Glial fibrillary acidic protein from bovin and rat brain Degradation in tissues and homogenates

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol. wt. polypeptide in bovine tissues incubated at 24 °C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000–40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 °C. At pH 8.0 proteolysis of the glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.     

Glial fibrillary acidic protein in the brain of prenatally stressed rats

The content of glial fibrillary acidic protein (GFAP) was studied in the brain structures of rats borne by intact females and females that underwent stress. In the offspring of stressed rats, the GFAP content in the brain gray and white matter on the 15th postnatal day noticeably dropped. On the 30th postnatal day, the GFAP content in the cortex and pons increased, while it somewhat decreased in the striatum and cerebellum. The results suggest that formation of the intermediate astrocyte filaments in the animals subjected to prenatal stress is markedly disturbed.     

Glial fibrillary acidic protein and differentiation of neonatal rat pituicytes in vitro

Summary The appearance and intracellular localisation of glial fibrillary acidic protein (GFAP) in pituicytes in neural lobe cultures of newborn rats aged 7 to 30 days were investigated by use of the indirect immunofluorescence method. GFAP-immunoreactive cells were observed mostly in the outgrowth zone. GFAP was localised in the perikaryal cytoplasm as well as in pituicyte processes. GFAP-positive pituicytes showed considerable morphological polymorphism. The presence of GFAP — astrocytic marker — in pituicytes in vitro and the evident morphological similarity to cultured astrocytes suggest the astroglial character of these cells.     

Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates.

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.     

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.     

Glial fibrillary acidic protein and desmin in salivary neoplasms

The presence of intermediate filament proteins (IFP) in normal salivary gland tissue and in a number of salivary gland neoplasms has been investigated by immunohistochemical techniques on frozen sections. Cytokeratins (CKs) were seen in almost all normal epithelial cells. In the parotid gland and in palatal gland tissue, a co-expression of cytokeratin and glial fibrillary acidic protein (GFAP) was seen in some myoepithelial cells, but this was not apparent in the submandibular gland. In some pleomorphic adenomas, carcinomas in pleomorphic adenomas, one mucoepidermoid carcinoma, one mucus-producing adenopapillary carcinoma and one adenoid cystic carcinoma, cells expressing three different IFP classes were found (CKs, vimentin, GFAP). These cells were most often situated peripherally in the tumour cords or ducts. The cytokeratin pattern in these cells, as revealed by mAbs PKK1-3, was similar to that in normal myoepithelial cells. Furthermore, reactivity for a fourth class of IFP, desmin, could be seen in this cell type in two carcinomas in pleomorphic adenomas, and also in a few cells in a pleomorphic adenoma and an adenoid cystic carcinoma. Thus the pattern of IFP expression in salivary gland neoplasms, is very complex, and cannot always be related to the normal tissue.     

Glial fibrillary acidic protein in regenerating teleost spinal cord

Immunohistological and ultrastructural studies were carried out on normal and regenerating spinal cord of the gymnotid Sternarchus albifrons, and in the brain and spinal cord of the goldfish Carassius auratus, to examine the distribution of glial fibrillary acidic protein (GFAP) in these tissues. Sections of normal goldfish brain and spinal cord exhibited positive staining for GFAP. In normal Sternarchus spinal cord, electron microscopy has revealed filament-filled astrocytic processes; however, such astrocytic profiles were more numerous in regenerated cord. Likewise, while normal Sternarchus spinal cord showed only a small amount of GFAP staining, regenerated cords were strongly positive for GFAP. Positive staining with anti-GFAP was observed along the entire length of the regenerated cord in Sternarchus, and was especially strong in the transition zone between regenerated and unregenerated cord. Both regeneration of neurites and production of new neuronal cell bodies occur readily in such regenerating Sternarchus spinal cords (Anderson MJ, Waxman SG: J Hirnforsch 24: 371, 1983). These results demonstrate that the presence of GFAP and reactive astrocytes in Sternarchus spinal cord does not prevent neuronal regeneration in this species.     

Glial fibrillary acidic protein is localized in the lens epithelium

The epithelium of the mouse lens stains intensely with antisera to glial fibrillary acidic protein (GFAP). A protein co-migrating with GFAP and immunoreactive with antisera to GFAP can be demonstrated in lens epithelium protein extracts by immunoblots. GFAP has previously been considered unique to cells of neural origin, but this study demonstrates that ectodermally derived cells express GFAP or a highly similar protein.     

Glial fibrillary acidic protein (GFAP)-like immunoreactivity in rat endocrine pancreas.

The study of intermediate filament expression in the pancreatic epithelium has been previously focused almost exclusively on cytokeratins. Transient vimentin immunoreactivity has also been detected in duct cells of rat fetal pancreas. Here we report that, in rat pancreas, intense GFAP-like immunoreactivity is detectable in a subpopulation of endocrine cells located in the periphery of the islet of Langerhans. In addition, staining appeared to be preferentially localized to the apical pole of the cells. Two different polyclonal antibodies were employed in this study, with analogous results. Staining of consecutive sections with anti-GFAP, anti-glucagon, and anti-somatostatin antibodies demonstrates that GFAP-like immunoreactivity is present in glucagon-secreting cells. The relevance of this finding is discussed. (J Histochem Cytochem 48:259-265, 2000)     

Glial fibrillary acidic protein is a major target of glycoxidative and lipoxidative damage in Pick's disease

Pick's disease is a subset of fronto-temporal dementia characterised by severe atrophy of the temporal and frontal lobes due to marked neuronal loss accompanied by astrocytic gliosis enriched in glial acidic protein. The remaining neurones have intracytoplasmic inclusions composed of hyperphosphorylated tau, called Pick bodies, in addition to hyperphosphorylated tau in astrocytes and oligodendrocytes. Gel electrophoresis and western blotting using markers of glycoxidation (advanced glycation end products, N-carboxyethyl-lysine and N-carboxymethyl-lysine: AGE, CEL, CML, respectively) and lipoxidation (4-hydroxy-2-nonenal: HNE, and malondialdehyde-lysine: MDAL) were used in the frontal and occipital cortex in three Pick's disease cases and three age-matched controls. In Pick's disease, increased AGE, CML, CEL, HNE and MDAL bands of about 50 kDa were observed in the frontal cortex (but not in the occipital cortex) in association with increased density of glial acidic protein bands. Bi-dimensional gel electrophoresis and western blotting also disclosed increased amounts and numbers of glial acidic protein isoforms in the frontal cortex in Pick's disease. Moreover, redox proteomics showed glycoxidation, as revealed with anti-CEL antibodies and lipoxidation using anti-HNE antibodies, of at least three glial acidic protein isoforms. The present results demonstrate that glial acidic protein is a target of oxidative damage in the frontal cortex in Pick's disease.     

Application of glial fibrillary acidic protein immunohistochemistry in the quantification of astrocytes in the rat brain

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was employed as a tool for quantification of astrocytes in the rat brain. One-micron-methacrylate sections were prepared from 70-micron slices stained for GFAP by using a preembedding staining procedure. Numbers/unit area of astrocytes and nonastrocytes were determined for cortex, corpus callosum, and hippocampal neuropil. In each, counts from GFAP-stained, toluidine-blue-counterstained sections were compared with counts obtained from sections stained with toluidine blue alone. Numbers of nonastrocytes and total glia in all three regions were comparable in both groups of sections. Astrocyte counts in the cortex and hippocampus also showed no significant differences between the two groups. In contrast, the number of astrocytes in the corpus callosum was significantly lower in GFAP-stained, toluidine-blue-counterstained sections than in sections stained with toluidine blue alone. GFAP immunohistochemistry is a useful tool for the quantification of astrocytes in semi-thin plastic sections of rat brain.     

The expression of glial fibrillary acidic protein in a rat cerebellar cell line

A rat cerebellar cell line, WC5, derived by transformation with Rous sarcoma virus, which is temperature-sensitive for transformation (ts-RSV), can be induced to express glial fibrillary acidic protein (GFAP). Immunofluorescence, radioimmune assay, and electron microscopy studies show that GFAP is expressed in WC5 cells grown at the nonpermissive temperature (NPT), but not at the permissive temperature (PT) for transformation. GFAP is first detectable about 3 days after incubating cells at the NPT, and reaches an apparent plateau by the seventh or eighth day. The expression of GFAP is reversible; shifting cells from the NPT to the PT causes a dramatic decrease in GFAP after 96 hr. In order to determine if the expression of GFAP is linked to the temperature-sensitive transforming activity of the viral src gene product, phenotype revertants of WC5 were established. By the criteria of morphology and growth in agar, the revertant lines, in contrast to the parent cell line WC5, were shown to exhibit a transformed phenotype at both the NPT and PT. Immunofluorescence studies on several of the revertant cell lines show that they do not express GFAP at either the PT or NPT. These findings suggest that the expression of GFAP in WC5 is linked to the expression of the src gene product. The advantage of using ts-RSV to derive neural cell lines which exhibit differentiated properties is discussed.     

Glial fibrillary acidic protein in the brain of the caecilian Typhlonectes natans (Amphibia,Gymnophiona): an immunocytochemical study

The astroglia of adult and juvenile (metamorphosed) Typhlonectes natans (Fischer) was investigated immunocytochemically with a monoclonal antibody directed against glial fibrillary acidic protein (GFAP). The astroglia of this member of the Order Gymnophiona of the class Amphibia is mainly composed of radial glial cells. Their somata limit the ventricles. They each give rise to a thick process that extends through the periventricular gray and arborizes within the neuropil. At the subpial surface, endfeet establish the membrana gliae limitans externa. Some extraependymal radial glial cells are immunoreactive, but no mammalian-like astrocytes are visualized. In the spinal cord, perikarya of radial glia are displaced from the GFAP-immunonegative ependyma. Perivascular endfeet and processes lining blood vessels are abundantly labeled. An increase in GFAP immunoreactivity extends from the exclusive labeling of subpial endfeet in newborn, recently metamorphosed animals, to the subsequent staining of distal processes and of the entire cell in older juveniles. The midline glia of the brainstem is immunoreactive at all ages examined. Strong glial wedges separate and delineate fiber tracts. Radial glial fibers in the habenulae are particularly thick and exhibit strong GFAP immunoreactivity, even in juveniles where GFAP immunoreactivity is otherwise minimal. The pattern of GFAP immunolabeling in the caecilian T. natans is similar to that in salamanders, but not to that in frogs.     

Developmental expression of the Glial fibrillary acidic protein (GFAP) gene in the mouse retina

1. In the nervous system, Glial fibrillary acidic protein (GFAP) is a well-known, cell type-specific marker for astrocytes. 2. In the mammalian retina, Muller cells, the major class of retinal glia, do not express GFAP or contain only low amounts of this protein. In retinas with photoreceptor degeneration, however, high levels of GFAP are found. It is possible that GFAP synthesis in these retinas could result from "dedifferentiation" of Muller cells as a consequence of disruption of normal neuron-glia interactions. 3. We have carried out immunocytochemical and in situ hybridization studies to examine whether GFAP or its mRNA is expressed by retinal cells early in embryonic development. 4. Our results show that GFAP-containing cells, which are probably astrocytes, are found only in the ganglion cell and nerve fiber layers and that these cells appear after postnatal day-1 (P-1) and continue to form until P-10. 5. Astrocyte formation starts from the optic disc and moves toward the periphery of the retina at a rate of approximately 160-200 microns per day. 6. An unexpected result from these studies is that GFAP mRNA levels are high in the first week of birth and decline rapidly as the animal develops. 7. Finally, we did not find either GFAP or GFAP mRNA in retinal cells other than astrocytes during normal development.     

Glial fibrillary acidic protein, J1-31 antigen and vimentin in adult hamster brain: an immunohistochemical study.

Different regions of the prosencephalon and mesencephalon of the adult hamster brain displayed differences in the immunofluorescence expression of astrocytic proteins, namely glial fibrillary acidic protein and J1-31 antigen (30 kD protein). Neither of these proteins could be detected in layers II-VI of the cerebral cortex. However, varying degrees of immunostaining were detectable in perivascular glia, stria medullaris thalamus, the basal cerebral peduncle and the dentate molecular layer of the hippocampus. Vimentin was conspicuous in neurons, particularly in the cerebral cortex and hippocampus, and in glial fibrillary acidic protein-positive astrocytes in major fibre tracts. These observations are discussed in relation to interspecies differences in the expression of intermediate filament proteins.     

Expression of glial fibrillary acidic protein in the developing human brain]

It was shown that the glial fibrillary acidic protein (GFAP) content in developing (fetal) human brain is sharply increased. The expression of GFAP was observed already on the 7th-8th week after gestation, the GFAP concentration being less than 0.05% in comparison with adult brain. GFAP can be immunohistochemically detected in radial glial cells. At early stages of development the presence of antigenic determinants of 68 kDa and 100 kDa polypeptides interacting with monoclonal antibodies alongside with native GFAP (51 kDa) and its low molecular weight forms was demonstrated. These antigenic determinants cannot be detected at later stages of development and are absent in adult brain. The data obtained testify to changes in the gene expression of intermediate filament proteins at early stages of human brain ontogenesis.     

Glial fibrillary acidic protein (GFAP) shows circadian oscillations in crayfish Procambarus clarkii putative pacemakers

Although several studies of glia have examined glial fibrillary acid protein (GFAP) and its relationship to the circadian rhythms of different organisms, they have not explored the daily GFAP oscillations in the putative pacemakers of the crayfish Procambarus clarkii or in other crustaceans. In this study we investigated the daily variations in GFAP concentrations in the eyestalk and brain, which are considered to be putative pacemakers in adult P. clarkii. In both structures, the glial GFAP was quantified using the indirect enzyme-linked immunosorbent assay (ELISA), and double labeling immunofluorescence was used to detect it and its co-localization with protein Period (PER), an important component of the circadian clock, in various regions of both structures. The ELISA results were analyzed using Cosinor and one-way ANOVA with Bonferroni and Scheffé’s post hoc tests. The results of this analysis showed that the GFAP levels present circadian oscillations in both structures. Moreover, GFAP was localized in different structures of the eyestalk and brain; however, co-localization with PER occurred only in the lamina ganglionaris, specifically in the cartridges of the eyestalk and in some of the cluster 9 brain cells. These results suggest that as in other invertebrates and vertebrates, glial cells could be involved in the circadian system of P. clarkii; however, thus far we cannot know whether the glial cells are only effectors, participate in afferent pathways, or are part of the circadian clock.