Negative regulation of oligodendrocyte differentiation by galactosphingolipids

Abstract

Galactocerebroside and sulfatide, major galactosphingolipid components of oligodendrocyte plasma membranes and myelin, are first expressed at a critical point, when progenitors cease to proliferate and commence terminal differentiation. We showed previously that an antibody to galactocerebroside/sulfatide arrested terminal differentiation, suggesting a role for these galactolipids in oligodendrocyte differentiation. We have now investigated the differentiation of oligodendrocytes (1) in response to other anti-galactolipid antibodies, showing that anti-sulfatide O4 but not anti-galactocerebroside O1 blocks terminal differentiation, perhaps by mimicking an endogenous ligand, and (2) in a transgenic mouse unable to synthesize these lipids because of mutation of the gene for ceramide galactosyltransferase, a key enzyme for galactosphingolipid synthesis. We find that galactosyltransferase mRNA expression begins at the late progenitor [pro-oligodendroblast (Pro-OL)] stage of the lineage and that the late progenitor marker pro-oligodendroblast antigen is not synthesized in the absence of galactosyltransferase. The principal outcome of the elimination of these galactolipids is a two- to threefold enhancement in the number of terminally differentiated oligodendrocytes both in culture and in vivo. Because the general pattern of differentiation and the level of progenitor proliferation and survival appear to be unaltered in the mutant cultures, we conclude that the increased number of oligodendrocytes is caused by an increased rate and probability of differentiation. In agreement with these two experimental approaches, we present a model in which galactosphingolipids (in particular galactocerebroside and/or sulfatide) act as sensors and/or transmitters of environmental information, interacting with endogenous ligands to function as negative regulators of oligodendrocyte differentiation, monitoring the timely progress of Pro-OLs into terminally differentiating, myelin-producing oligodendrocytes.

Fig. 1.

Reversible inhibition of OL differentiation by antibody O4 analyzed by immunofluorescence microscopy. Isolated rat OL progenitors were grown for 0–8 d in serum-free defined media alone (opentriangles) or with the addition of either O4 antibody (closedcircles), O1 antibody (closedsquares), or control serum components that are present in both antibody solutions (closedtriangles). In one group, cells grown in O4 were transferred to antibody-free media after 4 d and grown further in the absence of the antibody to test for the reversibility of the inhibition (dashedline,opencircles). The percent of total OL-lineage cells that became immunolabeled for O1 (i.e., GalC⁺) as they enter terminal differentiation has been plotted as a function of time. A representative experiment is shown. Error bars show the spread of the means. Note that although addition of serum components by themselves slightly retarded terminal differentiation, exposure to O4 antibody in addition totally inhibited it. Antibody O1, on the other hand, was unable to induce such an arrest.

Fig. 2.

Developmental expression of CGT mRNA during OL-lineage progression. Northern blot analysis of mRNA from purified populations of astrocytes (Ast; negative control), rat early progenitors (O2A), late progenitors (Pro-OL), and mature OLs (OL) grown for 2 d in the absence (−) or presence (+) of FGF is shown. Total RNA (20 μg/lane) was loaded, and the blots were first hybridized with a cDNA probe for CGT and then rehybridized with a probe for GAPDH. The mRNA levels were quantified and normalized for RNA loading (GAPDH), and values were expressed as relative mRNA levels; the highest level was set at 100%. Error bars represent SEM (n = 3–6). Note that CGT mRNA is first expressed at the Pro-OL stage in the OL lineage.

Fig. 3.

CGT-KO mice do not express POA, a marker of the Pro-OL stage (identified by O4 antibody). Parallel mixed primary cultures from control and CGT-KO were double-stained with antibodies O4 and Sulph-1 at a time in culture when both Pro-OLs and OLs are present. In the control cultures both Pro-OLs (arrows; expressing POA) and OLs (star; expressing sulfatide) were stained by O4, whereas only OLs (star) were labeled by Sulph-1. In the CGT-KO cultures, both OLs and Pro-OLs remain unstained by O4 as well as by Sulph-1, showing that not only sulfatide but also POA was absent, suggesting that CGT activity is required for the synthesis of POA. Scale bar, 50 μm.

Fig. 4.

Phenotype and morphology of mature OLs in CGT-KO cultures. Double-immunolabeling with anti-MBP and anti-GalC is shown. CGT-KO OLs express MBP, but not GalC, and produce extensive membranous sheaths comparable with control mouse OLs in culture. A single, typical OL (arrow) covering the entire 40× field is shown. Scale bar, 50 μm.

Fig. 5.

Analysis of PLP expression on the plasma membrane of CGT-KO OLs. Purified OL cultures from CGT-KO mice were double-immunolabeled with MBP and anti-PLP (O10) without previous fixation. PLP expression by CGT-KO OLs suggests that PLP was transported to the OL plasma membrane surface in the absence of sulfatide and GalC. Scale bar, 50 μm.

Fig. 6.

Analysis of OL differentiation in purified progenitor cultures from CGT-KO mice by immunofluorescent microscopy.A, Time course of OL differentiation. The percent of total cells immunolabeled at each time point for CNP (solidlines,circles) or MBP (dashedlines,triangles) from the control group (filledcircles, filled triangles) or CGT-KO mice (opencircles, opentriangles) is plotted as a function of time. Both weakly labeled immature OLs and mature OLs are included in the cell counts. At each point 400–600 cells were counted. Error bars represent SEM (n = 3–5). Note that cultures of CGT-KO progenitors yield a higher number of differentiated OLs compared with control cultures. B, Isolated OLs double-immunolabeled for MBP and a nuclear stain, Hoescht dye (H), after 4 d in culture. Note that in the fields shown, in the control group only two out of six cells shown in the field (arrows) are differentiated, whereas in the CGT-KO group three out of three cells are differentiated. Scale bar, 50 μm.

Fig. 7.

Analysis of OL differentiation in mixed primary cultures from CGT-KO by immunofluorescent microscopy. A,Mixed primary cultures of similar age from control and CGT-KO mice immunolabeled for MBP. Although both fields have a similar number of total cells determined by phase contrast (data not shown), note that in the control only one differentiated OL is seen, whereas in the CGT-KO cultures numerous mature OLs are already present. Scale bar, 50 μm.B, Biochemical analysis of CGT-KO OL differentiation in mixed primary cultures. MBP expression was measured by immunodot blotting followed by densitometric scanning as a function of time in culture. The MBP expression levels were comparable in the wild-type (filledcircles) and heterozygous (filledtriangles) groups, whereas the CGT-KO group (opencircles,dashedline) showed markedly elevated levels. Error bars represent SEM (n = 2 for wild type; n = 4 for heterozygous andn = 4 for homozygous groups from a litter of 10 pups). One representative experiment out of five is shown. Note that the extent of OL differentiation in mixed primary cultures is even more pronounced than that in the purified OL cultures. C,Western blot analysis of MBP isoform expression in CGT-KO (KO) and control (Cont) cultures, showing that all isoforms of MBP were elevated in the mutants.

Fig. 8.

Comparison of the proliferation and survival of Pro-OLs in purified cultures from CGT-KO and control mice.A, Cells were analyzed 2 and 3 d after plating in mN2 after exposure to BrdU for the last 24 hr. The incorporation of BrdU was analyzed by immunolabeling of cultures and counting the percent of total cells that were BrdU⁺. No increase in proliferation was seen in CGT-KO progenitors (stripedbars). In fact, an expected reduction is seen compared with control cultures (blackbars) as a result of entry into postmitotic terminal differentiation earlier than in control populations. Error bars represent SEM (n= 3). B, The number of total live cells per field was determined by counting the nonpyknotic nuclei labeled with the Hoescht dye as a function of time in culture. No differences were observed between the control (solidline) and CGT-KO (dashedline) groups. Error bars represent SEM (n = 4–6).

Fig. 9.

Enhancement of oligodendrocyte differentiationin vivo in CGT-KO mice. P7 forebrain sagittal sections taken from parallel regions of control (A, D, G) and CGT-KO (B, E, H) mice brains were analyzed by PLP mRNA in situ hybridization. PLP mRNA⁺cells in regions of the corpus callosum over the hippocampus are shown at low magnification (A–C) and high magnification (D–I). Sections hybridized with either antisense (A, B, D, E) or sense (C, F) PLP cRNA probes were costained with Hoescht dye (G, H, I) to show that similar brain regions of control and CGT-KO mice were analyzed. Representative sections show a higher number of PLP mRNA⁺ cells in CGT-KO (B, E) compared with control (A, D) mice of the same age. Sections hybridized with sense cRNA probes showed no labeling. Scale bars:A–C, 500 μm; D–I, 100 μm.

Fig. 10.

Quantification of the enhanced expression of PLP mRNA⁺ cells in vivo. P7 forebrain sagittal sections from control and CGT-KO mice were hybridized with an antisense PLP cRNA probe. All PLP mRNA positive cells in the cortical and corpus callosum region were counted for each section. Two to four sections each from 16 control (+/+, 5; +/−,12) and 9 homozygous mutant animals were counted. Comparisons of each control and CGT-KO were made on littermates. Control numbers (average of control for all +/+ and +/− animals) were set to 100%, and CGT-KO (−/−) levels are shown relative to that. Error bars represent SEM. The differences were statistically significant with 96% confidence limits using the Student’s t test.

Fig. 11.

A model for the role of galactosphingolipids as negative regulators of oligodendrocyte terminal differentiation. Based on previous studies (Bansal and Pfeiffer, 1989) and Figure 1, the model suggests that specific anti-galactosphingolipid antibodies, presumably mimicking an external ligand, either directly or indirectly continuously activate negative regulatory pathways, thus inhibiting terminal differentiation of oligodendrocyte progenitors. In the studies of oligodendrocyte differentiation in the CGT-KO mice, the model suggests that the absence of GalC/sulfatide precludes the instigation of the negative regulation, thus allowing terminal differentiation to proceed more efficiently. Sul, Sulfatide.