Glial Fibrillary Acidic Protein Synthesis Research Articles

Reduction in glial fibrillary acidic protein mRNA abundance induced by (-)-deprenyl and other monoamine oxidase B inhibitors in C6 glioma cells.

Gliosis is commonly observed in the CNS following tissue damage, and it also occurs in aging and in many neurodegenerative diseases. Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. An inhibition or delay in GFAP synthesis could mitigate scar formation and thus reduce the formation of a physical barrier. The consequence of this would be to allow neurons and oligodendrocytes to reestablish a functional environment. (-)-Deprenyl, a specific monoamine oxidase (MAO) B inhibitor, has been used as an effective antiparkinsonian drug, and it has been reported to possess neuroprotective and neurorescue properties. Using northern and slot blots to detect GFAP mRNA in C6 glioma cells, we have demonstrated that (-)-deprenyl decreases the abundance of GFAP mRNA in a time- and dose-dependent manner. The effect seems to be specific to MAO B inhibitors because (+)-deprenyl and clorgyline exhibit no effect. This study indicates therefore that (-)-deprenyl may be useful for regulating astrogliosis following CNS injury as well as in some neurodegenerative diseases.

Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis

Astrogliosis is a predictable response of astrocytes to various types of injury caused by physical, chemical, and pathological trauma. It is characterized by hyperplasia, hypertrophy, and an increase in immunodetectable glial fibrillary acidic protein (GFAP). As GFAP accumulation is one of the prominent features of astrogliosis, inhibition or delay in GFAP synthesis in damaged and reactive astrocytes might affect astrogliosis and delay scar formation. The aim of this study is to investigate the possibility of utilizing antisense oligonucleotides in controlling the response of astrocytes after mechanically induced injury. We scratched primary astrocyte cultures prepared from newborn rat cerebral cortex with a plastic pipette tip as an injury model and studied the astrogliotic responses in culture. Injured astrocytes became hyperplastic, hypertrophic, and had an increased GFAP content. These observations demonstrate that injured astrocytes in culture are capable of becoming reactive and exhibit gliotic behaviors in culture without neurons. The increase in GFAP content in injured astrocytes could be inhibited by incubating the scratched culture with commercially available liposome complexed with 3' or 5' antisense oligonucleotides (20 nt) in the coding region of mouse GFAP. The scratch model provides a simple system to examine in more detail the mechanisms involved in triggering glial reactivity and many of the cellular dynamics associated with scar formation. Antisense oligonucleotide treatment could inhibit the GFAP synthesis in injured astrocytes, hence it may be applicable in modifying scar formation in CNS injury in vivo.

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.

Inhibition of GFAP synthesis by antisense RNA in astrocytes

Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. The inhibition or delay in GFAP synthesis might delay scar formation resulting from an insult such as spinal cord injury or central nervous system (CNS) demyelination. The delay in the formation of a physical barrier might allow the neurons and oligodendrocytes to reestablish a functional environment. We delivered antisense GFAP RNA complexed with Lipofectin (LF), a cationic liposome, into cerebral astrocytes in culture and tested the feasibility of inhibiting GFAP synthesis. Our results demonstrate that LF facilitated antisense RNA uptake into astrocytes. Astrocytes took up 3H-antisense GFAP RNA alone and reached an equilibrium of 7-8.8 eta g per mg protein after 2.5 hr. When complexed with LF, astrocytes could increase the uptake to 14 eta g per mg protein and the time for reaching this quantity was shortened to 10 min. This uptake level was further enhanced if experiments were carried out in HEPES buffered saline (HBS). All uptake studies were dose- and time-dependent. Dibutyryl cyclic AMP (dBcAMP) is known to induce an increase of GFAP content in cultured astrocytes. We studied the effect of LF/antisense GFAP RNA on the GFAP content in dBcAMP (0.25 mM)-treated astrocytes. Cultures of astrocytes treated with dBcAMP contained almost twice as much GFAP as untreated cultures after 2 days. Similar cultures treated with LF/antisense RNA in HBS did not show an increase but a 30-40% decrease in GFAP content 2 days after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of human cDNA and genomic clones for glial fibrillary acidic protein

Both a partial cDNA clone and a complete genomic clone have been isolated for human gfa, the gene encoding the major component of astrocyte intermediate filaments, glial fibrillary acidic protein (GFAP). the nucleotide sequence of the entire coding region and 102 bp of the 5′ flanking DNA was determined. The mRNA start site was identified by primer extension and probe protection experiments, and a novel in vitro transcription and translation procedure was then used to established that the first ATG in the mRNA initiates GFAP synthesis. The predicted amino-terminal sequence for human GFAP differs greatly from that previously deduced for mouse GFAP from its gene sequence, despite otherwise high homology. This discrepancy was resolved by determining that the published mouse genomic sequence has an incorrect additional base. The corrected sequence produces strong homology between human and mouse GFAP in their predicted amino acid sequences, and suggests that human and mouse GFAP initiate at homologous positions. The beginning sequence deduced here for both proteins is matched closely by that previously obtained for porcine GFAP by direct sequencing of its amino-terminal end. This supports the protein initiation sites proposed, and also indicates that GFAP is not processed at its amino-terminal end.

Synthesis and phosphorylation of the glial fibrillary acidic protein during brain development: A tissue slice study

Brain slices were incubated with either [3H] amino acids or [32P] orthophosphate in order to characterize the synthesis and phosphorylation of the glial fibrillary acidic protein (GFAP) in the rat nervous system. The incorporation of [3H] amino acids into GFAP was found to increase significantly during early postnatal development, reaching a peak of activity on day 5 of life and then declining over the next 2 weeks. Concomitant with this peak of synthetic activity the content of GFAP in rat brain was also observed to increase dramatically. GFAP continued to accumulate in brain through postnatal day 30 despite a decrease in the synthesis of the protein. These results indicate that the increase in GFAP during the first month of life cannot be ascribed solely to the rate of GFAP synthesis. The findings are consistent with the hypothesis that during later stages of astrocytic development the accumulation of GFAP may be primarily dependent upon a low rate of protein degradation. The pattern of GFAP phosphorylation in the developing rat brain differed from that observed for the incorporation of [3H] amino acids. The peak incorporation of 32P into GFAP occurred on postnatal day 10 at a time when synthesis of the protein had declined by 43%. These findings suggest that during development phosphorylation of GFAP is mediated by factors different from those directing its synthesis. In addition, phosphorylation of GFAP did not alter its solubility in cytoskeletal preparations indicating that GFAP phosphorylation is probably not a major regulatory mechanism in disassembly of the astroglial filaments.

Neuronal modulation of Schwann cell glial fibrillary acidic protein (GFAP).

Adult rat sciatic nerves contain cytoskeletal peptides that resemble CNS glial fibrillary acidic protein (GFAP) in immunoreactivity and molecular weight. Immunohistological examination of teased nerve fascicles indicated that these peptides are expressed selectively by Schwann cells related to small axons. Radiolabelled mouse and rat CNS GFAP cDNA probes hybridized with a single, 2.7 kb RNA band in Northern blots prepared from total RNA from both rat sciatic nerve and rat brain. Sciatic nerve GFAP mRNA was detectable by this means in adult, 2 month, or 21 day postnatal rats, but not in 3,6, or 10 day postnatal rats. Sciatic nerve transection caused a marked reduction in the level of GFAP mRNA in the axotomized distal stump. We conclude that Schwann cell synthesis of GFAP is developmentally regulated and that Schwann cells, unlike astroglia, require continued trophic input from small axons in order to express GFAP.

The postmortem Alzheimer brain is a source of structurally and functionally intact astrocytic messenger RNA

Although the precise role of astrocytes in the pathogenesis of Alzheimer's disease (AD) is currently undefined, studies carried out at the molecular level may lead to new insights into the functioning of this class of brain cells in dementia. In order to facilitate such investigations, methods are described that establish that structurally and functionally intact messenger RNA (mRNA) for an astrocytic marker, glial fibrillary acidic protein (GFAP), is present in the postmortem Alzheimer's disease brain after long postmortem intervals. Rapid preparative procedures were used to obtain poly(A) + RNA from postmortem control and AD cortices. In vitro protein synthesis was carried out in a reticulocyte system. Relative to controls. AD mRNA synthesized a two-fold higher level of a 50,000 mol.wt. protein that was immunologically identified as GFAP. High levels of GFAP synthesis by purified mRNA from AD cortices was independent of age at death and postmortem interval up to 24 h. Northern blot hybridization using a cloned human GFAP riboprobe was used to evaluate postmortem GFAP mRNA stability. No appreciable degradation products of GFAP mRNA were observed on Northern blots for at least 10 h postmortem in poly(A) + RNA extracted from the AD brain. The described methodology demonstrates that the postmortem AD brain is an excellent source of functionally and structurally intact astrocyte-specific mRNA.

Increased glial fibrillary acidic protein synthesis in astrocytes during retrograde reaction of the rat facial nucleus.

Glial fibrillary acidic protein (GFAP) increases in astrocytes following axotomy of facial motoneurons. In the present study we quantified GFAP synthesis both in regenerating facial nuclei after nerve crush and in nonregenerating facial nuclei after nerve resection. An increase in GFAP synthesis during regeneration occurs as early as 24 h after the axotomy. Thus, the increase in the astrocytic GFAP synthesis seems to be the earliest glial response to retrograde changes in facial motoneurons.

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.

Chapter 37 Astrocyte activation and fibrous gliosis: glial fibrillary acidic protein immunostaining of astrocytes following intraspinal cord grafting of fetal CNS tissue

This chapter highlights some basic facts pertaining to glial intermediate filaments with emphasis on the chemistry and metabolism of their major constituent protein, glial fibrillary acidic protein (GFAP). Gliosis is usually characterized by extensive astroglial proliferation and hypertrophy. In addition, reactive astrocytes undergo numerous cytological and histochemical features, including increases in nuclear diameter, elevated DNA levels, an accumulation of intermediate filaments, heightened oxidoreductive enzyme activity, and increased synthesis of GFAP, vimentin, glutamine synthetase, and glycogen. In principle, fibrous gliosis can be considered as a part of an important healing response to a central nervous system (CNS) injury because astrocytes are thought to actively monitor and control the contents of the extracellular space of the CNS, including the amounts of ions, transmitters, trophic factors, nutrients, and waste materials. They also play a role in the removal of myelin and neuronal debris and encapsulate the regions of the CNS that are exposed to non-CNS tissue environments following trauma. The most prominent characteristic of fibrous gliosis is an extensive synthesis of intermediate filaments.

Astrocytic reactivity and intermediate filament metabolism in experimental autoimmune encephalomyelitis: The effect of suppression with prazosin

In either actively or passively transferred experimental autoimmune encephalomyelitis (EAE), increased immunocytochemical staining of glial fibrillary acidic protein (GFAP) in astrocytes was detected early in the disease process in both the gray and white matter of the spinal cord. Staining was not restricted to areas of perivascular mononuclear infiltration, and was observed at all levels of the cord. This enhanced staining pattern was delayed in rats in which clinical signs of EAE had been suppressed by treatment with theα 1-adrenoceptor antagonist prazosin. This glial reaction in EAE was not accompanied by increased GFAP synthesis, as measured by in vitro labeling of spinal cord slices, nor an increase in GFAP content, as measured by densitometry of intermediate filament fractions separated by polyacrylamide gel electrophoresis. Total protein synthesis was increased, with vimentin being labeled especially heavily; in prazosin-treated EAE animals, the increase in total protein synthesis was reduced and delayed.

Immunoblot identification of glial fibrillary acidic protein in rat sciatic nerve, brain, and spinal cord during development.

The appearance of the glial fibrillary acidic protein (GFAP) during embryonic and postnatal development of the rat brain and spinal cord and in rat sciatic nerve during postnatal development was examined by the immunoblot technique. Cytoskeletal proteins were isolated from the central and peripheral nervous system and separated by SDS slab gel electrophoresis or two-dimensional gel electrophoresis. Proteins from the acrylamide gels were transferred to nitrocellulose sheets which were treated with anti-bovine GFAP serum and GFAP was identified by the immunoblot technique. GFAP was present in the embryonic rat brain and spinal cord at 14 and 16 days of gestation respectively. The appearance of GFAP at this stage of neural development suggests that the synthesis of GFAP may be related to the proliferation of radial glial cells from which astrocytes are derived. It is also feasible that GFAP provides structural support for the radial glial cell processes analogous to its role in differentiated astrocytes. GFAP was found to be present in rat sciatic nerves at birth and at all subsequent stages of development. These results indicate that some cellular elements in the rat sciatic nerve, such as Schwann cells, are capable of synthesizing GFAP which is immunochemically indistinguishable from its counterpart in the central nervous system. Thus it appears that GFAP is present both in the central and peripheral nervous system of the rat when the glial cells synthesizing GFAP are still undergoing differentiation.

Regulation of glial fibrillary acidic protein (GFAP) expression in CNS development and in pathological states

Studies that have described changes in glial fibrillary acidic protein (GFAP) expression during CNS development and during pathological reactions are summarized. We describe our own studies examining GFAP expression in vitro. In primary cultures established from newborn rat forebrain and maintained in the presence of serum, GFAP expression is first observed in cells from the subventricular germinal zone. Glial precursor cells, at an uncommitted stage, can be induced by serum to begin GFAP synthesis. In astrocytes already established in culture, GFAP synthesis can be further increased by dibutyryl cyclic AMP (dbcAMP). By labelling astrocytes with radioactive methionine, we demonstrate increases of GFAP and vimentin synthesis within 48 h of exposure to dbcAMP. Continuous exposure results in a gradual rise in the cellular level of GFAP.

Fetal calf spinal cords as enriched source of mRNA encoding glial fibrillary acidic protein.

Total poly(A)+ RNA was isolated from fetal calf spinal cord, adult rat spinal cord, and young rat brain, and was translated using the rabbit reticulocyte lysate system. The amount of glial fibrillary acidic protein in the translation products was measured by immunoprecipitation with antiserum against glial fibrillary acidic protein. RNA from fetal calf spinal cord could direct glial fibrillary acidic protein synthesis such that this protein comprised approximately 1.4% of the total products. RNAs from adult rat spinal cord and brain could direct glial fibrillary acidic protein synthesis much less efficiently, with this protein comprising less than 0.3% of the total products. These results suggest that the gene for glial fibrillary acidic protein is strongly expressed in fetal calf spinal cord and that this tissue is an enriched source of mRNA encoding glial fibrillary acidic protein.

Immunoperoxidase study of astrocytic reaction in Junín virus encephalomyelitis of mice.

Intracerebral inoculation of strain XJ Clon 3 of Junín virus into 1-2-day-old mice resulted in the appearance of viral antigen, detectable by means of the peroxidase antiperoxidase (PAP) technique, within the cytoplasm of the neurons of the cerebral cortex, the basal nuclei, cerebellum, pons, medulla, spinal cord, and spinal ganglia; Junín antigen was likewise observed, although to a lesser extent, in the cytoplasm of astrocytes. The viral antigen was found in highest concentration at the cytoplasmic periphery, near the cell membrane, where complete virions are formed by budding. In some cells a "tigroid" distribution of the antigen was observed, suggestive of its production and concentration within the granular endoplasmic reticulum. In spite of the heavy infection of the neural structures, the neurons did not always show major alterations. By immunolabeling of the glial fibrillary acidic protein (GFAP) by the PAP method, a severe glial response could be seen in infected mice, featuring hyperplasia, hypertrophy, and shape distortion of the astrocytes. This easy labeling was not observed in normal mice of the same age, and suggests the accelerated maturation of the astrocytes and the increased GFAP synthesis by direct action of the virus upon such cells. In view of its specificity, use of the PAP technique for GFAP immunolabeling will most likely replace the traditional metal impregnation methods in the study of the astrocytes. Its utilization would be indicated whenever astroglial changes are suspected in any CNS pathologic condition.

The presence of glial fibrillary acidic protein in the human pituitary gland.

The presence of glial fibrillary acidic protein (GFAP) was studied in human pituitary glands with the peroxidase-antiperoxidase (PAP) method. Positive reaction was observed in cells and processes of the neurohypophysis, in occasional cells lining the Rathke's cysts of the pars intermedia, and in scattered star-shaped cells and small follicles of the pars distalis. GFAP immunoreactivity was sparse and variable in amount from case to case. An increase in GFAP-immunoreactivity was observed as a reaction to injury. GFAP-positive cells were seen within and around pituitary adenomas regardless of their secretory cell type. Evidence is presented to indicate that these cells do not contain conventional pituitary hormones. It is postulated that the GFAP-positive cells of the pars distalis are nonsecretory elements, identical to the folliculostellate cells. They may become visible by immunostaining following increased synthesis of GFAP. The latter may be a response to cell injury or metabolic changes in adjacent secretory elements. A similar reaction in pituicytes may explain the appearance of immunoreactive GFAP in the neural lobe. The presence of GFAP in the adenohypophysis suggests that some of their cells are neuroectodermal in origin.

Quantitative increase of neuroglia-specific GFA protein in rat C-6 glioma cells in vitro.

AMONG neural cell lines the C-6 glioma, a cell line cloned from an N-methylnitrosourea (MNU)-induced experimental glioma in rat, has generated considerable interest as a model system for the study of neuroglia1. The clone was originally selected on the basis of a high production of the protein S-100, a protein believed to be characteristic of glial cells in vivo2. Since its introduction, the line has been further characterised with regard to a number of other biochemical features3. Among these is the recent demonstration of the glial fibrillary acidic (GFA) protein by immunofluorescent staining of C-6 cells maintained in certain in vitro conditions4. The GFA protein, isolated from multiple sclerosis plaques and areas of sub-ependymal gliosis in the central nervous system, has been shown to be a specific marker for fibrillary astrocytes, is believed to be a component of the 80–90 A diameter glial filaments5–8, and has not been reported in non-glial neural cell lines. It is, therefore, a useful biochemical marker for the differentiation of a type of mature astrocyte. In previous work with the C-6 line, GFA protein was found to be present by immunofluorescence in only an occasional C-6 cell maintained in monolayer or suspension cultures, and to be markedly increased in many C-6 cells when they were maintained for 12–14 d in an organ culture system using Gelfoam matrices4. Such a system is known to favour structural differentiation in vitro9. No increase of filaments was observed, however, in any of the in vitro systems used, suggesting that the synthesis of GFA protein could proceed without the formation of intracytoplasmic glial filaments4. We compared the quantitative levels of GFA protein in C-6 and other cell types in various culture conditions, using a “two-site” immunoradiometric assay recently developed in this laboratory10.