Inhibitors of N‐linked oligosaccharide processing glucosidases interfere with oligodendrocyte differentiation in culture

Abstract

Previous studies have demonstrated that inhibitors of glycoprotein processing glucosidases interfere with the development of oligodendrocyte properties in primary cultures of embryonic rat brain cells (Bhat, J Neurosci Res 20:158-164, 1988). The present study examines the effect of castanospermine, an inhibitor of the processing glucosidases, on the development and differentiation of isolated oligodendrocyte progenitor cells. Treatment of oligodendrocyte progenitors with castanospermine did not affect the developmental progression of the precursors to become committed oligodendrocytes as revealed by comparable increases in the percentages of cells positive for galactocerebroside (a surface marker for terminally differentiated oligodendrocytes) in control and drug-treated cultures. On the other hand, there was an impairment of the expression of differentiated properties of oligodendrocytes [i.e., sulfolipid synthesis, myelin basic protein (MBP)] and 2'3'-cyclic nucleotide 3'-phosphohydrolase in the drug-treated cultures. Immunocytochemical analysis with anti-MBP antibodies revealed a reduced number of MBP-positive cells in inhibitor-treated cultures. Furthermore, a majority of MBP-positive cells in such cultures displayed immunoreactive MBP in their cell body and not the processes, unlike in control cultures where both cell body and the processes of oligodendrocytes stained intensely for MBP. The strong inhibitory effect of castanospermine on the expression of oligodendrocyte-specific activities was contrasted with a relatively smaller effect of swainsonine, a mannosidase inhibitor on oligodendrocyte differentiation. Both castanospermine and swainsonine, however, effectively blocked the formation of complex-type oligosaccharides, suggesting thereby a lack of correlation between the inhibition of the formation of complex-type oligosaccharides and oligodendrocyte differentiation. It is suggested, therefore, that early trimming reactions involving the removal of glucose residues from the high mannose oligosaccharides in the endoplasmic reticulum may be essential for the cell surface localization and function of glycoproteins critically involved in surface interactions of oligodendrocytes with each other and/or with the substratum.

The Expression of Myristoylated Alanine-Rich C-Kinase Substrate in Oligodendrocytes Is Developmentally Regulated

“…As reported previously [29], when derived from mixed glial cultures growing in the presence of CS (instead of FCS), a majority of the isolated oligodendro cyte progenitor cells express 0 4 antigen(s) and react only weakly with A2B5 monoclonal antibodies. How ever, when these cultures are grown in the presence of bFGF plus PDGF, a condition that induces the reversion of progenitor phenotype and promotes a continued cell proliferation, over 90% of these cells exhibit strong reac tivity against A2B5 antibodies and lose 0 4 antigen from the cell surface.…”

“…Cultures grown for different days were also analyzed for the expression of oligodendrocyte stage-specific antigens, as described before (29). The cells grown in chamber slides were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min, washed and incubated with 3% normal goat serum (NGS) in PBS for 30 min.…”

p38 MAP Kinase Regulation of Oligodendrocyte Differentiation with CREB as a Potential Target

“…Immunocytochemical analysis of oligodendrocyte-specific markers was performed as described before [33]. Briefly, the cultures grown in poly-D-lysine-coated chamber/slides were incubated for 30 min with the monoclonal antibodies A2B5, O4 and O1, diluted 1:25 in 3%NGS in PBS, washed 3 times with 1% NGS in PBS, incubated with TRITC-conjugated goat antimouse IgM at 1:50 dilution in 3% NGS for 15 min and washed 3 times with 1% NGS in PBS.…”

<i>Griffonia simplicifolia</i> II Lectin Labels a Population of Radial Glial Cells in the Embryonic Rat Brain

“…we suggest that this population of GSL Il+ radial glia represents oiigodendroglial precursors. Further evidence for our hypothesis is provided by the work of Bhat and Waechter [30], who have shown in vitro that N-linked glycoprotein synthesis corresponds with glial differentiation and more specifi cally, that interfering with post-translational N-giycosylation causes aberrant differentiation of oligodendrocytes [31]. The fact that GSL II binds to N-acetyl-D-glucosamine residues, which are prominent constituents of N-Iinked oligosaccharides, together with a glial localiza tion with GSL II binding in situ leads us to hypothesize that GSL II staining serves as a transient embryonic dif ferentiation marker for radial glia committing in situ to the oligodendroglial lineage.…”