Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

Analysis and Comparison of In Vitro Synthesized Glial Fibrillary Acidic Protein with Rat CNS Intermediate Filament Proteins

Intermediate filament (IF) proteins from rat spinal cord were analyzed by two-dimensional gel electrophoresis and compared with the in vitro translation products of a messenger RNA-dependent reticulocyte lysate system stimulated with 16-day-old rat brain polysomes. In two dimensions, the molecular weight 49,000 to 50,000 band of the IF preparation resolved to seven spots, whereas antiserum to glial fibrillary acidic (GFA) protein precipitated only two immediately adjacent radiolabeled in vitro synthesized products, with molecular weights of 49,000 to 50,000. Autoradiographs of two-dimensional gels of extracted IF proteins incubated with iodinated IgG fraction of GFA protein antiserum showed that all seven spots were recognized by the antiserum. These observations suggest that the primary gene product of GFA protein is modified either by post-translational processing or experimental artifact.     

Expression of Glial Fibrillary Acidic Protein in Rat C6 Glioma Relates to Vimentin and Is Independent of Cell-Cell Contact

Glial fibrillary acidic protein (GFAP) was induced in rat C6 glioma cells grown in M199 and HAM F10 media by addition of 1 mM dibutyryl cyclic AMP. The amount of GFAP per cell increased 7- and 33-fold in M199 and HAM F10 media, respectively. GFAP could be induced in each phase of the cell culture except for the lag phase, where GFAP synthesis was delayed until the onset of the logarithmic growth. The induction took place under conditions where the total protein content of the cell decreased. Measurement of the amount of vimentin indicated that GFAP was induced under conditions of low vimentin concentration. Our results do not support the hypothesis that GFAP induction depends on cell-cell contact or cell proliferation. They indicate a shift from vimentin to GFAP synthesis by an as yet unknown mechanism.     

Withaferin A Targets Intermediate Filaments Glial Fibrillary Acidic Protein and Vimentin in a Model of Retinal Gliosis

Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in the corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and down-regulates soluble vimentin and GFAP expression to cause cell cycle G₀/G₁ arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.     

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.     

Simultaneous Separation and Purification of Neurofilament and Glial Filament Proteins from Brain

Neurofilaments (NF) and glial filaments (GF) were purified from bovine brain by the axonal flotation method, followed by hydroxylapatite chromatography in 8 M-urea. The proteins were shown to be competent to reassemble into intermediate filaments with removal of the denaturant, and reassembly was used as the final step in the purification of the filament proteins. The reassembly was found to be dependent on ionic strength and pH. This dependence was greater for neurofilaments than for the glial filaments. The NF and GF preparations were found not to be contaminated with each other by their gel electrophoretic profile and their immunological distinctness. The filament proteins can be obtained in high yield, and remain in solution if the urea is removed by dialysis against a low-ionic-strength buffer. Hence, they can provide a source for further biochemical studies.     

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.     

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.     

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.     

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.     

Phorbol Myristate Acetate and 8-Bromo-Cyclic AMP-Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites

Both the protein kinase C (PK-C) activator, phorbol 12-myristate 13-acetate (PMA), and the cyclic AMP-dependent protein kinase (PK-A) activator, 8-bromo-cyclic AMP (8-BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8-BR results in the morphological transformation of flat, polygonal-shaped cells into stellate, process-bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8-BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8-BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8-BR treatment was restricted to serine residues in the N-terminal domain of these proteins. Studies were also conducted to compare the two-dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8-BR with those produced when the proteins were phosphorylated with purified PK-C or PK-A. PK-C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK-C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of Glial Fibrillary Acidic Protein in Rat C6 Glioma in Chemically Defined Medium: Cyclic AMP-Dependent Transcriptional and Translational Regulation

Glial fibrillary acidic protein (GFA) expression was induced in rat C6 glioma in chemically defined medium by the addition of N6, O2'-dibutyryl cyclic AMP (dbcAMP). Induction was dependent on the increase in intracellular cyclic AMP (cAMP), which was linearly correlated with added dbcAMP. Contrary to GFA mRNA synthesis, which can be obtained by cAMP-dependent and -independent pathways, translation of mRNA into GFA was observed only above a cellular cAMP concentration of approximately 0.2 fmol/cell. dbcAMP stimulation did not affect the vimentin concentration, which remained at a low level, but changed the cellular morphology from a bipolar to a stellate shape. A similar morphological change was observed after stimulation of C6 with lipopolysaccharide (LPS). However, LPS did not significantly increase the intracellular concentration of cAMP and the LPS-induced mRNA was not translated into GFA. Our results indicate that GFA synthesis is regulated at the mRNA level and at the translational level and that a cAMP-dependent mechanism determines the ultimate synthesis of GFA by a yet unknown mechanism.     

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.     

Study of Glial Fibrillary Acidic Protein in a Human Glioma Cell Line Grown in Culture and as a Solid Tumor

Abstract: A continuous human glioma cell line grown in culture and as a solid tumor was analyzed for glial fibrillary acidic (GFA) protein. This material provided a rich source for GFA protein that could also be manipulated and controlled. Immunoperoxidase staining at the light and electron microscopic levels revealed that the cell culture and tumor specimens were strongly positive for GFA protein. When aqueous soluble fractions of the cell culture and tumor were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose and stained immunochemically, they contained exclusively low molecular weight (41–43 K-dalton) GFA peptides. SDS (0.15%)-soluble fractions contained either low molecular weight only (culture) or a mixture of peptides ranging from 41 to 49K daltons. SDS (1%) extracts of either cell culture or tumor contained only 49K dalton GFA protein. Two-dimensional gel separation revealed that the GFA protein extracted from either the culture or tumor with 1% SDS resolved to two or three spots at pH 5.8. Low molecular weight GFA peptides (<49K daltons) in aqueous and 0.15% SDS-soluble extracts became increasingly more acidic with decreasing molecular weight. The extremely rapid degradation seen suggests that this cell line may be a valuable system for further study of intermediate filament protein turnover.     

Enzyme-Linked Immunosorbent Assay for the Human Glial Fibrillary Acidic Protein Using a Mouse Monoclonal Antibody

Two enzyme-linked immunosorbent assays (ELISAs) have been developed for the quantification of soluble human glial fibrillary acidic protein (GFAP). The specificity of the assays for GFAP is ensured by the use of a monoclonal antibody directed against a GFAP-specific antigenic determinant. One ELISA is a four-layer system working in the concentration range 5-600 ng GFAP/ml. The other ELISA is a five-layer system and includes a biotin/avidin binding reaction. The latter assay has a working range of 0.5-60 ng GFAP/ml. The assays may be used for quantification of GFAP in CSFs, amniotic fluids, and extracts or homogenates of normal and pathological brain material. GFAP in serum could not be quantified because of unidentified interference. CSFs from 18 nonneurological subjects were found to contain 2-14 ng GFAP/ml (mean 4.1 ng/ml), whereas amniotic fluids from 50 normal pregnant women contained up to 24 ng GFAP/ml (mean 12.4 ng/ml). GFAP concentrations in CSFs from 32 multiple sclerosis patients were found not to be elevated compared to the control group.