An in vitro model system of cultured oligodendrocytes was used to determine the susceptibility of these cells to oxidative stress induced by 15 min exposure to millimolar concentrations of hydrogen peroxide (H2O2). Following the exposure, the cells were incubated in normal growth medium, and analyzed at different time points. Although no cell loss was observed during the exposure period, there was a progressive depletion of adherent cells during the postexposure period as seen from either the number of recoverable nuclei, or from total RNA content of the cultures. Both the rate and the extent of cell deletion was directly dependent on H2O2 concentration. Cell death was preceded by structural alterations in the nuclear envelope resulting in “fragile” nuclei which disintegrated during isolation. Northern blot analysis showed that the expression of myelin-specific genes was rapidly downregulated in H2O2-treated cells. On the other hand, the expression of antiapoptotic gene, bcl-2 featured massive but transient upregulation. Oligodendrocyte degeneration also featured genomic DNA degradation into high molecular weight fragments, which are likely to represent cleaved chromosomal loops. The results demonstrate vulnerability of oligodendrocytes to oxidative stress that induces rapid degeneration and ultimately leads to delayed cell death. This feature is highly relevant to oligodendrocyte damage and depletion following ischemic, traumatic, or inflammatory insults to the central nervous system (CNS). J. Neurosci. Res. 55:303–310, 1999. © 1999 Wiley-Liss, Inc.

Myelin-associated glycoprotein (MAG) is emerging as an important molecule involved in the plasticity and regeneration of the central nervous system. In this study, the structure of MAG gene promoter was characterized in cultured rat oligodendrocyte lineage cells. Heterogeneous transcription initiation with five major and eight minor start sites scattered within 72 bp was shown by primer extension analysis. This TATA-less core promoter contains no prominent initiator (Inr) elements associated with the transcription initiation sites, and hence, appears to utilize novel positioning mechanisms. Genomic footprinting analysis revealed several putative protein-binding regions overlapping the initiation sites and containing a multitude of CG-rich sequences. However, no conspicuous alterations in the protein-binding pattern were evident between O2A progenitors in which the gene is inactive, and mature oligodendrocytes with fully upregulated gene. The core promoter DNA features a differentiation-dependent demethylation as shown by genomic sequencing analysis. Three of eight cytosines are totally demethylated in oligodendrocyte chromosomes, indicating that these unmodified bases may be critical for full activation of the promoter. The core promoter is located within an internucleosomal linker, and the upstream regulatory region appears to be organized into an array of nucleosomes with hypersensitive linkers. (C) 1997 Wiley-Liss, Inc.

The treatment of rat glioma C6 cells with 10 microM isoproterenol (Ipt) for 4 days upregulated the expression of the myelin-associated glycoprotein (MAG) gene by approximately 55-fold over the control value. The constant presence of Ipt in the medium was required for the maximal upregulation, as time-restricted exposures to the drug produced only partial, or no upregulation of the gene. No difference in the MAG mRNA stability could be detected in Ipt-treated vs untreated cells indicating that the drug upregulates the MAG gene at transcriptional level. Serum (FCS) strongly attenuated the response of the MAG gene to Ipt. The stimulatory effect of Ipt was profoundly reduced by spermine and H-89, indicating that protein kinase A-dependent protein phosphorylation is involved in the MAG gene activation. Within 30 min after Ipt administration, the c-fos gene was upregulated by 10-fold, and thereafter, its message level decreased and stabilized at approximately 3-fold over control. In contrast, the c-jun gene was downregulated to approximately 20% of control within 30 min after Ipt administration. Subsequently, its message level rose and fell once again within 12 h to approximately half of control, and returned to control level within 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

The methylation status of a 4.4-kb 5' end of the myelin-associated glycoprotein (MAG) gene was assessed in cells with different levels of transcriptional activity of the gene, i.e., liver, brain, O-2A oligodendrocyte precursors cells, mature oligodendrocytes, and glioma C6 cells. Purified DNA was digested with methylation-sensitive restriction enzymes, and the cuts were mapped by the indirect end-labeling technique. The restriction sites within the 4.4-kb fragment revealed a highly heterogenous methylation pattern among cells and tissues, and liver DNA was the most heavily methylated. Most of the restriction sites were partly demethylated in the nervous system cells. Notably, two adjacent Hha1 sites at +94 and +96 were fully methylated in liver, but partially demethylated in the brain, OL, and O2A. Two Hpa2 site located at -1836 and at -39 were progressively demethylated in oligodendrocyte lineage cells, indicating specific hypomethylation associated with the oligodendrocytic differentiation. Most of the restriction sites were weakly methylated in the DNA from neoplastic C6 cells, although the Hha1 sites were fully methylated. No clear-cut correlation between the extent of CpG dinucleotide methylation and the chromatin conformation was found. For example, out of four heavily methylated sites only two comapped with MNase hypersensitive sites. Also, the -1836 Hpa2 site whose demethylation is concomitant with oligodendrocytic differentiation seems to be localized within precisely positioned nucleosomal arrays of the MAG gene chromatin.The results indicate that the MAG gene undergoes progressive demethylation concomitant with the oligodendrocyte differentiation/maturation. However, certain CpG dinucleotides remain heavily methylated even in the fully active gene in mature oligodendrocytes, indicating that they may be essential in maintaining proper chromatin structure. (C) 1994 Wiley-Liss, Inc.

The effect of retinoic acid (RA) on the expression of myelin‐specific genes, i.e., proteolipid protein (PLP) and myelin‐associated glycoprotein (MAG) in rat glioma C6 cells, was analyzed by Northern blot hybridization. RA‐treatment increased the steady‐state level of the PLP‐specific messages within one day alter RA administration and the upregulation reached a maximum on the third day. Concomitantly, the expression of MAG‐specific messages in the RA‐treated C6 cells dropped below the detectability limit. The expression of the PLP gene was directly related to the RA concentration increasing to approximately 44‐ fold over the control (untreated cells) level at 10−6 RA. The stimulatory effect was vitiated by cyclohex‐imide indicating the involvement of intermediate genes in the PLP gene activation. The total cellular RNA content and the level of cyclophilin mRNA was not changed by the RA‐treatment. The present data indicate that RA can be a potent modulator of the myelin‐specific gene expression. Furthermore, the reciprocal response of PLP versus MAG genes to RA demonstrates that these two genes utilize different regulatory mechanisms.

The effect of retinoic acid (RA) on the expression of myelin-specific genes, i.e., proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) in rat glioma C6 cells, was analyzed by Northern blot hybridization. RA-treatment increased the steady-state level of the PLP-specific messages within one day after RA administration and the upregulation reached a maximum on the third day. Concomitantly, the expression of MAG-specific messages in the RA-treated C6 cells dropped below the detectability limit. The expression of the PLP gene was directly related to the RA concentration increasing to approximately 44-fold over the control (untreated cells) level at 10(-6) M RA. The stimulatory effect was vitiated by cycloheximide indicating the involvement of intermediate genes in the PLP gene activation. The total cellular RNA content and the level of cyclophilin mRNA was not changed by the RA-treatment. The present data indicate that RA can be a potent modulator of the myelin-specific gene expression. Furthermore, the reciprocal response of PLP versus MAG genes to RA demonstrates that these two genes utilize different regulatory mechanisms.

A model culture system of C6 rat glioma cells was used to test the involvement of cAMP in the regulation of the myelin PLP and MAG genes. The treatment of cells with isoproterenol (10(-5) to 10(-8) M) upregulated the expression of the PLP and MAG genes in a concentration-dependent manner. The mRNA for PLP reached a maximum (sevenfold higher than in control cells) after about 12-24 hr, then declined to approximately fourfold over the control level. The response of MAG gene was delayed by at least 36 hr, and the level of MAG mRNA reached a maximum of approximately 48-fold over the control level on the fourth day in culture. The co-administration of propranolol blocked the effect of isoproterenol, whereas 10(-5) M forskolin simulated the effect of isoproterenol, indicating a role of cAMP in the signal transduction cascades leading to upregulation of the myelin genes. However, the dissimilarity in the timing and the extent of upregulation of the PLP and MAG genes by cAMP-stimulating agents indicate the existence of different intracellular mechanisms for the activation of these two genes. Cycloheximide blocked the stimulatory effect of isoproterenol on both the PLP and MAG genes, indicating that the effect of cAMP on the myelin genes is mediated by protein product(s) of other cAMP-response gene(s).

The effect of ascorbic acid (AA) on rat glioma C6 cells was studied. At physiological AA concentrations of 0.1 and 1 mM, no morphological and no proliferative alterations in the C6 cultures were detectable. Although the total RNA content per cell was not affected by the AA-treatment, AA upregulated the expression of myelin-specific genes, i.e. proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) genes as assessed by northern blot analysis. The steady-state level of the specific mRNAs increased transiently in the AA-treated cells. Three days after AA administration the message level reached a maximum of 10- and 2-fold over control for the PLP and MAG genes, respectively. The upregulation of the genes was directly related to AA concentration. The present data indicate a possible involvement of AA in the regulation of myelin gene activity in the CNS.

A simple technique for the isolation of very high molecular weight genomic DNA from animal tissues and cells is described. The method involves rapid isolation of nuclei and their embedding in agarose beads followed by extraction of lipids and proteins with SDS. The protocol does not require proteolytic digestion and the whole procedure can be completed in 1 day. The isolated DNA is digestible by restriction enzymes and free of ligase inhibitors.