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Abstract Classic galactosemia (CG) arises from loss‐of‐function mutations in the Galt gene, which codes for the enzyme galactose‐1‐phosphate uridylyltransferase (GALT), a central component in galactose metabolism. The neonatal fatality associated with CG can be prevented by galactose dietary restriction, but for decades it has been known that limiting galactose intake is not a cure and patients often have lasting complications. Even on a low‐galactose diet, GALT's substrate galactose‐1‐phosphate (Gal1P) is elevated and one hypothesis is that elevated Gal1P is a driver of pathology. Here we show that Gal1P levels were elevated above wildtype (WT) in Galt mutant mice, while mice doubly mutant for Galt and the gene encoding galactokinase 1 (Galk1) had normal Gal1P levels. This indicates that GALK1 is necessary for the elevated Gal1P in CG. Another hypothesis to explain the pathology is that an inability to metabolize galactose leads to diminished or disrupted galactosylation of proteins or lipids. Our studies reveal that levels of a subset of cerebrosides—galactosylceramide 24:1, sulfatide 24:1, and glucosylceramide 24:1—were modestly decreased compared to WT. In contrast, gangliosides were unaltered. The observed reduction in these 24:1 cerebrosides may be relevant to the clinical pathology of CG, since the cerebroside galactosylceramide is an important structural component of myelin, the 24:1 species is the most abundant in myelin, and irregularities in white matter, of which myelin is a constituent, have been observed in patients with CG. Therefore, impaired cerebroside production may be a contributing factor to the brain damage that is a common clinical feature of the human disease.

Simple Summary: Galactosylceramide (GalCer) increases the resistance of breast cancer to anti-cancer drugs by acting as an anti-apoptotic molecule. This glycosphingolipid was found to specifically downregulate the levels of the pro-apoptotic TNFRSF1B and TNFRSF9 genes and upregulate the levels of the anti-apoptotic BCL2 gene, suggesting that GalCer regulates their expression at the transcriptional level. Consistent with this hypothesis, (1) MDA-MB-231 and MCF7 cell lines with increased GalCer content showed low activity of the TNFRSF1B and TNFRSF9 promoters; (2) in such MCF7 cells, the activity of the BCL2 promoter was high. However, no differences in BCL2 promoter activity were observed between MDA-MB-231 cells with low and high GalCer contents. Instead, GalCer increased the stability of Bcl-2 mRNA in the latter cells. The regulatory protein that simultaneously increases the expression of the TNFRSF1B and TNFRSF9 genes and decreases the expression of the BCL2 gene and the stability of Bcl-2 transcripts is most likely P53, the expression of which is GalCer dependent. Galactosylceramide (GalCer) increases the resistance of breast cancer cells to doxorubicin, paclitaxel, and cisplatin by acting as an anti-apoptotic molecule. GalCer was found to specifically downregulate the levels of the pro-apoptotic TNFRSF1B and TNFRSF9 genes and upregulate the levels of the anti-apoptotic BCL2 gene, suggesting that this glycosphingolipid regulates their expression at the transcriptional level. Consistent with this hypothesis, MDA-MB-231 and MCF7 breast cancer cells with high levels of GalCer showed lower activity of the TNFRSF1B and TNFRSF9 promoters than cells lacking GalCer. In contrast, the activity of the BCL2 promoter was higher in MCF7 cells overproducing GalCer than in MCF7 cells without GalCer. However, no difference in BCL2 promoter activity was observed between MDA-MB-231 cells with high and no GalCer content. Instead, we found that high levels of GalCer increased the stability of Bcl-2 mRNA. Subsequent studies showed that breast cancer cells with high levels of GalCer are characterized by significantly lower expression of P53. Importantly, inhibition of P53 expression by siRNA in MCF7 and MDA-MB-231 cells lacking GalCer resulted in decreased expression and promoter activity of the TNFRS1B and TNFRSF9 genes. On the other hand, increased expression and promoter activity of the BCL2 gene was found in such MCF7 cells, and increased stability of Bcl-2 transcripts was observed in such MDA-MB-231 cells. Taken together, these data strongly suggest that the regulatory protein that simultaneously increases the expression of the TNFRSF1B and TNFRSF9 genes and decreases the expression of the BCL2 gene and the stability of Bcl-2 transcripts is most likely P53, the expression of which is GalCer dependent. [ABSTRACT FROM AUTHOR]

Fungal glycosphingolipids (GSLs) are important membrane components which play a key role in vesicle trafficking. To assess the importance of GSLs in the fungal life cycle, we performed a mutant phenotypic study of the acidic and neutral GSL biosynthetic pathways in Neurospora crassa. GSL biosynthesis begins with two reactions leading up to the formation of dihydrosphingosine. The first of these reactions is catalyzed by serine palmitoyltransferase and generates 3-keto dihydrosphinganine. In N. crassa, this reaction is catalyzed by GSL-1 and GSL-2 and is required for viability. The second reaction is carried out by GSL-3, a 3-keto dihydrosphinoganine reductase to generate dihydrosphingosine, which is used for the synthesis of neutral and acidic GSLs. We found that deletion mutations in the acidic GSL pathway leading up to the formation of mannosylinositol-phosphoceramide are lethal, indicating that acidic GSLs are essential for viability in N. crassa. Once mannosylinositol-phosphoceramide is made, it is further modified by GSL-5, an inositol-phosphoceramide-B C26 hydroxylase, which adds a hydroxyl group to the amide-linked fatty acid. GSL-5 is not required for viability but gives a clear mutant phenotype affecting all stages of the life cycle. Our results show that the synthesis of mannosylinositol-phosphoceramide is required for viability and that the modification of the amide-linked fatty acid is important for acidic GSL functionality. We also examined the neutral GSL biosynthetic pathway and identified the presence of glucosylceramide. The deletion of neutral GSL biosynthetic genes affected hyphal morphology, vegetative growth rate, conidiation, and female development. Our results indicate that the synthesis of neutral GSLs is essential for normal growth and development of N. crassa. [ABSTRACT FROM AUTHOR]

Horse-derived ceramide (HC), which contains galactosylceramides as its main component, significantly improves skin symptoms when applied topically to patients with atopic dermatitis. We speculated that efficacy resulted from the amelioration of epidermal ceramide metabolism, and we characterized those effects using reconstructed human epidermal equivalents. Lipid analysis, RT-PCR and Western blotting revealed that HC significantly increased the total ceramide content of the stratum corneum (SC), accompanied by significantly increased gene and/or protein expression levels of ceramide synthase (CERS) 3, fatty acid elongase (ELOVL) 4, glucosylceramide synthase (GCS), β-glucocerebrosidase, sphingomyelin synthase and acid sphingomyelinase. Mechanistic analyses using cultures of primary human keratinocytes revealed the marked stimulatory effects of HC on the mRNA expression levels of CERS3, ELOVL4 and GCS under high calcium-derived differentiation conditions. Signaling analyses demonstrated that an antagonist of PPARβ/δ significantly abrogated the HC-stimulated mRNA expression levels of GCS, CERS3 and ELOVL4. GW9662, an antagonist of PPARγ, significantly abolished the HC-up-regulated mRNA expression levels of GCS and ELOVL4, but not of CERS3. These findings suggest that HC has the distinct potential to accentuate the expression of GCS, CERS3 and ELOVL4 via the activation of PPARβ/δ and/or PPARγ to accelerate ceramide synthesis in the SC. [ABSTRACT FROM AUTHOR]

Idiopathic normal pressure hydrocephalus usually exhibits triad of symptoms including gait disturbance, urinary incontinence, and dementia with ventriculomegaly. Currently, its pathogenesis remains to be fully elucidated. To provide a better understanding of this order, we examined whether dysmetabolism of sphingolipids as major lipid components in the brain present in cerebrospinal fluid (CSF) of the patients. Here, we measured various sphingolipidsincluding ceramide and sphingomyelin and glycolipids by electrospray ionization-tandem mass spectrometry in the cerebrospinal fluid of 19 consecutive idiopathic normal pressure hydrocephalus patients, 49 Parkinson's disease patients, and 17 neurologically normal controls. The data showed that there was a significant and specific reduction of all galactosylceramide subspecies levels in idiopathic normal pressure hydrocephalus patients compared with other groups, whereas ceramide and sphingomyelin levels as well as other neutral glycolipids such as glucosylceramide and lactosylceramide were similar in both disease states. Multiple regression analysis of sex and age did not show any correlation with galactosylceramide levels. We also examined whether MMSE scores are correlated with sphingolipid levels in iNPH patients. A specific subspecies of sphingomyelin (d18:1/18:0) only exhibited a statistically significant negative correlation (p = 0.0473, R = −0.4604) with MMSE scores but no other sphingolipids in iNPH patients. These data strongly suggest that myelin-rich galactosylceramide metabolism is severely impaired in idiopathic normal pressure hydrocephalus patients and might serve as the basis of biomarker for this disorder. • Pathogenesis of idiopathic normal pressure hydrocephalus (iNPH) is currently unknown. • Although clinical diagnostic criteria have been available, its excellent surrogate marker is still unavailable. • Brain white matter is usually involved in iNPH patients. • All galactosylceramide subspecies in CSF are severely reduced in iNPH compared with Parkinson's disease and normal subjects. • Whereas ceramides and sphingomyelin as well as other neutral glycolipids were unaltered. [ABSTRACT FROM AUTHOR]

Glycolipids are complex molecules involved in important cellular processes. Among them, the glycosphingolipid α-galactosylceramide has proven to be of interest in biomedicine for its immunostimulatory capabilities. Given its structural requirements, the use of ceramide glycosyltransferase enzymes capable of synthesizing this molecule under in vivo or in vitro conditions is a potential production strategy. Several GT4 enzymes from Bacteroides fragilis were considered as potential candidates in addition to the known BF9343_3149, but only this one showed glycolipid synthase activity. The enzyme was expressed as a SUMO fusion protein to produce soluble protein. It is a non-processive glycosyltransferase that prefers UDP-Gal over UDP-Glc as a donor substrate, and maximum activity was found at pH 7.3 and around 30–35 °C. It does not require metal cations for activity as other GT4 enzymes, but Zn2+ inactivates the enzyme. The reaction occurs when the ceramide lipid acceptor is solubilized with BSA (100% conversion) but not when it is presented in mixed micelles, and anionic lipids do not increase activity, as in other membrane-associated glycolipid synthases. Further protein engineering to increase stability and activity can make feasible the enzymatic synthesis of α-GalCer for biomedical applications. [ABSTRACT FROM AUTHOR]

Fungal glycosphingolipids (GSLs) are important membrane components which play a key role in vesicle trafficking. To assess the importance of GSLs in the fungal life cycle, we performed a mutant phenotypic study of the acidic and neutral GSL biosynthetic pathways in Neurospora crassa. GSL biosynthesis begins with two reactions leading up to the formation of dihydrosphingosine. The first of these reactions is catalyzed by serine palmitoyltransferase and generates 3-keto dihydrosphinganine. In N. crassa, this reaction is catalyzed by GSL-1 and GSL-2 and is required for viability. The second reaction is carried out by GSL-3, a 3-keto dihydrosphinoganine reductase to generate dihydrosphingosine, which is used for the synthesis of neutral and acidic GSLs. We found that deletion mutations in the acidic GSL pathway leading up to the formation of mannosylinositol-phosphoceramide are lethal, indicating that acidic GSLs are essential for viability in N. crassa. Once mannosylinositol-phosphoceramide is made, it is further modified by GSL-5, an inositol-phosphoceramide-B C26 hydroxylase, which adds a hydroxyl group to the amide-linked fatty acid. GSL-5 is not required for viability but gives a clear mutant phenotype affecting all stages of the life cycle. Our results show that the synthesis of mannosylinositol-phosphoceramide is required for viability and that the modification of the amide-linked fatty acid is important for acidic GSL functionality. We also examined the neutral GSL biosynthetic pathway and identified the presence of glucosylceramide. The deletion of neutral GSL biosynthetic genes affected hyphal morphology, vegetative growth rate, conidiation, and female development. Our results indicate that the synthesis of neutral GSLs is essential for normal growth and development of N. crassa.

Inositol phosphorylceramide (IPC) participates in hyphal growth and serves as a signaling molecule that enables fungi to adapt to diverse environments. Here, a gene, encodes IPC synthase, was identified from the Aspergillus oryzae 3.042 genome and designated AoAur1. The characteristics, phylogenetic evolution, and resistance to aureobasidin A of AoAur1 were analyzed. The expression pattern of AoAur1 was markedly downregulated under temperature stress. Additionally, an RNAi-AoAur1 strain in which the AoAur1 expression was inhibited had mycelial that grew more quickly, had a higher frequency of hyphal fusion, and was more resistant to high-temperature stress than the control. Gene expression profiles showed that the genes related to IPC biosynthesis were obviously downregulated, while AoCerS, which participates in dihydroceramide biosynthesis, increased in the RNAi-AoAur1 strain at the three temperature treatments. A metabolomic analysis revealed that the intracellular IPC content decreased, and the accumulation of dihydroceramide and galactosylceramide increased significantly in the RNAi-AoAur1 strain. Thus, the inhibition of AoAur1 reduced IPC level followed by an increase in the contents of dihydroceramide and galactosylceramide that promote mycelial growth and the formation of spores in the RNAi-AoAur1 strain. Interestingly, the inhibition of AoAur1 also induced the expression of hyphal fusion-related genes, which promote hyphal fusion, thus, contributing to the transduction of stress signal to enhance the ability of cells to adapt to temperature stress. Our results demonstrated that the downregulation of AoAur1 and a decrease in the accumulation of IPC is one of the mechanisms that enables A. oryzae to adapt low- and high-temperature stress. [ABSTRACT FROM AUTHOR]

Human brain lipidomics have elucidated structural lipids and lipid signal transduction pathways in neurologic diseases. Such studies have traditionally sourced tissue exclusively from brain bank biorepositories, however, limited inventories signal that these facilities may not be able to keep pace with this growing research domain. Formalin fixed, whole body donors willed to academic institutions offer a potential supplemental tissue source, the lipid profiles of which have yet to be described. To determine the potential of these subjects in lipid analysis, the lipid levels of fresh and fixed frontal cortical gray matter of human donors were compared using high resolution electrospray ionization mass spectrometry. Results revealed commensurate levels of specific triacylglycerols, diacylglycerols, hexosyl ceramides, and hydroxy hexosyl ceramides. Baseline levels of these lipid families in human fixed tissue were identified via a broader survey study covering six brain regions: cerebellar gray matter, superior cerebellar peduncle, gray and subcortical white matter of the precentral gyrus, periventricular white matter, and internal capsule. Whole body donors may therefore serve as supplemental tissue sources for lipid analysis in a variety of clinical contexts, including Parkinson's disease, Alzheimer's disease, Lewy body dementia, multiple sclerosis, and Gaucher's disease. [ABSTRACT FROM AUTHOR]

Human brain lipidomics have elucidated structural lipids and lipid signal transduction pathways in neurologic diseases. Such studies have traditionally sourced tissue exclusively from brain bank biorepositories, however, limited inventories signal that these facilities may not be able to keep pace with this growing research domain. Formalin fixed, whole body donors willed to academic institutions offer a potential supplemental tissue source, the lipid profiles of which have yet to be described. To determine the potential of these subjects in lipid analysis, the lipid levels of fresh and fixed frontal cortical gray matter of human donors were compared using high resolution electrospray ionization mass spectrometry. Results revealed commensurate levels of specific triacylglycerols, diacylglycerols, hexosyl ceramides, and hydroxy hexosyl ceramides. Baseline levels of these lipid families in human fixed tissue were identified via a broader survey study covering six brain regions: cerebellar gray matter, superior cerebellar peduncle, gray and subcortical white matter of the precentral gyrus, periventricular white matter, and internal capsule. Whole body donors may therefore serve as supplemental tissue sources for lipid analysis in a variety of clinical contexts, including Parkinson’s disease, Alzheimer’s disease, Lewy body dementia, multiple sclerosis, and Gaucher’s disease.

Krabbe disease is a lysosomal storage disease caused by mutations in the gene that encodes galactosylceramidase, in which galactosylsphingosine (psychosine) accumulation drives demyelination in the central and peripheral nervous systems, ultimately progressing to death in early childhood. Gene therapy, alone or in combination with transplant, has been developed for almost two decades in mouse models, with increasing therapeutic benefit paralleling the improvement of next-generation adeno-associated virus (AAV) vectors. This effort has recently shown remarkable efficacy in the canine model of the disease by two different groups that used either systemic or cerebrospinal fluid (CSF) administration of AAVrh10 or AAV9. Building on our experience developing CSF-delivered, AAV-based drug products for a variety of neurodegenerative disorders, we conducted efficacy, pharmacology, and safety studies of AAVhu68 delivered to the CSF in two relevant natural Krabbe animal models, and in nonhuman primates. In newborn Twitcher mice, the highest dose (1 × 1011 genome copies [GC]) of AAVhu68.hGALC injected into the lateral ventricle led to a median survival of 130 days compared to 40.5 days in vehicle-treated mice. When this dose was administered intravenously, the median survival was 49 days. A single intracisterna magna injection of AAVhu68.cGALC at 3 × 1013 GC into presymptomatic Krabbe dogs increased survival for up to 85 weeks compared to 12 weeks in controls. It prevented psychosine accumulation in the CSF, preserved peripheral nerve myelination, ambulation, and decreased brain neuroinflammation and demyelination, although some regions remained abnormal. In a Good Laboratory Practice-compliant toxicology study, we administered the clinical candidate into the cisterna magna of 18 juvenile rhesus macaques at 3 doses that displayed efficacy in mice. We observed no dose-limiting toxicity and sporadic minimal degeneration of dorsal root ganglia (DRG) neurons. Our studies demonstrate the efficacy, scalability, and safety of a single cisterna magna AAVhu68 administration to treat Krabbe disease. ClinicalTrials.Gov ID: NCT04771416. [ABSTRACT FROM AUTHOR]

Horse-derived ceramide (HC), which contains galactosylceramides as its main component, significantly improves skin symptoms when applied topically to patients with atopic dermatitis. We speculated that efficacy resulted from the amelioration of epidermal ceramide metabolism, and we characterized those effects using reconstructed human epidermal equivalents. Lipid analysis, RT-PCR and Western blotting revealed that HC significantly increased the total ceramide content of the stratum corneum (SC), accompanied by significantly increased gene and/or protein expression levels of ceramide synthase (CERS) 3, fatty acid elongase (ELOVL) 4, glucosylceramide synthase (GCS), β-glucocerebrosidase, sphingomyelin synthase and acid sphingomyelinase. Mechanistic analyses using cultures of primary human keratinocytes revealed the marked stimulatory effects of HC on the mRNA expression levels of CERS3, ELOVL4 and GCS under high calcium-derived differentiation conditions. Signaling analyses demonstrated that an antagonist of PPARβ/δ significantly abrogated the HC-stimulated mRNA expression levels of GCS, CERS3 and ELOVL4. GW9662, an antagonist of PPARγ, significantly abolished the HC-up-regulated mRNA expression levels of GCS and ELOVL4, but not of CERS3. These findings suggest that HC has the distinct potential to accentuate the expression of GCS, CERS3 and ELOVL4 via the activation of PPARβ/δ and/or PPARγ to accelerate ceramide synthesis in the SC.

Numerous clinical observations and exploitation of cellular and animal models indicate that glucosylceramide (GlcCer) and galactosylceramide (GalCer) are involved in many physiological and pathological phenomena. In many cases, the biological importance of these monohexosylcermides has been shown indirectly as the result of studies on enzymes involved in their synthesis and degradation. Under physiological conditions, GalCer plays a key role in the maintenance of proper structure and stability of myelin and differentiation of oligodendrocytes. On the other hand, GlcCer is necessary for the proper functions of epidermis. Such an important lysosomal storage disease as Gaucher disease (GD) and a neurodegenerative disorder as Parkinson's disease are characterized by mutations in the GBA1 gene, decreased activity of lysosomal GBA1 glucosylceramidase and accumulation of GlcCer. In contrast, another lysosomal disease, Krabbe disease, is associated with mutations in the GALC gene, resulting in deficiency or decreased activity of lysosomal galactosylceramidase and accumulation of GalCer and galactosylsphingosine. Little is known about the role of both monohexosylceramides in tumor progression; however, numerous studies indicate that GlcCer and GalCer play important roles in the development of multidrug-resistance by cancer cells. It was shown that GlcCer is able to provoke immune reaction and acts as a self-antigen in GD. On the other hand, GalCer was recognized as an important cellular receptor for HIV-1. Altogether, these two molecules are excellent examples of how slight differences in chemical composition and molecular conformation contribute to profound differences in their physicochemical properties and biological functions. [ABSTRACT FROM AUTHOR]

Abstract Very‐long‐chain fatty acids, with a chain length of >C20, are abundant in myelin sphingolipids. Recently, a de novo mutation in the ELOVL1 gene, which encodes fatty acid elongase, was identified in patients with neurocutaneous disorders involving skin ichthyosis and multiple neurological abnormalities, including hypomyelination, spastic paraplegia, and high‐frequency deafness. However, the consequences of ELOVL1 deficiency for lipid composition and detailed pathological changes in the brain remain unclear. Here, we analyzed Elovl1 mutant mice as a model of human ELOVL1 deficiency. The mice exhibited a decreased postnatal survival rate, and some died of startle epilepsy. The acyl chain length of sphingolipids such as galactosylceramides, sulfatides, sphingomyelins, and ceramides in the brains of these mice was markedly shortened. Moreover, the mice exhibited reduced levels of galactosylceramides, which are important for myelin formation and stability. Electron microscope analysis of the corpus callosum in Elovl1 mutant mice revealed modest hypomyelination, especially in large‐diameter axons. Furthermore, behavioral testing of the mice revealed deficits such as poorer motor coordination and reduced acoustic startle response to high‐intensity stimulus. These findings provide clues to the molecular mechanism of the neurological symptoms of patients with the ELOVL1 mutation.

A concise and stereoselective synthesis of α-galactosylceramide (α-GalCer) is described. The key features of the synthetic strategy are the use of a phytosphingosine in which the amine is masked as a tetrachlorophthalimide and the diol as an isopropylidene acetal, and the galactosyl donor is protected as a 4,6-benzylidene to improve the α selectivity of the glycosylation reaction. The pattern of protecting groups on the donor and the acceptor have proven to give an excellent match of reactivity, allowing the glycosylation reaction to take place stereoselectively. The overall synthesis gave α-GalCer in good yields and in few steps. [ABSTRACT FROM AUTHOR]

Plasma membranes of axon-wrapping glial cells develop specific cylindrical bilayer membranes that surround thin individual axons or axon bundles. Axons are wrapped with single layered glial cells in lower organisms whereas in the mammalian nervous system, axons are surrounded with a characteristic complex multilamellar myelin structure. The high content of lipids in myelin suggests that lipids play crucial roles in the structure and function of myelin. The most striking feature of myelin lipids is the high content of galactosylceramide (GalCer). Serological and genetic studies indicate that GalCer plays a key role in the formation and function of the myelin sheath in mammals. In contrast to mammals, Drosophila lacks GalCer. Instead of GalCer, ceramide phosphoethanolamine (CPE) has an important role to ensheath axons with glial cells in Drosophila. GalCer and CPE share similar physical properties: both lipids have a high phase transition temperature and high packing, are immiscible with cholesterol and form helical liposomes. These properties are caused by both the strong headgroup interactions and the tight packing resulting from the small size of the headgroup and the hydrogen bonds between lipid molecules. These results suggest that mammals and Drosophila wrap axons using different lipids but the same conserved principle. • Galactosylceramide plays a key role in the formation and function of the myelin sheath in mammals. • Ceramide phosphoethanolamine has an important role to ensheath axons by glial cells in Drosophila. • Galactosylceramide and ceramide phosphoethanolamine share similar physical properties. [ABSTRACT FROM AUTHOR]

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.

Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, β-galactosylceramide (GalCer) and β-glucosylceramide (GlcCer), which are involved in several diseases, such as sphingolipidosis, diabetes, chronic kidney diseases, or cancer. In contrast, Bacteroides fragilis, a member of the human gut microbiome, and the marine sponge, Agelas mauritianus, produce α-GalCer, one of the most potent stimulators for invariant natural killer T cells. To dissect the contribution of these individual stereoisomers to pathologies, we established a novel hydrophilic interaction chromatography-based LC-MS2 method and separated (R > 1.5) corresponding diastereomers from each other, independent of their lipid anchors. Testing various bacterial and mammalian samples, we could separate, identify (including the lipid anchor composition), and quantify endogenous β-GlcCer, β-GalCer, and α-GalCer isomers without additional derivatization steps. Thereby, we show a selective decrease of β-GlcCers versus β-GalCers in cell-specific models of GlcCer synthase-deficiency and an increase of specific β-GlcCers due to loss of β-glucoceramidase 2 activity. Vice versa, β-GalCer increased specifically when cerebroside sulfotransferase (Gal3st1) was deleted. We further confirm β-GalCer as substrate of globotriaosylceramide synthase for galabiaosylceramide synthesis and identify additional members of the human gut microbiome to contain immunogenic α-GalCers. Finally, this method is shown to separate corresponding hexosylsphingosine standards, promoting its applicability in further investigations.

Very‐long‐chain fatty acids, with a chain length of >C20, are abundant in myelin sphingolipids. Recently, a de novo mutation in the ELOVL1 gene, which encodes fatty acid elongase, was identified in patients with neurocutaneous disorders involving skin ichthyosis and multiple neurological abnormalities, including hypomyelination, spastic paraplegia, and high‐frequency deafness. However, the consequences of ELOVL1 deficiency for lipid composition and detailed pathological changes in the brain remain unclear. Here, we analyzed Elovl1 mutant mice as a model of human ELOVL1 deficiency. The mice exhibited a decreased postnatal survival rate, and some died of startle epilepsy. The acyl chain length of sphingolipids such as galactosylceramides, sulfatides, sphingomyelins, and ceramides in the brains of these mice was markedly shortened. Moreover, the mice exhibited reduced levels of galactosylceramides, which are important for myelin formation and stability. Electron microscope analysis of the corpus callosum in Elovl1 mutant mice revealed modest hypomyelination, especially in large‐diameter axons. Furthermore, behavioral testing of the mice revealed deficits such as poorer motor coordination and reduced acoustic startle response to high‐intensity stimulus. These findings provide clues to the molecular mechanism of the neurological symptoms of patients with the ELOVL1 mutation. [ABSTRACT FROM AUTHOR]

Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography–tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9–17.4 µg Crb/mL and 1.3–3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8–12.0 and 14.3–16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.

New derivatives of serinol, 3-[4-(2-chloroethoxy)benzyloxy]-2-phthalimidopropan-1-ol and 3-[4-(2-azidoethoxy)phenoxy]-2-phthalimidopropan-1-ol, were synthesized and tested as glycosyl acceptors in stereoselective 1,2-cis-galactosylation in the benzyl-free synthesis of analogs of α-linked galactosylceramides. It was found that NIS/AgOTf-promoted glycosylation of 3-[4-(2-chloroethoxy)benzyloxy]-2-phthalimidopropan-1-ol with ethyl 4,6-O-(di-tert-butylsilylene)-1-thio-β-D-galactoside resulted in both the expected α-linked diastereomeric glycosides and the product of iodination of the benzyl group of the aglycon. This process was avoided by using the corresponding galactosyl imidate under Et3SiOTf promotion to give exclusively α-linked diastereometic serinol glycosides. [ABSTRACT FROM AUTHOR]

Brain function relies on both rapid electrical communication in neural circuitry and appropriate patterns or synchrony of neural activity. Rapid communication between neurons is facilitated by wrapping nerve axons with insulation by a myelin sheath composed largely of different lipids. Recent evidence has indicated that the extent of myelination of nerve axons can adapt based on neural activity levels and this adaptive myelination is associated with improved learning of motor tasks, suggesting such plasticity may enhance effective learning. In this study, we examined whether another aspect of myelin plasticity-changes in myelin lipid synthesis and composition-may also be associated with motor learning. We combined a motor learning task in mice with in vivo two-photon imaging of neural activity in the primary motor cortex (M1) to distinguish early and late stages of learning and then probed levels of some key myelin lipids using mass spectrometry analysis. Sphingomyelin levels were elevated in the early stage of motor learning while galactosylceramide levels were elevated in the middle and late stages of motor learning, and these changes were correlated across individual mice with both learning performance and neural activity changes. Targeted inhibition of oligodendrocyte-specific galactosyltransferase expression, the enzyme that synthesizes myelin galactosylceramide, impaired motor learning. Our results suggest regulation of myelin lipid composition could be a novel facet of myelin adaptations associated with learning., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

Shiga toxin (Stx)-producing Escherichia coli are the leading cause of hemorrhagic colitis and life-threatening extraintestinal complications in humans. Stx1 and Stx2 are transferred by yet to be delineated mechanisms from the intestine to the circulation where they injure microvascular endothelial cells. The resulting vascular lesions cause renal failure and brain damage. Because lipoproteins are potential carriers of Stx through the circulation, we investigated human lipoprotein-associated neutral glycosphingolipids (GSLs) with emphasis on high (globotriaosylceramide) and low (globotetraosylceramide) affinity Stx-receptors. TLC overlay employing Stx1, Stx2, and anti-GSL antibodies demonstrated preferential distribution of globo-series GSLs to very low- and low-density lipoproteins compared with minor association with high-density lipoproteins. Electrospray ionization quadrupole time-of-flight mass spectrometry portrayed C24:0/C24:1 and C16:0 as the major fatty acid of the ceramide moieties of Stx-receptors carrying nonvarying d18:1 sphingosine. This structural heterogeneity was also found in precursor lactosylceramide, glucosylceramide, and galactosylceramide, the last showing an exceptionally high degree of hydroxylated C24 fatty acids. Our findings provide the basis for exploring the functional role of lipoprotein-associated Stx-receptors in human blood.

Myelin in the mammalian nervous system has a high concentration of galactolipids [galactosylceramide (GalCer) and sulfatide] with 2-hydroxy fatty acids. We recently reported that fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is the major fatty acid 2-hydroxylase in the mouse brain. In this report, we show that FA2H also plays a major role in the formation of 2-hydroxy galactolipids in the peripheral nervous system. FA2H mRNA and FA2H activity in the neonatal rat sciatic nerve increased rapidly during developmental myelination. The contents of 2-hydroxy fatty acids were ∼5% of total galactolipid fatty acids at 4 days of age and increased to 60% in GalCer and to 35% in sulfatides at 60 days of age. The chain length of galactolipid fatty acids also increased significantly during myelination. FA2H expression in cultured rat Schwann cells was highly increased in response to dibutyryl cyclic AMP, which stimulates Schwann cell differentiation and upregulates myelin genes, such as UDP-galactose:ceramide galactosyltransferase and protein zero. These observations indicate that FA2H is a myelination-associated gene. FA2H-directed RNA interference (RNAi) by short-hairpin RNA expression resulted in a reduction of cellular 2-hydroxy fatty acids and 2-hydroxy GalCer in D6P2T Schwannoma cells, providing direct evidence that FA2H-dependent fatty acid 2-hydroxylation is required for the formation of 2-hydroxy galactolipids in peripheral nerve myelin. Interestingly, FA2H-directed RNAi enhanced the migration of D6P2T cells, suggesting that, in addition to their structural role in myelin, 2-hydroxy lipids may greatly influence the migratory properties of Schwann cells.

The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2–3 fold higher than of GlcCer. Although both activities follow Michaelis–Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production. [ABSTRACT FROM AUTHOR]

2-Hydroxy fatty acids are relatively minor species of membrane lipids found almost exclusively as N-acyl chains of sphingolipids. In mammals, 2-hydroxy sphingolipids are uniquely abundant in myelin galactosylceramide and sulfatide. Despite the well-documented abundance of 2-hydroxy galactolipids in the nervous system, the enzymatic process of the 2-hydroxylation is not fully understood. To fill this gap, we have identified a human fatty acid 2-hydroxylase gene (FA2H) that is highly expressed in brain. In this report, we test the hypothesis that FA2H is the major fatty acid 2-hydroxylase in mouse brain and that free 2-hydroxy fatty acids are formed as precursors of myelin 2-hydroxy galactolipids. The fatty acid compositions of galactolipids in neonatal mouse brain gradually changed during the course of myelination. The relative ratio of 2-hydroxy versus nonhydroxy galactolipids was very low at 2 days of age (∼8% of total galactolipids) and increased 6- to 8-fold by 30 days of age. During this period, free 2-hydroxy fatty acid levels in mouse brain increased 5- to 9-fold, and their composition was reflected in the fatty acids in galactolipids, consistent with a precursor-product relationship. The changes in free 2-hydroxy fatty acid levels coincided with fatty acid 2-hydroxylase activity and with the upregulation of FA2H expression. Furthermore, mouse brain fatty acid 2-hydroxylase activity was inhibited by anti-FA2H antibodies. Together, these data provide evidence that FA2H is the major fatty acid 2-hydroxylase in brain and that 2-hydroxylation of free fatty acids is the first step in the synthesis of 2-hydroxy galactolipids.

The anatomical progression of neurofibrillary tangle pathology throughout Alzheimer's disease (AD) pathogenesis runs inverse to the pattern of developmental myelination, with the disease preferentially affecting thinly myelinated regions. Myelin is comprised 80% of lipids, and the prototypical myelin lipids, galactosylceramide, and sulfatide are critical for neurological function. We observed severe depletion of galactosylceramide and sulfatide in AD brain tissue, which can be traced metabolically to the loss of their biosynthetic precursor, very long chain ceramide. The synthesis of very long chain ceramides is catalyzed by ceramide synthase 2 (CERS2). We demonstrate a significant reduction in CERS2 activity as early as Braak stage I/II in temporal cortex, and Braak stage III/IV in hippocampus and frontal cortex, indicating that loss of myelin-specific ceramide synthase activity precedes neurofibrillary tangle pathology in cortical regions. These findings open a new vista on AD pathogenesis by demonstrating a defect in myelin lipid biosynthesis at the preclinical stages of the disease. We posit that, over time, this defect contributes significantly to myelin deterioration, synaptic dysfunction, and neurological decline. [ABSTRACT FROM AUTHOR]

By using shotgun lipidomics based on the separation of lipid classes in the electrospray ion source (intrasource separation) and two-dimensional (2D) MS techniques (Han, X., and R. W. Gross. 2004. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom. Rev. First published on June 18, 2004; doi: 10.1002/mas.20023, In press), individual molecular species of most major and many minor lipid classes can be quantitated directly from biological lipid extracts. Herein, we extended shotgun lipidomics to the characterization and quantitation of cerebroside molecular species in biological samples. By exploiting the differential fragmentation patterns of chlorine adducts using electrospray ionization (ESI) tandem mass spectrometry, hydroxy and nonhydroxy cerebroside species are readily identified. The hexose (either galactose or glucose) moiety of a cerebroside species can be distinguished by examination of the peak intensity ratio of its product ions at m/z 179 and 89 (i.e., 0.74 ± 0.10 and 4.8 ± 0.7 for galactose- and glucose-containing cerebroside species, respectively). Quantitation of cerebroside molecular species (as little as 10 fmol) from chloroform extracts of brain tissue samples was directly conducted by 2D ESI/MS after correction for differences in 13C-isotopomer intensities.This method was demonstrated to have a greater than 1,000-fold linear dynamic range in the low concentration region; therefore, it should have a wide range of applications in studies of the cellular sphingolipid lipidome.

We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [3H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an ∼40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation.The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.

Myelin is a multilayered membrane that tightly wraps neuronal axons, enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signaling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule neurofascin (NFASC), a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the NFASC isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferentially binds sulfatide-containing membranes in cis , with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.

UDP-galactose:ceramide galactosyltransferase (CGT) catalyzes the final step in the synthesis of galactosylceramide (GalCer). It has previously been shown that CGT-deficient mice do not synthesize GalCer and its sulfated derivative GalCer I3-sulfate (galactosylsulfatide, SM4s) but form myelin containing glucosylceramide (GlcCer) and sphingomyelin with 2-hydroxy fatty acids. Because relatively high concentrations of GalCer and SM4s are present also in mammalian kidney, we analyzed the composition of lipids in the kidney of Cgt−/− and, as a control, Cgt+/− and wild-type mice. The homozygous mutant mice lacked GalCer, galabiaosylceramide (Ga2Cer), and SM4s. Yet, they did not show any major morphological or functional defects in the kidney. A slight increase in GlcCer containing 4-hydroxysphinganine was evident among neutral glycolipids. Intriguingly, more polar sulfoglycolipids, that is, lactosylceramide II3-sulfate (SM3) and gangliotetraosylceramide II3,IV3-bis-sulfate (SB1a), were expressed at 2 to 3 times the normal levels in Cgt−/− mice, indicating upregulation of biosynthesis of SB1a from GlcCer via SM3. Given that SM4s is a major polar glycolipid constituting renal tubular membrane, the increase in SM3 and SB1a in the mice deficient in CGT and thus SM4s appears to be a compensatory process, which could partly restore kidney function in the knockout mice. —Tadano-Aritomi, K., T. Hikita, H. Fujimoto, K. Suzuki, K. Motegi, and I. Ishizuka. Kidney lipids in galactosylceramide synthase-deficient mice: absence of galactosylsulfatide and compensatory increase in more polar sulfoglycolipids. J. Lipid Res. 2000. 41: 1237–1243.

Galactosylceramide (GalCer) and lactosylceramide (LacCer) are structural and signaling lipids, playing important roles in signal transduction and cell adhesion. They are especially abundant in the nervous system and in important components of the myelin sheath. Although neurodegenerative disorders are associated with increased oxidative stress and lipid oxidation, the connection between oxidative stress and glycosphingolipid modification has been scarcely addressed. In this study, we aimed to characterize the structural changes caused by the hydroxyl radical to GalCer and LacCer molecular species using electrospray ionization mass spectrometry (ESI–MS and MS/MS) and high performance liquid chromatography–tandem mass spectrometry (HPLC-MS n ). ESI–MS and LC–MS spectra of 24:1GalCer and 24:1LacCer after free radical oxidation showed the formation of new species, which were identified as keto, hydroxyl and hydroperoxy derivatives, arising from modification in the mono unsaturated fatty acyl chain. Formation of ceramide and oxidized ceramides was also observed as a result of 24:1GalCer and 24:1LacCer radical oxidation. 24:1GlcCer (glucosylceramide) was detected after LacCer oxidation, probably due to oxidative cleavage of lactosyl moiety. This study shows that glycosphingolipids are prone to radical induced oxidation, which can be one of the causes of the increased ceramides content and pro apoptotic events during oxidative conditions and neurodegeneration. This MS study will support the future identification of oxidized galactosyl- and lactosylceramide species in sphingolipidomic studies applied to biological samples related with oxidative conditions. [ABSTRACT FROM AUTHOR]

Modified galactosylceramide with a long-chain cyclic acetal at the sugar moiety, plasmalogalactosylceramide, was isolated from equine brain. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from galactosylceramide through acetalization. The presence of cyclic acetal linkage, the linked position and length of the acetal chain of the synthesized and natural products were determined by proton nuclear magnetic resonance spectroscopy and fast-atom bombardment–mass spectrometry, as well as gas chromatography–mass spectrometry and gas–liquid chromatography. The orientation of the acetal chain linked to galactoside was characterized by connectivity between the cyclic acetal proton and ring proton(s) on the sugar moiety using the homonuclear Overhauser effect. This revealed that, of the two positional isomers of the acetal linkage with 4,6-O-acetal and 3,4-O-acetal derivatives obtained from the acetalization reaction, the former positional isomer, separated into two spots, was identified to 'endo'- and 'exo'-type acetal chains. In comparison to the NMR data of the synthesized derivative, equine brain acetalized lipid was found to be an 'endo'-type 4,6-O-acetal derivative.—Yachida, Y., M. Kashiwagi, T. Mikami, K. Tsuchihashi, T. Daino, T. Akino, and S. Gasa. Stereochemical structures of synthesized and natural plasmalogalactosylceramides from equine brain. J. Lipid Res. 1998. 39: 1039–1045.

High-performance thin layer chromatography (HPTLC) is a very robust, fast, and inexpensive technique that enables separation of complex mixtures. Here, we describe the analytical separation of glucosylceramide and galactosylceramide by HPTLC. This technique can be used for quantitation purposes but also with small modification for subsequent mass spectrum analysis for structural determination., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A not-stop-flow online two-dimensional (2D) liquid chromatography (LC) method was developed for comprehensive lipid profiling by coupling normal- and reversed-phase LC with quadrupole time-of-flight mass spectrometry (QToF-MS), which was then applied to separate and identify the lipid species in plasma, making its merits in quality and quantity of the detection of lipids. Total 540 endogenous lipid species from 17 classes were determined in human plasma, and the differences in lipid metabolism products in human plasma between atherosclerosis patients and control subjects were explored in detail. The limit of detections (LODs) of 19 validation standards could all reach ng/mL magnitude, and the RSDs of peak area and retention time ranged 0.4–8.0% and 0.010–0.47%, respectively. In addition, a pair of isomers, galactosylceramides (GalC) and glucosylceramides (GluC), was successfully separated, showing that only the levels of GalC in atherosclerosis patients were significantly increasing, rather than GluC, compared with the controls (controls vs. patients: the ratio was 1.5–2.8-fold increasing). It would be helpful to the further research of the atherosclerosis. [ABSTRACT FROM AUTHOR]

Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and H-NMR. We identified them as β-glucosylceramide and β-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of β-galactosylceramide in A. oryzae. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

M. Kuronen, M. Hermansson, O. Manninen, I. Zech, M. Talvitie, T. Laitinen, O. Gröhn, P. Somerharju, M. Eckhardt, J. D. Cooper, A.-E. Lehesjoki, U. Lahtinen and O. Kopra (2012) Neuropathology and Applied Neurobiology 38, 471-486 Galactolipid deficiency in the early pathogenesis of neuronal ceroid lipofuscinosis model Cln8mnd: implications to delayed myelination and oligodendrocyte maturation Aims: CLN8 deficiency underlies one of a group of devastating childhood neurodegenerative disorders, the neuronal ceroid lipofuscinoses. The function of the CLN8 protein is currently unknown, but a role in lipid metabolism has been proposed. In human CLN8 diseased brains, alterations in lipid composition have been detected. To further investigate the connection of CLN8 to lipid metabolism, we characterized the lipid composition of early symptomatic Cln8-deficient mouse ( Cln8mnd) brains. Methods: For lipid profiling, Cln8mnd cerebral cortical tissue was analysed by liquid chromatography/mass spectrometry. Galactolipid synthesis was measured through enzyme activity and real-time mRNA expression analyses. Based on the findings, myelination and white matter integrity were studied by immunohistochemistry, stereological methods, electron microscopy and magnetic resonance imaging. The development of myelin-forming oligodendrocytes was also studied in vitro. Results: Sphingolipid profiling showed a selective reduction in myelin-enriched galactolipids. The mRNA expression and activity of UDP-galactose:ceramide galactosyltransferase (CGT), the key enzyme in the galactolipid synthesis, was reduced in the Cln8mnd brain. Expression of oligodendrocyte markers suggests a maturation defect. The amount of myelin was reduced in 1-month-old Cln8mnd mice, but reached normal levels by 5 months of age. The level of Cln8 gene expression followed the developmental pattern of myelin formation and was high in primary oligodendrocytes. Conclusions: Taken together, these observations suggest that galactolipid deficiency and delayed myelin maturation characterize the early CLN8 disease pathogenesis through a maturation defect of oligodendrocytes. [ABSTRACT FROM AUTHOR]

The aim of this study is to illustrate the relationship between α-Galatosylceramide (α-GalCer) dose and α-GalCer induced anergy of immune system in vivo. α-GalCer, a glycolipid and a widely studied immune modulator extracted from a marine sponge, shows its effect in vivo via presentation on CD1d molecule, MHC I like molecule bound glycolipid antigens, to NKT cells, a lymphocyte has both TCR and NK1.1, marker of T cells and NK cells respectively. The contradictory reports about α-GalCer are focused on two opposite axes of immune system: cancer and autoimmunity, failure and exaggeration of Th1 type immune response, respectively. In this study, the relation between the α-GalCer dose and the anergy was considered to clarify this dilemma. The groups of BALB/c mice were injected with various titer of α-GalCer intravenous way repeatedly and serum level of IFN-γ was measured with mouse IFN-γ ELISA. The Dramatic difference was found between groups after 2nd application of α-GalCer: Low dose α-GalCer boosted IFN-γ; however, high dose, usual dose cited in literature, failed to induce IFN-γ production. Besides, low concentrations of _-GalCer succeeded considerable amount of IFN-γ in the serum after priming. This results show that minute level of α-GalCer can be recognized by NKT cells, and repeated in vivo application of low dose α-GalCer can improve Th1 capabilities of the immune system. [ABSTRACT FROM AUTHOR]

Abstract: A synthetic C-glycoside, α-C-galactosylceramide, is an active immunostimulant in mice. It displays better activity than α-O-galactosylceramide in several disease models. Syntheses of several α-C-galactosylceramides are described. Experiments that probe its immunostimulant activity are outlined. Possible explanations for its superior activity are discussed. [Copyright &y& Elsevier]

Abstract: Sulfatide is one of the most relevant endogenous acidic glycolipids found in mammal membranes, ubiquitously present in various tissues. The review will briefly introduce the physiopathological role of sulfatide, then focusing mainly on synthetic aspects related to the preparation of this molecule and of analogues thereof, as tools to better understand its biological role, its involvement in pathogenesis and to stimulate the research towards development of new derivatives for biomedical applications. [Copyright &y& Elsevier]