Sphingolipid Players in Multiple Sclerosis: Their Influence on the Initiation and Course of the Disease.

Sphingolipids (SLs) play a significant role in the nervous system, as major components of the myelin sheath, contributors to lipid raft formation that organize intracellular processes, as well as active mediators of transport, signaling and the survival of neurons and glial cells. Alterations in SL metabolism and content are observed in the course of central nervous system diseases, including multiple sclerosis (MS). In this review, we summarize the current evidence from studies on SLs (particularly gangliosides), which may shed new light upon processes underlying the MS background. The relevant aspects of these studies include alterations of the SL profile in MS, the role of antibodies against SLs and complexes of SL-ligand-invariant NKT cells in the autoimmune response as the core pathomechanism in MS. The contribution of lipid-raft-associated SLs and SL-laden extracellular vesicles to the disease etiology is also discussed. These findings may have diagnostic implications, with SLs and anti-SL antibodies as potential markers of MS activity and progression. Intriguing prospects of novel therapeutic options in MS are associated with SL potential for myelin repair and neuroprotective effects, which have not been yet addressed by the available treatment strategies. Overall, all these concepts are promising and encourage the further development of SL-based studies in the field of MS.

Distinctive sphingolipid patterns in chronic multiple sclerosis lesions.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Brain Gangliosides in Alzheimer's Disease: Increased Expression of Cholinergic Neuron-Specific Gangliosides.

BACKGROUND: Gangliosides are enriched in the neuronal membranes. Gangliosides are shown to interact with amyloid-ß proteins, leading to formation of amyloid fibrils in Alzheimer's disease (AD) brains. Several earlier studies indicated that the alterations of ganglioside metabolism could contribute the pathogenesis of AD. METHODS: Gangliosides were isolated from the frontal lobes in five patients with AD and three control subjects. Gangliosides were assessed by high performance thin-layer chromatography (HPTLC) with resorcinol staining and immunostaining using mouse monoclonal antibodies against cholinergic neuronspecific (Chol-1α) gangliosides. RESULTS: In all AD brains, not only the total sialic acid content but also a-series gangliosides, GM1 and GD1a, were dramatically reduced as compared with those in control subjects. These results are a hallmark of the pathogenesis in AD. In contrast, Chol-1α gangliosides, GT1aα and GQ1bα, which are specific markers of cholinergic neurons, were significantly increased in AD brains. CONCLUSION: The expression of Chol-1α gangliosides may be caused by a compensation to preserve the function of the cholinergic neuron and play an important role in cholinergic synaptic transmission.

The Pathogenic Role of Ganglioside Metabolism in Alzheimer's Disease-Cholinergic Neuron-Specific Gangliosides and Neurogenesis.

Alzheimer's disease (AD) is the most common type of dementia with clinical symptoms that include deficits in memory, judgment, thinking, and behavior. Gangliosides are present on the outer surface of plasma membranes and are especially abundant in the nervous tissues of vertebrates. Ganglioside metabolism, especially the cholinergic neuron-specific gangliosides, GQ1bα and GT1aα, is altered in mouse model of AD and patients with AD. Thus, alterations in ganglioside metabolism may participate in several events related to the pathogenesis of AD. Increased expressions of GT1aα may reflect cholinergic neurogenesis. Most changes in ganglioside metabolism occur in the specific brain areas and their lipid rafts. Targeting ganglioside metabolism in lipid rafts may represent an underexploited opportunity to design novel therapeutic strategies for AD.

Anti-Sulfoglucuronosyl Paragloboside Antibody: A Potential Serologic Marker of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons. Although the etiology of ALS is obscure, genetic studies of familiar ALS suggest a multifactorial etiology for this condition. Similarly, there probably are multiple causes for sporadic ALS. Autoimmune-mediated motor neuron dysfunction is one proposed etiology for sporadic ALS. In the present study, anti-glycolipid antibodies including GM1, GD1b, GD3, and sulfoglucuronosyl paragloboside (SGPG) were investigated in the sera of a large number of patient samples, including 113 ALS patients and 50 healthy controls, by means of enzyme-linked immunosorbent assay with affinity parametric complex criterion evaluation and thin-layer chromatography immunooverlay (immuno-TLC). Anti-SGPG antibodies were found in the sera of 13.3% ALS patients (15 out of 113). The highest titer reached 1:1600. The presence of anti-SGPG antibodies in the serum samples was also confirmed by immuno-TLC. Importantly, a multiple logistic regression analysis showed that the presence of anti-SGPG antibody was positively correlated with age (p < .01) and negatively correlated with ALS Functional Rating Scale score (p < .05). Moreover, the localization of SGPG-immunoreactivity on the motor neurons of rat spinal cord and a mouse motor neuronal cell line, NSC-34 was observed by an immunofluorescence method. These data suggest that SGPG could represent a specific pathogenic antigen in those ALS patients. The presence of anti-SGPG antibodies in the serum of ALS patients should represent a diagnostic biomarker of ALS, and it could reflect the severity of the disease.

Pathogenic role of ganglioside metabolism in neurodegenerative diseases.

Ganglioside metabolism is altered in several neurodegenerative diseases, and this may participate in several events related to the pathogenesis of these diseases. Most changes occur in specific areas of the brain and their distinct membrane microdomains or lipid rafts. Antiganglioside antibodies may be involved in dysfunction of the blood-brain barrier and disease progression in these diseases. In lipid rafts, interactions of glycosphingolipids, including ganglioside, with proteins may be responsible for the misfolding events that cause the fibril and/or aggregate processing of disease-specific proteins, such as α-synuclein, in Parkinson's disease, huntingtin protein in Huntington's disease, and copper-zinc superoxide dismutase in amyotrophic lateral sclerosis. Targeting ganglioside metabolism may represent an underexploited opportunity to design novel therapeutic strategies for neurodegeneration in these diseases.

Effects of amyloid ß-peptides and gangliosides on mouse neural stem cells.

The interaction of amyloid ß-proteins (Aßs) with membrane lipids has been postulated as an early event in Aß fibril formation in Alzheimer's disease. We evaluated the effects of several putative bioactive Aßs and gangliosides on neural stem cells (NSCs) isolated from embryonic mouse brains or the subventricular zone of adult mouse brains. Incubation of the isolated NSCs with soluble Aß1-40 alone did not cause any change in the number of NSCs, but soluble Aß1-42 increased their number. Aggregated Aß1-40 and Aß1-42 increased the number of NSCs but soluble and aggregated Aß25-35 decreased the number. Soluble Aß1-40 and Aß1-42 did not affect the number of apoptotic cells but aggregated Aß1-40 and Aß1-42 did. When NSCs were treated with a combination of GM1 or GD3 and soluble Aß1-42, cell proliferation was enhanced, indicating that both GM1 and GD3 as well as Aßs are involved in promoting cell proliferation and survival of NSCs. These observations suggest the potential of beneficial effects of using gangliosides and Aßs for promoting NSC proliferation.

Anti-Chol-1 antigen, GQ1bα, antibodies are associated with Alzheimer's disease.

The interaction of amyloid ß-proteins (Aß) with membrane gangliosides has been reported to be an early event in Aß fibril formation in Alzheimer's disease (AD). Neuronal degeneration in AD has been postulated to be associated with the presence of anti-ganglioside antibodies in patient sera. Using an enzyme-linked immunosorbent assay (ELISA) and high-performance thin-layer chromatography (HPTLC) immunostaining, sera from 27 individuals (10 with AD, 6 with vascular dementia (VD), and 11 non-demented age-matched pathological controls) were examined in order to detect anti-glycosphingolipid (GSL) antibodies, including anti-cholinergic-specific antigen (Chol-1α; GQ1bα) antibodies. All sera had natural antibodies against ganglio-N-tetraosyl gangliosides (brain-type gangliosides). However, sera of demented patients with AD and VD had significantly higher titers of anti-GSL antibodies than those in age-matched pathological controls. Although most serum antibodies, including anti- GM1, -GT1b, -GQ1b, -GQ1bα, were of the IgM type, the presence of the IgG type antibodies was also significantly elevated in the sera of demented patients with AD. Anti-GT1b antibodies of the IgG type were elevated in AD (90%, 9 of 10 cases) and VD (100%), respectively. Most surprisingly, anti-GQ1bα antibodies (IgM) were found in 90% (9/10) and 100% (6/6) in the sera of patients with AD and VD, respectively. Since GQ1bα is present in the cerebral cortex and hippocampus, the presence of anti-GQ1bα antibodies may play an important role in disrupting cholinergic synaptic transmission and may participate in the pathogenesis of dementia. We conclude that elevated anti-GSL antibody titers may be useful as an aid for clinical diagnosis of those dementias.

Brain gangliosides of a transgenic mouse model of Alzheimer's disease with deficiency in GD3-synthase: expression of elevated levels of a cholinergic-specific ganglioside, GT1aα.

In order to examine the potential involvement of gangliosides in AD (Alzheimer's disease), we compared the ganglioside compositions of the brains of a double-transgenic (Tg) mouse model [APP (amyloid precursor protein)/PSEN1 (presenilin)] of AD and a triple mutant mouse model with an additional deletion of the GD3S (GD3-synthase) gene (APP/PSEN1/GD3S(-/-)). These animals were chosen since it was previously reported that APP/PSEN1/GD3S(-/-) triple-mutant mice performed as well as WT (wild-type) control and GD3S(-/-) mice on a number of reference memory tasks. Cholinergic neuron-specific gangliosides, such as GT1aα and GQ1bα, were elevated in the brains of double-Tg mice (APP/PSEN1), as compared with those of WT mice. Remarkably, in the triple mutant mouse brains (APP/PSEN1/GD3S(-/-)), the concentration of GT1aα was elevated and as expected there was no expression of GQ1bα. On the other hand, the level of c-series gangliosides, including GT3, was significantly reduced in the double-Tg mouse brain as compared with the WT. Thus, the disruption of the gene of a specific ganglioside-synthase, GD3S, altered the expression of cholinergic neuron-specific gangliosides. Our data thus suggest the intriguing possibility that the elevated cholinergic-specific ganglioside, GT1aα, in the triple mutant mouse brains (APP/PSEN1/GD3S(-/-)) may contribute to the memory retention in these mice.

Functional roles of gangliosides in neurodevelopment: an overview of recent advances.

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nervous system. They are localized primarily in the outer leaflets of plasma membranes and participated in cell-cell recognition, adhesion, and signal transduction and are integral components of cell surface microdomains or lipid rafts along with proteins, sphingomyelin and cholesterol. Ganglioside-rich lipid rafts play an important role in signaling events affecting neural development and the pathogenesis of certain diseases. Disruption of gangloside synthase genes in mice induces developmental defects and neural degeneration. Targeting ganglioside metabolism may represent a novel therapeutic strategy for intervention in certain diseases. In this review, we focus on recent advances on metabolic and functional studies of gangliosides in normal brain development and in certain neurological disorders.

Structures, biosynthesis, and functions of gangliosides--an overview.

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nervous system. Heterogeneity and diversity of the structures in their carbohydrate chains are characteristic hallmarks of these lipids; so far, 188 gangliosides with different carbohydrate structures have been identified in vertebrates. The molecular structural complexity increases manifold if one considers heterogeneity in the lipophilic components. The expression levels and patterns of brain gangliosides are known to change drastically during development. In cells, gangliosides are primarily, but not exclusively, localized in the outer leaflets of plasma membranes and are integral components of cell surface microdomains with sphingomyelin and cholesterol from which they participate in cell-cell recognition, adhesion, and signal transduction. In this brief review, we discuss the structures, metabolism and functions of gangliosides.

The role of sulfoglucuronosyl glycosphingolipids in the pathogenesis of monoclonal IgM paraproteinemia and peripheral neuropathy.

In IgM paraproteinemia and peripheral neuropathy, IgM M-protein secretion by B cells leads to a T helper cell response, suggesting that it is antibody-mediated autoimmune disease involving carbohydrate epitopes in myelin sheaths. An immune response against sulfoglucuronosyl glycosphingolipids (SGGLs) is presumed to participate in demyelination or axonal degeneration in the peripheral nervous system (PNS). SGGLs contain a 3-sulfoglucuronic acid residue that interacts with anti-myelin-associated glycoprotein (MAG) and the monoclonal antibody anti-HNK-1. Immunization of animals with sulfoglucuronosyl paragloboside (SGPG) induced anti-SGPG antibodies and sensory neuropathy, which closely resembles the human disease. These animal models might help to understand the disease mechanism and lead to more specific therapeutic strategies. In an in vitro study, destruction or malfunction of the blood-nerve barrier (BNB) was found, resulting in the leakage of circulating antibodies into the PNS parenchyma, which may be considered as the initial key step for development of disease.

The pathological roles of ganglioside metabolism in Alzheimer's disease: effects of gangliosides on neurogenesis.

Conversion of the soluble, nontoxic amyloid ß-protein (Aß) into an aggregated, toxic form rich in ß-sheets is a key step in the onset of Alzheimer's disease (AD). It has been suggested that Aß induces changes in neuronal membrane fluidity as a result of its interactions with membrane components such as cholesterol, phospholipids, and gangliosides. Gangliosides are known to bind Aß. A complex of GM1 and Aß, termed "GAß", has been identified in AD brains. Abnormal ganglioside metabolism also may occur in AD brains. We have reported an increase of Chol-1α antigens, GQ1bα and GT1aα, in the brain of transgenic mouse AD model. GQ1bα and GT1aα exhibit high affinities to Aßs. The presence of Chol-1α gangliosides represents evidence for genesis of cholinergic neurons in AD brains. We evaluated the effects of GM1 and Aß1-40 on mouse neuroepithelial cells. Treatment of these cells simultaneously with GM1 and Aß1-40 caused a significant reduction of cell number, suggesting that Aß1-40 and GM1 cooperatively exert a cytotoxic effect on neuroepithelial cells. An understanding of the mechanism on the interaction of GM1 and Aßs in AD may contribute to the development of new neuroregenerative therapies for this disorder.

Histone acetylation-mediated glycosyltransferase gene regulation in mouse brain during development.

Gangliosides are sialic acid-containing glycosphingolipids abundant in the central nervous tissues. The quantity and expression pattern of gangliosides in brain change drastically during early development and are mainly regulated through stage-specific expression of glycosyltransferase (ganglioside synthase) genes. It is still unclear, however, how the transcriptional activation of glycosyltransferase genes is regulated during development. In this study, we investigated the epigenetic regulation of two key glycosyltransferases, N-acetylgalactosaminyltransferase I (GA2/GM2/GD2/GT2-synthase) and sialyltransferase II (GD3-synthase), in embryonic, postnatal, and adult mouse brains. Combined bisulfite restriction analysis assay showed that DNA methylation in the 5' regions of these glycosyltransferase genes was not associated with their expression patterns. On the other hand, chromatin immunoprecipitation assay of both glycosyltransferase genes showed that their histone H3 acetylation was highly correlated to their mRNA expression levels during development. In fact, we confirmed that the expression patterns of gangliosides and glycosyltransferases in neuroepithelial cells were changed after treatment with a histone deacetylase inhibitor, sodium butyrate. Our studies provide the first evidence that efficient histone acetylation of the glycosyltransferase genes in mouse brain contributes to the developmental alteration of ganglioside expression.

Ganglioside metabolism in a transgenic mouse model of Alzheimer's disease: expression of Chol-1α antigens in the brain.

The accumulation of Aß (amyloid ß-protein) is one of the major pathological hallmarks in AD (Alzheimer's disease). Gangliosides, sialic acid-containing glycosphingolipids enriched in the nervous system and frequently used as biomarkers associated with the biochemical pathology of neurological disorders, have been suggested to be involved in the initial aggregation of Aß. In the present study, we have examined ganglioside metabolism in the brain of a double-Tg (transgenic) mouse model of AD that co-expresses mouse/human chimaeric APP (amyloid precursor protein) with the Swedish mutation and human presenilin-1 with a deletion of exon 9. Although accumulation of Aß was confirmed in the double-Tg mouse brains and sera, no statistically significant change was detected in the concentration and composition of major ganglio-N-tetraosyl-series gangliosides in the double-Tg brain. Most interestingly, Chol-1α antigens (cholinergic neuron-specific gangliosides), such as GT1aα and GQ1bα, which are minor species in the brain, were found to be increased in the double-Tg mouse brain. We interpret that the occurrence of these gangliosides may represent evidence for generation of cholinergic neurons in the AD brain, as a result of compensatory neurogenesis activated by the presence of Aß.

HNK-1 epitope-carrying tenascin-C spliced variant regulates the proliferation of mouse embryonic neural stem cells.

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into neuronal and glial cells. NSCs are the source for neurogenesis during central nervous system development from fetal and adult stages. Although the human natural killer-1 (HNK-1) carbohydrate epitope is expressed predominantly in the nervous system and involved in intercellular adhesion, cell migration, and synaptic plasticity, the expression patterns and functional roles of HNK-1-containing glycoconjugates in NSCs have not been fully recognized. We found that HNK-1 was expressed in embryonic mouse NSCs and that this expression was lost during the process of differentiation. Based on proteomics analysis, it was revealed that the HNK-1 epitopes were almost exclusively displayed on an extracellular matrix protein, tenascin-C (TNC), in the mouse embryonic NSCs. Furthermore, the HNK-1 epitope was found to be present only on the largest isoform of the TNC molecules. In addition, the expression of HNK-1 was dependent on expression of the largest TNC variant but not by enzymes involved in the biosynthesis of HNK-1. By knocking down HNK-1 sulfotransferase or TNC by small interfering RNA, we further demonstrated that HNK-1 on TNC was involved in the proliferation of NSCs via modulation of the expression level of the epidermal growth factor receptor. Our finding provides insights into the function of HNK-1 carbohydrate epitopes in NSCs to maintain stemness during neural development.

Production and characterization of monoclonal antibodies specific to lactotriaosylceramide.

We have established hybridoma cell lines producing monoclonal antibodies (mAbs) directed to N-acetylglucosaminylß1-3galactose (GlcNAcß1-3Gal) residue by immunizing BALB/c mice with lactotriaosylceramide (Lc(3)Cer). These obtained hybridoma cells, specific to Lc(3)Cer, were dual immunoglobulin (Ig)-producing cells which secreted both IgM and IgG molecules as antibodies. The established mAbs are able to react with not only Lc(3)Cer but also GlcNAcß1-3-terminal glycosphingolipids (GSLs) despite branching or lactosamine chain lengths and human transferrin with terminal GlcNAc residues. Comparison of the variable regions of the cloned IgM and IgG by reversed transcription-polymerase chain reaction analysis confirmed that the variable regions determine the specificity, the other amino acids are conserved, and these mAbs are encoded by J558 and Vκ-21family genes. Furthermore, we have analyzed the expression of GSLs with GlcNAcß1-3 epitope in acute leukemia cell lines and mouse fetal tissues using these mAbs, in which antigens were distributed comparatively. These mAbs are useful for studying the precise distribution of GlcNAcß1-3Gal-terminating GSL expression in tissues as well as for detecting GSLs carrying terminal GlcNAcß1-3Gal carbohydrate structure.

Role of proteoglycans and glycosaminoglycans in the pathogenesis of Alzheimer's disease and related disorders: amyloidogenesis and therapeutic strategies--a review.

The extracellular accumulation of amyloid beta proteins (Abetas) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). The binding of Abetas to extracellular membranes (ECMs) is a critical step in developing AD. Abetas bind to many biomolecules, including lipids, proteins, and proteoglycans (PGs). PGs play several roles in amyloid formation, including promoting the aggregation of Abetas into insoluble amyloid fibrils, which contributes to the increased neurotoxicity of Abetas. Although Abetas readily self-aggregate to form amyloid fibrils in vitro, their binding to PGs and heparin enhances amyloid aggregation and fibril formation. The sulfate moiety in glycosaminoglycans (GAGs), the carbohydrate portion of PGs, is necessary for the formation of amyloid fibrils; no fibrils are observed in the presence of hyaluronic acid (HA), a nonsulfated GAG. PGs and Abetas are known to colocalize in senile plaques (SPs) and neurofibrillary tangles (NFTs) in the AD brain. The binding site of PGs to Abetas has been identified in the 13-16-amino-acid region (His-His-Gln-Lys) of Abetas and represents a unique target site for inhibition of amyloid fibril formation; His13 in particular is an important residue critical for interaction with GAGs. The sulfate moieties of GAGs play a critical role in the binding to Abetas and enhance Abeta fibril formation. Low-molecular-weight heparins (LMWHs) can reverse the process of amyloidosis to inhibit fibril formation by blocking the formation of beta-plated structures, suggesting a possible therapeutic approach using LMWHs to interfere with the interaction between PGs and Abetas and to arrest or prevent amyloidogenesis.