Glial fibrillary acidic protein is greatly modified by oxidative stress in aceruloplasminemia brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Decrease of Glial Fibrillary Acidic Protein in Rat Frontal Cortex Following Aluminum Treatment

Aluminum lactate was injected either intraperitoneally or stereotactically into the lateral cerebral ventricles of rats. Rats were killed at various times after treatment, and frontal cortex, hippocampus, and striatum were dissected out. Microtiter plate-based sandwich ELISA and immunohistochemistry were used to measure the glial fibrillary acidic protein (GFAP) concentration. GFAP levels were significantly decreased in frontal cortex 7 days after a single lateral ventricular injection of aluminum lactate and 14 days following systemic treatment. In contrast, neither hippocampus nor striatum exhibited any significant changes in the content of this astrocytic intermediate filament protein after aluminum treatment. Levels of a predominantly astroglial enzyme, glutamine synthetase, were also selectively reduced in the frontal cortex following intraventricular injection of aluminum. This depression exhibited a regional and temporal specificity similar to that found for GFAP. These results suggest a selective and progressive diminution of astrocytic responsivity in frontal cortex following either systemic or intraventricular aluminum dosing. The depression of GFAP levels reported here, which was found in the rat cerebral cortex 7-14 days after aluminum treatment in a species that does not form neurofilamentous aggregates, may reflect extended impairment of astrocytic function and suggests that these cells may be the primary targets of aluminum neurotoxicity.     

Mouse Monoclonal Antibodies Reacting with Human Brain Glial Fibrillary Acidic Protein

Abstract: Three different epitopes on the glial fibrillary acidic protein (GFAP) have been identified by means of three monoclonal antibodies. The antibodies were named anti-GFAP 1, anti-GFAP 2, and anti-GFAP 3. Antibody specificities were investigated by several techniques including indirect immunoprecipitation, immunoblotting, and immunohistochemistry. The anti-GFAP 1 antibodies recognized an epitope found on GFAP from all three species tested: human, rat, and ox, but in addition a reaction was observed with cells not containing GFAP. The epitope recognized by anti-GFAP 2 was present on GFAP from human and ox, but apparently not on rat GFAP; the anti-GFAP 2 antibodies also reacted with antigen(s) other than GFAP. In contrast, the epitope defined by anti-GFAP 3 has proved absolutely specific for GFAP in human, rat, and ox.     

Changes in the Solubility of Glial Fibrillary Acidic Protein After Ischemic Brain Damage in the Mouse

Abstract: In the present study, changes in the content of glial fibrillary acidic protein (GFAP) in mouse cortex were investigated at different time intervals after unilateral middle cerebral artery occlusion. The GFAP content was assessed semiquantitatively by ELISA and immunoblotting. GFAP immunoreactivity was determined for each animal separately in protein fractions obtained from the ipsilateral, lesioned cortex and the contralateral, unlesioned cortex. Changes in the GFAP content of the lesioned cortex with respect to that of the unlesioned cortex were calculated for each fraction individually. GFAP was detectable in all protein fractions with a significant amount recovered from the aqueous extracts. A pronounced increase in the GFAP content of the lesioned cortex was observed. As measured by ELISA, this increase was maximal 5 days after injury and significantly more pronounced for the soluble and the Triton X-100-soluble protein fractions (mean increase 7 days after lesion, 281.4 and 240.2%, respectively) than for the crude cytoskeletal fraction (mean increase, 153.3%). A small and transient increase in GFAP immunoreactivity was also found in all protein fractions prepared from the contralateral, unlesioned cortex. These results were confirmed by immunoblotting.     

Glial Fibrillary Acidic Protein: Norepinephrine Stimulated Phosphorylation in Intact C-6 Glioma Cells

Abstract: Coelectrophoresis in two-dimensional gels of rat glial fibrillary acidic protein (GFA) and ³²P-labeled whole cell extracts of rat C-6 glioma cells showed that the GFA migrated in close proximity to a previously noted phosphoprotein, 50K-6.1, of these cells. GFA electrophoresed as a 50K polypeptide with at least four charge variants, the most acidic of which coelectrophoresed with 50K-6.1. Exposure of the C-6 cultures to dibutyryl cyclic AMP (dbcAMP) for 48 h increased the relative abundance of the endogenous polypeptide associated with 50K-6.1 by threefold, consistent with the hypothesis that 50K-6.1 was GFA. Norepinephrine stimulated 50K-6.1 phosphorylation 3.2-fold in dbcAMP-induced cultures. Peptide mapping with V8 protease and subtilisin was used to test the hypothesis that GFA and 50K-6.1 were identical polypeptides. With V8 protease, the peptides generated from the [³⁵S]methionine labeled putative GFA spot of the C-6 cells were indistinguishable from the stained bands derived from authentic GFA in mixed samples of the two proteins. Likewise, the ³⁵S-labeled acidic satellite to the putative GFA spot also yielded a peptide map that matched that of the authentic GFA. ³²P-labeled peptides derived from the 50K-6.1 protein were a subset of those from authentic GFA. With three subtilisin concentrations, ³²P-labeled 50K-6.1 was degraded to peptides which were again a subset of the stained GFA peptides. A cytoskeletal fraction from ³²P-labeled C-6 cells contained a 50K phosphoprotein. Pep-, tide mapping with V8 protease produced a ³²P-peptide pattern which was a subset of that from authentic GFA. The pattern closely resembled the ³²P-peptide pattern for the 50K-6.1 protein from 2-dimensional gels of whole cell extract. It was concluded that the protein 50K-6.1 is a phosphorylated form of GFA and that GFA is a phosphoprotein whose phosphòrylation is stimulated by norepinephrine in C-6 glioma cells.     

Chronic Administration of Lithium Chloride Increases Immunodetectable Glial Fibrillary Acidic Protein in the Rat Hippocampus

Abstract: We studied the effect of treating rats with lithium salts on the content and in vitro phosphorylation rate of the astrocyte cell marker, glial fibrillary acidic protein (GFAP), in brain slices. Rats were fed a diet incorporating lithium chloride until the concentration of Li⁺ in serum reached 0.6–1.2 m M , a range similar to that achieved in clinical practice. Hippocampal tissue was analyzed for immunoreactive GFAP by a dot assay, and slices of hippocampus and caudate nucleus were labeled with [³²P]-phosphate to determine the in vitro rate of phosphorylation of GFAP. Compared with controls, the level of immunoreactive GFAP in the hippocampus from lithium-treated rats was increased 34%, and GFAP in hippocampal slices incorporated 39% more ³²P. This effect of lithium was apparently not confined to the hippocampus because the in vitro rate of phosphorylation of GFAP in caudate slices was also increased in the treated rats.     

Changes in Glial Fibrillary Acidic Protein (GFAP) Immonureactivity Reflect Neuronal States

The astroglial marker, glial fibrillary acidic protein (GFAP) was investigated by immunohistochemistry in various brain areas in order to see its fluctuations in various functional states. Different neuronal states were either experimentally induced or studied under physiological conditions. To produce experimental alterations the visual system was chosen as a model. Upon lesioning of the lateral geniculate body with the stereotaxic injection of ibotenic acid an increase in GFAP immunoreactivity could be induced in layers III and IV of the ipsilateral visual cortex where geniculo-cortical fibres terminate. Electron microscopy has revealed a synchronous degeneration of synaptic terminals and the hypertrophy of perisynaptic astrocyte processes. To study changes in the intact animal the effect of illumination was observed. In the lateral geniculate body the dorsal subnucleus was found immunonegative when studied at day and positive at night. Similar changes were observed in the suprachiasmatic nucleus. As to more generalized influences, the effect of gonadal steroids on the GFAP-reaction interpeduncular nucleus, an area not involved in hormonal regulatory mechanisms was studied. In males only castration could reduce constantly high GFAP immonoreactivity, whereas in females GFAP showed wide-range sexual cycle-related fluctuations. It was concluded that changes in GFAP immunoreactivity can indicate synaptic events whithin a circumscribed area of the brain. Special issue article in honor of Dr. Ricardo Tapia.     

Distribution of Glial Fibrillary Acidic Protein in Gliosed Human White Matter

Glial fibrillary acidic protein (GFAP) in gliosed white matter from multiple sclerosis plaques and cerebral infarcts was examined by polyacrylamide gel electrophoresis and immunoblotting. Using a monoclonal antibody raised against human GFAP, up to 11 GFAP polypeptide bands of molecular weight 37-49 kilodaltons were identified in particulate and supernatant fractions of CNS tissue homogenates. Soluble GFAP constituted about one-quarter of the total GFAP in normal cerebral white matter. In brain lesions in which reactive astrocytes were observed microscopically, the proportion of soluble GFAP was increased, with a greater representation of the lower-molecular-weight forms. In brain chronic sclerotic plaques, almost all of the GFAP was in the particulate form. Purified particulate GFAP was susceptible to proteolysis at acid but not at neutral pH in the presence of CNS homogenates. In tissue autolysis studies, GFAP was stable in situ for periods well in excess of average CNS postmortem times.     

Glial Fibrillary Acidic Protein Expression in Rat Brain and in Radial Glia Culture Is Delayed by Prenatal Ethanol Exposure

Abstract: The alterations in astrocyte proliferation and differentiation induced by prenatal exposure to alcohol (PEA) suggest that ethanol exposure affects the radial glial cells, the main astrocytic precursors. We have investigated the effects of ethanol on the early stages of astrogliogenesis by analyzing the developmental pattern of vimentin and glial fibrillary acidic protein (GFAP) immunoreactivity and their mRNA levels during embryonic/fetal brain development and in radial glia in primary culture. GFAP appeared late in gestation and at day 5 of culture of radial glial, whereas GFAP mRNA was first detected on fetal day 15 and increased in content on fetal day 21. In contrast, the levels of vimentin and its mRNA were high at fetal day 15 but decreased on day 21. Alcohol exposure delays the appearance of GFAP and its mRNA and significantly decreases the GFAP expression in fetal brain and in primary culture of radial glial. In addition, some morphological alterations were observed in PEA glial cells in culture. These results demonstrate that astroglial precursor cells are damaged by prenatal exposure to ethanol and suggest that abnormalities in the astrogliogenesis may underlie the disruption in neuronal migration and other CNS alterations observed after prenatal ethanol exposure.     

Cell Growth Suppression of Astrocytoma C6 Cells by Glial Fibrillary Acidic Protein cDNA Transfection

Abstract: The cellular functions of the intermediate filament family including glial fibrillary acidic protein (GFAP) are not well known yet beyond their roles as structural elements of cells. Expression of GFAP, which is specific in astrocytes and regulated developmentally, suggests its involvement in cell growth and differentiation of astrocytes. We transfected murine GFAP cDNA into a rat astrocytoma C6 cell line to assess the specific effect of GFAP on cells. Two stable GFAP-transfected cell lines, GFC6-5 and GFC6-6, exhibited a series of morphological and growth characteristics that distinguish them from their counterparts, i.e., NeoC6 cells transfected only with the neomycin-resistant gene, and native C6 cells. Both GFC6-5 and GFC6-6 cells showed elongated cell shapes with extended processes rich in GFAP, markedly suppressed cell growth, and decreased bromodeoxyuridine uptake. Western blot analysis revealed a remarkable increase of GFAP expression in GFC6-5 and GFC6-6 compared with that in NeoC6 and C6, in contrast to similar vimentin expression in all cell lines. The results indicate that the expression of GFAP has dramatic effects on cell morphology and cell growth suppression in C6 cells, suggesting that GFAP may function as a tumor suppressor in astrocytoma.     

Intragenic Sequences Affect the Expression of the Gene Encoding Glial Fibrillary Acidic Protein

We show that the expression of the gene encoding glial fibrillary acidic protein (GFAP) gene is affected by at least three cis-acting elements. A positive regulatory element that is located between nucleotides -1,631 and -1,479 can confer cell type-specific expression on a heterologous gene. A second regulatory element is located between nucleotides -97 and -80. The third is a negative regulatory element that is located within the first intron of the gene. Deletion of this element activates GFAP expression in HeLa cells, and affects promoter function in glioma cells.     

百草枯和代森锰所致帕金森病小鼠黑质GFAP的表达变化

目的:研究联合应用百草枯(Paraquat,PQ)和代森锰(Maneb,MB)对帕金森病(Parkinson′s disease,PD)小鼠脑黑质神经胶质原纤维酸性蛋白质(Glial fibrillary acldic protein,GFAP)的表达变化。方法:PQ/MB建立C57BL/6J小鼠PD模型,利用免疫组化方法观察不同时间点小鼠黑质区酪氨酸羟化酶(Tyrosine hydroxylase,TH)和GFAP表达变化及相互关系。结果:模型组黑质致密部(Substantial nigra pars compacta,SNpc)区残留的TH阳性神经元呈进行性下降趋势,显著低于对照组的相应时间点(P＜0．01),黑质区星形胶质细胞反应性增多并呈现明显活化状态。结论:PO/MB能诱导小鼠产生渐进性且不可逆的PD样改变,星形胶质细胞可能参与多巴胺能神经元的病理损伤过程。     

Rapid Increases in Glial Fibrillary Acidic Protein mRNA and Protein Levels in the Copper-Deficient, Brindled Mouse

The brindled mouse (MObr/y) carries an X-linked mutation that produces severe copper deficiency. Affected males suffer profound deficits in oxidative metabolism. We have examined astrocyte pathology in MObr/y during development and have found marked changes in the metabolism of glial fibrillary acidic protein (GFAP). Immunocytochemistry with anti-GFAP antisera revealed a marked increase in staining at postnatal day 12 (P12), compared to heterozygous female and unaffected male littermates, particularly in neocortex and thalamus. Septum, hypothalamus, and striatum showed little change. Western blot analysis revealed increased levels of GFAP in MObr/y forebrain and cerebellum. Levels of GFAP mRNA were determined by Northern blotting with a mouse GFAP cDNA probe. At P10, mRNA levels were normal, but increased to 8-10 times normal by P12. Levels at P15 remained similarly elevated. Thus, immunostaining and protein determinations correlate with mRNA elevations. Astrocytes can alter GFAP mRNA and protein levels over a relatively short time. Counts of neocortical cells did not reveal differences in cell numbers between MObr/y and controls, indicating that the observed changes reflect increased cellular levels and not a large increase in the numbers of astrocytes.     

Suppression of Glial Tumor Growth by Expression of Glial Fibrillary Acidic Protein

We examined the effect of expression of glial fibrillary acidic protein (GFAP) on the tumor growth of astrocytoma in vivo. When rat astrocytoma C6 cells were injected subcutaneously in athymic mice, the cells produced tumors that grew rapidly. The tumor growth of C6 cells transfected with GFAP cDNA was significantly reduced compared to that of control NeoC6 cells transfected only with the neomycin resistant gene. After implantation of C6 cells transfected with mutated GFAP cDNA at the phosphorylation sites, the tumor growth was suppressed similar to that of the wild GFAP transfectants. To determine whether the cell growth suppression by GFAP is specific for astroglial cells, we assessed the effect of GFAP on the cell growth of an L cell of fibroblast origin in vitro. By GFAP cDNA transfection, L cells showed morphological changes, but the cell growth was not reduced. These results suggest that GFAP is a critical regulator of the tumor growth of astrocytoma.