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Primary astrocytes were cultured from forebrains of 1-day-old rats. Immunofluorescence microscopy showed that approximately 80% of the cells were positive for glial fibrillary acidic protein (GFAP) and greater than 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 action. 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.

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.

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 electrophoresis, 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 greater than 8 mol% proline; and (3) no simple oligomeric relationship existed among the neurofilament triplets.

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.

Gomphoside, a 5 alpha-H cardiac glycoside isolated from Asclepias fructicosa, has an unique double glycosidic linkage to the aglycon through oxygen atoms at 2 alpha and 3 beta of the steroid. The 3'-axial hydroxyl of its conformationally rigid sugar residue appears to be the functional group responsible for its potent inotropic activity. With use of gomphoside as the model compound, the conformation of the flexible glycosidic linkage of the 5 beta-H cardenolides, digitoxigenin alpha-L-rhamnoside and digitoxigenin beta-D-digitoxoside, and the 5 alpha-H cardenolides, uzarigenin alpha-L-rhamnoside and uzarigenin beta-D-6-deoxyalloside, were investigated with the aid of computer graphics and conformational potential energy calculations. The relative inotropic potencies of these cardenolides can be accounted for by considering their active binding conformations with their potential energy distributions. The conformational distribution of the glycosidic moiety was postulated to be the major determinant of the biological activity of these cardenolides.

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.

A 66 kd protein, pl 5.4, was purified from the Triton-insoluble fraction of rat spinal cord. This protein formed 10 nm filaments in vitro. The 66 kd protein was unique, although it shared homology with the 70 kd neurofilament protein (NF-L) and vimentin. An antiserum (anti-66) specific to the 66 kd protein did not cross-react with any of the neurofilament triplet proteins. In the spinal cord, anti-66 intensely stained the axons of the anterior and lateral columns. However, afferents from dorsal root ganglia and the efferents from the motoneurons were negative. In the cerebellum, anti-66 intensely stained most axons. The 66 kd protein was readily detectable in homogenates of forebrain, cerebellum, brainstem, and spinal cord, but was found only in trace amounts in adult sciatic nerves and was not found in extraneural tissues. The 66 kd protein constituted 0.5% of total protein in the spinal cord, whereas NF-L constituted about 1.5%.

A method to purify glial fibrillary acidic protein (GFAP) from mouse spinal cord is described, which permits the measurement of GFAP in the sciatic nerve of the twitcher mutant and control mouse. Cytoskeletal proteins from sciatic nerves and purified GFAP standards were electrophoresed on gel, transferred to nitrocellulose paper, and immunostained with anti-GFAP antibody. From the immunostained, 51,000-dalton band, we estimated about 200 ng GFAP per 50 micrograms of cytoskeletal protein in the twitcher sciatic nerve. The control nerve showed no detectable GFAP. Double-labeled fluorescence immunocytochemistry showed that in the brainstem of twitcher mutant, GFAP and vimentin were coexpressed in the majority of astrocytes.

The Lewy body, a histological hallmark of Parkinson's disease, is a filamentous inclusion residing most prominently in pigmented neurons of the brainstem. Immunocytochemical reactions of Lewy bodies were examined with antisera to several filamentous proteins of the nervous system and positive reactions were found with those against neurofilaments. An abnormal organization of the neuronal cytoskeleton may be a pathological feature of Parkinson's disease.

A reverse-phase HPLC method was developed to separate individual neurofilament proteins (210,000, 160,000 and 70,000 Da) from the glial fibrillary acid protein. It is useful for analytical or preparative methods, with yields higher than 80%. The method represents improvement over previous methods in speed, efficiency, and purity. Combining this HPLC method with the conventional chromatographic method on DEAE-cellulose, highly purified individual neurofilament proteins can be obtained in large scale.

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 X 10(5)/cm2. At initial densities above 1 X 10(5) 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 X 10(5)/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.

The expression of glial fibrillary acidic protein (GFAP) was found to be markedly enhanced immunohistochemically and biochemically both in the central (CNS) and peripheral (PNS) nervous systems of the twitcher mutant, an authentic murine model of human globoid cell leukodystrophy. The astrocytes in the CNS, the unmyelinated Schwann cells in the sciatic nerve, and the satellite cells in the trigeminal ganglion stained heavily with anti-GFAP antiserum. These changes in GFAP expression occurred shortly before the initiation of demyelination and coincided chronologically and topographically with infiltration of macrophages, suggesting that the same or closely related factors trigger the infiltration of macrophages and activate expression of GFAP. Cytoskeletal protein preparations showed increases in GFAP as well as in vimentin in the brainstem, spinal cord, and sciatic nerve. These results demonstrate that at least two types of peripheral glia (the unmyelinated Schwann cell and the satellite cell), in addition to the astrocyte, respond to some pathologic stimuli with an increased expression of GFAP. However, two other GFAP-positive structures, the Bergmann and radial glia, showed no significant changes in their immunostaining.

To investigate a role for T lymphocytes in primary demyelination of central nervous system (CNS) tissue, antigen-specific T cell lines sensitized to myelin-associated and myelin-unrelated antigens were developed from SJL mice and tested on myelinated organotypic cultures of syngeneic spinal cord. Demyelination was assessed morphologically by electron microscopy. Antigen responsiveness and specificity, and the phenotypes of the cell lines, were determined by thymidine uptake (3H-TdR) assays and flow cytometry (FC), respectively. Although all T cell lines caused pathologic changes in myelin, the CNS-antigen-specific line induced the most pronounced effects. 3H-TdR uptake assays and FC showed that after three cycles of incubation in the presence of interleukin-2 (IL-2) or antigen, the T cell lines had increased specificity and responsiveness to the priming antigen and were enriched for the L3T4 (helper/inducer) phenotype. This represents the first direct demonstration of T-cell-mediated demyelination, supports a role for the helper/inducer subset in CNS lesion development, and may prove relevant to the human demyelinating disease multiple sclerosis.

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.

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.

Glial fibrillary acidic protein (GFAP) in the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) was quantitated by densitometry of both stained gels and immunoblots of electrophoretically separated cytoskeletal proteins. The experimental period ranged from 7 to 65 days postinoculation (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 histones 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 immunocytochemical 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.

Incubation of homogenates of rat, rabbit, and bovine spinal cord and of bovine brain white and gray matter in the presence of calcium (5 mM) produced an extensive degradation of the neurofilament triplet proteins (NFP; 200 K, 150 K, and 69 K). The breakdown products of the NFPs were identified by immunoblot. The glial fibrillary acidic protein (GFAP), microtubular proteins (MTP), and myelin proteins were also degraded. The 150 K NFP was more susceptible than the other NFPs. The extent of calcium-mediated degradation was slightly greater with rat spinal cord than the others. Bovine brain white matter had more activity than gray, which had no appreciable degradative activity. The breakdown was prevented by both EGTA and leupeptin but a similar concentration of MgCl2 (5 mM) had no effect. These results suggest that NFPs are degraded by a Ca2+-activated neutral proteinase in the central nervous system (CNS) of several species. The lesser activity in gray matter suggests that the enzyme is enriched in axons, myelin, and/or oligodendroglial cells.