricted to the rhombomere centers (Yoshida and Colman [2000] J. Comp. Neurol. 424:47-57). The present study reveals that vimentin and glial fibrillary acidic protein continue to display distinct expression domains throughout the developing Xenopus central nervous system. Although the precise function of the two intermediate filaments in glial cells has yet to be revealed, the observations presented here suggests that glial intermediate filament proteins demarcate different populations of glial cells during nervous system development and that the existence of different glial populations may define glial boundaries.

Characterization of the chicken transitin gene reveals a strong relationship to the nestin intermediate filament class.

Our laboratory previously reported that transitin is a radial glial intermediate filament protein sharing the basic structural features common to all intermediate filament (IF) proteins. It contains an alpha-helical core domain flanked by a short nonhelical head and a long COOH-terminal tail. The core sequence of transitin shows the greatest similarity to Xenopus tanabin and to rat and human nestin. We also reported that transitin has multiple splice variants derived from the deletion or inclusion of a leucine-zipper heptad repeat domain in the COOH-terminal tail. In the present study, we provide new evidence to support the classification of nestin and transitin in the same group of IF proteins based on the number and position of its introns. In addition, we suggest that the different isoforms of transitin are produced by a splicing mechanism that recognizes consensus 5' and 3' splice sites contained within the coding sequence of the leucine-zipper heptad repeat domain.

Immunocytochemical localization of a novel radial glial intermediate filament protein

We have examined by immunocytochemistry the subcellular localization of a chick radial glial protein, named transitin, that by molecular cloning has been shown to be a novel member of the intermediate filament protein superfamily. In astrocytes cultured from E10 chick brain, transitin is localized to the intermediate filament network in accordance with its structural properties. Using confocal microscopy we examined the expression of transitin, vimentin and glial fibrillary acidic protein (GFAP) in cultured astrocytes, and show that transitin co-distributes with these other glial intermediate filament proteins. The expression of transitin, vimentin and GFAP was also compared in embryonic chick spinal cord and brain radial glia, with these studies showing that these intermediate filament proteins display distinct expression patterns during CNS development. Of particular note is the absence of vimentin and GFAP in spinal cord midline radial glia that express transitin protein, and a transient expression of transitin in brain midline radial glia that continue to express vimentin. Our studies presented here therefore indicate that transitin, a novel radial glial intermediate filament protein, may have functions that are unrelated to GFAP or vimentin during CNS development, since transitin is localized to the processes of midline radial glia and is transiently expressed during chick CNS development.

Transient induction of the glial intermediate filament protein gene in Müller cells in the mouse retina.

The glial intermediate filament protein (GFAP) gene is not normally expressed by retinal Müller cells but it is transcriptionally activated following photoreceptor degeneration. In the present study, we have examined the relationship between progressive photoreceptor loss and changes in GFAP gene activity in Müller cells. In albino mice with light-induced photoreceptor degeneration, GFAP level was strongly elevated after 2 weeks. GFAP level remained high even after 3 months in light. In situ hybridization studies showed that GFAP transcripts were quite sparse in the first week but increased dramatically after 2 weeks of light exposure. After 4 weeks in constant light, however, little GFAP mRNA was detected in Müller cells. RNA blotting also showed that there was an approximately 20-fold increase in GFAP mRNA content at 2 weeks; but at 4 weeks, the RNA content fell to about four-fold higher than the basal level. These results show that GFAP level remains high long after its synthesis, probably as a consequence of low GFAP turnover in the Müller cell cytoskeleton, while GFAP mRNA level rises and declines rapidly due to transient activation of the GFAP gene in Müller cells.

Studies of childhood brain tumors using immunohistochemistry and microwave technology: methodological considerations

The immunohistochemical detection of antigens in archival tissue sections has been hampered by the poor reactivity of certain polypeptides in conventional formalin-fixed, paraffin-embedded material. For example, the poor reactivity of neurofilament proteins (NFPs) in surgical and autopsy specimens has been a major drawback of previous large, retrospective, clinicopathologic studies of pediatric primitive neuroectodermal tumors (PNETs), also known as medulloblastomas. We report our experience with a method of antigen retrieval which greatly enhanced the immunohistochemical detection of neuronal and glial intermediate filament proteins, retinal S-antigen (RSA), and the proliferating cell nuclear antigen (PCNA) in archival, paraffin-embedded, formalin or Bouin's-fixed, pediatric brain tumors, particularly PNETs. The technique involves adding a single brief step to an established avidin-biotin complex (ABC) immunohistochemical protocol (Vectastain Elite Kit). This step involves boiling tissue sections in distilled water for 5 min in a microwave oven. The specificity of staining was consistent with known cell and tissue specificities of the well-characterized monoclonal antibodies used and there was minimal background. Synaptophysin (SYN) staining was unaffected by heating and immunoreactivity of the low affinity nerve growth factor (p75NGFR) and the neural cell adhesion molecule (NCAM) were lost. The enhanced detection of neuronal and glial antigens in routinely prepared, formalin-fixed, archival material should facilitate large retrospective clinicopathologic studies designed to assess the prognostic implications of differentiation in PNETs and to better understand the biology of these tumors.

Expression of neural and glial intermediate filament proteins after rat brain injury

Expression of neural and glial intermediate filament proteins after rat brain injury

Transient ischemia stimulates glial fibrillary acid protein and vimentin gene expression in the gerbil neocortex, striatum and hippocampus

Astrocytic activation plays a major role in homeostatic maintenance of the central nervous system in response to neuronal damage. To assess the reactivity of astrocytes in transient cerebral ischemia of the gerbil, we studied the levels of glial fibrillary acidic protein (GFAP) and its mRNA. GFAP mRNA increased by 4 h after carotid artery occlusion, reached peak levels by 72 h with a 12-fold increase over control and then started declining as early as 96 h postischemia. An examination of the specific regions of the brain revealed an increase in GFAP mRNA associated with the forebrain, midbrain, hippocampus and striatum. GFAP mRNA in the non-ischemic cerebellum however, remained expressed at constitutively low levels. Immunoblot analysis with anti-GFAP antibodies demonstrated a 2- to 3-fold increase in the protein after 24 and 48 h of reperfusion. Pretreatment with pentobarbital and 1-(5′-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285), the drugs that have been shown to protect against ischemic damage, prevented the increase in GFAP mRNA in the cortex following ischemic injury. Forebrain ischemia also induced vimentin mRNA and protein quantities by 12 h of reperfusion in the cortex. The levels of c- fos and preproenkephalin mRNA increased rapidly within 1 h after ischemic injury, demonstrating a temporal difference in mRNA changes following ischemia. These results indicate that an increase in GFAP and vimentin, the two glial intermediate filament proteins in the area of the ischemic lesion may be associated with a glial response to injury.

Subcellular Localization of an Intermediate Filament Protein and Its mRNA in Glial Cells

Eucaryotic mRNAs are generally localized in the cell body, where most protein synthesis occurs. We have found that mRNAs encoding the glial intermediate filament protein are spatially distributed in the glial cell cytoplasm close to the location of the glial filaments. Whereas the glial filament protein mRNA was located predominantly in the distal process, actin mRNA was found almost exclusively in the apical portion of the glial cell. This pattern of mRNA localization might provide a mechanism for synthesis of proteins in specific subcellular compartments by mRNA translation locally.

Subcellular localization of an intermediate filament protein and its mRNA in glial cells.

Eucaryotic mRNAs are generally localized in the cell body, where most protein synthesis occurs. We have found that mRNAs encoding the glial intermediate filament protein are spatially distributed in the glial cell cytoplasm close to the location of the glial filaments. Whereas the glial filament protein mRNA was located predominantly in the distal process, actin mRNA was found almost exclusively in the apical portion of the glial cell. This pattern of mRNA localization might provide a mechanism for synthesis of proteins in specific subcellular compartments by mRNA translation locally.

Transcriptional activation of an intermediate filament protein gene in mice with retinal dystrophy.

We are interested in understanding neuronal interactions that regulate expression of specific genes in glial cells in the nervous system. In the normal mouse retina, the glial intermediate filament protein (GFAP) is not detectable in Müller cells, the predominant glial cells in the retina. Photoreceptor degeneration resulting from retinal degeneration (rd) mutation or environmental light damage, however, leads to the appearance of GFAP in Müller cells. We have investigated the mechanism underlying GFAP accumulation in these retinas. Western blotting analysis, steady-state mRNA level comparisons, and nuclear run-on assays show that transcription of the GFAP gene is activated in these retinas. In situ hybridizations with retinal sections and solitary Müller cells establish that GFAP mRNA levels are elevated in Müller cells. These results show that disruption of neuron-glia interactions resulting from photoreceptor degeneration leads to activation of the GFAP gene in glial cells of mice with retinal dystrophy. The functional significance of this glial response and the need for GFAP expression remain to be understood.

A type II keratin is expressed in glial cells of the goldfish visual pathway

The predominant intermediate filament proteins of the goldfish visual pathway consist of neuronal and nonneuronal isoelectric variants (58 kd). We have isolated a cDNA clone for the glial intermediate filament protein (ON 3) from an optic nerve expression library. The predicted amino acid sequence of this clone reveals that it codes for a type II keratin representing the goldfish equivalent of mammalian keratin K8. K8 has been shown to be associated with embryogenesis and development. Unlike the mammalian visual system, the goldfish visual pathway displays a remarkable capacity for functional regeneration. The expression of K8, a protein not usually expressed in glial cells but shown to be associated with development, in the goldfish optic nerve may be involved with the processes of growth and regeneration in the goldfish visual pathway.

An immunohistochemical quantification of fibrous astrocytes in the aging human cerebral cortex

In order to determine whether cortical fibrous astrocytes increase with age, we studied 25 patients ranging in age from 24 to 100 years with no clinical or pathological evidence of dementia or other cerebral disorder. Paraffin sections of mid-frontal cortex were obtained and stained with the avidin-biotin immunolabeling procedure for glial intermediate filament protein. The resulting immunolabeled fibrous astrocytes were then counted in the molecular and cellular (cortical laminae 2–6) layers. Populations of fibrous astrocytes in both layers varied widely among individuals, and in the molecular layer their numbers were not significantly correlated with advancing age. In the cellular layer, however, despite widely ranging cell counts among individuals within the same decades of life, there was a significant linear increase with age. Our data suggest that the increase occurs or accelerates significantly after age 70, but the case numbers preclude reaching such a conclusion with statistical confidence. However, when the patients are divided into those less than 70 and those older, fibrous astrocytes in the cellular layer are shown to be significantly increased in the latter group compared to the former.

Assembly of glial intermediate filament protein is initiated in the centriolar region

Assembly of glial intermediate filament protein (GFP) into intermediate filaments (IF) was first detected by immunofluorescence in the perinuclear region of astrocytes differentiating in colony cultures before the rest of the cytoplasm was labeled. Double labeling with antisera specific for centrioles indicated that this site corresponds to the centriolar region. These studies suggest that the centriolar region plays an important role in the assembly of some types of IF as well as microtubules.