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.     

Blood Levels of Glial Fibrillary Acidic Protein (GFAP) in Patients with Neurological Diseases

Background and Purpose

The brain-specific astroglial protein GFAP is a blood biomarker candidate indicative of intracerebral hemorrhage in patients with symptoms suspicious of acute stroke. Comparably little, however, is known about GFAP release in other neurological disorders. In order to identify potential “specificity gaps” of a future GFAP test used to diagnose intracerebral hemorrhage, we measured GFAP in the blood of a large and rather unselected collective of patients with neurological diseases.

Methods

Within a one-year period, we randomly selected in-patients of our university hospital for study inclusion. Patients with ischemic stroke, transient ischemic attack and intracerebral hemorrhage were excluded. Primary endpoint was the ICD-10 coded diagnosis reached at discharge. During hospital stay, blood was collected, and GFAP plasma levels were determined using an advanced prototype immunoassay at Roche Diagnostics.

Results

A total of 331 patients were included, covering a broad spectrum of neurological diseases. GFAP levels were low in the vast majority of patients, with 98.5% of cases lying below the cut-off that was previously defined for the differentiation of intracerebral hemorrhage and ischemic stroke. No diagnosis or group of diagnoses was identified that showed consistently increased GFAP values. No association with age and sex was found.

Conclusion

Most acute and chronic neurological diseases, including typical stroke mimics, are not associated with detectable GFAP levels in the bloodstream. Our findings underline the hypothesis that rapid astroglial destruction as in acute intracerebral hemorrhage is mandatory for GFAP increase. A future GFAP blood test applied to identify patients with intracerebral hemorrhage is likely to have a high specificity.     

应用ＣＡＴＳ法分离和鉴定猪ＧＦＡＰ基因的研究

根据比较锚定序列宗踪（ＣＡＴＳ）法,选择人和小鼠胶质细胞原纤维酸性蛋白（ＧＦＡＰ）基因的同源区域设计引,用ＰＣＲ方法从二花脸猪基因组中分离到４１２bp的基因片段,经与基因资料库中已训功能基因的同源性比较,该片段可鉴定为猪的ＧＦＡＰ基因,利用猪－啮齿类体细胞杂克隆板将ＧＦＡＰ基因定位于猪１２号染色体１２p11-(2/3)P13区域。     

Modelling and Characterization of Glial Fibrillary Acidic Protein

Glial Fibrillary Acidic Protein (GFAP) is an intermediate-filament (IF) protein that maintains the astrocytes of the Central Nervous System in Human. This is differentially expressed during serological studies in inflamed condition such as Rheumatoid Arthritis (RA). Therefore, it is of interest to glean molecular insight using a model of GFAP (49.88 kDa) due to its crystallographic nonavailability. The present study has been taken into consideration to construct computational protein model using Modeller 9.11. The structural relevance of the protein was verified using Gromacs 4.5 followed by validation through PROCHECK, Verify 3D, WHAT-IF, ERRAT and PROVE for reliability. The constructed three dimensional (3D) model of GFAP protein had been scrutinized to reveal the associated functions by identifying ligand binding sites and active sites. Molecular level interaction study revealed five possible surface cavities as active sites. The model finds application in further computational analysis towards drug discovery in order to minimize the effect of inflammation.     

Alterations in Glial Fibrillary Acidic Protein (GFAP) mRNA Levels in the Hamster Facial Motor Nucleus: Effects of Axotomy and Testosterone

Testosterone propionate (TP) administered at the time of facial nerve injury in the hamster accelerates the rate of regeneration. In this study, we tested the hypothesis that the mechanism by which TP augments peripheral nerve regeneration involves regulation of glial fibrillary acidic protein (GFAP) mRNA in the facial motor nucleus. Castrated male hamsters were subjected to right facial nerve transection, with half the animals implanted subcutaneously with Silastic capsules containing exogenous TP and the remainder sham implanted. Postoperative survival times were 0.25, 1, 2, 4, 7, and 14 d. Qualitative/quantitative analyses of both film and emulsion autoradiograms were accomplished. Axotomy, with or without TP, resulted in a dramatic increase in GFAP mRNA levels by 1 d postoperative on the axotomized side, relative to controls. GFAP mRNA levels remained elevated throughout all postoperative times in both the nonhormone- and TP-treated animals. Qualitative examination of the film autoradiograms indicated a generalized decrease in the amount of GFAP mRNA in the control and axotomized nuclei of TP-treated animals when compared to the control and axotomized nuclei, respectively, of nonhormone-treated animals. Statistical comparison of the values obtained for both the film and emulsion autoradiograms confirmed this impression. Thus, while the injury-induced increases in GFAP mRNA expression were not blocked by TP, the overall extent of the increase was significantly tempered by steroid treatment. These data suggest that hormonal modulation of the astrocytic response to peripheral nerve injury may be a contributing factor in the ability of steroids to enhance the regenerative capacities of injured motor neurons.     

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.     

Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease.     

Genetic Background Affects Human Glial Fibrillary Acidic Protein Promoter Activity

The human glial fibrillary acidic protein (hGFAP) promoter has been used to generate numerous transgenic mouse lines, which has facilitated the analysis of astrocyte function in health and disease. Here, we evaluated the expression levels of various hGFAP transgenes at different ages in the two most commonly used inbred mouse strains, FVB/N (FVB) and C57BL/6N (B6N). In general, transgenic mice maintained on the B6N background displayed weaker transgene expression compared with transgenic FVB mice. Higher level of transgene expression in B6N mice could be regained by crossbreeding to FVB wild type mice. However, the endogenous murine GFAP expression was equivalent in both strains. In addition, we found that endogenous GFAP expression was increased in transgenic mice in comparison to wild type mice. The activities of the hGFAP transgenes were not age-dependently regulated. Our data highlight the importance of proper expression analysis when non-homologous recombination transgenesis is used.     

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.     

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.     

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.     

人脑老化过程中胶质纤维酸性蛋白的表达变化

目的:研究人脑老化过程中胶质纤维酸性蛋白(GFAP)的表达变化.方法:两例人脑标本,样本一:女性,64岁,生前诊断有血管性淀粉病变;样本二:男性,16岁,死于急性心肌炎,两例标本均于死后12 h取脑,取皮质和海马部位,进行GFAP的常规免疫组化染色进行定量分析.结果:老年脑星形胶质细胞呈明显激活状态,GFAP阳性细胞数目显著增多,胞体增大,突起粗短,刚果红染色可见老年斑周围GFAP阳性细胞成簇聚集;年轻脑GFAP阳性细胞数目少,胞体瘦小,突起细长,未见GFAP阳性细胞成簇聚集.统计学分析表明GFAP阳性细胞数和蛋白质的表达变化在老年脑和年轻脑具有差别显著性(P＜0.Ol) .结论:衰老和血管淀粉样病变在人脑星形胶质细胞的增生和激活过程中具有重要的作用.     

Glial Fibrillary Acidic Protein in the Cytoskeletal and Soluble Protein Fractions of the Developing Rat Brain

The distribution of glial fibrillary acidic protein (GFAP) into cytoskeletal and soluble protein fractions during development of the rat brain has been studied by quantitative immunoblotting and enzyme-linked immunosorbent assay (ELISA). These assays indicate that cytoskeletal GFAP accounts for nearly all the total GFAP in the adult rat brain, and that the developmental increase in the GFAP content of the rat brain is due to accumulation of GFAP into the cytoskeleton. A small and constant amount of the total GFAP was detected in the soluble protein fraction. This GFAP had an apparent molecular mass (Mr) similar to that of the highest Mr form of GFAP detected in the cytoskeletal fraction. In contrast to the assays for cytoskeletal GFAP, no significant increase in the GFAP concentration of the soluble protein fraction could be measured during development. Sensitive, calibrated immunoblotting of cytoskeletal and soluble protein with [125I]protein A confirmed these findings, and showed that both cytoskeletal and soluble GFAP are first detected during the same period of foetal rat brain development. A finite and reproducible amount of lower Mr forms of GFAP were observed in the cytoskeletal fraction even when prepared in the presence of stringent proteolytic inhibitors. These presumed proteolytic degradation products of GFAP increased in abundance during development, parallel to the increase in cytoskeletal GFAP content of the rat brain. However, the abundant proteolytic degradation products of GFAP found in the cytoskeletal fraction were not detected in the soluble protein fraction at any age studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of Glial Fibrillary Acidic Protein Expression Following Kainate-Induced Cerebellar Lesion in Rats

In the present study glial fibrillary acidic protein (GFAP) expression was assessed following intravermian injection of kainic acid (KA) or physiological saline to adult rat cerebellum. After 2- to 30-day recovery period, free-floating sections cut with a microtome were obtained and were proccessed for immunocytochemistry against GFAP. Injection of both kainate and physiological saline elicited significant astrogliotic reaction, i.e. in the area around the lesion thick GFAP-positive Bergmann fibers with typical orientation appeared in the molecular and hypertrophied astrocytes abundantly appeared in the granular layer. However, following kainate intoxication lesion was not surrounded by typical demarcation glial scar during 30-day recovery period in contrast to the appearance of usual glial scar in the group injected with physiological saline, as early as 7-day postlesion. Preserved spatial organization of Bergmann fibers and the absence of typical demarcating glial scar after kainate-induced cerebellar lesion suggest distinct pattern of astrogliosis that presents an interesting model system to study the importance of glial scar in the recovery after ischemic brain insults.     

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.     

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.     

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.