The PGRMC1 Antagonist AG-205 Inhibits Synthesis of Galactosylceramide and Sulfatide.

Sulfatide synthesis in the human renal cancer cell line SMKT-R3 was strongly inhibited in the presence of low µM concentrations of AG-205, a progesterone receptor membrane component 1 (PGRMC1) antagonist. This was also the case in Chinese hamster ovary (CHO) cells stably transfected with UDP-galactose: ceramide galactosyltransferase and cerebroside sulfotransferase, the two enzymes required for sulfatide synthesis. In CHO cells synthesizing galactosylceramide but not sulfatide, galactosylceramide was also strongly reduced, suggesting an effect at the level of galactolipid synthesis. Notably, AG-205 inhibited galactosylceramide synthesis to a similar extent in wild type CHO cells and cells that lack PGRMC1 and/or PGRMC2. In vitro enzyme activity assays showed that AG-205 is an inhibitor of UDP-galactose: ceramide galactosyltransferase, but not cerebroside sulfotransferase. This study shows that PGRMC1 is only one of several targets of AG-205 and should be used with caution, especially in studies using cells synthesizing galactosylceramide and sulfatide.

Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and α-galactosylceramide.

Bacterial glycosphingolipids such as glucuronosylceramide and galactosylceramide have been identified as ligands for invariant natural killer T cells and play important roles in host defense. However, the glycosphingolipid synthases required for production of these ceramides have not been well-characterized. Here, we report the identification and characterization of glucuronosylceramide synthase (ceramide UDP-glucuronosyltransferase [Cer-GlcAT]) in Zymomonas mobilis, a Gram-negative bacterium whose cellular membranes contain glucuronosylceramide. On comparing the gene sequences that encode the diacylglycerol GlcAT in bacteria and plants, we found a homologous gene that is widely distributed in the order Sphingomonadales in the Z. mobilis genome. We first cloned the gene and expressed it in Escherichia coli, followed by protein purification using nickel-Sepharose affinity and gel filtration chromatography. Using the highly enriched enzyme, we observed that it has high glycosyltransferase activity with UDP-glucuronic acid and ceramide as sugar donor and acceptor substrate, respectively. Cer-GlcAT deletion resulted in a loss of glucuronosylceramide and increased the levels of ceramide phosphoglycerol, which was expressed in WT cells only at very low levels. Furthermore, we found sequences homologous to Cer-GlcAT in Sphingobium yanoikuyae and Bacteroides fragilis, which have been reported to produce glucuronosylceramide and α-galactosylceramide, respectively. We expressed the two homologs of the cer-glcat gene in E. coli and found that each gene encodes Cer-GlcAT and Cer-galactosyltransferase, respectively. These results contribute to the understanding of the roles of bacterial glycosphingolipids in host-bacteria interactions and the function of bacterial glycosphingolipids in bacterial physiology.

Ceramide galactosyltransferase expression is regulated positively by Nkx2.2 and negatively by OLIG2.

Myelin, a multilamellar structure extended from oligodendrocytes or Schwann cells, plays a critical role in maintenance of neuronal function, and damage or loss of myelin causes demyelinating diseases such as multiple sclerosis. For precise alignment of the myelin sheath, there is a requirement for expression of galactosylceramide (GalCer), a major glycosphingolipid in myelin. Synthesis of GalCer is strictly limited in oligodendrocytes in a developmental stage-specific manner. Ceramide galactosyltransferase (CGT), a key enzyme for biosynthesis of GalCer, exhibits restricted expression in oligodendrocytes but the mechanism is poorly understood. Based on our assumption that particular oligodendrocyte-lineage-specific transcription factors regulate CGT expression, we co-expressed a series of candidate transcription factors with the human CGT promoter-driving luciferase expression in oligodendroglioma cells to measure the promoter activity. We found that Nkx2.2 strongly activated the CGT promoter. In addition, we identified a novel repressive DNA element in the first intron of CGT and OLIG2, an oligodendrocyte-specific transcription factor, as a binding protein of this element. Moreover, overexpression of OLIG2 completely canceled the activating effect of Nkx2.2 on CGT promoter activity. Expression of CGT mRNA was also upregulated by Nkx2.2, but this upregulation was cancelled by co-expression of OLIG2 with Nkx2.2. Our study suggests that CGT expression is controlled by balanced expression of the negative modulator OLIG2 and positive regulator Nkx2.2, providing new insights into how expression of GalCer is tightly regulated in cell-type- and stage-specific manners.

Inefficient fusion due to a lack of attachment receptor/co-receptor restricts productive human immunodeficiency virus type 1 infection in human hepatoma Huh7.5 cells.

Since the widespread use of the highly active antiretroviral therapy, the incidence of liver disease has increased to become a leading cause of death among human immunodeficiency virus type 1 (HIV-1)-infected individuals. It can be proposed that the ability of HIV-1 to infect hepatocytes could influence liver diseases. Although the presence of HIV-1 was identified in hepatocytes from HIV-1 seropositive patients, the susceptibility of hepatocytes to HIV-1 infection in vitro remains controversial. We present evidence here that human hepatoma cells are not productively infected with CD4-dependent HIV-1 strains because of inefficient fusion related to an absence of cell surface CD4 and CXCR4. However, these cells display an increased susceptibility to infection with a CD4-independent viral isolate through an interaction with galactosyl ceramide, an alternate receptor for HIV-1. This study provides further understanding of the susceptibility of human hepatocytes to HIV-1 infection. However, in vivo investigations are recommended to consolidate these data.

Galactosyl ceramide expressed on dendritic cells can mediate HIV-1 transfer from monocyte derived dendritic cells to autologous T cells.

Mucosa, comprising epithelial and dendritic cells, are the major sites for Human Immunodeficiency Virus type 1 (HIV-1) transmission. There, DCs can capture incoming HIV-1 and in turn transfer virus to CD4(+) T lymphocytes in a two-phase process, thereby initiating HIV-1 dissemination. We show that the glycosphingolipid Galactosyl Ceramide (GalCer), acting as mucosal epithelial receptor for HIV-1, was expressed by human monocyte derived immature DCs (iDCs), human primary DCs isolated from blood and mucosal tissue and in situ on mucosal tissue and acts as HIV-1-gp41 receptor. Blocking both GalCer and CD4 with specific mAbs results in a >95% transfer inhibition of HIV-1 from human monocyte-derived iDCs to autologous resting T cells. GalCer interaction with HIV-1 controls the early infection-independent phase of HIV-1 transfer to T cells. Thus, GalCer appears as an initial receptor for HIV-1, common to both mucosal epithelial cells and iDCs.

Discrepancies of NKT cells expression in peripheral blood and in cerebrospinal fluid from Behçet's disease.

The precise role of natural killer T (NKT) cells in the pathogenesis of Behçet's disease (BD) remains unknown. The frequency, cytokine profile and heterogeneity of NKT cells were studied in peripheral blood mononuclear cells (PBMC) from 42BD patients and in cerebrospinal fluid (CSF) samples from 9 neuro-BD patients. Flow cytometry revealed a decreased frequency of NKT cells in PBMC from BD patients (median: 0.06%; range: 0%-0.3%) when compared to healthy controls (median: 0.23%; range: 0.1%-0.7%; P<0.01). NKT cells were biased toward a Th(1)-like phenotype, with a significant decrease of IL-4/IFN-gamma ratio in BD (median: 0.049; range: 0.01-0.13) vs. healthy controls (median: 0.82; range: 0.4-1.33; P<0.01). NKT cells were increased in CSF-BD samples (median: 0.18%; range: 0.1%-0.4%), when compared to CSF-NIND patients (median: 0.05%; range: 0.01%-0.09%; P<0.01). Based on the reactivity of PBMC-derived NKT cells toward alpha-galactosylceramide (alpha-GalCer), 80% of BD patients were non-responsive. At the opposite, the reactivity of NKT cells in CSF from BD patients was not impaired. BD-CSF NKT cells exhibited an increased expression of IFN-gamma-producing cells, demonstrating that CSF-NKT cells were functional, and biased toward a Th(1)-like phenotype. These data suggest that functional NKT cells are recruited into BD inflammatory sites contributing to BD pathogenesis.

A mammalian fatty acid hydroxylase responsible for the formation of alpha-hydroxylated galactosylceramide in myelin.

Hydroxylation is an abundant modification of the ceramides in brain, skin, intestinal tract and kidney. Hydroxylation occurs at the sphingosine base at C-4 or within the amide-linked fatty acid. In myelin, hydroxylation of ceramide is exclusively found at the alpha-C atom of the fatty acid moiety. alpha-Hydroxylated cerebrosides are the most abundant lipids in the myelin sheath. The functional role of this modification, however, is not known. On the basis of sequence similarity to a yeast C26 fatty acid hydroxylase, we have identified a murine cDNA encoding FA2H (fatty acid 2-hydroxylase). Transfection of FA2H cDNA in CHO cells (Chinese-hamster ovary cells) led to the formation of alpha-hydroxylated fatty acid containing hexosylceramide. An EGFP (enhanced green fluorescent protein)-FA2H fusion protein co-localized with calnexin, indicating that the enzyme resides in the endoplasmic reticulum. FA2H is expressed in brain, stomach, skin, kidney and testis, i.e. in tissues known to synthesize fatty acid alpha-hydroxylated sphingolipids. The time course of its expression in brain closely follows the expression of myelin-specific genes, reaching a maximum at 2-3 weeks of age. This is in agreement with the reported time course of fatty acid alpha-hydroxylase activity in the developing brain. In situ hybridization of brain sections showed expression of FA2H in the white matter. Our results thus strongly suggest that FA2H is the enzyme responsible for the formation of alpha-hydroxylated ceramide in oligodendrocytes of the mammalian brain. Its further characterization will provide insight into the functional role of alpha-hydroxylation modification in myelin, skin and other organs.

Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth.

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non-pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3',4'-ethylenedioxy-P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans.

Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due to uncoupling of glycolipid biosynthesis in the Golgi apparatus.

Multidrug-resistant tumor cells display enhanced levels of glucosylceramide. In this study, we investigated how this relates to the overall sphingolipid composition of multidrug-resistant ovarian carcinoma cells and which mechanisms are responsible for adapted sphingolipid metabolism. We found in multidrug-resistant cells substantially lower levels of lactosylceramide and gangliosides in sharp contrast to glucosylceramide, galactosylceramide, and sphingomyelin levels. This indicates a block in the glycolipid biosynthetic pathway at the level of lactosylceramide formation, with concomitant accumulation of glucosylceramide. A series of observations exclude regulation at the enzyme level as the underlying mechanism. First, reduced lactosylceramide formation occurred only in intact resistant cells whereas cell-free activity of lactosylceramide synthase was higher compared with the parental cells. Second, the level of lactosylceramide synthase gene expression was equal in both phenotypes. Third, glucosylceramide synthase (mRNA and protein) expression and activity were equal or lower in resistant cells. Based on the kinetics of sphingolipid metabolism, the observation that brefeldin A does not restore lactosylceramide synthesis, and altered localization of lactosylceramide synthase fused to green fluorescent protein, we conclude that lactosylceramide biosynthesis is highly uncoupled from glucosylceramide biosynthesis in the Golgi apparatus of resistant cells.

Lead exposure delays the differentiation of oligodendroglial progenitors in vitro.

Lead (Pb) is an environmental neurotoxicant that can cause hypo- and demyelination. Oligodendrocytes (OLs), the myelin-forming cells in the central nervous system, may be a possible target for Pb toxicity. The present study describes the effect of Pb on the maturation of rat OL progenitor (OP) cells and the developmental expression of myelin-specific galactolipids. Dose-response studies showed that OP cultures were more sensitive to Pb than mature OLs. Pb delayed the differentiation of OL progenitors, as demonstrated by cell morphology and immunostaining with a panel of stage-specific differentiation markers. Pb given prior to and during differentiation caused a decrease in the biosynthesis of galactolipids in both undifferentiated and differentiated OLs, as detected by metabolic radiolabeling with 3H-D-galactose. While the ratios of galacto/gluco-cerebrosides, hydroxy fatty acid/nonhydroxy fatty acid galactolipids, and galactocerebrosides/sulfatides increased in control cultures during cell differentiation, Pb treatment prevented these changes. The results suggest that chronic Pb exposure may impact brain development by interfering with the timely developmental maturation of OL progenitors.

Direct cell-cell interactions control apoptosis and oligodendrocyte marker expression of neuroepithelial cells.

During brain development, the neuroepithelium generates neurons and glial cells. Proliferation and differentiation of neuroepithelial cells are controlled by a complex combination of secreted factors and more intrinsic or local mechanisms, such as lateral inhibition and asymmetric division. To obtain further insights into the signals governing neuroepithelial cell fate, we used the immortomouse to derive conditionally immortalised cell lines from mouse E10 neuroepithelium. We isolated a nestin-positive basic fibroblast growth factor (bFGF)-responsive cell line (SVE10-23) which mostly differentiate into astrocytes when cocultured with primary cortical cells. We found that, by simply lowering the cell density, SVE10-23 cells embarked on oligodendrocytic differentiation as indicated by the strong expression of galactocerebroside C and 2'3'-cyclic nucleotide 3'-phosphodiesterase. Apoptosis accompanied the differentiation, and all cells died within 1 week. We present here evidence that direct interactions between cells are the main mechanism regulating this oligodendrocytic differentiation. We demonstrate that SVE10-23 cells contact or proximity inhibit their differentiation, prevent apoptosis, and promote their proliferation. Similarly, others nestin-positive precursor cell lines and nonimmortalised bFGF-grown E10 cells were found to spontaneously differentiate at low density, thus generalising the idea that neural precursor fate is regulated by direct cell-cell interactions. The SVE10-23 cell line provides a valuable tool with which to study further the molecular components implicated in this mode of regulation.

Variable galactocerebroside expression by human Schwann cells in dissociated and peripheral nerve explant cultures.

It has been well established that rat Schwann cells down regulate their cell-surface expression of galactocerebroside (GalC) in vitro under normal cell culture conditions. To determine whether human Schwann cells exhibit a similar down-regulation of GalC in vitro we examined GalC expression in dissociated human Schwann cell cultures derived from normal adult peripheral nerve. Twenty-four hours post-dissociation up to 63% of human Schwann cells were found to express detectable levels of GalC on their surface whereas less than 8% of the Schwann cells expressed detectable levels of GalC at 14 days post-dissociation. In contrast, after nearly 3 months of peripheral nerve explant culture, greater than 30% of human Schwann cells still retained their GalC expression. A similar pattern was also observed when analyzing Schwann cell purity with dissociated cultures exhibiting a rapid decrease in Schwann cell purity under normal culturing conditions although Schwann cell purity was found to be largely unaffected during the period of peripheral nerve explant culture. In summary, we found there was less variation in both GalC expression and Schwann cell purity with time in peripheral nerve explant cultures than dissociated cultures.

Differential expression of sphingolipids in MRP1 overexpressing HT29 cells.

We have obtained a novel multidrug resistant cell line, derived from HT29 G(+) human colon carcinoma cells, by selection with gradually increasing concentrations of the anti-mitotic, microtubule-disrupting agent colchicine. This HT29(col) cell line displayed a 25-fold increase in colchicine resistance and exhibited cross-resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT-PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29(col) cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5-carboxyfluorescein, an increased efflux activity of HT29(col) cells was measured, as compared to the wild-type HT29 G(+) cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5-carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29(col) cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P-glycoprotein-specific substrate. Colchicine-induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis-related signal transduction processes, was much more abundant in HT29(col) cells, which is indicative of a stress response.

Biochemistry and neuropathology of mice doubly deficient in synthesis and degradation of galactosylceramide.

We have generated mice doubly deficient in both synthesis and degradation of galactosylceramide by cross-breeding twitcher mice and galactosylceramide synthase (UDP-galactose:ceramide galactosyltransferase, CGT) knockout mice. The prediction that the phenotype of the doubly deficient mice should be the same as the cgt -/- mice, since the degrading enzyme should not be necessary if the substrate is not synthesized, proved to be only partially correct. In early stages of the disease, the doubly deficient mice (galc -/-, cgt -/-) were essentially indistinguishable from the cgt -/- mice. However, the doubly deficient mice had a much shorter life span than cgt -/- mice. Both galactosylceramide and galactosylsphingosine (psychosine), were undetectable in the brain of the cgt -/- and the doubly deficient mice. The characteristic twitcher pathology was never seen in the galc -/-, cgt -/- mice. However, after 43 days, neuronal pathology was observed in the brainstem and spinal cord. This late neuronal pathology has not been seen in the CGT knockout mice but has been described in some long surviving bone marrow-transplanted twitcher mice. Furthermore, the motor segment of the trigeminal nerve of the galc -/-, cgt -/- mice showed severe degeneration not seen in either twitcher or CGT knockout mice. Thus, the galc -/-, cgt -/- mice, while primarily showing the cgt -/- phenotype as predicted, develop late pathology that is seen only in twitcher mouse and also a unique pathology in the trigeminal nerve. These observations indicate that the functional relationship between galactosylceramidase and galactosylceramide synthase is complex.

Temporal and quantitative expression of the myelin-associated lipids, ethanolamine plasmalogen, galactocerebroside, and sulfatide, in the differentiating CG-4 glial cell line.

We determined the expression of three myelin-typical lipids in the continuous CG-4 glial cell line of oligodendrocyte progenitor cells, as the cells differentiated into oligodendrocytes. On 6 different days during the first 9 days of oligodendrocyte development, cells were labeled for 24 h with [3H]ethanolamine to label ethanolamine plasmalogens or with [3H]galactose to label the galactocerebroside and sulfogalactocerebroside; and the amount of labeled lipid expressed on each day was determined. Each labeled lipid was expressed with its own specific time course and in a defined amount on each day of differentiation. Increased labeling of plasmalogens and sulfogalactocerebroside started at early developmental stages, and increased labeling of galactocerebroside started at later stages. The results indicate that the differentiating CG-4 cell line provides a valuable system to investigate factors affecting the early time course of myelin-lipid expression and the amounts expressed.

Non-myelin-forming perisynaptic schwann cells express protein zero and myelin-associated glycoprotein.

Perisynaptic Schwann cells (PSCs) envelop axonal terminals and are physiologically distinct from the nearby myelinating Schwann cells (MSCs), which surround the same innervating motor axons. PSCs have special functions at the neuromuscular synapse, where they detect and can modulate neurotransmitter release. Although PSCs are similar to non-myelinating Schwann cells in that they do not form multiple myelin wrappings around nerve terminals, they do wrap around single nerve terminals. These differences, as well as others, lead us to question whether PSCs are truly of Schwann cell origin. We thus characterized the expression of molecules, classically associated with myelin and Schwann cells, in PSCs at the frog neuromuscular junction. We wondered whether PSCs express the Schwann cell marker protein zero (P(0)) and whether their lack of myelination was related to an absence of myelin-associated glycoprotein (MAG), a protein found in myelinating cells that is considered important in myelination. Instead, we found that PSCs express both P(0) and MAG, and other myelinating glial markers such as galactocerebroside and 2',3'-cyclic nucleotide 3'-phosphodiesterase. In denervated preparations, P(0) and MAG expression persisted, including at newly formed PSC extensions. Because PSCs do not myelinate, it is clear that expression of these proteins alone is not sufficient for myelin formation. It is possible that factors present at synapses may prevent myelination, while P(0) and MAG may mediate adhesion between nerve terminals and the surrounding PSCs. The results indicate that PSCs are of Schwann cell origin.

Temporal expression pattern of peripheral myelin protein 22 during in vivo and in vitro myelination.

Peripheral myelin protein 22 (PMP22) was initially described as a minor component of peripheral myelin. Mutations affecting the PMP22 gene cause demyelinating neuropathies, supporting a role for the protein in PNS myelination. Furthermore, PMP22 carries the L2/HNK-1 carbohydrate epitope suggesting an adhesion/recognition function. Despite advances in characterizing the PMP22 gene, the specific role(s) of the protein in myelin remains unknown. In this study we determined the temporal expression pattern of PMP22 in comparison to galactocerebroside (GalC) and myelin associated glycoprotein (MAG), early constituents of PNS myelin, and to protein zero (P0) and myelin basic protein (MBP), late components of myelin. In sciatic nerve lysates, PMP22 was detected at postnatal day 3, after MAG, but before MBP expression. The same results were obtained in cocultures of dorsal root ganglion neurons and Schwann cells (SCs). Low levels of PMP22 were found in early, anti-MAG and anti-GalC immunoreactive, myelinating cocultures. However, PMP22 could only be detected in the SC plasma membrane after basal lamina formation. In long-term myelinating cocultures PMP22 levels continued to increase and the protein was found in anti-P0 and anti-MBP immunoreactive myelin segments. Furthermore, PMP22, MBP, and P0 protein levels were greatly enhanced by progesterone treatment of the cocultures. The highest levels of PMP22 expression were associated with late stages of myelination; however the presence of the protein in nonmyelinating SCs and in SCs commencing myelination supports multiple roles for PMP22 in peripheral nerve biology.

UDP-galactose:ceramide galactosyltransferase is a class I integral membrane protein of the endoplasmic reticulum.

UDP-galactose:ceramide galactosyltransferase (CGalT) transfers UDP-galactose to ceramide to form the glycosphingolipid galactosylceramide. Galactosylceramide is the major constituent of myelin and is also highly enriched in many epithelial cells, where it is thought to play an important role in lipid and protein sorting. Although the biochemical pathways of glycosphingolipid biosynthesis are relatively well understood, the localization of the enzymes involved in these processes has remained controversial. We here have raised antibodies against CGalT and shown by immunocytochemistry on ultrathin cryosections that the enzyme is localized to the endoplasmic reticulum and nuclear envelope but not to the Golgi apparatus or the plasma membrane. In pulse-chase experiments, we have observed that newly synthesized CGalT remains sensitive to endoglycosidase H, confirming the results of the morphological localization experiments. In protease protection assays, we show that the largest part of the protein, including the amino terminus, is oriented toward the lumen of the endoplasmic reticulum. CGalT enzyme activity required import of UDP-galactose into the lumen of the endoplasmic reticulum by a UDP-galactose translocator that is present in the Golgi apparatus of CHO cells but absent in CHOlec8 cells. Finally, we show that CGalT activity previously observed in Golgi membrane fractions in vitro, in the absence of UDP-glucose, is caused by UDP-glucose:ceramide glucosyltransferase. Therefore all galactosylceramide synthesis occurs by CGalT in vivo in the lumen of the endoplasmic reticulum.

Effects of cyclic AMP on expression of myelin genes in the N20.1 oligodendroglial cell line.

The N20.1 immortalized cell line has several characteristics of differentiating oligodendrocytes (OLs), including expression of the glycolipids galactocerebroside (GalC) and sulfatide, and the myelin proteins CNPase and myelin basic protein (MBP) (1,2). Addition of 1-100 microM forskolin to elevate cyclic AMP (cAMP) levels changed cell morphology from irregular and flattened to a more rounded birefringent cell with multiple branched processes. GalC and sulfatide were detected immunocytochemically after permeabilization in the untreated cells and levels appeared to increase slightly following exposure to forskolin. Further analysis showed that most of the glycolipid was internal, with virtually no detectable levels on the cell surface in untreated cells and a very slight change following treatment with forskolin. Synthesis of the two lipids as measured by [H3]galactose incorporation doubled within 24 hours of treatment with forskolin. Levels of message for UDP-galactose: ceramide galactosyl transferase (CGT), a key enzyme in the synthesis of GalC and sulfatide, were compared with those of MBP and proteolipid protein (PLP), before and after elevation of cAMP. No changes were observed in levels of mRNA for CGT and PLP after 24 hours, with a possible increase by 48 hours. In contrast, levels of MBP message dropped precipitously by 24 hours; this was accompanied by an increase in levels of message for suppressed cAMP-inducible POU (SCIP). Thus CGT transcription is regulated independently of MBP and SCIP in N20.1 cells. Analysis of MBP levels by immunocytochemistry and Western blot showed little or no change in protein levels at 24 and 48 hours, in contrast to the sharp decrease in message levels by 24 hours, indicating a relatively long half life for MBP in this cell line. Thus, the N20.1 cells are an informative model for examining regulation of expression of myelinotypic proteins and GalC, as well as the transport of this lipid to the plasma membrane.

Inhibition of glycosphingolipid biosynthesis does not impair growth or morphogenesis of the postimplantation mouse embryo.

Whole embryo culture (WEC) of organogenesis-stage mouse embryos was adapted for glycosphingolipid (GSL) metabolic studies to evaluate the hypothesis that de novo GSL biosynthesis is a prerequisite for growth and morphogenesis of the early postimplantation embryo. WEC supports the growth and development of postimplantation mouse embryos to stages that are indistinguishable from those achieved in vivo. N-Butyldeoxygalactonojirimycin (NB-DGJ) is an N-alkylated imino sugar that specifically inhibits biosynthesis of all glucosylceramide-based GSLs. NB-DGJ inhibited glucosylceramide and lactosylceramide biosynthesis nearly completely and inhibited ganglioside biosynthesis approximately 90% in both the embryo and visceral yolk sac. NB-DGJ also significantly reduced total ganglioside content in both the embryo and visceral yolk sac as estimated by the cholera toxin immunooverlay technique. A shift in expression from the structurally simple to the structurally complex gangliosides was also observed in NB-DGJ-treated embryos and yolk sacs. Despite causing major changes in GSL biosynthesis and composition, NB-DGJ had no effect on embryo viability, growth, or morphology. The findings suggest that de novo GSL biosynthesis may not be a prerequisite for the growth and morphogenesis of the organogenesis-stage mouse embryo.