i(3)so3-galactosylceramide and Disease-Models--Animal

In 1961, Wago et al. reported a potential anticoagulant role for sulfatide using animal experiments. Since then there have been many studies of sulfatide in the field of thrombogenesis/haemostasis, yielding contradictory conclusions. Some report that sulfatide has anti-thrombotic activity because it prolongs clotting time, inhibits platelet adhesion, and prolongs bleeding. Others report that sulfatide induces thrombosis in animal models. This mini-review is a chronologic review of reports examining the role of sulfatide in thrombogenesis/haemostasis together with the introduction of data from our laboratory and a discussion of the possible mechanisms underlying these curious phenomena.

Topics: Animals; Anticoagulants; Blood Coagulation; Disease Models, Animal; Hemostasis; Humans; Platelet Adhesiveness; Sulfoglycosphingolipids; Thrombosis; Venous Thrombosis

Topics: Animals; Body Weight; Brain; Disease Models, Animal; DNA; Dopamine; Female; Gangliosides; Gestational Age; Humans; Intelligence; Myelin Sheath; Neuroglia; Neurons; Norepinephrine; Nutrition Disorders; Organ Size; Pregnancy; Proteins; Rats; Sulfoglycosphingolipids; Thyroid Gland; Time Factors

Aging and neurodegenerative disease are accompanied by lipid perturbations in the brain. Understanding the changes in the contents and functional activity of lipids remains a challenge not only because of the many areas in which lipids perform bioactivities but also because of the technical limitations in identifying lipids and their metabolites. In the present study, we aimed to evaluate how brain lipids are altered in Alzheimer's disease (AD)-like pathology by using mass spectrometry imaging (MSI). The spatial distributions and relative abundances of lipids in the brains were compared between APP/PS1 mice and their age-matched wild-type (WT) mice by matrix-assisted laser desorption ionization (MALDI) MSI assays. The comparisons were correlated with the analysis using a spectrophotometric method to determine the relative contents of sulfatides in different brain regions. Significant changes of brain lipids between APP/PS1 and WT mice were identified: eight sulfoglycosphingolipid species, namely, sulfatides/sulfated hexosyl ceramides (ShexCer) and two glycerophosphoinositol (GroPIn) species, PI 36:4 and PI 38:4. The declines in the spatial distributions of these ShexCer and GroPIn species in the APP/PS1 mice brains were associated with learning- and memory-related brain regions. Compared with young WT mice, aged WT mice showed significant decreases in the levels of these ShexCer and GroPIn species. Our results provide technical clues for assessing the impact of brain lipid metabolism on the senescent and neurodegenerative brain. The decline in sulfatides and GroPIns may be crucial markers during brain senescence and AD pathology. Appropriate lipid complementation might be important potentials as a therapeutic strategy for AD.

Topics: Alzheimer Disease; Animals; Brain; Ceramides; Cognition; Disease Models, Animal; Mice; Neurodegenerative Diseases; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfates; Sulfoglycosphingolipids

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by an arylsulfatase A (ARSA) deficiency and characterized by severe neurological symptoms resulting from demyelination within the central and peripheral nervous systems. We investigated the feasibility and efficacy of intrathecal administration of a type 9 adeno-associated viral vector encoding ARSA (AAV9/ARSA) for the treatment of 6-week-old MLD model mice, which are presymptomatic, and 1-year-old mice, which exhibit neurological abnormalities. Immunohistochemical analysis following AAV9/ARSA administration showed ARSA expression within the brain, with highest activities in the cerebellum and olfactory bulbs. In mice treated at 1 year, alcian blue staining and quantitative analysis revealed significant decreases in stored sulfatide. Behaviorally, mice treated at 1 year showed no improvement in their ability to traverse narrow balance beams as compared to untreated mice. By contrast, MLD mice treated at 6 weeks showed significant decreases in stored sulfatide throughout the entire brain and improved ability to traverse narrow balance beams. These findings suggest intrathecal administration of an AAV9/ARSA vector is a promising approach to treating genetic diseases of the central nervous system, including MLD, though it may be essential to begin therapy before the onset of neurological symptoms.

Topics: Age Factors; Animals; Cerebellum; Cerebroside-Sulfatase; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Injections, Spinal; Leukodystrophy, Metachromatic; Mice, Knockout; Spinal Cord; Sulfoglycosphingolipids

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized-GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.

Topics: Acidic Glycosphingolipids; Aging; Animals; Animals, Genetically Modified; Brain; Chromatography, Liquid; Disease Models, Animal; G(M3) Ganglioside; Humans; Hydrophobic and Hydrophilic Interactions; Neurofibrils; Rats; Sulfoglycosphingolipids; tau Proteins; Tauopathies

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.

Topics: Amidohydrolases; Animals; Cell Line; Cerebroside-Sulfatase; Disease Models, Animal; Female; Leukodystrophy, Metachromatic; Lysosomal Storage Diseases; Mice; Mice, Knockout; Microglia; Primary Cell Culture; Psychosine; Sulfoglycosphingolipids

Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.

Topics: Animals; Ceramides; Corpus Callosum; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Immunohistochemistry; Lipid Metabolism; Male; Mice; Mice, Transgenic; Myelin Sheath; Phosphatidylcholines; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfoglycosphingolipids; White Matter

Alport syndrome is caused by mutations in collagen IV that alter the morphology of renal glomerular basement membrane. Mutations result in proteinuria, tubulointerstitial fibrosis, and renal failure but the pathogenic mechanisms are not fully understood. Using imaging mass spectrometry, we aimed to determine whether the spatial and/or temporal patterns of renal lipids are perturbed during the development of Alport syndrome in the mouse model. Our results show that most sulfatides are present at similar levels in both the wild-type (WT) and the Alport kidneys, with the exception of two specific sulfatide species, SulfoHex-Cer(d18:2/24:0) and SulfoHex-Cer(d18:2/16:0). In the Alport but not in WT kidneys, the levels of these species mirror the previously described abnormal laminin expression in Alport syndrome. The presence of these sulfatides in renal tubules but not in glomeruli suggests that this specific aberrant lipid pattern may be related to the development of tubulointerstitial fibrosis in Alport disease.

Topics: Animals; Disease Models, Animal; Kidney Tubules; Lipid Metabolism; Mice; Nephritis, Hereditary; Sulfoglycosphingolipids

Sphingolipids play important roles in beta cell physiology, by regulating proinsulin folding and insulin secretion and in controlling apoptosis, as studied in animal models and cell cultures. Here we investigate whether sphingolipid metabolism may contribute to the pathogenesis of human type 1 diabetes and whether increasing the levels of the sphingolipid sulfatide would prevent models of diabetes in NOD mice.. We examined the amount and distribution of sulfatide in human pancreatic islets by immunohistochemistry, immunofluorescence and electron microscopy. Transcriptional analysis was used to evaluate expression of sphingolipid-related genes in isolated human islets. Genome-wide association studies (GWAS) and a T cell proliferation assay were used to identify type 1 diabetes related polymorphisms and test how these affect cellular islet autoimmunity. Finally, we treated NOD mice with fenofibrate, a known activator of sulfatide biosynthesis, to evaluate the effect on experimental autoimmune diabetes development.. We found reduced amounts of sulfatide, 23% of the levels in control participants, in pancreatic islets of individuals with newly diagnosed type 1 diabetes, which were associated with reduced expression of enzymes involved in sphingolipid metabolism. Next, we discovered eight gene polymorphisms (ORMDL3, SPHK2, B4GALNT1, SLC1A5, GALC, PPARD, PPARG and B4GALT1) involved in sphingolipid metabolism that contribute to the genetic predisposition to type 1 diabetes. These gene polymorphisms correlated with the degree of cellular islet autoimmunity in a cohort of individuals with type 1 diabetes. Finally, using fenofibrate, which activates sulfatide biosynthesis, we completely prevented diabetes in NOD mice and even reversed the disease in half of otherwise diabetic animals.. These results indicate that islet sphingolipid metabolism is abnormal in type 1 diabetes and suggest that modulation may represent a novel therapeutic approach.. The RNA expression data is available online at https://www.dropbox.com/s/93mk5tzl5fdyo6b/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%2C%20RNA%20expression.xlsx?dl=0 . A list of SNPs identified is available at https://www.dropbox.com/s/yfojma9xanpp2ju/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%20SNP.xlsx?dl=0 .

Topics: Adult; Animals; Autoimmunity; Case-Control Studies; Cell Proliferation; Cells, Cultured; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Fenofibrate; Gene Expression Regulation, Enzymologic; Humans; Islets of Langerhans; Lipid Metabolism; Lymphocyte Activation; Male; Mice, Inbred NOD; Polymorphism, Genetic; Sulfoglycosphingolipids; T-Lymphocytes

Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells Translational Medicine 2017;6:352-368.

Topics: Animals; Cell Differentiation; Cell Line; Cell Movement; Cellular Reprogramming; Cellular Reprogramming Techniques; Cerebroside-Sulfatase; Coculture Techniques; Disease Models, Animal; Enzyme Induction; Gene Expression Regulation, Developmental; Genetic Therapy; Humans; Induced Pluripotent Stem Cells; Leukodystrophy, Metachromatic; Mice, Inbred NOD; Mice, SCID; Nerve Regeneration; Neural Stem Cells; Phenotype; Stem Cell Transplantation; Sulfoglycosphingolipids; Transcriptome

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder resulting from a functional deficiency of arylsulfatase A (ARSA), an enzyme that catalyzes desulfation of 3-O-sulfogalactosylceramide (sulfatide). Lack of active ARSA leads to the accumulation of sulfatide in oligodendrocytes, Schwann cells and some neurons and triggers progressive demyelination, the neuropathological hallmark of MLD. Several therapeutic approaches have been explored, including enzyme replacement, autologous hematopoietic stem cell-based gene therapy, intracerebral gene therapy or cell-based gene delivery into the central nervous system (CNS). However, long-term treatment of the blood-brain-barrier protected CNS remains challenging. Here we used MLD patient-derived induced pluripotent stem cells (iPSCs) to generate long-term self-renewing neuroepithelial stem cells and astroglial progenitors for cell-based ARSA replacement. Following transplantation of ARSA-overexpressing precursors into ARSA-deficient mice we observed a significant reduction of sulfatide storage up to a distance of 300 µm from grafted cells. Our data indicate that neural precursors generated via reprogramming from MLD patients can be engineered to ameliorate sulfatide accumulation and may thus serve as autologous cell-based vehicle for continuous ARSA supply in MLD-affected brain tissue.

Topics: Animals; Axons; Brain; Cell Differentiation; Cell Survival; Cell- and Tissue-Based Therapy; Central Nervous System; Cerebroside-Sulfatase; Disease Models, Animal; DNA-Binding Proteins; Gene Expression; Gene Order; Genetic Therapy; Genetic Vectors; Humans; Induced Pluripotent Stem Cells; Lentivirus; Leukodystrophy, Metachromatic; Mice; Mice, Knockout; Neuroglia; Neurons; Sulfoglycosphingolipids; Transduction, Genetic

The endogenous glycosphingolipid sulfatide is a ligand for CD1d-restricted type II natural killer T (NKT) lymphocytes. Through the action of these cells,sulfatide treatment has been shown to modulate the immune response in mouse models for autoimmune diseases, infections and tumour immunity. Sulfatide exists naturally in different organs including the pancreas, where sulfatide colocalizes with insulin within the Langerhans islet b-cells, targets for the immune destruction in type 1 diabetes (T1D). Human T1D patients, but not patients with type 2 diabetes nor healthy individuals, have autoantibodies against sulfatide in serum, suggesting that sulfatide induces an immune response in the natural course of T1D in humans. Here, we investigate sulfatide as an autoantigen and a modulator of autoimmune disease in the murine model forT1D, the non-obese diabetic (NOD) mice. We demonstrate that aged NOD mice displayed serum autoantibody reactivity to sulfatide; however, this reactivity did not correlate with onset of T1D. Repeated administration of sulfatide did not result in an increase in serum reactivity to sulfatide. Moreover, a multidose sulfatide treatment of female NOD mice initiated at an early (5 weeks of age),intermediate (8 weeks of age) or late (12 weeks of age) phase of T1D progression did not influence the incidence of disease. Thus, we demonstrate that a fraction of NOD mice develop autoantibody reactivity to sulfatide; however, we fail to demonstrate that sulfatide treatment reduces the incidence of T1D in this mouse strain.

Topics: Animals; Antigens, CD1d; Autoantibodies; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Disease Models, Animal; Disease Progression; Female; Galactosylceramides; Humans; Islets of Langerhans; Mice; Mice, Inbred NOD; Natural Killer T-Cells; Sulfoglycosphingolipids

As a glycosphingolipid that can bind to several extracellular matrix proteins, sulfatide has the potential to become an effective targeting agent for tumors overexpressing tenasin-C in their microenvironment. To overcome the dose-limiting toxicity of doxorubicin (DOX), a sulfatide-containing nanoliposome (SCN) encapsulation approach was employed to improve treatment efficacy and reduce side effects of free DOX. This study analysed in vitro characteristics of sulfatide-containing nanoliposomal DOX (SCN-DOX) and assessed its cytotoxicity in vitro, as well as biodistribution, therapeutic efficacy, and systemic toxicity in a human glioblastoma U-118MG xenograft model. SCN-DOX was shown to achieve highest drug to lipid ratio (0.5∶1) and a remarkable in vitro stability. Moreover, DOX encapsulated in SCN was shown to be delivered into the nuclei and displayed prolonged retention over free DOX in U-118MG cells. This simple two-lipid SCN-DOX nanodrug has favourable pharmacokinetic attributes in terms of prolonged circulation time, reduced volume of distribution and enhanced bioavailability in healthy rats. As a result of the improved biodistribution, an enhanced treatment efficacy of SCN-DOX was found in glioma-bearing mice compared to the free drug. Finally, a reduction in the accumulation of DOX in the drug's principal toxicity organs achieved by SCN-DOX led to the diminished systemic toxicity as evident from the plasma biochemical analyses. Thus, SCN has the potential to be an effective and safer nano-carrier for targeted delivery of therapeutic agents to tumors with elevated expression of tenascin-C in their microenvironment.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Line, Tumor; Disease Models, Animal; Doxorubicin; Female; Glioma; Humans; Kinetics; Liposomes; Male; MCF-7 Cells; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Nanoparticles; Particle Size; Rats; Rats, Sprague-Dawley; Sulfoglycosphingolipids; Tissue Distribution; Transplantation, Heterologous

The gracile axonal dystrophy (gad) mutation in Uch-l1, the gene encoding the ubiquitin carboxy-terminal hydrolase isozyme L1 (UCH-L1), causes selective dying back degeneration of dorsal root ganglion neuron in the medulla oblongata along with progressive sensory-motor ataxia. Axonal spheroids are observed within degenerating axons, and their contents may illuminate the pathogenic mechanisms leading to neurodegeneration in gad mice. To analyze changes in negatively charged lipid molecules in dystrophic axons of gad mice, we performed matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS), electron microscopy, and fluorescence immunohistochemistry on tissue sections from gad and wild-type mouse medulla. MALDI-IMS revealed that m/z 806.68 and 822.68 molecules, assigned to sulfatide (ST) C18:0 and ST C18:0(OH), respectively, were concentrated in the dorsomedial medulla. This spatial distribution overlapped significantly with that of axonal spheroids. Immunostaining revealed that spheroids accumulated myelin and lymphocyte protein, a known ST binding protein. Sulfatides with short-chain fatty acids (C16-C20) are generally localized in intracellular vesicles; therefore, ST C18:0 accumulation may reflect intracellular vesicle aggregation within spheroids. Ubiquitin system disruption apparently alters lipid metabolism, membrane organization, protein turnover, and axonal transport. Changes in membrane organization, particularly STs within lipid rafts, may disrupt cellular signaling pathways necessary for neuronal viability.

Topics: Animals; Axons; Disease Models, Animal; Medulla Oblongata; Mice; Mice, Transgenic; Neuroaxonal Dystrophies; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfoglycosphingolipids

Serum sulfatides are the major glycosphingolipids in lipoproteins. Although serum sulfatides are mainly synthesized and secreted by the liver, they are significantly decreased when the kidneys are impaired. Our recent experimental study using a murine protein-overload nephropathy model suggested a hypothetical mechanism whereby serum sulfatides were reduced due to kidney dysfunction. This was the result of decreased hepatic expression of a sulfatide synthetic enzyme, cerebroside sulfotransferase (CST), which is associated with systemic enhancement of oxidative stress. However, there is a possibility that the experimental process, protein-overload itself, directly affected the sulfatide metabolism and oxidative stress in the liver. To determine whether kidney dysfunction actually reduces the hepatic synthesis of sulfatides via oxidative stress, we examined sulfatide levels, the hepatic content of metabolic sulfatide enzymes, and the degree of oxidative stress in protein-overload mice subjected to renoprotective therapy using clofibrate, a representative hypolipidemic medicine. Protein-overload mice exhibited marked kidney injuries, enhancement of hepatic oxidative stress, decreased levels of serum and hepatic sulfatides, and decreased expression of hepatic CST. The clofibrate treatment attenuated kidney damage and hepatic oxidative stress while maintaining serum/hepatic sulfatide levels and hepatic CST content in the mice. Because clofibrate monotherapy without protein-overload treatment only minimally affected these hepatic parameters, the hepatic synthesis of sulfatides appeared to be strongly influenced by kidney dysfunction and subsequent oxidative stress. This study suggests that the crosstalk between kidney dysfunction and hepatic sulfatide metabolism is mediated by oxidative stress. These results should help to understand the phenomenon in patients with end-stage kidney disease.

Topics: Acute Disease; Animals; Clofibrate; Disease Models, Animal; Drug Antagonism; Female; Gene Expression Regulation, Enzymologic; Hypolipidemic Agents; Kidney; Kidney Diseases; Liver; Mice; Mice, 129 Strain; Oxidative Stress; Serum Albumin, Bovine; Sulfoglycosphingolipids; Sulfotransferases

Arylsulfatase A-deficient (ASA(-/-)) mice constitute an animal model for metachromatic leukodystrophy, a lysosomal storage disorder. We had previously examined the behavioural phenotype of these mice, but were unable to distinguish between proper cognitive symptoms and potentially interfering, solely neuromotor impairments. In the present study, T-maze delayed alternation (TMDA) showed that ASA(-/-) mice perform worse than controls already at the age of 6 months in a hippocampus-dependent task that does not require motor proficiency. In addition, long term potentiation (LTP) in the CA1 region of the hippocampus, a cellular correlate of learning and memory, was also impaired in ASA(-/-) mice. Finally, histological analysis of previously unexamined telencephalic and diencephalic structures illustrated sulfatide accumulation in brain areas that are important for cognitive functioning. These include the hippocampus, striatum, internal capsule and diencephalon as well as prefrontal, insular, and motor and somatosensory cortices. Together these data corroborate the usefulness of the model in preclinical evaluations of therapeutic strategies that aim to reverse cognitive defects in the human disease.

Topics: Animals; Brain; Cerebroside-Sulfatase; Disease Models, Animal; Hippocampus; Leukodystrophy, Metachromatic; Long-Term Potentiation; Maze Learning; Mice; Mice, Knockout; Neuronal Plasticity; Sulfoglycosphingolipids

CNS myelin is strongly inhibitory to growing axons and is thought to be a major contributor to CNS axon regenerative failure. Although a number of proteins present in myelin, including Nogo, MAG, and oligodendrocyte-myelin glycoprotein (OMgp), have been identified as myelin-associated inhibitors, studies of mice lacking these genes suggest that additional inhibitors present in CNS myelin remain to be identified. Here we have investigated the hypothesis that myelin lipids contribute to CNS regenerative failure. We identified sulfatide, a major constituent of CNS myelin, as a novel myelin-associated inhibitor of neurite outgrowth. Sulfatide, but not galactocerebroside or ceramide, strongly inhibited the neurite outgrowth of retinal ganglion cells (RGCs) when used as a purified lipid substrate. The mechanism involved in sulfatide-mediated inhibition may share features with other known inhibitors, because the Rho inhibitor C3 transferase lessened these effects. Myelin in which sulfatide was lacking or blocked using specific antibodies was significantly less inhibitory to RGC neurite outgrowth in vitro than was wild-type myelin, indicating that sulfatide is a major component of the inhibitory activity of CNS myelin. Mice unable to make sulfatide did not regenerate RGC axons more robustly after optic nerve crush than wild-type littermates under normal conditions but did exhibit a small but significant enhancement in the extent of zymosan-induced regeneration. These results demonstrate that specific lipids can powerfully inhibit axon growth, identify sulfatide as a novel myelin-associated axon growth inhibitor, and provide evidence that sulfatide inhibition contributes to axon regenerative failure in vivo.

Topics: Animals; Animals, Newborn; Antibodies; Axons; Cell Survival; Cells, Cultured; Central Nervous System Diseases; Disease Models, Animal; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Myelin Proteins; Nerve Regeneration; Neural Inhibition; Optic Nerve Injuries; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells; rhoA GTP-Binding Protein; Sulfoglycosphingolipids; Sulfotransferases; Sulfurtransferases; Transfection; Zymosan

Vaccinia virus (VACV) was used as a surrogate of variola virus (genus Orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection. VACV infects cells via attachment and fusion of the viral membrane with the host cell membrane. Glycosphingolipids, expressed in multiple organs, are major components of lipid rafts and have been associated with the infectious route of several pathogens.. We demonstrate that the VACV-WR (VACV Western-Reserve strain) displays no binding to Cer (ceramide) or to Gal-Cer (galactosylceramide), but binds to a natural sulfated derivative of these molecules: the Sulf (sulfatide) 3' sulfogalactosylceramide. The interaction between Sulf and VACV-WR resulted in a time-dependent inhibition of virus infection. Virus cell attachment was the crucial step inhibited by Sulf. Electron microscopy showed that SUVs (small unilamellar vesicles) enriched in Sulf bound to VACV particles. Both the A27 and L5 viral membrane proteins were shown to interact with Sulf, indicating that they could be the major viral ligands for Sulf. Soluble Sulf was successful in preventing mortality, but not morbidity, in a lethal mouse model infection with VACV-WR.. Together the results suggest that Sulf could play a role as an alternate receptor for VACV-WR and probably other Orthopoxviruses.

Topics: Animals; Cell Line, Tumor; Ceramides; Cricetinae; Disease Models, Animal; Epithelial Cells; Galactosylceramides; Humans; Ligands; Membrane Proteins; Mice; Structure-Activity Relationship; Sulfoglycosphingolipids; Vaccinia; Vaccinia virus; Variola virus; Viral Proteins

Herein, we tested a recently proposed working model of apolipoprotein E (apoE)-mediated sulfatide metabolism/trafficking/homeostasis with two well-characterized amyloid precursor protein (APP) transgenic (Tg) animal models of Alzheimer's disease (AD) (i.e., APP(V717F) and APPsw) on a wild-type murine apoE background or after being bred onto an Apoe(-/-) background. As anticipated, lipidomics analysis demonstrated that the sulfatide levels in brain tissues were reduced beginning at approximately 6 months of age in APP(V717F) Tg, Apoe(+/+) mice and at 9 months of age in APPsw Tg, Apoe(+/+) mice relative to their respective non-APP Tg littermates. This reduction increased in both APP Tg mice as they aged. In contrast, sulfatide depletion did not occur in APP Tg, Apoe(-/-) animals relative to the Apoe(-/-) littermates. The lack of sulfatide depletion in APP Tg, Apoe(-/-) mice strongly supports the role of apoE in the deficient sulfatide content in APP Tg, Apoe(+/+) mice. Collectively, through different animal models of AD, this study provides evidence for an identified biochemical mechanism that may be responsible for the sulfatide depletion at the earliest stages of AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Apolipoproteins E; Brain; Brain Chemistry; Disease Models, Animal; Down-Regulation; Homeostasis; Lipid Metabolism; Lipoproteins; Mice; Mice, Knockout; Mice, Transgenic; Models, Biological; Myelin Sheath; Sphingomyelins; Sulfoglycosphingolipids

Altered sphingolipid metabolism plays an emergent role in the etiology of Alzheimer's disease (AD). In this study, we determined the levels of ceramides and other related-sphingolipids (sphingomyelins, sulfatides and galactosylceramides) in the cerebral cortex of an APP(SL)/PS1Ki mouse model of AD. The results demonstrate that ceramides accumulated in the cortex of APP(SL)/PS1Ki mice, but not in PS1Ki mice, whereas all others major sphingolipids (except galactosylceramides) were not altered in comparison with those from age-matched wild-type mice. Furthermore, as early as 3 months of age, female mice but not males, exhibit a strong increase in 2-hydroxy fatty acid-containing ceramides, whereas males display a significant elevation of non-hydroxy fatty acid ceramide species. Therefore, the gender differences in ceramide accumulation in the brain of mice expressing APP(SL) suggest that additional factors like modified ceramide metabolism may contribute to the increased propensity of females to develop AD.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Ceramides; Cerebral Cortex; Disease Models, Animal; Female; Galactosylceramides; Gene Knock-In Techniques; Male; Mice; Mice, Transgenic; Sex Factors; Sphingomyelins; Sulfoglycosphingolipids

The galactolipids galactocerebroside and sulfatide have been implicated in oligodendrocyte (OL) development and myelin formation. Much of the early evidence for myelin galactolipid function has been derived from antibody and chemical perturbation of OLs in vitro. To determine the role of these lipids in vivo, we previously characterized mice lacking galactocerebroside and sulfatide and observed abundant, unstable myelin and an increased number of OLs. We have also reported that mice incapable of synthesizing sulfatide (CST-null) while maintaining normal levels of galactocerebroside generate relatively stable myelin with unstable paranodes. Additionally, Hirahara et al. (2004; Glia 45:269-277) reported that these CST-null mice also contain an increased number of OLs in the forebrain, medulla, and cerebellum at 7 days of age. Here, we further the findings of Hirahara et al. by demonstrating that the number of OLs in the CST-null mice is also increased in the spinal cord and that this elevated OL population is maintained through, at least, 7 months of age. Moreover, we show that the enhanced OL population is accompanied by increased proliferation and decreased apoptosis of oligodendrocytic-lineage cells. Finally, through ultrastructural analysis, we show that the CST-null OLs exhibit decreased morphological complexity, a feature that may result in decreased OL competition and increased OL survival.

Topics: Aging; Animals; Apoptosis; Cell Count; Cell Differentiation; Cell Lineage; Cell Proliferation; Cell Survival; Disease Models, Animal; Gene Expression Regulation; Hereditary Central Nervous System Demyelinating Diseases; Mice; Mice, Knockout; Myelin Sheath; Nerve Fibers, Myelinated; Oligodendroglia; Spinal Cord; Stem Cells; Sulfoglycosphingolipids; Sulfotransferases; Up-Regulation

We recently proposed serum sulfatides as a novel biomarker for cardiovascular disease in patients with end-stage renal failure (ESRF), based on the possible antithrombotic properties of this molecule. In this earlier study, the level of serum sulfatides was gradually decreased in parallel with kidney dysfunction; however the precise mechanism underlying this decrease was unknown. The aim of the present study was to investigate the mechanism underlying the decrease in serum sulfatide levels caused by kidney dysfunction in an experimental animal model. To produce a kidney dysfunction animal model, we prepared a mouse model of protein overload nephropathy. Using high-throughput analysis combined with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry, we measured the levels of sulfatides in the sera, livers, small intestines and kidneys of protein overload nephropathy mice. As the disease progressed, the levels of sulfatides in sera decreased. Also, the levels in livers and small intestines decreased in a similar manner to those in sera, to approximately 60% of the original levels. On the contrary, those in kidneys increased by approximately 1.4-fold. Our results indicate that kidney dysfunction affects the levels of sulfatides in lipoprotein-producing organs, such as livers and small intestines, and lowers the levels of sulfatides in sera.

Topics: Animals; Cardiovascular Diseases; Disease Models, Animal; Intestine, Small; Kidney Diseases; Liver; Male; Mice; Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfoglycosphingolipids

Sulfatides, possible antithrombotic factors belonging to sphingoglycolipids, are widely distributed in mammalian tissues and serum. We recently found that the level of serum sulfatides was significantly lower in hemodialysis patients than that in normal subjects, and that the serum level closely correlated to the incidence of cardiovascular disease. These findings suggest a relationship between the level of serum sulfatides and kidney function; however, the molecular mechanism underlying this relationship remains unclear. In the present study, the influence of kidney dysfunction on the metabolism of sulfatides was examined using an established murine model of acute kidney injury, protein-overload nephropathy in mice. Protein-overload treatment caused severe proximal tubular injuries within 4days, and this treatment obviously decreased both serum and hepatic sulfatide levels. The sphingoid composition of serum sulfatides was very similar to that of hepatic ones at each time point, suggesting that the serum sulfatide level is dependent on the hepatic secretory ability of sulfatides. The treatment also decreased hepatic expression of cerebroside sulfotransferase (CST), a key enzyme in sulfatide metabolism, while it scarcely influenced the expression of the other sulfatide-metabolizing enzymes, including arylsulfatase A, ceramide galactosyltransferase, and galactosylceramidase. Pro-inflammatory responses were not detected in the liver of these mice; however, potential oxidative stress was increased. These results suggest that down-regulation of hepatic CST expression, probably affected by oxidative stress from kidney injury, causes reduction in liver and serum sulfatide levels. This novel mechanism, indicating the crosstalk between kidney injury and specific liver function, may prove useful for helping to understand the situation where human hemodialysis patients have low levels of serum sulfatides.

Topics: Acute Disease; Animals; Disease Models, Animal; Down-Regulation; Gene Expression Regulation; Kidney Diseases; Liver; Male; Mice; Mice, Mutant Strains; Oxidative Stress; PPAR alpha; Proteins; Sulfoglycosphingolipids; Sulfotransferases

The developing brain is uniquely susceptible to injury after exposure to hypoxia-ischemia (H-I). Lecticans are developmentally regulated in formative white matter and exert growth-inhibitory effects in several adult injury models, yet little is known regarding their role in neonatal H-I injury. The main objectives of this study were to examine the expression profiles of brevican and versican in rat using a standard H-I model and to determine whether altered expression was associated with distinct components of white and gray matter pathology. The H-I procedure in postnatal day 7 rats produced progressive injury limited to the ipsilateral hemisphere. Cresyl violet staining revealed severe cavitary infarctions at 14 and 21 days that were absent at 4 days. Cellular damage, as measured by glial fibrillary acidic protein and fractin immunoreactivity, occurred in cortical and subcortical gray matter at all end points. O4 sulfatide immunoreactivity was reduced in the external capsule, hippocampal fimbria, and corpus striatum at 4 days relative to that contralaterally, suggesting the loss of preoligodendrocytes. Brevican expression was reduced in the cortex and hippocampus at 4 days but was markedly elevated at later end points, localizing to regions of cellular damage both in and proximal to the lesion core. However, versican was reduced in the external capsule 4 days after H-I, a reduction that was sustained up to 21 days in white matter. These data demonstrate unique expression profiles for lecticans after neonatal H-I, suggesting brevican deposition is elevated in response to progressive gray matter injury, whereas diminished versican expression may be associated with deep cerebral white matter injury.

Topics: Animals; Animals, Newborn; Brain; Brain Infarction; Brevican; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Down-Regulation; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Lectins, C-Type; Nerve Fibers, Myelinated; Nerve Tissue Proteins; Neuroglia; Rats; Rats, Sprague-Dawley; Ribulose-Bisphosphate Carboxylase; Stem Cells; Sulfoglycosphingolipids; Versicans

The quality of tissue imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) depends on the effectiveness of the matrix deposition, especially for lipids that may dissolve in the solvent used for the matrix application. This article describes the use of an oscillating capillary nebulizer (OCN) to spray small droplets of matrix aerosol onto the sample surface for improved matrix homogeneity, reduced crystal size, and controlled solvent effects. This system was then applied to the analysis of histological slices of brains from mice with homozygous disruption of the hexb gene (hexb-/-), a model of Tay-Sachs and Sandhoff disease, versus the functionally normal heterozygote (hexb+/-) by imaging MALDI-MS. This allowed profiling and localization of many different lipid species, and of particular interest, ganglioside GM2, asialo-GM2 (GA2), and sulfatides (ST). The presence of these compounds was confirmed by analysis of brain extracts using electrospray ionization in conjunction with tandem mass spectrometry (MS/MS). The major fatty acid of the ceramide backbone of both GM2 and GA2 was identified as stearic acid (18:0) versus nervonic acid (24:1) for ST by both tissue-imaging MS and ESI-MS/MS. GM2 and GA2 were highly elevated in hexb-/- and were both localized in the granular cell region of the cerebellum. ST, however, was localized mainly in myelinated fiber (white matter) region of the cerebellum as well as in the brain stem with a relatively uniform distribution and had similar relative signal intensity for both hexb+/- and hexb-/- brain. It was also observed that there were distinct localizations for numerous other lipid subclasses; hence, imaging MALDI-MS could be used for "lipidomic" studies. These results illustrate the usefulness of tissue-imaging MALDI-MS with matrix deposition by OCN for histologic comparison of lipids in tissues such as brains from this mouse model of Tay-Sachs and Sandhoff disease.

Topics: Animals; Brain; Brain Chemistry; Disease Models, Animal; G(M2) Ganglioside; Gangliosides; Lipid Metabolism; Lipids; Mice; Nebulizers and Vaporizers; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingolipids; Sulfoglycosphingolipids; Tay-Sachs Disease

Sulfatide (galactocylceramide-3'-sulfate), a cell surface glycosphingolipid interacts with several cell adhesion molecules including fibrinogen, von Willebrand factor (VWF), P-selectin, thrombospondin (TSP) and laminin, which are involved in haemostasis. We have used a sulfatide-specific single-chain fragment variable (scFv) antibody probe PA38 and sulfatide-deficient mice to investigate the role of membrane sulfatide in platelet function. PA38 bound to platelets and binding increased following platelet activation. Sulfatide was localized as a large cluster towards the center of the platelet surface when examined in a confocal microscope. PA38 (20 microg/ml) inhibited the adhesion of activated platelets to fibrinogen, VWF, P-selectin, TSP1 and laminin by 30%, 30%, 75%, 20% and 35%, respectively, compared to a control scFv (p < 0.05). Furthermore, PA38 inhibited collagen, ADP, thrombin and ristocetin-induced platelet aggregation in PRP by 25%, 30%, 18% and 20%, respectively, compared to the control scFv (p < 0.05). In a PFA-100 platelet function assay, PA38 prolonged the occlusion time by 25% (p < 0.05). Under flow PA38 decreased the thrombus formation on collagen by 31%, (p < 0.01). Sulfatide-deficient mice displayed an extended lag-phase in collagen-induced platelet aggregation compared to wild type (p < 0.05), though in-vivo haemostasis did not differ significantly. Thus, this study provides new evidence for a role for membrane sulfatide in platelet function.

Topics: Animals; Blood Platelets; Cell Membrane; Cerebroside-Sulfatase; Disease Models, Animal; Hemorheology; Hemostasis; Humans; Immunoglobulin Variable Region; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Platelet Adhesiveness; Platelet Aggregation; Platelet Function Tests; Sulfoglycosphingolipids; Thrombosis

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA) and is characterized by deposition of sulfatide in all organs, particularly the nervous system. Recently, formylglycine-generating enzyme (FGE) was found to be essential for activation of sulfatases. This study examined the utility of FGE co-expression in AAV type 1 vector (AAV1)-mediated gene therapy of ASA knockout (MLD) mice. AAV1-ASA alone or AAV1-ASA and AAV1-FGE were co-injected into a single site of the hippocampus. Enzyme assay and immunohistochemical analysis showed that ASA was detected not only in the injected hemisphere but also in the non-injected hemisphere by 7 months after injection. Level of ASA activity and extent of ASA distribution were significantly enhanced by co-introduction of AAV1-FGE. Marked reductions in sulfatide levels were observed throughout the entire brain. The unexpectedly widespread distribution of ASA may be due to a combination of diffusion in extracellular spaces, transport through axons, and circulation in cerebrospinal fluid. The rotarod test revealed improvement of neurological functions. These results demonstrate that direct injection of AAV1 vectors expressing ASA and FGE represents a highly promising approach with significant implications for the development of clinical protocols for MLD gene therapy.

Topics: Animals; Arylsulfatases; Dependovirus; Disease Models, Animal; Gene Expression Regulation, Enzymologic; Genetic Therapy; Genetic Vectors; Glycine; Humans; Leukodystrophy, Metachromatic; Mice; Sulfoglycosphingolipids

In an effort to develop an encapsulated cell-based system to deliver arylsulfatase A (ARSA) to the central nervous system of metachromatic leukodystrophy (MLD) patients, we engineered C2C12 mouse myoblasts with a retroviral vector containing a full-length human ARSA cDNA and evaluated the efficacy of the recombinant secreted enzyme to revert the MLD phenotype in oligodendrocytes (OL) of the As2-/- mouse model. After transduction, C2C12 cells showed a fifteen-fold increase in intracellular ARSA activity and five-fold increase in ARSA secretion. The secreted hARSA collected from transduced cells encapsulated in polyether-sulfone polymer, was taken up by enzyme-deficient OL derived from MLD mice and normally sorted to the lysosomal compartment, where transferred enzyme reached 80% of physiological levels, restoring the metabolism of sulfatide. To evaluate whether secreted enzyme could restore metabolic function in the brain, encapsulated cells and secreted ARSA were shown to be stable in CSF in vitro. Further, to test cell viability and enzyme release in vivo, encapsulated cells were implanted subcutaneously on the dorsal flank of DBA/2J mice. One month later, all retrieved implants released hARSA at rates similar to unencapsulated cells and contained well preserved myoblasts, demonstrating that encapsulation maintains differentiation of C2C12 cells, stable transgene expression and long-term cell viability in vivo. Thus, these results show the promising potential of developing an ARSA delivery system to the CNS based on the use of a polymer-encapsulated transduced xenogenic cell line for gene therapy of MLD.

Topics: Animals; Arylsulfatases; Capsules; Cell Line; Cell Survival; Disease Models, Animal; Genetic Vectors; Graft Survival; Humans; Leukodystrophy, Metachromatic; Mice; Mice, Knockout; Myoblasts; Nerve Regeneration; Oligodendroglia; Polymers; Sulfoglycosphingolipids; Transduction, Genetic; Transgenes; Transplantation, Heterologous; Treatment Outcome; Up-Regulation

In this study we show the effect of anti-sulfatide (RmAb) antibodies and inflammatory cytokines, TNF-alpha and IFN-gamma in inducing myelin basic protein (MBP) degradation in myelin isolated from control wild type (WT) and glia maturation factor (GMF)-deficient (GMF-KO) mice. GMF was not detected in isolated myelin from WT and GMF-KO mice although it is present in brains of WT mice. Our results show that calcium-dependent neutral protease activity caused significantly elevated degradation of 18.5 and/or 17.5kDa isoforms of MBP in WT myelin treated with RmAb or IFN-gamma. In contrast, MBP degradation in isolated myelin from GMF-KO mice remained unaffected following treatment with RmAb, IFN-gamma, or GM-CSF. Neither the 14kDa isoform of MBP nor proteolipid protein (PLP) showed an elevated degradation compared to controls. A virtual absence of GM-CSF, TNF-alpha and IFN-gamma in GMF-KO brain compared to WT was also evident when the animals were challenged with MOG 35-55. Additionally, the myelin from GMF-KO mice showed difference in distribution of myelin oligodendrocyte glycoprotein (MOG) and beta-tubulin in a sucrose density gradient myelin-axolemmal fractions compared to WT. Taken together, our data suggests a role for GMF in the biochemical organization of myelin and thereby its effect on MBP degradation induced by RmAb and IFN-gamma.

Topics: Animals; Antibodies; Calcium; Chelating Agents; Disease Models, Animal; Drug Interactions; Egtazic Acid; Gene Expression; Glia Maturation Factor; Interferon-gamma; Male; Mice; Mice, Knockout; Myelin Basic Protein; Myelin Proteins; Myelin Sheath; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; Neuritis, Autoimmune, Experimental; Sulfoglycosphingolipids

Metachromatic leukodystrophy is a lysosomal storage disorder caused by deficiency in the sulfolipid degrading enzyme arylsulfatase A (ASA). In the absence of a functional ASA gene, 3-O-sulfogalactosylceramide (sulfatide; SGalCer) and other sulfolipids accumulate. The storage is associated with progressive demyelination and various finally lethal neurological symptoms. Lipid storage, however, is not restricted to myelin-producing cells but also occurs in neurons. It is unclear whether neuronal storage contributes to symptoms of the patients. Therefore, we have generated transgenic ASA-deficient [ASA(-/-)] mice overexpressing the sulfatide synthesizing enzymes UDP-galactose:ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST) in neurons to provoke neuronal lipid storage. CGT-transgenic ASA(-/-) [CGT/ASA(-/-)] mice showed an accumulation of C18:0 fatty acid-containing SGalCer in the brain. Histochemically, an increase in sulfolipid storage could be detected in central and peripheral neurons of both CGT/ASA(-/-) and CST/ASA(-/-) mice compared with ASA(-/-) mice. CGT/ASA(-/-) mice developed severe neuromotor coordination deficits and weakness of hindlimbs and forelimbs. Light and electron microscopic analyses demonstrated nerve fiber degeneration in the spinal cord of CGT/ASA(-/-) mice. CGT/ASA(-/-) and, to a lesser extent, young ASA(-/-) mice exhibited cortical hyperexcitability, with recurrent spontaneous cortical EEG discharges lasting 5-15 s. These observations suggest that SGalCer accumulation in neurons contributes to disease phenotype.

Topics: Analysis of Variance; Animals; Behavior, Animal; Cerebral Cortex; Cerebroside-Sulfatase; Disease Models, Animal; Electroencephalography; In Situ Hybridization; Leukodystrophy, Metachromatic; Lipids; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Motor Skills; N-Acylsphingosine Galactosyltransferase; Nerve Degeneration; Neurons; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spinal Cord; Sulfoglycosphingolipids; Sulfotransferases

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ASA). This results in accumulation of sulfated glycosphingolipids, mainly 3-O-sulfogalactosylceramide (sulfatide), in the nervous system and various other organs. In patients, lipid storage causes a progressive loss of myelin leading to various neurological symptoms. The sulfatide storage pattern in ASA-deficient [ASA(-/-)] mice is comparable to humans, but regrettably, the mice do not mimic the myelin pathology. We reasoned that increasing sulfatide storage in this animal model might provoke demyelination. Therefore, we generated transgenic ASA(-/-) [tg/ASA(-/-)] mice overexpressing the sulfatide-synthesizing enzyme galactose-3-O-sulfotransferase-1 in myelinating cells. Indeed, these tg/ASA(-/-) mice displayed a significant increase in sulfatide storage in brain and peripheral nerves. Mice older than 1 year developed severe neurological symptoms. Nerve conduction velocity was significantly reduced in tg/ASA(-/-) mice because of a peripheral neuropathy characterized by hypomyelinated and demyelinated axons. Inhomogeneous myelin thickness in the corpus callosum, increased frequency of hypomyelinated and demyelinated axons in corpus callosum and optic nerve, and substantially reduced myelin basic protein levels are in accordance with loss of myelin in the CNS. Thus, increasing sulfatide storage in ASA(-/-) mice leads to neurological symptoms and morphological alterations that are reminiscent of human MLD. The approach described here may also be applicable to improve other mouse models of lysosomal as well as nonlysosomal disorders.

Topics: Age Factors; Animals; Cerebroside-Sulfatase; Demyelinating Diseases; Disease Models, Animal; Electromyography; Hindlimb Suspension; Humans; Leukodystrophy, Metachromatic; Lipids; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Motor Activity; Myelin Basic Protein; Myelin Sheath; Neural Conduction; Peripheral Nerves; Rotarod Performance Test; Sciatic Nerve; Spinal Cord; Sulfoglycosphingolipids

Recent studies suggest that increased T-cell and autoantibody reactivity to lipids may be present in the autoimmune demyelinating disease multiple sclerosis. To perform large-scale multiplex analysis of antibody responses to lipids in multiple sclerosis, we developed microarrays composed of lipids present in the myelin sheath, including ganglioside, sulfatide, cerebroside, sphingomyelin and total brain lipid fractions. Lipid-array analysis showed lipid-specific antibodies against sulfatide, sphingomyelin and oxidized lipids in cerebrospinal fluid (CSF) derived from individuals with multiple sclerosis. Sulfatide-specific antibodies were also detected in SJL/J mice with acute experimental autoimmune encephalomyelitis (EAE). Immunization of mice with sulfatide plus myelin peptide resulted in a more severe disease course of EAE, and administration of sulfatide-specific antibody exacerbated EAE. Thus, autoimmune responses to sulfatide and other lipids are present in individuals with multiple sclerosis and in EAE, and may contribute to the pathogenesis of autoimmune demyelination.

Topics: Animals; Autoimmune Diseases; Brain; Disease Models, Animal; Encephalitis; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Humans; Lipids; Mice; Microarray Analysis; Multiple Sclerosis; Reproducibility of Results; Sensitivity and Specificity; Sulfoglycosphingolipids; T-Lymphocytes

This work describes the first successful oligodendrocyte-based cell therapy for presymptomatic arylsulfatase A (ARSA) null neonate mice, a murine model for human metachromatic leukodystrophy (MLD). We found that oligodendrocyte progenitors (OLPs) engrafted and survived into adulthood when transplanted in the neonatal MLD brain. Transplanted cells integrated nondisruptively, did not produce tumors, and survived as proteolipid protein- and MBP-positive postmitotic myelinating oligodendrocytes (OLs) intermingled with endogenous MLD OLs within the adult MLD white matter. Transplanted MLD mice had reduced sulfatide accumulation in the CNS, increased brain ARSA activity, and full prevention of the electrophysiological and motor deficits that characterize untreated MLD mice. Our results provide direct evidence that healthy OLPs can tolerate the neurotoxic accumulation of sulfatides that evolves during the postnatal development of the MLD brain and contribute to OL cell replacement to limit the accumulation of sulfatides and the evolution of CNS defects in this lysosomal storage disease mouse model.

Topics: Animals; Animals, Newborn; Brain Tissue Transplantation; Cell Differentiation; Cell Movement; Cells, Cultured; Cerebroside-Sulfatase; Disease Models, Animal; Graft Survival; Leukodystrophy, Metachromatic; Mice; Mice, Knockout; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Nerve Fibers, Myelinated; Oligodendroglia; Stem Cell Transplantation; Sulfoglycosphingolipids; Treatment Outcome

Arylsulfatase A (ASA)-deficient mice are a model for the lysosomal storage disorder metachromatic leukodystrophy. This lipidosis is characterised by the lysosomal accumulation of the sphingolipid sulfatide. Storage of this lipid is associated with progressive demyelination. We have mated ASA-deficient mice with mice heterozygous for a non-functional allele of UDP-galactose:ceramide-galactosyltransferase (CGT). This deficiency is known to lead to a decreased synthesis of galactosylceramide and sulfatide, which should reduce sulfatide storage and improve pathology in ASA-deficient mice.. ASA-/- CGT+/- mice, however, showed no detectable decrease in sulfatide storage. Neuronal degeneration of cells in the spiral ganglion of the inner ear, however, was decreased. Behavioural tests showed small but clear improvements of the phenotype in ASA-/- CGT+/- mice.. Thus the reduction of galactosylceramide and sulfatide biosynthesis by genetic means overall causes modest improvements of pathology.

Topics: Analysis of Variance; Animals; Behavior, Animal; Brain; Breeding; Cerebroside-Sulfatase; Disease Models, Animal; Ear, Inner; Female; Galactosylceramides; Genotype; Leukodystrophy, Metachromatic; Male; Mice; Mice, Knockout; Motor Activity; N-Acylsphingosine Galactosyltransferase; Neurons; Phenotype; Sulfoglycosphingolipids; Time Factors; Uridine Diphosphate Galactose

Gastric sulfatide, whose carbohydrate moiety resembles that of the anti-ulcer drug sucralfate, has been shown to play a role in mucosal protection in an experimental ulcer model. To elucidate the functional significance of gastric lipids, precise determination of the lipids in human gastric fluid and epithelium was performed, and the anti-ulcer effects of all lipids in the fluid were measured in mouse ulcer models.. The lipids in human gastric fluid and epithelium were analyzed by thin layer chromatography and immunostaining, and the anti-ulcer effects of gastric lipids were determined using mouse ulcer models.. Human gastric epithelium contained both sulfatide and cholesterol sulfate (CS) as sulfolipids, which were also detected in gastric fluid, showing their stable natures in the gastric fluid. Hemorrhaging in HCl-induced gastric lesions was suppressed in a dose-dependent manner by the administration of sulfolipid-containing liposomes, but suppression of stress ulcers was only accomplished with CS-containing liposomes, ie, not with sulfatide-containing ones, due to the longer retainment of CS than sulfatide in the stomach.. Among the lipids in human gastric fluid, CS was revealed to exhibit a gastroprotective activity, which was more effective than that of sulfatide.

Topics: Adult; Aged; Aged, 80 and over; Animals; Anticarcinogenic Agents; Cholesterol Esters; Chromatography, Thin Layer; Cytoprotection; Disease Models, Animal; Female; Gastric Juice; Gastric Mucosa; Humans; Hydrochloric Acid; Lipid Metabolism; Lipids; Liposomes; Mice; Mice, Hairless; Mice, Inbred BALB C; Middle Aged; Stomach Ulcer; Sulfoglycosphingolipids

Leukocytes may be important in the development of intimal hyperplasia, but little is known about the participation of sulfatides (3-sulfated galactosyl ceramides) which are native ligands of L- and P-selectin. This study was designed to determine whether sulfatides affect the development of intimal hyperplasia. ICR mice were randomized to receive vehicle or sulfatides intravenously either at 1, 3, or 10 mg/kg/day for 7 days, or at 10 mg/kg/day for 1, 3, or 7 days. Endothelial damage was inflicted on the femoral artery via the photochemical reaction between rose bengal and green light. Scanning electron and light microscopic observations 3 days after the injury indicated that sulfatides-treated animals had more neutrophils adhering to the injury site than vehicle-treated controls. At 21 days, sulfatides-treated animals had a greater neointimal area than controls. In in vitro studies, sulfatides (i) increased cytosolic free calcium in mouse neutrophils, (ii) caused increases in expression of Mac-1 (CD 11 b/CD 18) on the neutrophil membrane surface in mouse whole blood. These findings suggest that neutrophil accumulation on the subendothelial matrix or adherence of platelets mediated by adhesive interactions between L- or P-selectin and sulfatides may contribute to the development of intimal hyperplasia. The neutrophil accumulation may be mediated by an increase in Mac-1 caused by the agonistic effects of sulfatides on the neutrophil membrane surface, or by an increase in L- and P-selectin ligands resulting from the binding of sulfatides onto the exposed subendothelial matrix.

Topics: Animals; Antibodies, Monoclonal; Calcium; CD18 Antigens; Cell Adhesion; Disease Models, Animal; Endothelium, Vascular; Femoral Artery; Hyperplasia; L-Selectin; Leukocytes; Ligands; Male; Mice; Mice, Inbred ICR; P-Selectin; Peripheral Vascular Diseases; Peritonitis; Platelet Adhesiveness; Random Allocation; Sulfoglycosphingolipids; Thioglycolates; Time Factors

Inherited deficiency for the lysosomal enzyme arylsulfatase A (ASA) leads to lysosomal storage of sulfatides and to dramatic demyelination in the CNS of humans (metachromatic leukodystrophy, MLD). As an animal model, ASA(-/-) mice have previously been generated by disruption of the ASA gene and are known to develop lysosomal sulfatide storage similar to that in human MLD, and, moreover, to become deaf because of degeneration of the primary neurons of the auditory pathway. The present study deals with the cellular and topographic distribution of sulfatide storage throughout the CNS of ASA(-/-) mice between a few days and 24 months of age. Sulfatide accumulation was detected on the ultrastructural level and by histochemical staining with alcian blue. Sulfatide storage was found in oligodendroglia and neurons in young mice, and in activated microglia (phagocytes) in adult mice. Neuronal sulfatide storage was most prominent in many nuclei of the medulla oblongata and pons, and in several nuclei of midbrain and forebrain. Sulfatide-storing phagocytes were most frequent in the white matter tracts of aged ASA(-/-) mice, whereas no widespread demyelination was obvious. Loss of neurons was found in two nuclei of the auditory pathway of aged ASA(-/-) mice (ventral cochlear nucleus and nucleus of trapezoid body). The distributional pattern of sulfatide storage throughout the CNS of ASA(-/-) mice largely corresponds to data reported for human MLD. An important difference, however, which remains unexplained at present, is the absence of obvious demyelination from the CNS of ASA(-/-) mice up to the age of 2 years.

Topics: Age Factors; Animals; Auditory Pathways; Brain; Cerebroside-Sulfatase; Disease Models, Animal; Female; Humans; Immunohistochemistry; Leukodystrophy, Metachromatic; Lysosomes; Male; Mice; Microglia; Microscopy, Electron, Transmission; Neurons; Oligodendroglia; Spinal Cord; Sulfoglycosphingolipids

Previous studies have shown that sulfatide is present and functionally involved in beta cells, and that anti-sulfatide antibodies (ASA) exist during development of type I diabetes mellitus. To further explore the possible role of sulfatide in type I diabetes, developmental expression was examined in human pancreas and in pancreas of the type I diabetes models BB rat and NOD mouse compared to Lewis rat and BALB/c mouse, respectively. Sulfatide was not only expressed in adult pancreas, but also in human fetal and rodent neonatal pancreas, i.e., during the growing period of the immunological self. Sulfatide had a different expression pattern in human beings and rodents, concerning both the amounts of sulfatide and expression during development. There was no change in the sulfatide fatty acid isoform expression during development. The pancreatic expression of another sulfated glycosphingolipid, sulfated lactosylceramide, indicated that this molecule is a potential fetal/neonatal marker, which was further expressed in the type I diabetic models. In conclusion, these findings give further support to the possibility that sulfatide is a relevant autoantigen in type I diabetes and that sulfated lactosylceramide might function as a potential risk factor for disease development, at least in the animal models.

Topics: Animals; Antigens, CD; Chromatography, Thin Layer; Diabetes Mellitus, Type 1; Disease Models, Animal; Humans; Lactosylceramides; Mice; Pancreas; Rats; Species Specificity; Spectrometry, Mass, Electrospray Ionization; Sulfoglycosphingolipids

We showed previously that spinal cord implants of hybridoma cells (O1) that secrete an IgM antigalactocerebroside cause focal multiple-sclerosis-like plaques of demyelination followed by remyelination to form "shadow plaques" (Rosenbluth et al., 1999). The antibody in that case was directed against a glycolipid present in mature oligodendrocytes and myelin but not in precursor cells. We now report the effects of implanting a different hybridoma (O4) that secretes IgM antibodies directed against sulfatide, a constituent not only of mature myelin and oligodendrocytes but also of late precursor cells, in order to determine whether this hybridoma too would generate focal demyelination and would, in addition, block remyelination. Our results show that focal plaques of demyelination indeed appear after O4 implantation, and that remyelination does occur, but only in cases where the hybridoma cells have degenerated, probably through host rejection. The occurrence of remyelination suggests that oligodendrocyte precursor cells are capable of migrating in rapidly from adjacent areas or that early precursors, not yet expressing sulfatide, remain undamaged within the lesions. In cases where intact hybridoma cells persist at lesion sites, remyelination does not occur. Failure of remyelination in this model thus appears to result from the continuing presence of antimyelin antibodies rather than from depletion of oligodendrocyte precursors.

Topics: Animals; Animals, Newborn; Autoantibodies; Axons; Cell Movement; Demyelinating Diseases; Disease Models, Animal; Female; Graft Survival; Hybridomas; Immunoglobulin M; Male; Mice; Microscopy, Electron; Myelin Sheath; Oligodendroglia; Rats; Rats, Wistar; Regeneration; Spinal Cord; Stem Cells; Sulfoglycosphingolipids; Tumor Cells, Cultured

Sulfatides show structural, and possibly physiological similarities to gangliosides. Kidney dysfunction might be correlated with changes in sulfatides, the major acidic glycosphingolipids in this organ. To elucidate their in vivo metabolic pathway these compounds were analyzed in mice afflicted with inherited glycosphingolipid disorders. The mice under study lacked the genes encoding either beta-hexosaminidase alpha-subunit (Hexa-/-), the beta-hexosaminidase beta-subunit (Hexb-/-), both beta-hexosaminidase alpha and beta-subunits (Hexa-/- and Hexb-/-), GD3 synthase (GD3S-/-), GD3 synthase and GalNAc transferase (GD3S-/- and GalNAcT-/-), GM2 activator protein (Gm2a-/-), or arylsulfatase A (ASA-/-). Quantification of the sulfatides, I(3)SO(3)(-)-GalCer (SM4s), II(3)SO(3)(-)-LacCer (SM3), II(3)SO(3)(-)-Gg(3)Cer (SM2a), and IV(3,) II(3)-(SO(3)(-))(2)-Gg(4)Cer (SB1a), was performed by nano-electrospray tandem mass spectrometry. We conclude for the in vivo situation in mouse kidneys that: 1) a single enzyme (GalNAc transferase) is responsible for the synthesis of SM2a and GM2 from SM3 and GM3, respectively. 2) In analogy to GD1a, SB1a is degraded via SM2a. 3) SM2a is hydrolyzed to SM3 by beta-hexosaminidase S (Hex S) and Hex A, but not Hex B. Both enzymes are supported by GM2-activator protein. 4) Arylsulfatase A is required to degrade SB1a. It is probably the sole sphingolipid-sulfatase cleaving the galactosyl-3-sulfate bond. In addition, a human Tay-Sachs patient's liver was investigated, which showed accumulation of SM2a along with GM2 storage. The different ceramide compositions of both compounds indicated they were probably derived from different cell types. These data demonstrate that in vivo the sulfatides of the ganglio-series follow the same metabolic pathways as the gangliosides with the replacement of sulfotransferases and sulfatases by sialyltransferases and sialidases. Furthermore, a novel neutral GSL, IV(6)GlcNAcbeta-Gb(4)Cer, was found to accumulate only in Hexa-/- and Hexb-/- mouse kidneys. From this we conclude that Hex S also efficiently cleaves terminal beta1-6-linked HexNAc residues from neutral GSLs in vivo.

Topics: Animals; Chromatography, Thin Layer; Disease Models, Animal; Gangliosides; Kidney; Lipid Metabolism, Inborn Errors; Mice; Mice, Mutant Strains; Nanotechnology; Reference Standards; Spectrometry, Mass, Electrospray Ionization; Sulfoglycosphingolipids

The Mongolian gerbil has been used as an excellent experimental animal model for studying Helicobacter pylori infection because it can stably colonize and induce severe chronic gastritis, ulceration, and cancer-simulating human diseases in this animal. In contrast, H. pylori can only induce mild inflammation in many mouse models. The aim in this study is to clarify the difference of induction of pathological lesions in the two animal models. SPF ICR mice and Mongolian gerbils were inoculated with a clinically isolated strain of H. pylori. Six weeks after inoculation, bacteria colonizing the stomach were counted. Immunohistochemical staining and biochemical analyses of three putative receptor glycolipids were performed with monoclonal antibodies to the respective glycolipids. Significantly higher numbers of H. pylori were recovered from the stomachs of Mongolian gerbils than mice (5.77 +/- 0.46 log CFU vs 4.17 +/- 0.55 log CFU, P < 0.01). Immunohistochemical studies showed that sulfatide expression in the gastric mucosa of Mongolian gerbils was much stronger than that in mice, whereas the expression of Lewis(b) glycolipid and GM3 were almost equal. Quantitative analysis of each glycolipid by thin-layer chromatography confirmed the results of immunohistochemical study, showing 4.1 times higher sulfatide content in the Mongolian gerbil stomach. The content of both Lewis(b) and GM3 was almost equivalent in these two animals. In conclusions, higher levels of sulfatide expression, a putative adhesion receptor, in the gastric mucosa of Mongolian gerbils may allow abundant colonization by H. pylori, resulting in the development of gastric lesions in this animal model.

Topics: Animals; Chromatography, Thin Layer; Disease Models, Animal; Gerbillinae; Helicobacter pylori; Immunohistochemistry; Male; Mice; Mice, Inbred ICR; Receptors, Cell Surface; Specific Pathogen-Free Organisms; Sulfoglycosphingolipids

The inherited deficiency of arylsulfatase A (ASA) in humans causes lysosomal accumulation of sulfatides in visceral organs and in the nervous system and leads to wide-spread demyelination (metachromatic leukodystrophy, MLD). ASA-deficient mice have previously been generated by means of targeted gene disruption. In the present study, visceral organs of ASA-deficient mice were investigated. A simple technique for the histochemical detection of accumulated sulfatides was elaborated using pre-embedding staining with alcian blue. The gall bladder, intrahepatic bile ducts, exocrine pancreatic ducts, respiratory epithelium and, with low degree, testicular Sertoli cells, showed sulfolipid storage. The storage pattern in the kidney will be described in a separate publication. Hepatocytes, pancreatic islets, adrenal glands, and gastric epithelium were unaffected. Ultrastructurally, the intralysosomal storage material displayed parallel and concentric lamellar patterns. Apart from some differences, the topographic distribution of the sulfatide storage resembled that in human MLD. In addition to being an animal model of the human disease, the ASA-deficient mouse may be useful for investigating the cell biology of sulfolipids in visceral organs.

Topics: Animals; Cerebroside-Sulfatase; Child, Preschool; Disease Models, Animal; Histocytochemistry; Humans; Leukodystrophy, Metachromatic; Lysosomes; Male; Mice; Mice, Knockout; Microscopy, Electron; Sulfoglycosphingolipids; Tissue Distribution; Viscera

We previously reported that the sulfatide (galactosylceramide I3-sulfate) may have contradictory functions, namely both coagulant and anticoagulant roles in vivo: sulfatide induced giant thrombi formation when injected into rats with vein ligation, whereas no thrombi were formed when sulfatide was injected into rats without vein ligation. Rather it prolonged bleeding time. To investigate the structural features of sulfatide for both functions, a synthetic sulfatide (galactosylceramide I6-sulfate) which does not occur naturally, cholesterol 3-sulfate and ganglioside GM4 were examined together with naturally occurring sulfatide. Both sulfatides and cholesterol 3-sulfate induced giant thrombi in the rats with vein ligation within ten minutes of injection, although cholesterol 3-sulfate exhibited weaker coagulant activity than the sulfatides. On the contrary, both sulfatides significantly prolonged bleeding time but cholesterol 3-sulfate barely prolonged it when injected without vein ligation. GM4 exhibited neither coagulant nor anticoagulant activity. These results suggested that sulfate moiety in the sulfatides is essential for coagulant activity and that galactose residue enhances the activity, whereas both galactose and sulfate residues seem to be important for anticoagulant activity. This is because the sulfatides possess both residues but GM4 possesses galactose without sulfate and cholesterol 3-sulfate possesses sulfate without galactose. We previously reported that the possible mechanism of anticoagulation by sulfatide was due to its binding to fibrinogen, thereby inhibiting the conversion to fibrin. In this paper we reveal that both sulfatides inhibited thrombin activity independent of heparin cofactor II, thus providing evidence of another anticoagulation mechanism for the sulfatides.

Topics: Animals; Anticoagulants; Blood Coagulation; Calcium Chloride; Cattle; Cholesterol; Coagulants; Disease Models, Animal; Dose-Response Relationship, Drug; Galactose; Gangliosides; Humans; Male; Plasma; Rats; Rats, Sprague-Dawley; Sulfates; Sulfoglycosphingolipids; Swine; Thrombin; Venous Thrombosis

Although sulfatide (galactosylceramide I3-sulfate) has been reported to activate blood coagulation factor XII (Hageman factor), it has been administered to animals without subsequent thrombus formation. We recently found that sulfatide binds to fibrinogen and thus disturbs fibrin formation in vitro, suggesting its possible role as an anticoagulant rather than as a coagulant. We therefore examined the in vivo effects of sulfatide on thrombogenesis by using a rat deep vein thrombosis model in which thrombus is induced by ligating the inferior vena cava. Sulfatide and gangliosides were each separately administered to rats 1 min before the vein ligation, and after 3 h, sulfatide but not gangliosides markedly (P < .001) enhanced the thrombogenesis. A kinetic turbidmetric assay of plasma coagulation initiated by CaCl2 in the wells of a microtiter plate revealed that coagulation was also markedly accelerated in the presence of sulfatide but not gangliosides, the results of which seemed to be very consistent with those of the in vivo experiments. Because sulfatide could not induce thrombosis without vein ligation in rats, the enhancement of thrombogenesis by sulfatide in the in vivo model might require endothelial damage and/or venous congestion, both of which could be induced by vein ligation.

Topics: Animals; Blood Coagulation; Calcium Chloride; Disease Models, Animal; Factor XII; Fibrinogen; Gangliosides; Kinetics; Male; Protein Binding; Prothrombin Time; Rats; Rats, Sprague-Dawley; Sulfoglycosphingolipids; Venous Thrombosis

Polycystic kidney disease is a disorder marked by aberrant renal tubular epithelial cell proliferation and transport abnormalities. Sphingolipids are ubiquitous membrane components implicated in several cellular functions including cell membrane sorting, signaling, growth, ion transport, and adhesion. To investigate a potential pathogenic role for sphingolipids in cystic kidney disease, we studied the sphingolipid content and associated enzymatic activities of the kidneys from cpk/cpk mice and their phenotypically normal litter mates. The neutral glycolipids, including glucosylceramide and lactosylceramide, displayed a striking increase in 3-week-old cpk/cpk mice as did the acidic lipid, ganglioside GM3. However, a correspondingly significant decrease in sulfoglycolipid and ceramide concentration was observed in the cpk/cpk kidneys. Glucosylceramide synthase activity was higher in the kidneys of the cpk/cpk mice than in those of the controls. Kinetic analysis of the glucosylceramide synthase revealed the presence of an endogenous activator in the cystic kidney. A marked decrease in sulfotransferase activity was observed in both whole kidney homogenates and in microsomal preparations that was consistent with the decrement in sulfolipid content. The increase in GM3, glucosyl- and lactosylceramide may therefore be the result of impaired sulfolipid synthesis at the 3-week time point. While sulfolipid and glucosylceramide concentrations are not different at 1 and 2 weeks of age, ceramide concentrations in cystic kidneys are significantly reduced compared to kidneys from phenotypically normal mice. These results suggest that sphingolipids may play a potential role in the proliferative and transport abnormalities associated with cystic renal disease and the development of azotemia.

Topics: Animals; Ceramides; Disease Models, Animal; Female; Glucosyltransferases; Glycosphingolipids; Kidney; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Polycystic Kidney, Autosomal Recessive; Sulfoglycosphingolipids; Sulfotransferases

Sulphatide is found to be a major glycosphingolipid in serum lipoproteins of rabbit and its content is markedly elevated in serum of WHHL rabbit, an animal model for human familial hypercholesterolemia. On analysis of tissue sulphatide contents, serum appears to derive its sulphatide from liver (90%) and small intestine (10%) and passes on to aorta of WHHL rabbit which is found to have a large amount of sulphatide while none is found in normal aorta. Thus it seems that sulphatide finally accumulates in arterial walls along with the progression of atherosclerosis in WHHL rabbit. Since sulphatide at median concentration (8 nmole/ml serum) in various mammals is found to increase activated partial thromboplastin time by 25%, it is suggested that anticoagulant activity may be one of the physiological functions of sulphatide in serum. The observation of an increase in activated partial thromboplastin time by 2.5-fold on injection of sulphatide (10 mg/kg body wt) into rabbit suggests that sulphatide may be an effective and safe antithrombotic agent.

Topics: Animals; Anticoagulants; Aorta; Disease Models, Animal; Glycosphingolipids; Humans; Hypercholesterolemia; Intestine, Small; Lipoproteins; Liver; Rabbits; Reference Values; Sulfoglycosphingolipids

Glycosphingolipids in serum and lipoproteins from Watanabe hereditable hyperlipidemic rabbit (WHHL rabbit), which is an animal model for human familial hypercholesterolemia (FH), were analyzed for the first time in this study. Chylomicrons and very low density, low density, and high density lipoproteins contained sulfatide as a major glycosphingolipid (12 nmol/mumol total phospholipids (PL) in chylomicrons, 19 nmol/mumol PL in VLDL, 18 nmol/mumol PL in LDL, and 14 nmol/mumol PL in HDL) with other minor glycosphingolipids such as glucosylceramide, galactosylceramide, GM3 ganglioside, lactosylceramide, and globotriaosylceramide. The concentration of sulfatide as a major glycosphingolipid in WHHL rabbit serum (121 nmol/ml) was much higher than that in normal rabbit serum (3 nmol/ml). Fatty acids of the sulfatides comprised mainly nonhydroxy fatty acids (C22, 23, and 24) and significant amounts of hydroxy fatty acids (about 10%) whereas long chain bases of the sulfatides comprised mostly (4E)-sphingenine with a significant amount of 4D-hydroxysphinganine (about 10%). Furthermore, sulfatides in the liver and small intestine from normal and WHHL rabbits (where serum lipoproteins are produced) were determined to amount to 260 nmol/g liver in WHHL rabbit, 104 nmol/g liver in control rabbit, 99.6 nmol/g small intestine in WHHL rabbit, and 31.2 nmol/g small intestine in control rabbit. Ceramide portions of the sulfatides in the liver were mainly composed of (4E)-sphingenine and nonhydroxy fatty acids, while those in the small intestine were mainly composed of 4D-hydroxysphinganine and hydroxy fatty acids. These results indicated that the sulfatides of serum lipoproteins were mostly derived from the liver (90% of the total), and that the remaining sulfatides (10% of the total) might be derived from the small intestine. These two sulfatides, which have different ceramide portions, could be useful markers for metabolic and biosynthetic studies of various lipoproteins in WHHL rabbit, and thus would be helpful to further elucidate the relationship between hypercholesterolemia and atherosclerosis in the rabbit.

Topics: Animals; Chylomicrons; Disease Models, Animal; Fatty Acids; Glycosphingolipids; Hyperlipidemias; Lipoproteins; Lipoproteins, HDL; Lipoproteins, LDL; Lipoproteins, VLDL; Rabbits; Sulfoglycosphingolipids

Rats with experimental allergic encephalomyelitis (EAE) induced with myelin or spinal cord show decreases in the content of sulphatides and cerebrosides and increases in the level of esterified cholesterol in the CNS. In this work it is shown that brain sulphatide changes can be obtained by injection of mixtures containing glycosphingolipids. Alterations in the content of cerebrosides occur when the injection mixture contains cerebrosides. The alterations of sulphatides and cholesterol ester induced by injection of spinal cord could be suppressed by treatment with immunosuppressive drugs (dexamethasone, cyclophosphamide and 6-mercaptopurine) able to prevent clinical signs of EAE.

Topics: Animals; Brain; Cerebrosides; Cholesterol Esters; Cyclophosphamide; Dexamethasone; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Immunosuppressive Agents; Lipid Metabolism; Male; Mercaptopurine; Myelin Sheath; Rats; Spinal Cord; Sulfoglycosphingolipids

Topics: Animals; Brain; Brain Diseases; Cerebrosides; Cholesterol; Disease Models, Animal; DNA; Female; Lead Poisoning; Microscopy, Electron; Myelin Sheath; Nerve Tissue Proteins; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Rats; Sphingomyelins; Sulfoglycosphingolipids

Topics: Animals; Brain; Brain Stem; Carbon Radioisotopes; Central Nervous System; Ceramides; Cerebellum; Cerebrosides; Cholesterol; Chromatography, Thin Layer; Copper; Deficiency Diseases; Disease Models, Animal; Female; Galactose; Gangliosides; Hexosyltransferases; Male; Movement Disorders; Myelin Sheath; Phosphatidylcholines; Phosphatidylethanolamines; Rats; Sphingomyelins; Sulfoglycosphingolipids

Topics: Animals; Brain; Cell Differentiation; Central Nervous System Diseases; Cholesterol; Chromatography, Thin Layer; Demyelinating Diseases; Diffuse Cerebral Sclerosis of Schilder; Disease Models, Animal; DNA; Electrophoresis, Polyacrylamide Gel; Male; Mice; Mice, Inbred Strains; Myelin Basic Protein; Myelin Sheath; Nerve Fibers, Myelinated; Nerve Tissue Proteins; Neuroglia; Phospholipids; RNA; Sulfoglycosphingolipids

Topics: Animals; Centrifugation, Density Gradient; Demyelinating Diseases; Disease Models, Animal; Dose-Response Relationship, Drug; Evoked Potentials; Hexachlorophene; Myelin Sheath; Neural Conduction; Peripheral Nervous System Diseases; Rats; Sciatic Nerve; Sulfoglycosphingolipids