Specific N-Linked Oligosaccharides Are Required for Oligodendroglial Differentiation but Probably Not for Astrocytic Differentiation

Abstract

Primary cultures of cerebral glia derived from neonatal rat brain were utilized to determine whether specific glycoproteins are involved in oligodendroglial and astrocytic differentiation. Specific emphasis was placed on the oligosaccharide portion of glycoproteins, and inhibitors of glycoprotein processing were studied. Castanospermine, an inhibitor of glucosidase I, and thereby formation of both complex glycoproteins and high mannose glycoproteins, and deoxymannojirimycin (DMM), an inhibitor of mannosidase I and thereby formation of complex glycoproteins, were utilized. Castanospermine exposure prevented the developmental inductions of the two oligodendroglial markers, 2'',3''-cyclic nucleotide 3''-phosphohydrolase and glycerol-3-phosphate dehydrogenase. The effect of castanospermine on oligodendroglial differentiation was reversible. In contrast, castanospermine had no effect on the developmental inductions of the two astrocytic markers, glutamine synthetase and lactate dehydrogenase. DMM exposure had no effect on either oligodendroglial or astrocytic differentiation. Although both inhibitors caused a marked decrease in the formation of complex glycoproteins and an increase in high mannose structures, the oligosaccharide composition of these high mannose structures differed markedly. Castanospermine caused an increase in ''abnormal'', apparently glucosylated high mannose structures and a decrease in all other ''normal'' high mannose oligosaccharides, whereas DMM caused an increase in most high mannose structures, especially those migrating in the region of the Man₇GlcNAc standard. The data indicate that oligodendroglial differentiation requires specific N-linked oligosaccharides, probably principally of the high mannose type, and that astrocytic differentiation can proceed normally despite marked alterations in both complex and high mannose glycoproteins.

Glycoprotein Processing Is Required for Completion But Not Initiation of Oligodendroglial Differentiation from Its Bipotential Progenitor Cell

“…tri-and tetraantennary complex glycopeptides) and weakly bound radioactivity (bi-antennary complex glycopeptides), and a corresponding increase in strongly bound radioactivity (high mannose-and hybrid-type glycopeptides). These changes are qualitatively similar to our findings with castanospermine-treated mixed glial cultures [14] and ex pected from the site of action of this inhibitor [52][53][54],…”

“…Inhibitions of the inductions of CNP and GPDH were clearly apparent after 6 days of subculture, and the inhibitions were similar and marked after 9 days, when specific activities were approximately 50°/o lower in castanospermine-treated than in control cultures. The required duration of castanospermine exposure and the magnitude of the effect of the inhibitor in these progeni tor subcultures were similar to the duration and the mag nitude that we have reported previously with mixed glial primary cultures [14], Castanospermine had no effect on cell number or to tal protein content of the cultures. Thus, after 6 days cell number in control cultures was 53 ± 4 (cells x lOVwell) Fig.…”

“…The current findings indicate that the inhibitory effect of perturbation of glycoprotein processing on the devel opmental inductions of CNP and GPDH, shown previ ously in mixed glial cultures [14], was related to a direct effect on oligodendroglia and was not an indirect effect on oligodendroglia mediated by a perturbation in surface glycoproteins of the bed layer of type 1 astrocytes. Thus, in the present studies, the progenitor cells were separated from the bed layer of astrocytes by the selective detach ment technique [33] and grown in subculture.…”

“…Glycopeplides from control and castanospermine-treated cells were prepared by pronasc treatment of delipidated glycoprotein as described previ ously [14], Glycopeptides were fractionated by chromatography on a column (4 ml bed volume) of concanavalin A (Con A)-Sepharose 4B equilibrated with lOmA/Tris-HCl/1 m M CaCL/l mA/MgCL/ 1 mM MnCb/0.1 M NaCl/0.02% NaN.u pH 7.5. After columns were washed with the equilibrating buffer (unbound glycopep tides), loosely bound glycopeptides were eluted with 10 m M methyl-a-glucoside in the same buffer.…”

“…Second, N-linked glycoprotein synthesis is necessary for peripheral nervous system myelination [11][12][13], Third, in a previous study of mixed glial primary cultures derived from neonatal rat cerebrum, we showed that the developmental induction of the oligodendroglial enzymatic markers, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and gIycerol-3-phosphaíe dehydroge nase (CPDH), was prevented by exposure to an inhibitor of N-linked glycoprotein processing, i.e. castanospermine [14], This compound is an inhibitor of glucosidase 1, the enzyme responsible for initial trimming of glucose residues from the core oligosaccharide synthesized on the dolichyl phosphate carrier (Glc^MaruGlcNAcj) [15][16][17], Prevention of the induction of CNP activity by exposure to another glucosidase inhibitor, 1-deoxynojirimycin, was also shown in mixed glial primary cultures of fetal rat brain [18]. However, conclusive interpretation of these studies with mixed glial primary cultures is hin dered by several questions raised by the cellular hetero geneity of the system, particularly the marked predomi nance of astrocytes (approximately 95%) over Oligo dendroglia.…”