In the present paper, we report on the properties of sphingolipid-enriched domains of rat cerebellar granule cells in culture at different stages of neuronal development. The major lipid components of these domains were glycerophospholipids and cholesterol. Glycerophospholipids were 45-75% and cholesterol 15-45% of total lipids of the domains. This corresponded to 5-17% of total cell glycerophospholipids and 15-45% of total cell cholesterol. Phosphatidylcholine, mainly dipalmitoylphosphatidylcholine, was 66 -85% of all the glycerophospholipids associated with these domains. Consequently, the palmitoyl residue was significantly enriched in the domains. The surface occupied by these structures increased during development. 40 -70% of cell sphingolipids segregated in sphingolipid-enriched membrane domains, with the maximum ganglioside density in fully differentiated neurons. A high content of ceramide was found in the domains of aging neurons. Then, the sphingolipid/glycerophospholipid molar ratio was more than doubled during the initial stage of development, whereas the cholesterol/glycerophospholipid molar ratio gradually decreased during in vitro differentiation. Phosphorylated phosphoinositides, which were scant in the domains of undifferentiated cells, dramatically increased during differentiation and aging in culture. Proteins were minor components of the domains (0.1-2.8% of all domain components). Phosphotyrosine-containing proteins were selectively recovered in the sphingolipid-enriched domain. Among these, Src family protein-tyrosine kinases, known to participate to the process of neuronal differentiation, were associated with the sphingolipid-enriched domains in a way specific for the type of kinase and for the developmental stage of the cell. Proteins belonging to other signaling pathways, such as phosphoinositide 3-kinase and its downstream target, Akt, were not associated with the domains.

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-3 H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-3 H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-3 H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions. Glycosphingolipids (GSLs)1 expressed at the cell surface are well known as modulators of several aspects of signal transduction processes involved in the control of cell proliferation, survival, and differentiation (1). GSLs in the plasma membrane are able to interact laterally with other membrane molecules modulating their properties (cis-interactions). Lipid rafts or membrane microdomains result from dynamic clustering of sphingolipids and cholesterol to form the so-called sphingolipid-enriched domain (SED) or lipid rafts (2). These structures move within the fluid bilayer and function as platforms for the attachment of proteins when membranes are moved around the cell and during signal transduction (3, 4). In addition, the expression pattern of GSLs in several cells and tissues undergoes deep changes during development and neoplastic transformation (5). These events are usually characterized by dramatic changes in cell recognition, suggesting that GSLs as cell surface antigens could play relevant roles as receptor sites in cell-cell recognition (trans-interaction). The receptor role of GSLs has been hypothesized in the case of microbial infections based on their ability to interact with bacterial toxins and microbial lectins (6, 7). Conformational analysis confirms that the orientation of the oligosaccharide chains of glycolipids at the cell surface complies wit...

The central nervous system is characterized by a high content of sphingolipids and by a high diversity in terms of different structures. Stage- and cell-specific sphingolipid metabolism and expression are crucial for brain development and maintenance toward adult age. On the other hand, deep dysregulation of sphingolipid metabolism, leading to altered sphingolipid pattern, is associated with the majority of neurological and neurodegenerative diseases, even those totally lacking a common etiological background. Thus, sphingolipid metabolism has always been regarded as a promising pharmacological target for the treatment of brain disorders. However, any therapeutic hypothesis applied to complex amphipathic sphingolipids, components of cellular membranes, has so far failed probably because of the high regional complexity and specificity of the different biological roles of these structures. Simpler sphingosine-based lipids, including ceramide and sphingosine 1-phosphate, are important regulators of brain homeostasis, and, thanks to the relative simplicity of their metabolic network, they seem a feasible druggable target for the treatment of brain diseases. The enzymes involved in the control of the levels of bioactive sphingoids, as well as the receptors engaged by these molecules, have increasingly allured pharmacologists and clinicians, and eventually fingolimod, a functional antagonist of sphingosine 1-phosphate receptors with immunomodulatory properties, was approved for the therapy of relapsing–remitting multiple sclerosis. Considering the importance of neuroinflammation in many other brain diseases, we would expect an extension of the use of such analogs for the treatment of other ailments in the future. Nevertheless, many aspects other than neuroinflammation are regulated by bioactive sphingoids in healthy brain and dysregulated in brain disease. In this review, we are addressing the multifaceted possibility to address the metabolism and biology of bioactive sphingosine 1-phosphate as novel targets for the development of therapeutic paradigms and the discovery of new drugs.

Sphingolipids are polar membrane lipids present as minor components in eukaryotic cell membranes. Sphingolipids are highly enriched in nervous cells, where they exert important biological functions. They deeply affect the structural and geometrical properties and the lateral order of cellular membranes, modulate the function of several membrane-associated proteins, and give rise to important intra- and extracellular lipid mediators. Sphingolipid metabolism is regulated along the differentiation and development of the nervous system, and the expression of a peculiar spatially and temporarily regulated sphingolipid pattern is essential for the maintenance of the functional integrity of the nervous system: sphingolipids in the nervous system participate to several signaling pathways controlling neuronal survival, migration, and differentiation, responsiveness to trophic factors, synaptic stability and synaptic transmission, and neuron-glia interactions, including the formation and stability of central and peripheral myelin. In several neurodegenerative diseases, sphingolipid metabolism is deeply deregulated, leading to the expression of abnormal sphingolipid patterns and altered membrane organization that participate to several events related to the pathogenesis of these diseases. The most impressive consequence of this deregulation is represented by anomalous sphingolipid-protein interactions that are at least, in part, responsible for the misfolding events that cause the fibrillogenic and amyloidogenic processing of disease-specific protein isoforms, such as amyloid beta peptide in Alzheimer's disease, huntingtin in Huntington's disease, alpha-synuclein in Parkinson's disease, and prions in transmissible encephalopathies. Targeting sphingolipid metabolism represents today an underexploited but realistic opportunity to design novel therapeutic strategies for the intervention in these diseases.