Sphingolipids: Less Enigmatic but Still Many Questions about the Role(s) of Ceramide in the Synthesis/Function of the Ganglioside Class of Glycosphingolipids.

While much has been learned about sphingolipids, originally named for their sphinx-like enigmatic properties, there are still many unanswered questions about the possible effect(s) of the composition of ceramide on the synthesis and/or behavior of a glycosphingolipid (GSL). Over time, studies of their ceramide component, the sphingoid base containing the lipid moiety of GSLs, were frequently distinct from those performed to ascertain the roles of the carbohydrate moieties. Due to the number of classes of GSLs that can be derived from ceramide, this review focuses on the possible role(s) of ceramide in the synthesis/function of just one GSL class, derived from glucosylceramide (Glc-Cer), namely sialylated ganglio derivatives, initially characterized and named gangliosides (GGs) due to their presence in ganglion cells. While much is known about their synthesis and function, much is still being learned. For example, it is only within the last 15-20 years or so that the mechanism by which the fatty acyl component of ceramide affected its transport to different sites in the Golgi, where it is used for the synthesis of Glu- or galactosyl-Cer (Gal-Cer) and more complex GSLs, was defined. Still to be fully addressed are questions such as (1) whether ceramide composition affects the transport of partially glycosylated GSLs to sites where their carbohydrate chain can be elongated or affects the activity of glycosyl transferases catalyzing that elongation; (2) what controls the differences seen in the ceramide composition of GGs that have identical carbohydrate compositions but vary in that of their ceramide and vice versa; (3) how alterations in ceramide composition affect the function of membrane GGs; and (4) how this knowledge might be applied to the development of therapies for treating diseases that correlate with abnormal expression of GGs. The availability of an updatable data bank of complete structures for individual classes of GSLs found in normal tissues as well as those associated with disease would facilitate research in this area.

The Ying and Yang of Ganglioside Function in Cancer.

The plethora of information about the expression of cancer cell-associated gangliosides, their role(s) in signal transduction, and their potential usefulness in the development of cancer treatments makes this an appropriate time to review these enigmatic glycosphingolipids. Evidence, reflecting the work of many, indicates that (1) expression of specific gangliosides, not generally found in high concentrations in most normal human cells, can be linked to certain types of cancer. (2) Gangliosides can affect the ability of cells to interact either directly or indirectly with growth factor receptors, thereby changing such things as a cell's mobility, rate of proliferation, and metastatic ability. (3) Anti-ganglioside antibodies have been tested, with some success, as potential treatments for certain cancers. (4) Cancer-associated gangliosides shed into the circulation can (a) affect immune cell responsiveness either positively or negatively, (b) be considered as diagnostic markers, and (c) be used to look for recurrence. (5) Cancer registries enable investigators to evaluate data from sufficient numbers of patients to obtain information about potential therapies. Despite advances that have been made, a discussion of possible approaches to identifying additional treatment strategies to inhibit metastasis, responsible for the majority of deaths of cancer patients, as well as for treating therapy-resistant tumors, is included.

Carbohydrates: Binding Sites and Potential Drug Targets for Neural-Affecting Pathogens.

A number of viruses that have caused wide spread concern e.g. Ebola, Zika, and SARS-CoV2 (severe acute respiratory syndrome coronavirus 2 also known as COVID 19) have at various times, become newsworthy as a result of being newly discovered, mutations enabling them to more efficiently infect humans or modern modes of transportation moving them to areas with naive, susceptible populations. As more is learned about the mechanisms whereby these pathogens enter human cells it has become increasingly evident that carbohydrates expressed on the surface of either target cells or the pathogens themselves are essential. Variability in carbohydrate structures as well as the presence of carbohydrate binding receptors (lectins) provides a plethora of potential binding interactions by which infection of cells can occur. Identification of specific lipid- or protein-associated carbohydrates essential for infection provides support for research being done to develop carbohydrate related inhibitors of those interactions. This chapter (1) discusses scenarios for how carbohydrates affect the ability of specific infectious agents to interact with neural cells, (2) gives examples of problems that may result from development of antibodies to carbohydrate antigens found on pathogens that are similar to epitopes expressed on mammalian cells, and (3) provides examples of approaches either in use or under consideration for translational uses of this information.

Association Between Iron and Cholesterol in Neuroblastomas.

BACKGROUND/AIM: Neuroblastoma is the most common childhood extracranial solid malignancy. Although cancer cells need iron and lipids for active cell division, possible links between iron and lipid metabolism in neuroblastomas have not been studied. MATERIALS AND METHODS: We evaluated the levels and association between iron and cholesterol on in vitro neuroblastoma cancer models. RESULTS: We found that the levels of iron and cholesterol are diverse among neuroblastoma cell lines. There is a bi-directional association between iron and cholesterol in drug-resistant neuroblastoma SK-N-AS cells. In drug-resistant neuroblastoma cells, low concentration of an iron chelator did not have an impact on iron levels, but on cellular cholesterol levels. Furthermore, a cholesterol decreasing agent, simvastatin, influenced both iron and cholesterol levels in drug-resistant neuroblastoma cells. CONCLUSION: Cholesterol decreasing agents may be more effective than iron chelators for drug-resistant neuroblastoma treatment.

Gangliosides and Neuroblastomas.

The focus of this review is the ganglio-series of glycosphingolipids found in neuroblastoma (NB) and the myriad of unanswered questions associated with their possible role(s) in this cancer. NB is one of the more common solid malignancies of children. Five-year survival for those diagnosed with low risk NB is 90-95%, while that for children with high-risk NB is around 40-50%. Much of the survival rate reflects age of diagnosis with children under a year having a much better prognosis than those over two. Identification of expression of GD2 on the surface of most NB cells led to studies of the effectiveness and subsequent approval of anti-GD2 antibodies as a treatment modality. Despite much success, a subset of patients, possibly those whose tumors fail to express concentrations of gangliosides such as GD1b and GT1b found in tumors from patients with a good prognosis, have tumors refractory to treatment. These observations support discussion of what is known about control of ganglioside synthesis, and their actual functions in NB, as well as their possible relationship to treatment response.

Gangliosides: glycosphingolipids essential for normal neural development and function.

Lipid rafts, sites of signal transduction, are enriched in glycosphingolipids (GSLs). Gangliosides, a class of GSLs found in greatest concentration in the grey matter of the brain, can affect neuronal function by modulating cell signaling. This review summarizes changes in ganglioside expression during brain development, the specific effects they induce, and makes observations about their possible role(s) in dementing diseases. Given that the average lifespan of individuals in many countries has increased, and that aging is accompanied by an increasing probability of dementia, understanding how changes in the GSL composition of lipid rafts may contribute to the cell biological basis of a specific dementing phenotype is an important area of study.

Roles of carbohydrates in the interaction of pathogens with neural cells.

Numerous pathogens that can affect neural function utilize oligosaccharide-protein interactions as a first step in the infection process. The variability in carbohydrate structures as well as the presence of carbohydrate binding receptors on the surface of cells provides a plethora of potential binding sites for viruses, bacteria, and bacterial toxins. This chapter discusses scenarios for how carbohydrates may affect the ability of infectious agents to interact with neural cells, provides examples of problems that may result from development of antibodies to carbohydrate antigens found on pathogens that are similar to epitopes expressed on mammalian cells, and presents approaches either in use or under consideration for translational uses of this information.

HFE gene variants, iron, and lipids: a novel connection in Alzheimer's disease.

Iron accumulation and associated oxidative stress in the brain have been consistently found in several neurodegenerative diseases. Multiple genetic studies have been undertaken to try to identify a cause of neurodegenerative diseases but direct connections have been rare. In the iron field, variants in the HFE gene that give rise to a protein involved in cellular iron regulation, are associated with iron accumulation in multiple organs including the brain. There is also substantial epidemiological, genetic, and molecular evidence of disruption of cholesterol homeostasis in several neurodegenerative diseases, in particular Alzheimer's disease (AD). Despite the efforts that have been made to identify factors that can trigger the pathological events associated with neurodegenerative diseases they remain mostly unknown. Because molecular phenotypes such as oxidative stress, synaptic failure, neuronal loss, and cognitive decline, characteristics associated with AD, have been shown to result from disruption of a number of pathways, one can easily argue that the phenotype seen may not arise from a linear sequence of events. Therefore, a multi-targeted approach is needed to understand a complex disorder like AD. This can be achieved only when knowledge about interactions between the different pathways and the potential influence of environmental factors on them becomes available. Toward this end, this review discusses what is known about the roles and interactions of iron and cholesterol in neurodegenerative diseases. It highlights the effects of gene variants of HFE (H63D- and C282Y-HFE) on iron and cholesterol metabolism and how they may contribute to understanding the etiology of complex neurodegenerative diseases.

H63D mutation in hemochromatosis alters cholesterol metabolism and induces memory impairment.

The H63D variant of the hemochromatosis (HFE) gene, when expressed in carriers of the apolipoprotein E4 allele, is implicated as a risk factor for earlier onset of Alzheimer's disease (AD). We tested the hypothesis that like expression of apolipoprotein E4, expression of H63D-HFE disrupts cholesterol metabolism contributing to an increase in neurodegeneration and memory deficits. Analysis of SH-SY5Y human neuroblastoma cells transfected to stably express either wild type- (WT) or H63D-HFE indicated about a 50% reduction in cholesterol content in cells expressing H63D-HFE. This was accompanied by a significant decrease in expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase, and a significant increase in expression of cholesterol 24-hydroxylase. Consistent with these studies, H67D-HFE (orthologous to human H63D-HFE) knock-in mice, showed a greater age dependent decline in brain cholesterol than WT-HFE animals and changes in expression of proteins regulating cholesterol metabolism. Brains of aged H67D-HFE mice also exhibited a significant decrease in expression of synapse proteins and a significant increase in caspase-3 expression relative to WT-HFE controls. H67D-HFE mice also had a greater reduction in brain volume and poorer recognition and spatial memory than WT-HFE mice, symptoms associated with AD. These results indicate that the alterations in cholesterol metabolism associated with expression of H63D-HFE may contribute to the development of AD.

Cholesterol, GM1, and autism.

Disruption of cholesterol metabolism has been hypothesized to contribute to dementia, possibly due to its role in maintaining membrane fluidity as well as the integrity of lipid rafts. Previously, we reported an apparent inverse relationship between membrane cholesterol levels and those of GM1, another lipid that can be found in rafts. This paper describes the observation that red blood cell (RBC) membranes isolated from blood drawn from children diagnosed with autism have on the average significantly less cholesterol and significantly more GM1 than RBC membranes isolated from blood obtained from control children. While cholesterol in the circulation does not cross the blood brain barrier, a generalized defect in its synthesis could affect its concentration in the central nervous system and that, coupled with a change in ganglioside expression, could contribute to development of the behaviors associated with autism.

Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1.

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3'-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC(50)s ranging from 0.1 to 7.4 µg/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions.

Lipid rafts: keys to neurodegeneration.

The increase in life expectancy seen in many countries has been accompanied by an increase in the number of people living with dementia and a growing need for health care. The large number of affected individuals emphasizes the need to identify causes for the phenotypes associated with diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, Huntington's, and those caused by prions. This review addresses the hypothesis that changes in lipid rafts induced by alterations in their ganglioside and/or cholesterol content or the interaction of mutant proteins with them provide the keys to understanding the onset of neurodegeneration that can lead to dementia. The biological function(s) of raft-associated gangliosides and cholesterol are discussed prior to reviewing what is known about their roles in lipid rafts in the aforementioned diseases. It concludes with some questions that need to be addressed in order to provide investigators with the basis for identifying small molecule agonists or antagonists to test as potential therapeutics.

Surface plasmon resonance analysis of ricin binding to plasma membranes isolated from NIH 3T3 cells.

As the potential for bioterrorism has appeared to increase, the need for simple systems for identifying potential inhibitors of the binding of such biological agents to cell membranes has increased. In this work, surface plasmon resonance (SPR) was used to monitor binding of ricin, a ribosome-inactivating protein, to the plasma membranes of NIH 3T3 cells. Once conditions were established, efficacy of the system for monitoring effectiveness of compounds at inhibiting ricin binding was ascertained by determining the IC(50) values for asialofetuin (ASF) and for bovine serum albumin derivatized with an average of 34 lactosyl moieties (BSA-Lac(34)). Results indicated that SPR is an efficient method for measuring adherence of a toxin to isolated cell plasma membranes. SPR can also indicate whether a compound that is an effective inhibitor of binding when a single ligand such as ASF is used will be as effective when used in studies with cells that may express multiple cell surface ligands for ricin and/or the inhibitor.

Membrane raft disruption promotes axonogenesis in n2a neuroblastoma cells.

Membrane rafts are discrete microdomains found in cell membranes that contain cholesterol and glycosphingolipids such as gangliosides. As cholesterol is a major component of membrane rafts, its sequestration by the polyene filipin can be used to disrupt them. In previous work we observed that membrane raft disruption by filipin treatment of murine neuroblastoma N2a cells led to changes in expression of cell processes. In this study, we determined the type of process formation induced by filipin treatment as well as whether their expression was accompanied by changes in ganglioside content or subcellular distribution. The results indicate that the processes formed were axonal in nature and their expression was accompanied by changes in both ganglioside content as well as the subcellular localization of GM1.

Multivalent binding of ricin to bovine serum albumin-based neoglycoconjugates.

Ricin, a ribosome-inactivating protein from the plant Ricinus communis, is a heterodimeric protein. The A chain is a N-glycosidase and the B chain (RTB) is a lectin with two carbohydrate binding sites. RTB has been shown to bind asialofetuin with much greater affinity than it does galactose, supporting the hypothesis that it may exhibit multivalency. To test this, neoglycoconjugates were prepared and tested for their ability to function as ligands for ricin binding. Because the two carbohydrate binding sites on RTB are approximately 70A apart, bovine serum albumin (BSA) was used as the carbohydrate carrier. It was derivatized with either the oligosaccharide portion of asialo-GM1 or with lactose. These sugars were used because ricin was found to adhere more effectively to asialo-GM1 and LacCer immobilized on plastic than to the other glycosphingolipids tested. Results of binding studies done using surface plasmon resonance indicated that the RTB subunit of ricin exhibited a multivalent effect when it bound to the neoglycoconjugates.

Disruption of lipid rafts enhances activity of botulinum neurotoxin serotype A.

Botulinum neurotoxin serotype A (BoNT/A), one of seven serotypes of botulinum neurotoxin, is taken up by neurons of the peripheral nervous system. Within the neurons it catalyzes cleavage of the synaptosomal-associated protein having a mass of 25kDa, SNAP-25, thereby blocking neurotransmission. BoNT/A has been shown to interact with SV2, as well as gangliosides that are often found in lipid rafts. Lipid rafts are microdomains that can be found on the outer leaflet of the plasma membrane and are enriched in cholesterol and glycosphingolipids. To determine whether lipid rafts are needed for BoNT/A activity, those associated with the plasma membranes of murine N2a neuroblastoma cells were disrupted using either methyl-beta-cyclodextrin or filipin. Disruption of cholesterol-containing lipid rafts by either reagent did not prevent the action of BoNT/A on N2a cells, in fact activity was enhanced. While our results indicate that disruption of lipid rafts enhances BoNT/A activity, disruption of clathrin-dependent endocytosis appeared to be inhibitory.

Glycoconjugates: roles in neural diseases caused by exogenous pathogens.

Numerous reports indicate that lipid or protein associated carbohydrates are essential for infection of cells by various viruses, bacteria, or bacterial toxins, some of which affect the nervous system. Examples of such pathogens include tetanus and botulinum neurotoxin, Shiga and Shiga-like toxins, Borrelia burgdorferi, Mycobacterium leprae, and human immunodeficiency virus. This review discusses evidence indicating that carbohydrates are essential for these pathogens to induce their deleterious effects, the putative function of the carbohydrates, and how this knowledge might be used to combat the effects of the pathogen.

Biochemical analysis of human milk treated with sodium dodecyl sulfate, an alkyl sulfate microbicide that inactivates human immunodeficiency virus type 1.

Reduction of transmission of human immunodeficiency virus type 1 (HIV-1) through human milk is needed. Alkyl sulfates such as sodium dodecyl sulfate (SDS) are microbicidal against HIV-1 at low concentrations, have little to no toxicity, and are inexpensive. The authors have reported that treatment of HIV-1-infected human milk with < or = 1% (10 mg/mL) SDS for 10 minutes inactivates cell-free and cell-associated virus. The SDS can be removed with a commercially available resin after treatment without recovery of viral infectivity. In this article, the authors report results of selective biochemical analyses (ie, protein, immunoglobulins, lipids, cells, and electrolytes) of human milk subjected to SDS treatment and removal. The SDS treatment or removal had no significant effects on the milk components studied. Therefore, the use of alkyl sulfate microbicides to treat milk from HIV-1-positive women may be a simple, practical, and nutritionally sound way to prevent or reduce transmission of HIV-1 while still feeding with mother's own milk.

Inactivation of HIV-1 in breast milk by treatment with the alkyl sulfate microbicide sodium dodecyl sulfate (SDS).

BACKGROUND: Reducing transmission of HIV-1 through breast milk is needed to help decrease the burden of pediatric HIV/AIDS in society. We have previously reported that alkyl sulfates (i.e., sodium dodecyl sulfate, SDS) are microbicidal against HIV-1 at low concentrations, are biodegradable, have little/no toxicity and are inexpensive. Therefore, they may be used for treatment of HIV-1 infected breast milk. In this report, human milk was artificially infected by adding to it HIV-1 (cell-free or cell-associated) and treated with or=0.1%) was virucidal against cell-free and cell-associated HIV-1 in breast milk. SDS could be substantially removed from breast milk, without recovery of viral infectivity. Viral load in artificially infected milk was reduced to undetectable levels after treatment with 0.1% SDS. SDS was virucidal against HIV-1 in human milk and could be removed from breast milk if necessary. Milk was not infectious after SDS removal. CONCLUSION: The proposed treatment concentrations are within reported safe limits for ingestion of SDS by children of 1 g/kg/day. Therefore, use of alkyl sulfate microbicides, such as SDS, to treat HIV1-infected breast milk may be a novel alternative to help prevent/reduce transmission of HIV-1 through breastfeeding.

Comparison of glycosphingolipids and antibodies as receptor molecules for ricin detection.

Glycosphingolipids (GSLs) have been shown to undergo strong interactions with a number of protein toxins, including potential bioterrorism agents such as ricin and botulinum neurotoxin. Characterization of this interaction in recent years has led to a number of studies where GSLs were used as the recognition molecules for biosensing applications. Here, we offer a comparison of quartz crystal microbalance (QCM) sensors for the detection of ricin using antibodies and the GSLs GM1 and asialoGM1, which have been shown to undergo strong interactions with ricin. The presence, orientation, and activity of the GSL and antibody films were confirmed using ellipsometry, Fourier transform infrared spectroscopy (FT-IR), and QCM. It was found that the GSLs offered more sensitive detection limits when directly compared with antibodies. Both GSLs had lower detection limits at 5 microg/mL, approximately 5 times lower than were found for antibodies (25 microg/mL), and their linear detection range extended to the highest concentrations tested (100 microg/mL), almost an order of magnitude beyond the saturation point for the antibody sensors. Potential sites for nonspecific adsorption were blocked using serum albumin without sacrificing toxin specificity.