Cytoskeletal Structures and Oligodendroglial Differentiation in C-6 Glial Cells

Abstract: The relationship of the cytoskeleton to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Specifically, we investigated the effect of the cytoskeletal perturbants, colchicine and cytochalasin D, on the induction of the oligodendroglial marker enzyme. 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), caused by removal of serum from the culture medium. Each drug inhibited CNP induction in a concentration-dependent manner, and essentially complete inhibition of induction was observed with 0.25 μ M colchicine or 2.0μ M cytochalasin D. Detailed study of the effect of colchicine was carried out. This antimicrotubular agent not only totally prevented induction if added at the onset of serum removal, but also prevented further induction when added at various times after serum removal. That the effect of colchicine related to the drug's effect on microtubules was supported by the demonstration that lumicolchicine, a colchicine isomer which has no effect on microtubules, had no effect on the CNP induction. Moreover, colchicine, but not lumicolchicine, prevented the morphological signs of differentiation provoked by serum removal. The effect of colchicine was reversible and relatively specific. Thus, no concomitant effect of colchicine on the activity of another plasma membrane enzyme of C-6 cells, i.e., (Na⁺+ K ⁺)-acti-vated ATPase, or on the rate of incorporation of [³H]leucine into total protein of intact cells could be discerned. The possibility that the site of the effect of colchicine is on intracellular events was suggested by the observation that the drug inhibited the induction of CNP by dibutyryl cyclic AMP. The data suggest that the cytoskeleton is involved in oligodendroglial differentiation.     

Relation of Cellular Phospholipid Composition to Oligodendroglial Differentiation in C-6 Glial Cells

The relation of the polar head group composition of cellular phospholipids to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Induction of the oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), was determined after alteration of the polar head group composition of phospholipids by exposure of the cells to choline analogues, especially N,N'-dimethylethanolamine. To accomplish the phospholipid alteration, cells were grown in the presence of the analogue in medium free of exogenous lipid, i.e., first for 24 h in 10% delipidated serum and then for 48 h in serum-free medium. The 48-h exposure to serum-free medium resulted in untreated C-6 cells in a several fold increase in CNP activity, but in cells treated with 2.5 mM N,N'-dimethylethanolamine, total inhibition of this induction was observed. A graded, concentration-dependent inhibitory effect of the analogue on the induction of CNP was defined. The effect of the analogue was relatively specific, e.g., the activity of another plasma membrane enzyme of C-6 cells, (Na+ + K+)-activated ATPase, was not affected. Morever, there was no evidence of a toxic effect of the analogue; thus, total protein synthesis and cell growth were not altered, and the induction of CNP in serum-free medium recurred after removal of the analogue. N,N'-Dimethylethanolamine was shown to be incorporated into cellular phospholipids, primarily at the expense of phosphatidylcholine. The data define an important role for the polar head group composition of membrane phospholipids in oligodendroglial differentiation in this model system.     

Relation of Cholesterol to Astrocytic Differentiation in C-6 Glial Cells

Abstract: The relation of cellular cholesterol content to a biochemical expression of astrocytic differentiation was investigated in cultured C-6 glial cells. The astrocytic marker, glutamine synthetase, was studied. Cellular sterol content was perturbed with compactin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase and, thereby, cholesterol biosynthesis. Depletion of cellular sterol resulted in 72 h in a more than twofold increase in glutamine synthetase activity. Production of various degrees of sterol depletion with different concentrations of compactin demonstrated a striking inverse relationship between glutamine synthetase activity and the cellular sterol/phospholipid molar ratio. That the effect of compactin, in fact, is mediated by depletion of sterol was shown further by prevention of the compactin-induced increase in synthetase activity by simultaneous addition of exogenous cholesterol. Moreover, addition of cholesterol alone to the culture medium led to both a decrease in glutamine synthetase activity and an increase in the sterol/phospholipid molar ratio. The possibility that the compactin-induced increase in glutamine synthetase activity is caused by an increase in synthesis of the enzyme was suggested by prevention of the increase by cycloheximide. The data suggest that astrocytic differentiation is stimulated by a decrease in cellular sterol content. When considered with our previous observation that oli-godendroglial differentiation is inhibited by such a decrease, the findings suggest that cellular sterol content is a critical determinant of the direction of glial differentiation, i.e., whether along astrocytic or oligodendroglial lines.     

Oligodendroglial Differentiation in Glial Primary Cultures: Requirement for Mevalonate

The oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), is a valuable marker for expression of oligodendroglial differentiation in glial primary cultures, and the inducibility of this enzyme by dibutyryl-3',5'-cyclic AMP (dBcAMP) appears to be limited to immature or developing oligodendroglia. To investigate the relationship between the induction of CNP and the sterol biosynthetic pathway, primary cultures of glia dissociated from the brains of newborn rats were maintained in 10% fetal calf serum (FCS) and exposed to 1 mM dBcAMP on day 7 in culture. Cultures so treated for either 48 h or 72 h demonstrated a three- to fourfold induction of CNP specific activity. The magnitude of this induction was not affected when the cholesterol content of the culture medium was reduced by greater than 95% by placing the cultures in 10% lipoprotein-poor serum rather than 10% FCS during the exposure to dBcAMP. Mevinolin (10 microM), a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of the sterol biosynthetic pathway, completely inhibited the induction of CNP by dBcAMP, while not affecting either the accumulation of cellular protein per flask or rate of protein synthesis. Simultaneous addition of mevalonate (20 mM) prevented the inhibition of the induction of CNP by mevinolin. However, simultaneous addition of low-density lipoprotein sufficient to increase the cholesterol content of the medium 80-fold failed to correct mevinolin's inhibition of the induction of CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of Hyaluronectin by C-6 Glial Cells at High Density

Hyaluronectin, a brain glycoprotein that has been localized to the nodes of Ranvier in vivo and to oligodendrocytes in primary cultures of neonatal rat brain cells, was shown by using an unlabeled immunoperoxidase method to be present in C-6 glial cells grown to high density. The density-dependent expression of this glycoprotein is in accordance with the known properties of the glial stem cells, i.e., induction of differentiated properties such as 2',3'-cyclic nucleotide-3'-phosphohydrolase, glutamine synthetase, S-100 protein, and glial fibrillary acidic protein.     

Demonstration of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Cultured Human Schwann Cells

Abstract: Schwann cell cultures were established from adult human sural nerve biopsies. 2'3'-Cyclic nucleotide 3'-phosphohydrolase (CNPase) activity was estimated in the homogenates of those cells by a sensitive isotope assay using [³H]2',3'-cyclic AMP as substrate. A high level of CNPase activity was observed in cultured Schwann cells, whereas cultured human muscle and skin fibroblasts contained negligible levels of CNPase activity. CNPase of human Schwann cells followed typical enzyme-substrate kinetics, with an apparent K _m of 1.6 m M for 2',3'-cyclic AMP, and the enzyme was stimulated by detergents such as Triton X-100 and deoxycholate. It was inhibited by p -chloromercuricbenzoate and 2'-AMP. These properties are typical of CNPase isolated from adult brain and spinal cord. CNPase can serve as a new biochemical marker of normal cultured human Schwann cells and can be useful in analyzing the properties of cultured Schwann cells from patients with dysschwannian neuropathies.     

Antibodies to Myelin/Oligodendrocyte-Specific Protein and Myelin/Oligodendrocyte Glycoprotein Signal Distinct Changes in the Organization of Cultured Oligodendroglial Membrane Sheets

Abstract: Cultured murine oligodendrocytes elaborate extensive membrane sheets that, unlike multilamellar myelin in vivo, allow the study of interactions between myelin proteins and cytoskeletal elements. This article describes the events that occur due to the interaction of specific antibodies with their respective antigens, myelin/oligodendrocyte-specific protein (MOSP) and myelin/oligodendrocyte glycoprotein (MOG), which are expressed uniquely by oligodendrocytes. After antibody binding, surface anti-MOSP:MOSP complexes redistribute over those cytoplasmic microtubular veins that have 2',3'-cyclic nucleotide 3'-phosphohydrolase colocalized along them. In contrast, surface anti-MOG-MOG complexes redistribute over internal myelin basic protein domains. Long-term anti-MOSP IgM exposure results in an apparent increase in number as well as thickness of microtubular structures in oligodendrocyte membrane sheets, whereas long-term anti-MOG exposure causes depolymerization of microtubular veins in membrane sheets. These data suggest that antibody binding to these two surface proteins elicits signals that have opposite effects on the cytoskeleton in oligodendroglial membrane sheets. Thus, it is possible that signals transduced via antibody binding may contribute to the pathogenesis of diseases affecting CNS myelin.     

Cholesterol Biosynthesis and Its Regulation in Dissociated Cell Cultures of Fetal Rat Brain: Developmental Changes and the Role of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15-16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.     

Glial Fibrillary Acidic Protein: Norepinephrine Stimulated Phosphorylation in Intact C-6 Glioma Cells

Abstract: Coelectrophoresis in two-dimensional gels of rat glial fibrillary acidic protein (GFA) and ³²P-labeled whole cell extracts of rat C-6 glioma cells showed that the GFA migrated in close proximity to a previously noted phosphoprotein, 50K-6.1, of these cells. GFA electrophoresed as a 50K polypeptide with at least four charge variants, the most acidic of which coelectrophoresed with 50K-6.1. Exposure of the C-6 cultures to dibutyryl cyclic AMP (dbcAMP) for 48 h increased the relative abundance of the endogenous polypeptide associated with 50K-6.1 by threefold, consistent with the hypothesis that 50K-6.1 was GFA. Norepinephrine stimulated 50K-6.1 phosphorylation 3.2-fold in dbcAMP-induced cultures. Peptide mapping with V8 protease and subtilisin was used to test the hypothesis that GFA and 50K-6.1 were identical polypeptides. With V8 protease, the peptides generated from the [³⁵S]methionine labeled putative GFA spot of the C-6 cells were indistinguishable from the stained bands derived from authentic GFA in mixed samples of the two proteins. Likewise, the ³⁵S-labeled acidic satellite to the putative GFA spot also yielded a peptide map that matched that of the authentic GFA. ³²P-labeled peptides derived from the 50K-6.1 protein were a subset of those from authentic GFA. With three subtilisin concentrations, ³²P-labeled 50K-6.1 was degraded to peptides which were again a subset of the stained GFA peptides. A cytoskeletal fraction from ³²P-labeled C-6 cells contained a 50K phosphoprotein. Pep-, tide mapping with V8 protease produced a ³²P-peptide pattern which was a subset of that from authentic GFA. The pattern closely resembled the ³²P-peptide pattern for the 50K-6.1 protein from 2-dimensional gels of whole cell extract. It was concluded that the protein 50K-6.1 is a phosphorylated form of GFA and that GFA is a phosphoprotein whose phosphòrylation is stimulated by norepinephrine in C-6 glioma cells.     

Induction of Myelin Components: Cyclic AMP Increases the Synthesis Rate of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in C6 Glioma Cells

In an effort to determine the factors that stimulate myelin synthesis, we investigated the mechanism by which dibutyryl cyclic AMP induces the activity of the myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), in C6 glioma cells. Immunotitration experiments and measurements of the accumulation of [35S]methionine-labeled CNP showed that dibutyryl cyclic AMP increased the amount of CNP in the cells but not the catalytic activity per molecule of the enzyme. Moreover, inhibition of protein synthesis with cycloheximide abolished induction of enzyme activity. Dibutyryl cyclic AMP doubled the rate of CNP synthesis but had no effect on the half-life of the enzyme (approximately 33 h). The induction was partially blocked by the inhibitors of mRNA synthesis, cordycepin or alpha-amanitin. Thus, cyclic AMP induces the synthesis of CNP.     

Induction of Glutamine Synthetase by Dibutyryl Cyclic AMP in C-6 Glioma Cells

Glutamine synthetase was found to be increased in C-6 glioma cells as a result of increasing culture passage and N-6,2'-O-dibutyryl cyclic AMP (dbcAMP) treatment. At low passage dbcAMP produced a 2.5-fold increase in glutamine synthetase activity per unit of cellular protein. At high passage control glutamine synthetase was approximately double that seen at low passage, but dbcAMP produced an additional 65% increase. Lactate dehydrogenase activity was also increased by dbcAMP treatment at both low and high passage, but culture passage produced no change in the lactate dehydrogenase. With increasing culture passage, the ratio of cellular protein to DNA doubled. Therefore, expression of data per unit of protein tended to minimize the apparent changes in activity. The maximum increase in glutamine synthetase activity produced by both dbcAMP and increasing culture passage and expressed on a DNA basis was 5.6-fold. The increase in glutamine synthetase activity was generally linear during the first 20 h of drug treatment, after which enzyme activity remained nearly constant up to 72 h. Ninety percent or more of the dbcAMP remained in the medium at the end of 48-h exposure of cells to dbcAMP. 8-br-Cyclic AMP also increased glutamine synthetase activity of C-6-cels, but n-butyrate did not. Isoproterenol, which increases cyclic AMP in C-6-cells, increased glutamine synthetase activity. The effect of isoproterenol on glutamine synthetase was inhibited by the beta-adrenergic blocking agent sotalol. Cycloheximide (10 micrograms/ml) inhibited the dbcAMP effect on glutamine synthetase activity and also decreased the control enzyme activity by 60%.     

Hypomyelination in the Cerebrum of the Congenitally Hypothyroid Mouse (hyt)

2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the cerebrum of the inherited primary hypothyroid mouse (hyt/hyt) is reduced in comparison with the normal heterozygate (hyt/+). No differences were observed with regard to DNA and RNA content and the RNA/DNA ratio. The results of this study indicate that hypomyelination in the hypothyroid mouse is restricted to the cerebrum, and is not related to arrested glial proliferation.     

Factors Contributing to the Poor Myelination in the Brain of the Snell Dwarf Mouse

Abstract: Conventional histological examination of the pituitary does not distinguish Snell dwarf mutants (dw/dw) from their normal littermates (+/?) in the neonatal stage. However, immunohistochemical examination of pituitaries of litters born to heterozygous Snell parents revealed that in approximately 25% of the glands examined, the number of positive cells was very low in the neonatal stage. We attempted to delineate the events resulting in the poor myelination in the brain of the Snell dwarf mouse, and to devise an immunohistochemical method for identifying the mutant neonate. Differences in the brain weights of the dw/dw and +/? mice first became apparent on the 10th day of age, and from this time on no further increase in the weight of the dwarf mouse brain was recorded. Increase in CNPase activity was found to be suppressed in the cerebrum and brain stem throughout the developmental stage, but not in the other parts of the brain. The yield of isolated myelin decreased by 58% in the mutant mouse, but CNPase activity was equivalent to that of control myelin. Differences in DNA content per cerebrum from the dw/dw and +/? mice first became apparent on the 10th day of age. Henceforth, the dw/dw mice showed no further increase, although the +/? mice continued to increase. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the cerebrum is most active, was suppressed to about 50% of the control level in all parts of the dwarf brain. These findings indicate that the poor myelination found in the mutant cerebrum is a hypomyelination due to reduced oligodendroglial proliferation caused by lack of circulating growth hormone.     

Uptake of Glutamate and Cystine in C-6 Glioma Cells and in Cultured Astrocytes

The uptake of glutamate in rat glioma C-6 cells and cultured astrocytes derived from rat cerebral hemispheres was found to be mediated by a Na(+)-dependent and a Na(+)-independent system. The Na(+)-dependent system was inhibited by aspartate and was consistent with the commonly occurring system designated system X-AG. The Na(+)-independent system was inhibited by cystine and was consistent with system x-c described in various types of cells in the periphery. It was also found that quisqualate selectively and competitively interfered with the Na(+)-independent glutamate uptake. In C-6 cells, the glutamate uptake via systems X-AG and x-c accounted for approximately 35% and 55% of the total uptake, respectively, at 0.05 mM glutamate. In cultured astrocytes, the glutamate uptake via system X-AG was very potent, whereas the uptake via system xc- was relatively weak and its contribution to the total uptake of glutamate seemed almost negligible. However, in both C-6 cells and astrocytes, system xc- was necessary for the uptake of cystine, another substrate of system xc-. Cystine in the culture medium was an essential precursor of glutathione, and the inhibition of the cystine uptake by excess glutamate as a competitor led to a severe deficiency in glutathione, followed by cell degeneration.     

Induction of N-Glycosylation Activity in Cultured Embryonic Rat Brain Cells

Developmental changes in protein N-glycosylation activity have been studied using cultures of dissociated fetal rat brain cells as an in vitro model system. These cultures undergo an initial phase of neurite outgrowth and cell proliferation (4-6 days in culture), followed by a period of cellular differentiation. N-Glycosylation activity has been measured by assaying the incorporation of [2-3H]mannose into dolichol-linked oligosaccharides and glycoprotein over a period of 1-25 days in culture. This study revealed a marked induction of N-glycosylation activity beginning at approximately 1 week of culture. [2-3H]Mannose incorporation into the oligosaccharide-lipid intermediate fraction and glycoprotein reached maximal values between 12 and 16 days of culture and declined thereafter. The major dolichol-linked oligosaccharide labeled by the brain cell cultures was shown to be Glc3Man9GlcNAc2 by HPLC analysis. Parallel incorporation studies with [3H]leucine showed that the increase in protein N-glycosylation was relatively higher than a concurrent increase in cellular protein synthesis observed during the induction period. Maximal labeling of glycoprotein corresponded to the period of glial differentiation, as indicated by a sharp rise in the marker enzymes, 2',3'-cyclic nucleotide 3'-phosphohydrolase (an oligodendroglial marker) and glutamine synthetase (an astroglial marker). The results describe a developmental activation of the N-glycosylation pathway and suggest a possible relationship between N-linked glycoprotein assembly and the growth and differentiation of glial cells.     

Glial Cell Markers in the Reeler Mutant Mouse: A Biochemical and Immunohistological Study

The glial cell contents of S100 protein, 2',3'-cyclic AMP, 3'-phosphohydrolase (CNP), isoenzyme II of carbonic anhydrase (CAII) and butyrylcholinesterase (BuChE) were biochemically determined in the cerebellum and cerebrum of the reeler mutant mouse. Astrocytes and oligodendrocytes, shown by this study, contain abnormal amounts of these components. The CAII concentration was significantly increased in the particulate fraction of the reeler cerebellum and cerebrum (by 50% and 89%, respectively). The BuChE specific activity was greatly increased in the reeler, by 120% for cerebellum and by 40% in cerebrum. In contrast, the S100 protein concentration was reduced in the reeler cerebellum by 40% and by 25% in cerebrum, while the CNP specific activity increased by 30% in the reeler cerebellum. In addition, the glial cell distribution was studied by immunohistological techniques with antibodies directed against S100 protein, glial fibrillary acidic protein (GFA) and CAII. Apparently the density of glial cells is not significantly affected. However, the Golgi epithelial cells were usually abnormally placed and their Bergmann fibres were less well developed.     

Morphological and Biochemical Studies on the Cerebral Cortex from Reeler Mutant Mice: Development of Cortical Layers and Metabolic Mapping by the Deoxyglucose Method

Abstract: Cerebral cortex from reeler mutant mice was examined morphologically and biochemically. The sequential process of postnatal cell migration in the cerebral cortex of reeler (rl/rl) was examined morphologically. The dense cellular cortical plate lies below the molecular layer near the cerebral surface just after birth in normal mice while in reeler most of the cells are concentrated in the center of the cortex. In the cortex of adult reeler, the broad laminar structure of the neurons could be seen to form inverted positions in the cortical layers. The total wet weight, and the concentration of DNA and RNA in the pallium cerebri from reeler did not differ significantly from those in the control. As to the protein profiles of the pallium cerebri detected by SDS- polyacrylamide gel electrophoresis, no significant differences were observed. Activities of CNPase (2',3'-cyclic nucleotide 3'-phosphohydrolase), which is a myelin enzyme of CNS, and choline acetyltransferase were at the same level in both the reeler and the control. Therefore, reeler mutation does not appear to affect the genetically determined cell numbers, number of cholinergic fibers, and myelination. By autoradiographic observation of the cerebral cortex after intraperitoneal injection of [¹⁴C]2-deoxyglucose, it was revealed that 2-deoxyglucose was incorporated intensively into the fourth layer (granular layer) of the cerebrum from the control. In reeler it was also incorporated into the granular layer but in a more widespread distribution. We conclude that terminals to the granular layer make metabolically active synapse, perhaps even in a manner inverted from normal.     

Activation of Dopamine D2 Receptors Linked to Voltage-Sensitive Potassium Channels Reduces Forskolin-Induced Cyclic AMP Formation in Rat Pituitary Cells

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptor agonists, including BHT 920 and bromocriptine, and the potassium channel opener minoxidil share the property of hyperpolarizing the plasma membrane by activating voltage-dependent potassium channels. These drugs were tested for their ability to inhibit the cyclic AMP formation induced by forskolin either in intact or in broken pituitary cells. In contrast to bromocriptine, which was active in both experimental systems, BHT 920 and minoxidil inhibited the forskolin-induced cyclic AMP formation in intact-cell but not in broken-cell preparations. The effects of BHT 920 were (a) concentration dependent, with a calculated IC50 of 0.7 microM, (b) dopaminergic in nature, being specifically antagonized by sulpiride, (c) not additive with those induced by minoxidil, and (d) less effective in the presence of potassium channel blockers, such as 4-aminopyridine and tetraethylammonium. These data indicate that the inhibition of forskolin-induced cyclic AMP formation by BHT 920 in intact pituitary cells is not a primary consequence of receptor occupation, but a late event, possibly related to the opening of voltage-dependent potassium channels elicited by this drug through the activation of a subtype of dopamine D2 receptors uncoupled to adenylyl cyclase.