Fung-Chow Chiu

Quantitative aspects of reactive gliosis a review

Recent studies of gliosis in a variety of animal models are reviewed. The models include brain injury, neurotoxic damage, genetic diseases and inflammatory demyelination. These studies show that reactive gliosis is not a stereotypic response, but varies widely in duration, degree of hyperplasia, and time course of expression of GFAP immunostaining, content and mRNA. We conclude that there are different biological mechanisms for induction and maintenance of reactive gliosis, which, depending on the kind of tissue damage, result in different expressions of the gliotic response.

The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acidic protein, and the fibroblast-type filament protein, vimentin.

Abstract: Primary astrocytes were cultured from the forebrains of 1‐day‐old rats. Immunofluorescence microscopy showed that approximately 80% of the cells were positive for glial fibrillary acidic protein (GFAP) and >80% were stained with an antiserum to the molecular weight 58,000 fibroblast intermediate filament protein (vimentin). Gel electrophoresis of Triton‐insoluble cytoskeleton preparations from these cultures revealed three major bands having molecular weights of 58,000, 51,000, and 42,000, together with some prominent lower‐molecular‐weight species. The protein of molecular weight 51,000 was not present in preparations from fibroblasts. Each of the three major astrocyte proteins was subjected to limited proteolysis, while two of the proteins were cleaved by cyanogen bromide. The electrophoretic peptide patterns of the 58,000 protein were similar to those of vimentin isolated from NIL‐8 fibroblasts, and the patterns of the 51,000 protein were similar to those of GFAP isolated from rat spinal cord. The patterns of the protein of molecular weight 42,000 resembled those of muscle actin. Rocket immunoelectrophoresis showed that the 51,000 astrocyte protein reacted with an antiserum to bovine GFAP, but the 58,000 and 42,000 proteins failed to react. We conclude that the major proteins of cytoskeleton preparations from cultured primary astrocytes are vimentin (58,000), GFAP (51,000), and actin (42,000), and that our data show no obvious structural relationship among them.

Synthesis and Turnover of Cytoskeletal Proteins in Cultured Astrocytes

Abstract: We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X‐100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short‐term incorporation experiments vimentin, GFAP, and actin in the Triton‐insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast‐decaying pool with a half‐life of 12–18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half‐life of about 8 days.

Bulk Preparation of CNS Cytoskeleton and the Separation of Individual Neurofilament Proteins by Gel Filtration: Dye-Binding Characteristics and Amino Acid Compositions

Abstract: The three major proteins of mammalian neurofilaments, of molecular weight 70,000, 160,000, and 210,000, have been resolved by sodium dodecyl sulfate (SDS)‐polyacrylamide gel eJectrophoresis, and more recently, by ion‐exchange chromatography in urea solution. We describe here a method to separate the neurofilament proteins by gel filtration without the use of SDS. A bulk preparation of cytoskeleton from rat spinal cord was first characterized. This preparation was then solubilized in a buffer containing 8 M urea and subjected to gel filtration. Individual neurofilament proteins, in milligram quantities, were harvested following the pooling of appropriate fractions. Gel electrophoresis showed a high degree of homogeneity in each of the three pooled fractions. Dye binding studies demonstrated that the protein of molecular weight 210,000 was relatively underrepresented when stained with Coomassie Blue, while all three neurofilament proteins showed similar dye binding properties with Fast Green. Amino acid analysis indicated that (1) all three neurofilament proteins contained a high content of acidic residues; (2) the molecular weight 210.000 protein contained >8 mol% proline; and (3) no simple oligomeric relationship existed among the neurofilament triplets.

Biochemical and immunocytochemical changes in glial fibrillary acidic protein after sab wounds

Changes of glial fibrillary acidic protein (GFAP) in the forebrain of rats with stab wounds were determined by quantitative immunoblots and by immunohistochemistry. Bilateral stab wounds were made stereotaxically in the cortex and hippocampus. In control rats, the scalp was retracted and depressions were etched on the intact skull. At various times up to 21 days postoperation, one cerebral hemisphere was homogenized, proteins were separated by polyacrylamide gel electrophoresis and immunoblots were quantitated by densitometry. The contralateral hemisphere was immunostained for GFAP. Three hours postoperation GFAP+ cells were detected around the wound but there was no increase of total GFAP. At 6 h postoperation total GFAP in the forebrain decreased to 80% of the sham-operated control value and the number of GFAP+ cells was lower, compared to the controls, in layer 1 of the cortex, corpus callosum, cingulum, external capsule, internal capsule, hippocampus, optic tracts and around blood vessels. This early relative decrease in GFAP levels was actually due to an increase in GFAP in the sham-operated controls, which mounted a stronger gliotic response during the first 24 h. In neither group of animals did the GFAP levels drops below those of intact unoperated animals. At 24 h total GFAP began to increase. The number and intensity of reactive glia in the vicinity of the wound increased steadily, appearing to reach a maximum at about 7 days, then declining significantly by 21 days. The glial reaction was most pronounced in the hippocampus. Total GFAP reached 180% of the control value by 7 days and then declined to 117% by 21 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Dibutyryl cyclic AMP causes intermediate filament accumulation and actin reorganization in astrocytes

We have examined the effects of dibutyryl-cyclic AMP (dBcAMP) on the organization and expression of filamentous proteins in astroglia. The drug produced several effects on astrocytes grown in primary cultures. Cultures ceased to grow, and cells changed shape to a contracted form, displaying thin cytoplasmic processes. Cellular levels of the intermediate filament (IF) proteins, vimentin and glial fibrillary acidic protein (GFAP), and actin, insoluble in Triton X-100, were examined by polyacrylamide gel electrophoretic analysis. The cellular content of both of the IF proteins increased concurrently, approximately doubling during a 2-week course of treatment. The content of actin associated with the Triton residue decreased, however, a biochemical alteration which correlated with a loss of stress fibers in treated cells. Treatment with sodium butyrate did not change either cell shape or cytoskeletal protein content. Filament protein expression in astrocytes can, therefore, be modulated via cAMP-dependent mechanisms. The effects do not, however, appear specific for the GFAP-type of intermediate filament.

Glial fibrillary acidic protein increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis.

Abstract: Glial fibrillary acidic protein (GFAP) in the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) was quantitated by densi‐tometry of both stained gels and immunoblots of electro‐phoretically separated cytoskeletal proteins. The experimental period ranged from 7 to 65 days postinocula‐tion (dpi). Greater than 92% of the total spinal cord GFAP was recovered in the Triton‐insoluble cytoskeletal pellet; less than 2% was truly soluble. GFAP increased gradually and significantly with time, reaching a level one‐and‐a‐half to two times greater than that of controls by 35 dpi and remaining elevated at 65 dpi. In EAE animals, GFAP was 33% of the total Triton‐insoluble protein (excluding his‐tones and other small basic proteins) at 7 dpi, rising to 48% at 65 dpi. Increases in vimentin were also noted, following a time course similar to that of GFAP. An increase in immu‐nocytochemical staining of GFAP was noticeable at 10 dpi and became marked at 14 dpi, a time before GFAP levels had increased significantly. Thus, enhanced staining at the peak of the disease cannot be explained simply by an increase in antigen protein. Other possible explanations, such as an increase in soluble GFAP content, proteolytic degradation, or modifications in the immunochemical properties of GFAP in EAE animals, were ruled out. Both the biochemical and immunocytochemical increases in GFAP persisted through 65 dpi, even though the animals recovered from clinical signs at approximately 18 dpi.

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.

Intermediate Filaments from Bovine, Rat, and Human CNS: Mapping Analysis of the Major Proteins

Abstract: Intermediate filaments were isolated by an axon‐flotation method from bovine, rat, and human CNS. Gel electrophoresis showed four major proteins, having molecular weights of about 50,000, 70,000, 160,000, and 210,000, to be present in filaments of all three species. Small differences in molecular weights and major differences in relative distribution of the filament proteins were observed among species. In bovine and rat brain the predominant protein was the 50,000 band, but in human brain the 70,000 band was present in greatest amount. Each filament protein of the three species was studied by peptide mapping using limited proteolysis and cyanogen bromide cleavage. Within the same molecular weight group, filament proteins from different species gave similar maps with both techniques. Some degree of heterogeneity was also observed. However, filament proteins of different molecular weights of the same species gave distinctly different maps. These studies rule out the possibility that filament proteins from different molecular weight groups are related to each other by oligomerization; nor is it likely that the lower molecular weight proteins are derived from the subunit of molecular weight 210,000.

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 the alpha 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.