Rat Glial Fibrillary Acidic Protein Research Articles

The influence of human umbilical cord mesenchymal stem cells transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression of glial fibrillary acidic protein

Objective To observe the influence of human umbilical cord mesenchymal stem cells(hUCMSC) transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression level of glial fibrillary acidic protein (GFAP). Methods Seventy clean male Sprague-Dawley rats were randomly divided into the normal control group (group A), diabetes mellitus (DM) only group (group B), DM + balanced salt solution (BSS) group (group C), DM + hUCMSC group (group D), with 10 rats in each group. DM rats were induced by intraperitoneal injection of streptozotocin. Apoptosis of retinal cells was assayed by dUTP nick end labeling. Immunohistochemistry and Western blot was performed to detect the retinal expressions of GFAP in rats. Results Compared with group A, large numbers of apoptotic cells could be found in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) of group B and group C, however the apoptotic cells in group D were significantly reduced than group B and C. The expression of GFAP was mainly located in the retinal GCL and retinal nerve fibre layer (RNFL) in group A, throughout the inner plexiform layer(IPL) in group B and C, only distributed in RNFL and GCL in group D. It was obvious that the expression of GFAP in group B and C was higher than group A. Compared with group B and C, the expression of GFAP in group D was significantly reduced. The difference of GFAP expression among the 4 groups was significant(F=79.635, P<0.05). Conclusion hUCMSC could inhibit the apoptosis of retinal cells and activation of glial cells in early DM rats. Key words: Diabetic retinopathy; Mesenchymal stem cells; Neurons; Apoptosis

Ablating adult neurogenesis in the rat has no effect on spatial processing: evidence from a novel pharmacogenetic model.

The function of adult neurogenesis in the rodent brain remains unclear. Ablation of adult born neurons has yielded conflicting results about emotional and cognitive impairments. One hypothesis is that adult neurogenesis in the hippocampus enables spatial pattern separation, allowing animals to distinguish between similar stimuli. We investigated whether spatial pattern separation and other putative hippocampal functions of adult neurogenesis were altered in a novel genetic model of neurogenesis ablation in the rat. In rats engineered to express thymidine kinase (TK) from a promoter of the rat glial fibrillary acidic protein (GFAP), ganciclovir treatment reduced new neurons by 98%. GFAP-TK rats showed no significant difference from controls in spatial pattern separation on the radial maze, spatial learning in the water maze, contextual or cued fear conditioning. Meta-analysis of all published studies found no significant effects for ablation of adult neurogenesis on spatial memory, cue conditioning or ethological measures of anxiety. An effect on contextual freezing was significant at a threshold of 5% (P = 0.04), but not at a threshold corrected for multiple testing. The meta-analysis revealed remarkably high levels of heterogeneity among studies of hippocampal function. The source of this heterogeneity remains unclear and poses a challenge for studies of the function of adult neurogenesis.

Effect of Neuregulin on Apoptosis and Expressions of STAT3 and GFAP in Rats Following Cerebral Ischemic Reperfusion

The aim is to investigate the effect of neuregulin-1beta (NRG-1beta) on the neuronal apoptosis and the expressions of signal transducer and activator of transcription (STAT3) and glial fibrillary acidic protein (GFAP) in rats following cerebral ischemia/reperfusion. The animal models of middle cerebral artery occlusion/reperfusion (MCAO/R) were established by an intraluminal filament method from left external-internal carotid artery in 100 cases of adult healthy male Wister rats. NRG-1beta was administered from the internal carotid artery (ICA) into MCA in the treatment group. The neuronal apoptosis was detected by terminal deoxynucleotidyl transference-mediated biotinylated deoxyuridine triphosphate nick-end labeling technique. The expression alternations of STAT3 and GFAP proteins were determined by fluorescent labeling analysis and Western blotting assay. Ischemic cerebral injury could induce neuronal apoptosis. Furthermore, with the duration of ischemia, the amount of apoptotic cells increased in the control group. These apoptotic cells distributed in various brain regions, especially the cortex, striatum, and hippocampus, while only a small amount of apoptotic cells could be observed in the treatment group, and there were significant differences compared with that in the control group (P < 0.01). The expressions of STAT3 and GFAP proteins in brain tissue gradually increased in the control group with the duration of ischemia. And NRG-1beta could elevate the expressional level of STAT3 and GFAP proteins in contrast to the control group (P < 0.05). NRG-1beta may play a neuroprotective role in cerebral ischemic insult by activating JAK/STAT signal transduction pathway, promoting the astrocyte gumnosis and regulating the anti-apoptosis mechanism in neurocytes.

Future Contrast Agents for Molecular Imaging in Stroke

The objective of this article is to illustrate both the potential and the limitations of molecular imaging in stroke research. By molecular imaging we mean the visual representation of biological processes at the cellular and molecular level. The use of molecular imaging for stroke diagnosis is still at a very preliminary stage and many of these procedures have only been tested in animals. In rats, stroke therapy using stem cells can be monitored by magnetic resonance imaging (MRI), green fluorescent protein (GFP) or luciferase (LUC) imaging. The migration of macrophages, which take up intravenously administered iron-based contrast agents and then migrate to the area of infarction, can already be observed in stroke patients. With MRI, the new agent Gd-DTPA-sLexA that binds to E- and P-selectin can specifically visualize selectin-mediated early endothelial activation after transient focal ischemia "in vivo". Decreased glial fibrillary acidic protein (GFAP) gene expression can be imaged in vivo by scintigraphy 24 hours after cerebral ischemia using a peptide nucleic acid antisense conjugate labeled with 111In and that hybridizes to the rat GFAP mRNA. Technetium-99m hydrazine nicotinamide-labeled HYNIC-annexin V SPECT can not only detect sites of neuronal injury in stroke patients but also can monitor the effects of neuroprotective therapy with a monoclonal antibody raised against FasLigand (FasL) in rats. Finally, information about cell metabolism in the infarct region can be gained using certain intracellular tracers [e.g. 18F-fluoromisonidazole (FMISO)]. Imaging benzodiazepine receptors with 11C-flumazenil (FMZ) can distinguish between irreversibly damaged and viable penumbra tissue early after stroke.

Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts

The expression of glial fibrillary acidic protein (GFAP) by perivascular cells of many mammalian organs suggests an as yet unknown function of this intermediate filament protein in the maintenance of homeostasis and vascular permeability at the blood-tissue interface. Although a similar situation may exist at the air-tissue interface, the cellular distribution of GFAP in skin tissue has never been demonstrated. To approach this issue, we have employed immunofluorescence and Western blotting techniques to detect GFAP in skin sections of young and adult humans, normal rodents, and two types of mutant mice, as well as in rat lung sections, and in cultured human keratinocytes and fibroblasts. Colocalization with antigens known to be associated with GFAP in other tissues was also tested. Epidermal and hair follicle keratinocytes and dermal fibroblasts showed distinct staining for GFAP as well as colocalization with alpha-actin, metallothionein, and antigens of the class-II major histocompatibility complex (MHC II). GFAP was also identified in rat alveolar fibroblasts which, in common with keratinocytes, form part of the air-tissue interface. GFAP was upregulated together with MHC II in nude mice but was barely detectable in the skin of non-obese diabetic severe combined immunodeficiency mice, suggesting a possible involvement in antigen-presenting functions. The intriguing distribution of a common set of antigens both in certain cells of the integumentary system and at the blood-tissue interfaces of internal organs suggests the involvement of these proteins in universal mechanisms controlling tissue homeostasis and protection.

Redistribution of GFAP and αB-crystallin after thermal stress in C6 glioma cell line

Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein alphaB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, alphaB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein alphaB-crystallin may play a functional role regulating the cytoarchitecture of GFAP.

Imaging Gene Expression in Regional Brain Ischemia in Vivo with a Targeted [111In]-Antisense Radiopharmaceutical

The gene encoding glial fibrillary acidic protein (GFAP) is downregulated 24 hr after reversible brain ischemia, such as with a middle cerebral artery occlusion (MCAO). The in vivo imaging of decreased GFAP gene expression in cerebral ischemia was examined in the present studies using a targeted peptide nucleic acid (PNA), which was labeled with (111)In, and which hybridized to nucleotides 20-37 of the rat GFAP mRNA. The PNA was monobiotinylated, and was attached to a monoclonal antibody (MAb) to the transferrin receptor (TfR) via a biotin-streptavidin linkage. The TfR MAb enables trans-membrane transport of the PNA antisense radiopharmaceutical from blood to the cytosol of brain cells. The decreased GFAP gene expression at 24 hr after a 1-hr reversible MCAO was confirmed by immunocytochemistry. The [(111)In]-labeled PNA - MAb conjugate was administered intravenously to anesthetized rats at 24 hr after the 1-hr reversible MCAO, and the brain uptake of the targeted antisense imaging agent was decreased relative to brain regions outside of the infarct zone. These studies provide evidence that decreased expression of a target gene in brain can be imaged in vivo with a sequence-specific PNA, provided the antisense radiopharmaceutical is delivered across cell membranes with a receptor-specific targeting agent.

Imaging Gene Expression in Regional Brain Ischemia In Vivo with a Targeted [&lt;SUP&gt;111&lt;/SUP&gt;In]-Antisense Radiopharmaceutical

The gene encoding glial fibrillary acidic protein (GFAP) is downregulated 24 hr after reversible brain ischemia, such as with a middle cerebral artery occlusion (MCAO). The in vivo imaging of decreased GFAP gene expression in cerebral ischemia was examined in the present studies using a targeted peptide nucleic acid (PNA), which was labeled with (111)In, and which hybridized to nucleotides 20-37 of the rat GFAP mRNA. The PNA was monobiotinylated, and was attached to a monoclonal antibody (MAb) to the transferrin receptor (TfR) via a biotin-streptavidin linkage. The TfR MAb enables trans-membrane transport of the PNA antisense radiopharmaceutical from blood to the cytosol of brain cells. The decreased GFAP gene expression at 24 hr after a 1-hr reversible MCAO was confirmed by immunocytochemistry. The [(111)In]-labeled PNA - MAb conjugate was administered intravenously to anesthetized rats at 24 hr after the 1-hr reversible MCAO, and the brain uptake of the targeted antisense imaging agent was decreased relative to brain regions outside of the infarct zone. These studies provide evidence that decreased expression of a target gene in brain can be imaged in vivo with a sequence-specific PNA, provided the antisense radiopharmaceutical is delivered across cell membranes with a receptor-specific targeting agent.

Estradiol (E2) Enhances Neurite Outgrowth by Repressing Glial Fibrillary Acidic Protein Expression and Reorganizing Laminin

Neuronal remodeling in response to deafferenting lesions in the brain can be enhanced by estradiol (E2). Astrocytes are among the targets of E2 in complex interactions with neurons and may support or inhibit neuronal remodeling. In ovariectomized female rats given entorhinal cortex lesions, E2 replacement inhibited the increase of glial fibrillary acidic protein (GFAP) protein. To model the role of E2 in these complex processes, we used the “wounding-in-a-dish” of astrocyte-neuron cocultures. Low physiological E2 (1 pm) blocks the wound-induced increase of GFAP expression (transcription and protein) and enhances neurite outgrowth. The transcriptional responses to E2 during wounding are mediated by sequences in the 5′-upstream region of the rat GFAP promoter. Concurrently, E2 reorganized astrocytic laminin into extracellular fibrillar arrays, which others have shown support neurite outgrowth. The inhibition of GFAP expression by E2 in this model is consistent with in vivo findings that E2 enhanced recovery from deafferenting cortical lesions by increased neurite outgrowth in association with decreased GFAP expression. More generally, we hypothesize that physiological variations in E2 levels modulate neuronal plasticity through direct effects on GFAP transcription that, in turn, modify GFAP-containing intermediate filaments and reorganize astrocytic laminin.

Estradiol (E2) enhances neurite outgrowth by repressing glial fibrillary acidic protein expression and reorganizing laminin.

Neuronal remodeling in response to deafferenting lesions in the brain can be enhanced by estradiol (E2). Astrocytes are among the targets of E2 in complex interactions with neurons and may support or inhibit neuronal remodeling. In ovariectomized female rats given entorhinal cortex lesions, E2 replacement inhibited the increase of glial fibrillary acidic protein (GFAP) protein. To model the role of E2 in these complex processes, we used the "wounding-in-a-dish" of astrocyte-neuron cocultures. Low physiological E2 (1 pM) blocks the wound-induced increase of GFAP expression (transcription and protein) and enhances neurite outgrowth. The transcriptional responses to E2 during wounding are mediated by sequences in the 5'-upstream region of the rat GFAP promoter. Concurrently, E2 reorganized astrocytic laminin into extracellular fibrillar arrays, which others have shown support neurite outgrowth. The inhibition of GFAP expression by E2 in this model is consistent with in vivo findings that E2 enhanced recovery from deafferenting cortical lesions by increased neurite outgrowth in association with decreased GFAP expression. More generally, we hypothesize that physiological variations in E2 levels modulate neuronal plasticity through direct effects on GFAP transcription that, in turn, modify GFAP-containing intermediate filaments and reorganize astrocytic laminin.

GFAP gene methylation in different neural cell types from rat brain

It is generally believed that specific demethylation processes take place in the promoter of tissue-specific genes during development. It has been suggested that hypomethylation of the −1500/−1100 domain of the 5′ flanking regulatory region of the rat glial fibrillary acidic protein gene may be specific for neuroectodermal derivatives such as neurons and astrocytes. In the present work the methylation status of one of those seven CG sites (the −1176) of the ‘neuroectoderm-specific domain’ was analyzed. In agreement with the neuroectoderm hypothesis, the −1176 site is highly demethylated in astroglial, oligodendroglial and neuronal cells, but heavily methylated in microglial and fibroblast cells. The three different glial population are derived from the same tissue (cerebral hemispheres of newborn rats) but have a different embryological origin: oligodendrocytes and astrocytes originate from neuroectoderm, while microglia is of mesodermal origin.It is not clear if GFAP-negative neuronal cells maintain such demethylation in the advanced stage of maturation or if they undergo a second phase of de novo methylation. In order to clarify this point we used a subcellular fractionation method which allowed us to separate two different nuclear populations from adult rat cerebral hemispheres: one enriched in neuronal nuclei (called N1) and the other enriched in glial nuclei (N2). A higher methylation level of the −1176 site was detected in the N1 fraction, suggesting the GFAP gene undergo a de novo methylation process during neuronal maturation. This observation is in agreement with recent results showing a de novo methylation of the −1176 site during postnatal brain development. We hypothesize that a DNA demethylation process takes place in neuroectodermal precursor cells and that the −1176 site persists demethylated at the earlier stages of neuronal differentiation (immature neurons) and becomes fully methylated at more advanced stages of differentiation.

Structural features of the rat GFAP gene and identification of a novel alternative transcript

The glial fibrillary acidic protein (GFAP) is expressed in a cell-specific manner and represents the major subunit of intermediate filaments of astroglial cells. The knowledge of the gene structure is an important step for further understanding the mechanisms of cell-specific expression. In the present study, we report the complete sequence of the rat GFAP gene and provide evidence for the existence, in the rat brain, of a novel alternative transcript. Since three different transcripts, indicated as GFAPα, β, and γ, have been previously reported (Feinstein et al. [1992] J. Neurosci. Res. 32:1–14; Zelenika et al. [1995] Mol. Brain Res. 30:251–258), we called this novel mRNA isoform GFAPδ. It is generated by the alternative splicing of a novel exon located in the classic seventh intron. This alternative exon (called VII+) contains a 101-bp coding sequence in frame with exon VII and interrupted by a stop codon TAA at position +5451. Therefore, the novel GFAPδ transcript encodes for an hypothetical GFAP where the forty-two carboxy-terminal amino acids encoded by exon VIII and IX are replaced by thirty-three amino acids encoded by exon VII+. Northern blot analysis with a specific probe for exon VII+ revealed a 4.2-kb mRNA, expressed in several brain areas, but absent in extracerebral tissues (lung, heart, kidney, liver, spleen). The previously discovered GFAP isoforms (α, β, and γ) produce hypothetical translation products differing in the aminoterminal Head domain. The present data suggest, for the first time, the possible existence of GFAP isoforms differing in the carboxy-terminal Tail domain. J. Neurosci. Res. 56:219–228, 1999. © 1999 Wiley-Liss, Inc.

Structural features of the rat GFAP gene and identification of a novel alternative transcript.

The glial fibrillary acidic protein (GFAP) is expressed in a cell-specific manner and represents the major subunit of intermediate filaments of astroglial cells. The knowledge of the gene structure is an important step for further understanding the mechanisms of cell-specific expression. In the present study, we report the complete sequence of the rat GFAP gene and provide evidence for the existence, in the rat brain, of a novel alternative transcript. Since three different transcripts, indicated as GFAPalpha, beta, and gamma, have been previously reported (Feinstein et al. [1992] J. Neurosci. Res. 32:1-14; Zelenika et al. [1995] Mol. Brain Res. 30:251-258), we called this novel mRNA isoform GFAPdelta. It is generated by the alternative splicing of a novel exon located in the classic seventh intron. This alternative exon (called VII+) contains a 101-bp coding sequence in frame with exon VII and interrupted by a stop codon TAA at position +5451. Therefore, the novel GFAPdelta transcript encodes for an hypothetical GFAP where the forty-two carboxy-terminal amino acids encoded by exon VIII and IX are replaced by thirty-three amino acids encoded by exon VII+. Northern blot analysis with a specific probe for exon VII+ revealed a 4.2-kb mRNA, expressed in several brain areas, but absent in extracerebral tissues (lung, heart, kidney, liver, spleen). The previously discovered GFAP isoforms (alpha, beta, and gamma) produce hypothetical translation products differing in the amino-terminal Head domain. The present data suggest, for the first time, the possible existence of GFAP isoforms differing in the carboxy-terminal Tail domain.

Glial fibrillary acidic protein transcription responses to transforming growth factor-beta1 and interleukin-1beta are mediated by a nuclear factor-1-like site in the near-upstream promoter.

Elevated expression of glial fibrillary acidic protein (GFAP) is associated with astrocyte activation during responses to injury in the CNS. Because transforming growth factor-beta1 (TGF-beta1) and interleukin-1beta (IL-1beta) are released during neural responses to injury and because these cytokines also modulate GFAP mRNA levels, it is of interest to define their role in GFAP transcription. The increases of GFAP mRNA in response to TGF-beta1 and decreases in response to IL-1beta were shown to be transcriptionally mediated in rat astrocytes transfected with a luciferase-reporter construct containing 1.9 kb of 5'-upstream rat genomic DNA. Constructs containing sequential deletions of the rat GFAP 5'-upstream promoter identified a short region proximal to the transcription start (-106 to -53 bp) that provides full responses to TGF-beta1 and IL-1beta. This region contains an unusual sequence motif with overlapping nuclear factor-1 (NF-1)- and nuclear factor-kappaB (NF-kappaB)-like binding sites and homology to known TGF-beta response elements. Mutagenesis (3-bp exchanges) in -70 to -68 bp blocked the induction of GFAP by TGF-beta1 and the repression by IL-1beta. Gel shift experiments showed that the DNA segment -85 to -63 bp was bound by a factor(s) in nuclear extracts from astrocytes. The concentrations of these DNA binding factors were increased by treatment of astrocytes with TGF-beta1 and decreased by IL-1beta. Binding of these nuclear factors was blocked by mutation of -70 to -68 bp. Despite homology to NF-1 or NF-kappaB binding sites in the GFAP promoter at segment -79 to -67 bp, anti-NF-kappaB or anti-NF-1 antibodies did not further retard the gel shift of the nuclear factors/DNA complex. Moreover, astrocytic nuclear proteins do not compete for the specific binding to NF-1 consensus sequence. Thus, nuclear factors from astrocytes that bind to the -85- to -63-bp promoter segment might be only distantly related to NF-1 or NF-kappaB. These findings are pertinent to the use of GFAP promoter constructs in transgenic animals, because cisacting elements in the GFAP promoter are sensitive to cytokines that may be elaborated in response to expression of transgene products.

Ciliary neurotrophic factor activates JAK/Stat signal transduction cascade and induces transcriptional expression of glial fibrillary acidic protein in glial cells.

In recent reports, ciliary neurotrophic factor (CNTF) has been implicated as an injury factor involved in regulating astrogliosis in the CNS. In this study, we used a rat oligodendroglial progenitor cell line that is highly responsive to CNTF to examine CNTF-induced alterations that may play a role in activation of the glial fibrillary acidic protein (GFAP) gene. We determined that CNTF induces the transient translocation of Stat1 alpha/p91 to the nucleus. This nuclear translocation was followed by GFAP promoter activation and an up-regulation of GFAP mRNA and protein. Level of CNTF-alpha receptor mRNA, however, were unaffected by addition of the ligand. Transfection studies using an upstream 5'-flanking, 1.9-kb rat GFAP promoter linked to a luciferase reporter gene revealed CNTF-induced transcriptional activation within 1 h of ligand exposure. Moreover, serial-deleted constructs identified a distal (-1,857 to -1,546 bp) and a proximal (-384 to -106 bp) region as being important for CNTF-induced GFAP promoter activation. These two regions showed a strong degree of overlap for CNTF- and serum-induced activation of the GFAP gene. Analysis of the two regions revealed several cis-elements that are thought to be involved in GFAP regulation and/or the regulation of other genes by members of the interleukin-6 family of cytokines. Moreover, we are the first to report the presence of several putative CNTF-responsive elements within our identified distal and proximal regions in the GFAP gene promoter.

Immunocytochemical analyses of astrocyte development in the olfactory bulb

Several lines of evidence suggest an important role for glia in establishing boundaries during development of mammalian cortex and insect olfactory lobe. In the adult rat olfactory bulb distinct morphological categories of astroglial cells with clear laminar specificity are easily recognized following immunocytochemical staining of glial fibrillary acidic protein (GFAP). To explore the developmental distribution of olfactory bulb astrocytes and their possible role in establishing the segregation of neurons in specific olfactory bulb laminae, we used immunocytochemical localization of GFAP in rats at 0, 6, 9, 12, 15 and 21 days postnatal plus the adult. In the adult we confirmed prior observations and identified five morphological categories of astrocytes: linear, wedge, elongate, semicircular, and circular. Each category had a unique sublaminar distribution across the olfactory bulb, although categories could occur in more than one lamina. Between 0 and 21 days postnatal a 6th category was apparent, radial glial cells. The mature astrocyte morphologies did not emerge uniformly. Astrocytes found in the outermost glomerular layer developed first with the appearance of the linear, wedge and elongate morphologies. Deeper laminae of the olfactory bulb followed in a successive fashion until the adult pattern was evident around 15 days postnatal. As radial glia disappeared, the mature morphologies assumed their final position. The data suggest that the maturation of olfactory bulb astrocytes may be linked to the final migration and maturation of olfactory bulb neurons.

Transcriptional regulation of glial fibrillary acidic protein by corticosterone in rat astrocytes in vitro is influenced by the duration of time in culture and by astrocyte-neuron interactions.

In the rat hippocampus and cortex, the transcription of glial fibrillary acidic protein (GFAP), an astrocyte intermediate filament protein, is inhibited by glucocorticoids. The present study examined the regulation of GFAP expression by glucocorticoids in astrocytes in vitro. Corticosterone (CORT) increased GFAP messenger RNA, protein, and transcription rates in cultured primary neonatal astrocytes, responses opposite the GFAP responses to CORT in vivo. The direction of GFAP regulation by corticosterone in vitro is reversed by coculture with neurons or by extended culture for 3 months. The switch in the direction of GFAP regulation by CORT during prolonged culture is associated with a 3-fold increased prevalence of type II glucocorticoid receptor (GR). These findings were corroborated with a promoter construct that contained 1.9 kilobases of 5'-up-stream rat GFAP DNA with a luciferase reporter. Thus, the direction of GFAP transcription to CORT is subject to the postreplicative time in culture and to interactions with neurons, in which 5'-up-stream sequences contain sufficient information to mediate the switch in the direction of the response to CORT. This in vitro model may be used to analyze how interactions of astrocytes with neurons or other cell types influence the hormonal regulation of GFAP.

Tissue-specific DNA methylation patterns of the rat glial fibrillary acidic protein gene.

The glial fibrillary acidic protein (GFAP) is an intermediate filament protein, specific of the cytoskeleton of astrocytes in the central nervous system. In the present work, as a preliminary step to the study of glial-specific gene expression, we cloned the rat GFAP gene, and we report the sequence of 1.9 kb of the 5' flanking region, exon 1, and the majority of the first intron. By digestion with methylation-sensitive restriction enzymes followed by Southern blot analysis, the methylation status of various CpG sites was examined in this genomic segment. We tested whether structural modification of the GFAP gene, such as DNA methylation, could be related to its tissue-specific transcriptional activity. Therefore, we compared a GFAP-expressing cell population (primary culture of astroglial cells), a mixed population of GFAP-expressing and -nonexpressing cells (adult rat cerebral hemispheres), and a GFAP-nonexpressing tissue (liver). In the 5' flanking region we identified a CpG site at position -1176 whose level of methylation is inversely correlated to GFAP expression. In primary cultured astrocytes, 75% of the GFAP gene alleles were demethylated at this site, while the corresponding value obtained for the cerebral hemispheres was 45%, and for liver only 9%. On the basis of the sequence data, a CpG-rich region (putative CpG island) was identified extending from -38 to +347 and overlapping 80% of the first exon. HhaI and HpaII sites located in the putative CpG island showed a relatively high level of methylation in all the cell populations examined, and did not show any clear correlation with the level of GFAP gene expression or with the methylation status of the -1176 site. Further in vivo developmental studies and in vitro differentiation studies are necessary to better understand the functional differences of the various methylatable CpG sites in the 5' end of the GFAP gene.

Methylation of the rat glial fibrillary acidic protein gene shows tissue‐specific domains

The gene for glial fibrillary acidic protein (GFAP) was compared for CpG sites that are potential locations of methylated cytosine (mC). GFAP sequences in the 5'-upstream promoter and in exon 1 of rat, mouse, and human showed extensive similarity in the locations of CpG sites in the promoter and in exon 1, implying conservation. The methylation of mC at 9 CpG sites in the promoter and 10 sites in exon 1 was analyzed in F344 male rats by a quantitative application of ligation-mediated polymerase chain reaction (LMPCR). CpG sites with varying mC in different tissues were found in the GFAP promoter and in a CpG island in exon 1. In the brain, the promoter had about 40% less mC than in testis and liver. The degree of methylation varied strikingly between adjacent sites within and between tissues. Testis GFAP exon 1 had a gradient of mC from 5' to 3' across the exon that was absent in liver, brain, and cultured neurons and astrocytes. Among brain regions, the hippocampus had 10-40% less mC at 12 CpG sites than in hypothalamus; the other sites (7/19) showed smaller differences between these brain regions. In DNA from primary cultures, astrocytes had slightly less mC than neurons at all sites. Because neuron-rich hippocampal subregions and primary neurons cultures had less methylation than nonneural tissues, we hypothesize that neuroectodermal derivatives tend to be less methylated, whether or not GFAP is expressed. Four domains of methylated CpG sites are proposed on the basis of tissue and cell-type distribution: I) a constitutively methylated domain in the mid-upstream promoter; II) a testis-specific gradient of methylation in exon 1; III) a hypomethylated domain found in neuroectodermal derivatives; and IV) subsets of sites in the promoter and in exon 1 that have the least methylation in astrocytes, and therefore may be astrocyte-specific domains.

Reexpression of glial fibrillary acidic protein rescues the ability of astrocytoma cells to form processes in response to neurons.

Astroglial cells play an important role in orchestrating the migration and positioning of neurons during central nervous system development. Primary astroglia, as well as astrocytoma cells will extend long stable processes when co-cultured with granule neurons. In order to determine the function of the glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astroglia and astrocytoma cells, we suppressed the expression of GFAP by stable transfection of an anti-sense GFAP construct in human astrocytoma U251MG cells. The resulting AS2-U251 cells can no longer extend stable processes in the presence of granule neurons. To show that this effect is due specifically to the absence of GFAP, we reintroduced a fully encoding rat brain GFAP cDNA into these AS2-U251 cells. The resulting rat GFAP appeared as a filamentous network and the reexpression of GFAP rescued the ability of these astrocytoma cells to form stable processes when co-cultured with neurons. From these results, it is clear that the glial specific intermediate filament protein, GFAP, is required for process extension of these astrocytoma cells in response to granule neurons.