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1.
Methods Mol Biol ; 2761: 149-157, 2024.
Article in English | MEDLINE | ID: mdl-38427236

ABSTRACT

Chromatographic separation and purification of an individual lipid to homogeneity have long been introduced. Using this concept, a more precise method has been developed to identify and characterize the sphingolipid composition(s) using a small amount (30 mg) of biological sample. Sphingolipids (lipids containing sphingosine or dihydrosphingosine) are well-known regulators of the central nervous system development and play a critical role in neurodegenerative diseases. Introducing a silicic acid column chromatography, sphingolipid components have been separated to individual fractions such as ceramide, glucosyl/galactosylceramide, other neutral and acidic glycosphingolipids, including (dihydro)sphingosine and psychosine; as well as phospholipids from which individual components are quantified employing a single or combination of other advanced chromatography procedures such as thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography-mass spectrometry.


Subject(s)
Sphingolipids , Sphingosine , Sphingolipids/chemistry , Sphingosine/analysis , Ceramides/analysis , Chromatography, Thin Layer/methods , Central Nervous System/chemistry
2.
Int J Mol Sci ; 20(20)2019 Oct 11.
Article in English | MEDLINE | ID: mdl-31614447

ABSTRACT

Ceramide and sphingosine display a unique profile during brain development, indicating their critical role in myelinogenesis. Employing advanced technology such as gas chromatography-mass spectrometry, high performance liquid chromatography, and immunocytochemistry, along with cell culture and molecular biology, we have found an accumulation of sphingosine in brain tissues of patients with multiple sclerosis (MS) and in the spinal cord of rats induced with experimental autoimmune encephalomyelitis. The elevated sphingosine leads to oligodendrocyte death and fosters demyelination. Ceramide elevation by serine palmitoyltransferse (SPT) activation was the primary source of the sphingosine elevation as myriocin, an inhibitor of SPT, prevented sphingosine elevation and protected oligodendrocytes. Supporting this view, fingolimod, a drug used for MS therapy, reduced ceramide generation, thus offering partial protection to oligodendrocytes. Sphingolipid synthesis and degradation in normal development is regulated by a series of microRNAs (miRNAs), and hence, accumulation of sphingosine in MS may be prevented by employing miRNA technology. This review will discuss the current knowledge of ceramide and sphingosine metabolism (synthesis and breakdown), and how their biosynthesis can be regulated by miRNA, which can be used as a therapeutic approach for MS.


Subject(s)
Ceramides/biosynthesis , MicroRNAs/genetics , Multiple Sclerosis/genetics , Serine C-Palmitoyltransferase/metabolism , Sphingosine/biosynthesis , Animals , Brain/metabolism , Fingolimod Hydrochloride/pharmacology , Fingolimod Hydrochloride/therapeutic use , Humans , MicroRNAs/antagonists & inhibitors , Molecular Targeted Therapy , Multiple Sclerosis/drug therapy , Multiple Sclerosis/metabolism , Myelin Sheath/metabolism , Rats , Serine C-Palmitoyltransferase/antagonists & inhibitors
3.
Neural Regen Res ; 14(11): 1880-1892, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31290437

ABSTRACT

All retinoids, which can be natural and synthetic, are chemically related to vitamin A. Both natural and synthetic retinoids use specific nuclear receptors such as retinoic acid receptors and retinoid X receptors to activate specific signaling pathways in the cells. Retinoic acid signaling is extremely important in the central nervous system. Impairment of retinoic acid signaling pathways causes severe pathological processes in the central nervous system, especially in the adult brain. Retinoids have major roles in neural patterning, differentiation, axon outgrowth in normal development, and function of the brain. Impaired retinoic acid signaling results in neuroinflammation, oxidative stress, mitochondrial malfunction, and neurodegeneration leading to progressive Alzheimer's disease, which is pathologically characterized by extra-neuronal accumulation of amyloid plaques (aggregated amyloid-beta) and intra-neurofibrillary tangles (hyperphosphorylated tau protein) in the temporal lobe of the brain. Alzheimer's disease is the most common cause of dementia and loss of memory in old adults. Inactive cholinergic neurotransmission is responsible for cognitive deficits in Alzheimer's disease patients. Deficiency or deprivation of retinoic acid in mice is associated with loss of spatial learning and memory. Retinoids inhibit expression of chemokines and neuroinflammatory cytokines in microglia and astrocytes, which are activated in Alzheimer's disease. Stimulation of retinoic acid receptors and retinoid X receptors slows down accumulation of amyloids, reduces neurodegeneration, and thereby prevents pathogenesis of Alzheimer's disease in mice. In this review, we described chemistry and biochemistry of some natural and synthetic retinoids and potentials of retinoids for prevention of neuroinflammation and neurodegeneration in Alzheimer's disease.

4.
Neurochem Res ; 42(10): 2755-2768, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28474276

ABSTRACT

Multiple sclerosis (MS) is a demyelinating disorder characterized by massive neurodegeneration and profound axonal loss. Since myelin is enriched with sphingolipids and some of them display toxicity, biological function of sphingolipids in demyelination has been investigated in MS brain tissues. An elevation of sphingosine with a decrease in monoglycosylceramide and psychosine (myelin markers) was observed in MS white matter and plaque compared to normal brain tissue. This indicated that sphingosine toxicity might mediate oligodendrocyte degeneration. To explain the source of sphingosine accumulation, total sphingolipid profile was investigated in Lewis rats after inducing experimental autoimmune encephalomyelitis (EAE) and also in human oligodendrocytes in culture. An intermittent increase in ceramide followed by sphingosine accumulation in EAE spinal cord along with a stimulation of serine-palmitoyltransferase (SPT) activity was observed. Apoptosis was identified in the lumbar spinal cord, the most prominent demyelinating area, in the EAE rats. TNFα and IFNγ stimulation of oligodendrocytes in culture also led to an accumulation of ceramide with an elevation of sphingosine. Ceramide elevation was drastically blocked by myriocin, an inhibitor of SPT, and also by FTY720. Myriocin treatment also protected oligodendrocytes from cytokine mediated apoptosis or programmed cell death. Hence, we propose that sphingosine toxicity may contribute to demyelination in both EAE and MS, and the intermittent ceramide accumulation in EAE may, at least partly, be mediated via SPT activation, which is a novel observation that has not been previously reported.


Subject(s)
Ceramides/pharmacology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Multiple Sclerosis/drug therapy , Serine C-Palmitoyltransferase/metabolism , Sphingosine/toxicity , Animals , Disease Models, Animal , Fingolimod Hydrochloride/pharmacology , Humans , Myelin Sheath/metabolism , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Rats , Spinal Cord/metabolism
5.
J Neurol Psychol ; 5(1)2017 Dec.
Article in English | MEDLINE | ID: mdl-30338269

ABSTRACT

Sphingolipids are enriched in the Central Nervous System (CNS) and display multiple biological functions. They participate in tissue development, cell recognition and adhesion, and act as receptors for toxins. During myelination, a variety of interactive molecules such as myelin basic protein, myelin associated glycoprotein, phospholipids, cholesterol, sphingolipids, etc., participate in a complex fashion. Precise roles of some sphingolipids in myelination still remain unexplored. Our investigation delineated participation of several sphingolipids in myelination during rat brain development as well as in human brain demyelination during pathogenesis of Multiple Sclerosis (MS). These sphingolipids included Ceramide (Cer)/dihydroceramide (dhCer), Sphingosine (Sph)/dihydrosphingosine (dhSph), and glucosyl/galactosylceramide (glc/galCer) as we detected these by column chromatography, high performance thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography. Cer/dhCer level rises during rat brain development starting at Embryonic stage (E) until postnatal day (P21), then gradually falls until the maturity (P30 and onwards), and remains steady maintaining a constant ratio (4-4.5:1) throughout the brain development. GlcCer is the initial Monoglycosylceramide (MGC) that appears at early Postnatal stage (P8) and then GalCer appears at P10 with an increasing trend until P21 and its concentration remains unaltered. Sph and dhSph profiles show a similar trend with an initial peak at P10 and then a comparatively smaller peak at P21 maintaining a ratio of (2-2.5:1) of Sph:dhSph. The profiles of all these sphingolipids, specifically at P21, clearly indicate their importance during rat brain development but somewhat unspecified roles in myelination. While Cer has been reported to involve in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, Sph being a potent inhibitor of protein kinase C has recently been implicated in CNS demyelination due to MS. Inflammatory cytokines stimulate Sph elevation in MS brains and lead to demyelination due to oligodendrocyte death as we examined by using human oligodendroglioma culture. In conclusions, sphingolipids are essential for brain development but they have deleterious effects in demyelinating diseases such as MS.

6.
Article in English | MEDLINE | ID: mdl-29732412

ABSTRACT

Multiple sclerosis (MS) is a fatal demyelinating disease that primarily affects axons leading to massive neurodegeneration. Many studies have reported the causes and drawn the conclusions that multiple factors such as recurrent viral infections, hereditary link, and environmental condition are involved in the pathogenesis of MS. In essence, all these reports indicate a severe change in the biochemical milieu in the central nervous system (CNS) leading to inflammation and neurodegeneration. Recent studies in our laboratory revealed aberrant sphingolipid metabolism and accumulation of toxic sphingosine in the CNS tissues in MS patients. An elevation in sphingosine in MS brain white matter and plaque indicated that sphingosine toxicity might mediate degeneration of oligodendrocytes contributing to demyelination. An intermittent increase in ceramide followed by sphingosine accumulation in spinal cords from Lewis rats with experimental autoimmune encephalitis (EAE) and also stimulation of serine-palmitoyltransferase (SPT) activity correlated with induction of apoptosis in the lumbar spinal cord in EAE animals. Cytokine-stimulated ceramide elevation in cultured human oligodendrocytes was almost completely blocked by myriocin, an inhibitor of SPT. Myriocin exposure also protected oligodendrocytes from induction of apoptosis. Sphingosine toxicity via ceramide biosynthesis contributed to oligodendrocyte degeneration in both EAE and MS. Although many clinical trials are being conducted for MS, to the best of our knowledge, there is still no sphingolipid-targeted therapy available. Hence, we propose that sphingosine toxicity via ceramide generation may be a potential therapeutic target in both EAE and MS.

7.
J Nutr ; 146(2): 200-8, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26701794

ABSTRACT

BACKGROUND: Sialyllactose is a key human milk oligosaccharide and consists of sialic acid (SA) bound to a lactose molecule. Breastfed infants have increased accumulation of ganglioside-bound SA compared with formula-fed infants. OBJECTIVE: This study aimed to determine whether different isomers of sialyllactose enrich brain SA and modulate the microbiome of developing neonatal piglets. METHODS: Day-old pigs were randomly allocated to 6 diets (control, 2 or 4 g 3'-sialyllactose/L, 2 or 4 g 6'-sialyllactose/L, or 2 g polydextrose/L + 2 g galacto-oligosaccharides/L; n = 9) and fed 3 times/d for 21 d. Pigs were killed, and the left hemisphere of the brain was dissected into cerebrum, cerebellum, corpus callosum, and hippocampus regions. SA was determined by using a modified periodic acid-resorcinol reaction. Microbial composition of the intestinal digesta was analyzed with the use of 16S ribosomal DNA Illumina sequencing. RESULTS: Dietary sialyllactose did not affect feed intake, growth, or fecal consistency. Ganglioside-bound SA in the corpus callosum of pigs fed 2 g 3'-sialyllactose or 6'-sialyllactose/L increased by 15% in comparison with control pigs. Similarly, ganglioside-bound SA in the cerebellum of pigs fed 4 g 3'-sialyllactose/L increased by 10% in comparison with control pigs. Significant (P < 0.05, Adonis Test) microbiome differences were observed in the proximal and distal colons of piglets fed control compared with 4-g 6'-sialyllactose/L formulas. Differences were attributed to an increase in bacterial taxa belonging to species Collinsella aerofaciens (phylum Actinobacteria), genera Ruminococcus and Faecalibacterium (phylum Firmicutes), and genus Prevotella (phylum Bacteroidetes) (Wald test, P < 0.05, DeSeq2) compared with piglets fed the control diet. Taxa belonging to families Enterobacteriaceae and Enterococcaceae (phylum Proteobacteria), as well as taxa belonging to family Lachnospiraceae and order Lactobacillales (phylum Firmicutes), were 2.3- and 4-fold lower, respectively, in 6'-sialyllactose-fed piglets than in controls. CONCLUSIONS: Supplementation of formula with 3'- or 6'-sialyllactose can enrich ganglioside SA in the brain and modulate gut-associated microbiota in neonatal pigs. We propose 2 potential routes by which sialyllactose may positively affect the neonate: serving as a source of SA for neurologic development and promoting beneficial microbiota.


Subject(s)
Brain/drug effects , Colon/drug effects , Dietary Supplements , Gangliosides/metabolism , Gastrointestinal Microbiome/drug effects , Infant Formula , Lactose/analogs & derivatives , Sialic Acids/pharmacology , Animals , Bacteria/growth & development , Brain/metabolism , Cerebellum/drug effects , Cerebellum/metabolism , Colon/microbiology , Corpus Callosum/drug effects , Corpus Callosum/metabolism , Diet , Isomerism , Lactose/pharmacology , Milk, Human/chemistry , Oligosaccharides/pharmacology , Swine
8.
Int J Cancer ; 137(7): 1610-20, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-25833198

ABSTRACT

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA-MB-231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP-associated exosomes, while siRNA-mediated knockdown or GW4869-mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor-labeled breast cancer stem-like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than fivefold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Breast Neoplasms/drug therapy , Doxorubicin/pharmacology , Neoplasm Proteins/metabolism , Pregnenediones/pharmacology , Tetrahydronaphthalenes/pharmacology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Bexarotene , Breast Neoplasms/pathology , Cell Death/drug effects , Cell Line, Tumor , Ceramides/biosynthesis , Down-Regulation/drug effects , Drug Resistance, Neoplasm , Drug Synergism , Exosomes/drug effects , Exosomes/metabolism , Humans
9.
J Alzheimers Dis ; 46(1): 55-61, 2015.
Article in English | MEDLINE | ID: mdl-25720409

ABSTRACT

We present evidence that 5XFAD Alzheimer's disease model mice develop an age-dependent increase in antibodies against ceramide, suggesting involvement of autoimmunity against ceramide in Alzheimer's disease pathology. To test this, we increased serum anti-ceramide IgG (2-fold) by ceramide administration and analyzed amyloid plaque formation in 5XFAD mice. There were no differences in soluble or total amyloid-ß levels. However, females receiving ceramide had increased plaque burden (number, area, and size) compared to controls. Ceramide-treated mice showed an increase of serum exosomes (up to 3-fold using Alix as marker), suggesting that systemic anti-ceramide IgG and exosome levels are correlated with enhanced plaque formation.


Subject(s)
Aging , Alzheimer Disease/blood , Alzheimer Disease/drug therapy , Ceramides/administration & dosage , Ceramides/immunology , Immunoglobulin G/blood , Plaque, Amyloid/pathology , Alzheimer Disease/genetics , Alzheimer Disease/immunology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Analysis of Variance , Animals , Calcium-Binding Proteins/metabolism , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Exosomes/metabolism , Female , Humans , Mice , Mice, Transgenic , Mutation/genetics , Plaque, Amyloid/physiopathology , Presenilin-1/genetics , Presenilin-1/metabolism
10.
PLoS One ; 9(10): e110119, 2014.
Article in English | MEDLINE | ID: mdl-25330231

ABSTRACT

The sphingosine-1-phosphate (S1P) transporter Spns2 regulates myocardial precursor migration in zebrafish and lymphocyte trafficking in mice. However, its function in cancer has not been investigated. We show here that ectopic Spns2 expression induced apoptosis and its knockdown enhanced cell migration in non-small cell lung cancer (NSCLC) cells. Metabolically, Spns2 expression increased the extracellular S1P level while its knockdown the intracellular. Pharmacological inhibition of S1P synthesis abolished the augmented cell migration mediated by Spns2 knockdown, indicating that intracellular S1P plays a key role in this process. Cell signaling studies indicated that Spns2 expression impaired GSK-3ß and Stat3 mediated pro-survival pathways. Conversely, these pathways were activated by Spns2 knockdown, which explains the increased cell migration since they are also crucial for migration. Alterations of Spns2 were found to affect several enzymes involved in S1P metabolism, including sphingosine kinases, S1P phosphatases, and S1P lyase 1. Genetically, Spns2 mRNA level was found to be reduced in advanced lung cancer (LC) patients as quantified by using a small scale qPCR array. These data show for the first time that Spns2 plays key roles in regulating the cellular functions in NSCLC cells, and that its down-regulation is a potential risk factor for LC.


Subject(s)
Anion Transport Proteins/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Movement , Lung Neoplasms/pathology , Lysophospholipids/metabolism , Sphingosine/analogs & derivatives , Animals , Anion Transport Proteins/deficiency , Anion Transport Proteins/genetics , Apoptosis , Biological Transport , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Survival , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Intracellular Space/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Lysosphingolipid/metabolism , Sphingosine/metabolism
11.
Mol Biol Cell ; 25(11): 1715-29, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24694597

ABSTRACT

We show here that human embryonic stem (ES) and induced pluripotent stem cell-derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC (aPKC), both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes. Neural differentiation of human ES cells to NPs is concurrent with a threefold elevation of ceramide-in particular, saturated, long-chain C16:0 ceramide (N-palmitoyl sphingosine) and nonsaturated, very long chain C24:1 ceramide (N-nervonoyl sphingosine). Decreasing ceramide levels by inhibiting ceramide synthase or neutral sphingomyelinase 2 leads to translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora kinase A (AurA). Inhibition of aPKC, AurA, or a downstream target of AurA, HDAC6, restores ciliogenesis in ceramide-depleted cells. Of importance, addition of exogenous C24:1 ceramide reestablishes membrane association of aPKC, restores primary cilia, and accelerates neural process formation. Taken together, these results suggest that ceramide prevents activation of HDAC6 by cytosolic aPKC and AurA, which promotes acetylation of tubulin in primary cilia and, potentially, neural processes. This is the first report on the critical role of ceramide generated by nSMase2 in stem cell ciliogenesis and differentiation.


Subject(s)
Ceramides/pharmacology , Cilia/metabolism , Embryonic Stem Cells/metabolism , Neural Stem Cells/metabolism , Sphingomyelin Phosphodiesterase/metabolism , Acetylation/drug effects , Animals , Aurora Kinase A/metabolism , Cell Differentiation/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Ceramides/metabolism , Cilia/drug effects , Cytosol/drug effects , Cytosol/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Enzyme Activation/drug effects , Histone Deacetylase 6 , Histone Deacetylases/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Mice , Neural Stem Cells/cytology , Neural Stem Cells/enzymology , Organogenesis/drug effects , Protein Kinase C/metabolism , Protein Transport/drug effects , Proteolysis/drug effects , Spindle Apparatus/drug effects , Spindle Apparatus/metabolism , Tubulin/metabolism
12.
Neurobiol Aging ; 35(8): 1792-800, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24650793

ABSTRACT

We present evidence here that exosomes stimulate aggregation of amyloid beta (Aß)1-42 in vitro and in vivo and interfere with uptake of Aß by primary cultured astrocytes and microglia in vitro. Exosome secretion is prevented by the inhibition of neutral sphingomyelinase 2 (nSMase2), a key regulatory enzyme generating ceramide from sphingomyelin, with GW4869. Using the 5XFAD mouse, we show that intraperitoneal injection of GW4869 reduces the levels of brain and serum exosomes, brain ceramide, and Aß1-42 plaque load. Reduction of total Aß1-42 as well as number of plaques in brain sections was significantly greater (40% reduction) in male than female mice. Our results suggest that GW4869 reduces amyloid plaque formation in vivo by preventing exosome secretion and identifies nSMase2 as a potential drug target in AD by interfering with exosome secretion.


Subject(s)
Alzheimer Disease/etiology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Brain/metabolism , Exosomes/metabolism , Peptide Fragments/metabolism , Plaque, Amyloid/metabolism , Alzheimer Disease/therapy , Aniline Compounds/pharmacology , Animals , Astrocytes/metabolism , Benzylidene Compounds/pharmacology , Cells, Cultured , Ceramides/metabolism , Disease Models, Animal , Female , Male , Mice, Inbred C57BL , Microglia/metabolism , Molecular Targeted Therapy , Protein Aggregation, Pathological , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelin Phosphodiesterase/physiology
13.
PLoS One ; 8(11): e80841, 2013.
Article in English | MEDLINE | ID: mdl-24312247

ABSTRACT

Ceramide is a precursor for complex sphingolipids in vertebrates, while plants contain phytoceramide. By using a novel chromatography purification method we show that phytoceramide comprises a significant proportion of animal sphingolipids. Total ceramide including phytoceramide from mouse tissue (brain, heart, liver) lipid extracts and cell culture (mouse primary astrocytes, human oligodendroglioma cells) was eluted as a single homogenous fraction, and then analyzed by thin layer chromatography, and further characterized by gas chromatography-mass spectrometry (GC-MS). We detected a unique band that migrated between non-hydroxy fatty acyl ceramide and hydroxy fatty acyl ceramide, and identified it as phytoceramide. Using RT-PCR, we confirmed that mouse tissues expressed desaturase 2, an enzyme that has been reported to generate phytoceramide from dihydroceramide. Previously, only trace amounts of phytoceramide were reported in vertebrate intestine, kidney, and skin. While its function is still elusive, this is the first report of phytoceramide characterization in glial cells and vertebrate brain, heart, and liver.


Subject(s)
Ceramides/chemistry , Ceramides/metabolism , Metabolomics , Animals , Metabolomics/methods , Mice , Organ Specificity , Vertebrates
14.
Mol Biol Cell ; 23(16): 3156-66, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22718902

ABSTRACT

We show that in Madin-Darby canine kidney (MDCK) cells, an apical ceramide-enriched compartment (ACEC) at the base of primary cilia is colocalized with Rab11a. Ceramide and Rab11a vesicles isolated by magnetic sorting contain a highly similar profile of proteins (atypical protein kinase C [aPKC], Cdc42, Sec8, Rab11a, and Rab8) and ceramide species, suggesting the presence of a ciliogenic protein complex associated with ceramide at the ACEC. It is intriguing that C16 and C18 ceramide, although less abundant ceramide species in MDCK cells, are highly enriched in ceramide and Rab11a vesicles. Expression of a ceramide-binding but dominant-negative mutant of aPKC suppresses ciliogenesis, indicating that the association of ceramide with aPKC is critical for the formation of this complex. Our results indicate that ciliogenic ceramide is derived from apical sphingomyelin (SM) that is endocytosed and then converted to the ACEC. Consistently, inhibition of acid sphingomyelinase with imipramine disrupts ACEC formation, association of ciliogenic proteins with Rab11a vesicles, and cilium formation. Ciliogenesis is rescued by the histone deacetylase (HDAC) inhibitor trichostatin A, indicating that ceramide promotes tubulin acetylation in cilia. Taken together, our results suggest that the ACEC is a novel compartment in which SM-derived ceramide induces formation of a ciliogenic lipid-protein complex that sustains primary cilia by preventing deacetylation of microtubules.


Subject(s)
Ceramides/metabolism , Cilia/physiology , Acetylation , Animals , Cell Polarity , Cells, Cultured , Cilia/enzymology , Cilia/metabolism , Cytoplasmic Vesicles/metabolism , Dogs , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Hydroxamic Acids/pharmacology , Microscopy, Fluorescence , Protein Kinase C/metabolism , Protein Processing, Post-Translational , Protein Transport , Sphingomyelins/metabolism , Tubulin/metabolism , rab GTP-Binding Proteins/metabolism
15.
J Neurochem ; 119(4): 749-59, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21916893

ABSTRACT

Sulfoglucuronosyl paragloboside (SGPG), a minor glycosphingolipid of endothelial cells, is a ligand for L-selectin and has been implicated in neuro-inflammatory diseases, such as Guillian-Barré syndrome. Inflammatory cytokines, such as TNFα and IL-1ß, up-regulate SGPG expression by stimulating gene expression for glucuronosyltransferases, both P and S forms (GlcATp and GlcATs), and the human natural killer antigen (HNK-1) sulfotransferase (HNK-1 ST). Transfection of a human cerebromicrovascular endothelial cell (SV-HCEC) line with HNK-1 ST siRNA down-regulated SGPG expression, inhibited cytokine-stimulated T-cell adhesion, and offered protection against apoptosis. However, the precise mechanisms of SGPG elevation in endothelial cell apoptosis and the maintenance of blood-brain or blood-nerve barrier integrity in inflammation have not been elucidated. Blocking SGPG expression inhibited cytokine-mediated stimulation of NF-κB activity but stimulated MAP kinase activity. Furthermore, elevation of SGPG by over-expression of GlcATp and GlcATs triggered endothelial cell apoptosis, with GlcATs being more potent than GlcATp. Although SGPG-mediated endothelial cell apoptosis was preceded by inhibiting the intracellular NF-κB activity, interfering with Akt and ERK activation and stimulating caspase 3 in SV-HCECs, HNK-1ST siRNA transfection also interfered with IκB phosphorylation but stimulated ERK activation. Our data indicate that SGPG is a critical regulatory molecule for maintaining endothelial cell survival and blood-brain or blood-nerve barrier function.


Subject(s)
Apoptosis/drug effects , Endothelial Cells/drug effects , Gene Expression Regulation/drug effects , Globosides/pharmacology , Glycosphingolipids/metabolism , Signal Transduction/drug effects , Caspases/metabolism , Cell Line , Cerebral Cortex/anatomy & histology , Enzyme Activation , Gene Expression Regulation/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Microvessels/cytology , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction/physiology , Sulfotransferases/genetics , Sulfotransferases/metabolism , Transfection/methods
16.
J Lipid Res ; 51(6): 1394-406, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20154333

ABSTRACT

Fast migrating cerebrosides (FMC) are derivatives of galactosylceramide (GalCer). The structures of the most hydrophobic FMC-5, FMC-6, and FMC-7 were determined by electrospray ionization linear ion-trap mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy complementing previous NMR spectroscopy and gas chromatography-mass spectrometry to be 3-O-acetyl-sphingosine-GalCer derivatives with galactose O-acetyl modifications. FMC-5 and FMC-6 are 3-O-acetyl-sphingosine-2,3,4,6-tetra-O-acetyl-GalCer with nonhydroxy and hydroxy-N-fatty-acids, while FMC-7 has an additional O-acetylation of the 2-hydroxy-fatty acid. The immuno-reactivity in human cerebrospinal fluid (CSF) to these acetylated glycolipids was examined in central nervous system (CNS) infectious disease, noninflammatory disorders, and multiple sclerosis (MS). Screening for lipid binding in MS and other neurological disease groups revealed that the greatest anti-hydrophobic FMC reactivity was observed in the inflammatory CNS diseases (meningitis, meningo-encephalitis, and subacute sclerosing panencephalitis). Some MS patients had increased reactivity with the hydrophobic FMCs and with glycoglycerophospholipid MfGL-II from Mycoplasma fermentans. The cross-reactivity of highly acetylated GalCer with microbial acyl-glycolipid raises the possibility that myelin-O-acetyl-cerebrosides, bacterial infection, and neurological disease are linked.


Subject(s)
Cerebrospinal Fluid/immunology , Galactosylceramides/chemistry , Galactosylceramides/immunology , Myelin Sheath/chemistry , Acetylation , Animals , Antibodies/immunology , Brain/cytology , Cattle , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Female , Galactosylceramides/analysis , Glycolipids/immunology , Humans , Hydrophobic and Hydrophilic Interactions , Lipopolysaccharides/immunology , Magnetic Resonance Spectroscopy , Male , Mass Spectrometry , Mycoplasma fermentans , Rats , Sphingosine/chemistry
17.
J Neurosci Res ; 87(16): 3591-9, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19533738

ABSTRACT

Inflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta stimulate glucuronosyltransferase genes (S and P) in endothelial cells (ECs) and up-regulate sulfoglucuronosyl paragloboside (SGPG) expression, which serves as a ligand for T cell adhesion. However, the mechanism of cytokine-mediated gene up-regulation has not been elucidated. To evaluate the precise mechanism of SGPG up-regulation, we have specifically inhibited the SGPG synthesis in the cerebromicrovascular EC line (SV-HCECs), a transformed brain ECs of human origin. SV-HCECs were transfected with small interfering RNA designed to mimic the human natural killer epitope-1 sulfotransferase (HNK-1ST), the ultimate enzyme that transfers the sulfate group to glucuronic acid for SGPG synthesis. An inhibition of SGPG expression along with a reduction of human CD4(+) cell adhesion was observed in siRNA HNK-1ST (siHNK-1)-transfected cells after TNFalpha stimulation. A thorough screening of the signaling system confirmed that TNFalpha/IL-1beta stimulation up-regulated nuclear factor kappaB (NFkappaB) signaling in SV-HCECs. siHNK-1 transfection interfered with the SGPG up-regulation after TNFalpha/IL-1beta stimulation in transfected cells and reduced the T cell adhesion. Hence, our study indicates that T cell-SGPG adhesion in SV-HCECs may proceed through NFkappaB activation. In addition, siHNK-1 transfection reduced the NFkappaB activity compared with cells that were transfected with scrambled siRNA, before and after TNFalpha/IL-1beta stimulation. This is the first report indicating that NFkappaB signaling is involved in SGPG gene expression in brain ECs by an unknown mechanism. Its down-regulation by inhibiting HNK-1ST expression may have a potential use in preventing the T cell invasion and consequently nerve damage during inflammation.


Subject(s)
Brain/metabolism , Globosides/metabolism , NF-kappa B/metabolism , T-Lymphocytes/metabolism , Brain/cytology , Brain/drug effects , CD4 Antigens , Cell Adhesion/drug effects , Cell Line , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Globosides/genetics , Humans , Immunohistochemistry , Interleukin-1beta/metabolism , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Sulfotransferases/genetics , T-Lymphocytes/drug effects , Transfection , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation/drug effects , Up-Regulation/genetics
18.
J Lipid Res ; 49(10): 2188-96, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18587070

ABSTRACT

Previous studies from our laboratory indicated that pre-germinated brown rice (PR) contained certain unknown bioactive lipids that activated two enzymes related to diabetes: Na+/K+ATPase and homocysteine-thiolactonase. In this paper, we report on the isolation and structural characterization of the activator lipids from PR bran as acylated steryl glucosides (ASGs). The activator lipid was isolated by silica gel column chromatography, and its chemical structure was determined by NMR, GC-MS, and tandem mass spectrometry. We demonstrated that the bioactive component consists of a mixture of acylated steryl beta-glucosides. Delta8-cholesterol and 2-hydroxyl stearic acid were identified as constituents of ASGs. The steryl glucosides (SGs) subsequent to alkaline hydrolysis lost this enzyme activator activity. Soybean-derived ASGs were not active. This activity may be quite peculiar to PR-derived ASGs. Our findings suggest that the molecular species of ASG may play an important contributing role in the anti-diabetic properties of a PR diet.


Subject(s)
Dietary Fiber/analysis , Germination , Glucosides/chemistry , Oryza/chemistry , Acylation , Animals , Carboxylesterase/metabolism , Cholesterol, HDL/metabolism , Chromatography, Liquid , Chromatography, Thin Layer , Gas Chromatography-Mass Spectrometry , Glucosides/metabolism , Hydrolysis , Lipid Metabolism , Lipids/chemistry , Lipids/isolation & purification , Magnetic Resonance Spectroscopy , Male , Oryza/physiology , Rats , Sodium-Potassium-Exchanging ATPase/metabolism
19.
J Neurochem ; 106(2): 718-33, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18466329

ABSTRACT

We provide evidence that the sphingolipid ceramide, in addition to its pro-apoptotic function, regulates neural progenitor (NP) motility in vitro and brain development in vivo. Ceramide (N-palmitoyl d-erythro sphingosine and N-oleoyl d-erythro sphingosine) and the ceramide analog N-oleoyl serinol (S18) stimulate migration of NPs in scratch (wounding) migration assays. Sphingolipid depletion by inhibition of de novo ceramide biosynthesis, or ceramide inactivation using an anti-ceramide antibody, obliterates NP motility, which is restored by ceramide or S18. These results suggest that ceramide is crucial for NP motility. Wounding of the NP monolayer activates neutral sphingomyelinase indicating that ceramide is generated from sphingomyelin. In membrane processes, ceramide is co-distributed with its binding partner atypical protein kinase C zeta/lambda (aPKC), and Cdc42, alpha/beta-tubulin, and beta-catenin, three proteins involved in aPKC-dependent regulation of cell polarity and motility. Sphingolipid depletion by myriocin prevents membrane translocation of aPKC and Cdc42, which is restored by ceramide or S18. These results suggest that ceramide-mediated membrane association of aPKC/Cdc42 is important for NP motility. In vivo, sphingolipid depletion leads to ectopic localization of mitotic or post-mitotic neural cells in the embryonic brain, while S18 restores the normal brain organization. In summary, our study provides novel evidence that ceramide is critical for NP motility and polarity in vitro and in vivo.


Subject(s)
Cell Movement/drug effects , Ceramides/pharmacology , Neurons/physiology , Stem Cells/drug effects , Actins/metabolism , Animals , Antibodies/pharmacology , Cell Differentiation/drug effects , Cell Polarity/drug effects , Cells, Cultured , Ceramides/immunology , Embryo, Mammalian , Fatty Acids, Monounsaturated/pharmacology , Immunosuppressive Agents/pharmacology , Mice , Oleic Acids/pharmacology , Propylene Glycols/pharmacology , Protein Binding/drug effects , Protein Binding/physiology , Protein Kinase C/metabolism , Protein Transport/drug effects , Tubulin/metabolism , beta Catenin/metabolism , cdc42 GTP-Binding Protein/metabolism
20.
Glia ; 56(4): 449-56, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18205190

ABSTRACT

The pro-apoptotic sphingolipid ceramide plays an emergent role in the etiology of Alzheimer's disease (AD), although its function for neurodegeneration is not known. We determined the concentration and composition of ceramide in hippocampal tissue from newborn presenilin 1 (PS1) knock-in (PS1M146V) mice, a mouse model for early-onset familial AD. We found that PS1 tissue contains 3.1 (+/-0.5)-fold more total ceramide than wild-type tissue. In particular, the proportion of C20 and C24 ceramide is increased by 4.0- or 8.5-fold, respectively. The ceramide elevation in PS1 brain is consistent with a 3.7 (+/-0.5)-fold increase of the protein level of the neurotrophin receptor p75NTR, which has been suggested to stimulate the hydrolysis of sphingomyelin to generate ceramide. The predominance of C20 and C24 ceramide is concurrent with the elevated gene expression of lass 2 and lass 4, two isoforms of ceramide synthase that generate dihydroceramide with long-chain fatty acid. Our study indicates that primary cultured astrocytes but not neurons from PS1 mice undergo apoptosis when incubated with C20 ceramide. In contrast, wild-type astrocytes remain unaffected. The sensitivity of PS1 astrocytes is most likely due to the 9.5 (+/-0.4)-fold elevated expression of PAR-4 (prostate apoptosis response-4), a protein that inhibits atypical PKC zeta/lambda in the presence of ceramide. Our results suggest that astroglial death due to ceramide/PAR-4-induced apoptosis may critically contribute to the etiology of AD.


Subject(s)
Apoptosis/genetics , Astrocytes/physiology , Brain/cytology , Brain/metabolism , Ceramides/metabolism , Presenilin-1/genetics , Animals , Animals, Newborn , Ceramides/genetics , Chromatography, High Pressure Liquid/methods , Gas Chromatography-Mass Spectrometry/methods , Humans , In Situ Nick-End Labeling , Mice , Mice, Transgenic , Mutation/genetics , Neurons/physiology , Receptors, Proteinase-Activated/genetics , Receptors, Proteinase-Activated/metabolism , Serine C-Palmitoyltransferase/genetics , Serine C-Palmitoyltransferase/metabolism
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