Search

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed at the apical plasma membrane (PM) of different epithelial cells. The most common mutation responsible for the onset of cystic fibrosis (CF), F508del, inhibits the biosynthesis and transport of the protein at PM, and also presents gating and stability defects of the membrane anion channel upon its rescue by the use of correctors and potentiators. This prompted a multiple drug strategy for F508delCFTR aimed simultaneously at its rescue, functional potentiation and PM stabilization. Since ganglioside GM1 is involved in the functional stabilization of transmembrane proteins, we investigated its role as an adjuvant to increase the effectiveness of CFTR modulators. According to our results, we found that GM1 resides in the same PM microenvironment as CFTR. In CF cells, the expression of the mutated channel is accompanied by a decrease in the PM GM1 content. Interestingly, by the exogenous administration of GM1, it becomes a component of the PM, reducing the destabilizing effect of the potentiator VX-770 on rescued CFTR protein expression/function and improving its stabilization. This evidence could represent a starting point for developing innovative therapeutic strategies based on the co-administration of GM1, correctors and potentiators, with the aim of improving F508del CFTR function.

Human primary bronchial epithelial cells differentiated in vitro represent a valuable tool to study lung diseases such as cystic fibrosis (CF), an inherited disorder caused by mutations in the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator. In CF, sphingolipids, a ubiquitous class of bioactive lipids mainly associated with the outer layer of the plasma membrane, seem to play a crucial role in the establishment of the severe lung complications. Nevertheless, no information on the involvement of sphingolipids and their metabolism in the differentiation of primary bronchial epithelial cells are available so far. Here we show that ceramide and globotriaosylceramide increased during cell differentiation, whereas glucosylceramide and gangliosides content decreased. In addition, we found that apical plasma membrane of differentiated bronchial cells is characterized by a higher content of sphingolipids in comparison to the other cell membranes and that activity of sphingolipids catabolic enzymes associated with this membrane results altered with respect to the total cell activities. In particular, the apical membrane of CF cells was characterized by high levels of ceramide and glucosylceramide, known to have proinflammatory activity. On this basis, our data further support the role of sphingolipids in the onset of CF lung pathology. Electronic supplementary material The online version of this article (10.1007/s10719-020-09935-x) contains supplementary material, which is available to authorized users.

We show that glioblastoma multiform (GBM) cells overexpressing the constitutively active form of the epidermal growth factor receptor [epidermal growth factor receptor variant III (EGFRvIII) and U87MG human GBM cell line overexpressing EGFRvIII (EGFR+) cells] possess greater invasive properties and have higher levels of extracellular sphingosine-1-phosphate (S1P) and increased sphingosine kinase-1 (SK1) activity than the empty vector-expressing cells. Notably, the inhibition of SK1 or S1P receptors decreases the invasiveness of EGFR+ cells. Moreover, EGFR and MEK1 inhibitors reduce both SK1 activation and cell invasion, suggesting that the enhanced invasiveness observed in the EGFR+ cells depends on the increased S1P secretion, downstream of the EGFRvIII-ERK-SK1-S1P pathway. Altogether, the results of the present study indicate that, in GBM cells, EGFRvIII is connected with the S1P signaling pathway to enhance cell invasiveness and tumor progression.

Human and mouse chronic lymphocytic leukemia (CLL) develops from CD5+ B cells that in mice and macaques are known to define the distinct B1a B-cell lineage. B1a cells are characterized by lack of germinal center (GC) development, and the B1a cell population is increased in mice with reduced GC formation. As a major mediator of follicular B-cell migration, the G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) directs B-cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B cells toward the extrafollicular area, whereas downregulation is essential for GC formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to CLL. Here, we demonstrate that B-cell-targeted expression of human EBI2 (hEBI2) in mice reduces GC-dependent immune responses, reduces total immunoglobulin M (IgM) and IgG levels, and leads to increased proliferation and upregulation of cellular oncogenes. Furthermore, hEBI2 overexpression leads to an abnormally expanded CD5+ B1a B-cell subset (present as early as 4 days after birth), late-onset lymphoid cancer development, and premature death. These findings are highly similar to those observed in CLL patients and identify EBI2 as a promoter of B-cell malignancies.

Lysosomal accumulation of undegraded materials is a common feature of lysosomal storage diseases, neurodegenerative disorders, and the aging process. To better understand the role of lysosomal storage in the onset of cell damage, we used human fibroblasts loaded with sucrose as a model of lysosomal accumulation. Sucrose-loaded fibroblasts displayed increased lysosomal biogenesis followed by arrested cell proliferation. Notably, we found that reduced lysosomal catabolism and autophagy impairment led to an increase in sphingolipids ( i.e., sphingomyelin, glucosylceramide, ceramide, and the gangliosides GM3 and GD3), at both intracellular and plasma membrane (PM) levels. In addition, we observed an increase in the lysosomal membrane protein Lamp-1 on the PM of sucrose-loaded fibroblasts and a greater release of the soluble lysosomal protein cathepsin D in their extracellular medium compared with controls. These results indicate increased fusion between lysosomes and the PM, as also suggested by the increased activity of lysosomal glycosphingolipid hydrolases on the PM of sucrose-loaded fibroblasts. The inhibition of β-glucocerebrosidase and nonlysosomal glucosylceramidase, both involved in ceramide production resulting from glycosphingolipid catabolism on the PM, partially restored cell proliferation. Our findings indicate the existence of a new molecular mechanism underlying cell damage triggered by lysosomal impairment.-Samarani, M., Loberto, N., Solda, G., Straniero, L., Asselta, R., Duga, S., Lunghi, G., Zucca, F. A., Mauri, L., Ciampa, M. G., Schiumarini, D., Bassi, R., Giussani, P., Chiricozzi, E., Prinetti, A., Aureli, M., Sonnino, S. A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

Sphingolipids (SLs) are amphiphilic molecules mainly associated with the external leaflet of eukaryotic plasma membrane, and are structural membrane components with key signaling properties. Since the beginning of the last century, a large number of papers described the involvement of these molecules in several aspects of cell physiology and pathology. Several lines of evidence support the critical role of SLs in inflammatory diseases, by acting as anti- or pro-inflammatory mediators. They are involved in control of leukocyte activation and migration, and are recognized as essential players in host response to pathogenic infection. We propose here a critical overview of current knowledge on involvement of different classes of SLs in inflammation, focusing on the role of simple and complex SLs in pathogen-mediated inflammatory response.

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p amp;lt; 0.01) Gb3 ganglioside (-50 +/- 3%) and sphingosine 1-phosphate (S1P, -40 +/- 4%), which ended up to reduction in cell motility (-46 +/- 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility. Funding Agencies|Lions Club Milano ai Cenacoli

Cystic fibrosis (CF) is the most common autosomal genetic recessive disease caused by mutations of gene encoding for the cystic fibrosis transmembrane conductance regulator. Patients with CF display a wide spectrum of symptoms, the most severe being chronic lung infection and inflammation, which lead to onset of cystic fibrosis lung disease. Several studies indicate that sphingolipids play a regulatory role in airway inflammation. The inhibition and downregulation of GBA2, the enzyme catabolizing glucosylceramide to ceramide, are associated with a significant reduction of IL-8 production in CF bronchial epithelial cells. Herein, we demonstrate that GBA2 plays a role in the proinflammatory state characterizing CF cells. We also report for the first time that Pseudomonas aeruginosa infection causes a recruitment of plasma membrane-associated glycosphingolipid hydrolases into lipid rafts of CuFi-1-infected cells. This reorganization of cell membrane may be responsible for activation of a signaling cascade, culminating in aberrant inflammatory response in CF bronchial epithelial cells upon bacterial infection. Taken together, the presented data further support the role of sphingolipids and their metabolic enzymes in controlling the inflammatory response in CF.

The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (˜66 kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitro sumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Nitroso-N-methylurea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.

The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested., Author Summary Live attenuated yellow fever vaccine (YF-17D) is an efficient and generally safe vaccine. Nevertheless, in recent years the reporting of serious adverse effects together with the given limitations in the use of this live vaccine in certain risk groups has spurred an interest in developing a more generally applicable and safer alternative. Using an adenovector platform and recombinant vaccines targeting both structural and non-structural YF antigens, we now demonstrate that non-replicating adenobased vaccines may be used to induce a state of host immunity, which like YF-17D vaccination encompasses both major arms of the adaptive immune system. Furthermore, in a murine challenge model, adenovector induced protection fully matched that induced by the current vaccine. Taken together our results strongly suggest that adenovectored vaccines targeting structural and non-structural viral antigens represent a viable and safe alternative to the existing live, attenuated YF vaccine.

We detected significant levels of β-glucosidase, β-galactosidase, sialidase Neu3 and sphingomyelinase activities associated with the plasma membrane of fibroblasts from normal and Niemann-Pick subjects and of cells from breast, ovary, colon and neuroblastoma tumors in culture. All of the cells subjected to ionizing radiations showed an increase of the activity of plasma membrane β-glucosidase, β-galactosidase and sialidase Neu3, in addition of the well known increase of activity of plasma membrane sphingomyelinase, under similar conditions. Human breast cancer cell line T47D was studied in detail. In these cells the increase of activity of β-glucosidase and β-galactosidase was parallel to the increase of irradiation dose up to 60 Gy and continued with time, at least up to 72 h from irradiation. β-glucosidase increased up to 17 times and β-galactosidase up to 40 times with respect to control. Sialidase Neu3 and sphingomyelinase increased about 2 times at a dose of 20 Gy but no further significant differences were observed with increase of radiation dose and time. After irradiation, we observed a reduction of cell proliferation, an increase of apoptotic cell death and an increase of plasma membrane ceramide up to 3 times, with respect to control cells. Tritiated GM3 ganglioside has been administered to T47D cells under conditions that prevented the lysosomal catabolism. GM3 became component of the plasma membranes and was transformed into LacCer, GlcCer and ceramide. The quantity of ceramide produced in irradiated cells was about two times that of control cells.

The activities of plasma membrane associated sialidase Neu3, total β-glucosidase, CBE-sensitive β-glucosidase, non-lysosomal β-glucosyl ceramidase GBA2, β-galactosidase, β-hexosaminidase and sphingomyelinase were determined at three different stages of differentiation of murine neural stem cell cultures, corresponding to precursors, commited progenitors, and differentiated cells. Cell immunostaining for specific markers of the differentiation process, performed after 7 days in culture in presence of differentiating agents, clearly showed the presence of oligodendrocytes, astrocytes and neurons. Glial cells were the most abundant. Sialidase Neu3 after a decrease from progenitors to precursors, showed an increase parallel to the differentiation process. All the other glycosidases increased their activity along differentiation. The activity of CBE-sensitive β-glucosidase and GBA2 were very similar at the precursor stage, but CBE-sensitive β-glucosidase increased 7 times while GBA2 only two in the differentiated cells. In addition, we analysed also sphingomyelinase as enzyme specifically associated to sphingolipids. The activity of this enzyme increased from precursors to differentiated cells.

Glioblastoma is a deadly cancer with intrinsic chemoresistance. Understanding this property will aid in therapy. Glucosylceramide synthase (GCS) is associated with resistance and poor outcome; little is known about glioblastomas. In glioblastoma cells, temozolomide and paclitaxel induce ceramide increase, which in turn promotes cytotoxicity. In drug-resistant cells, both drugs are unable to accumulate ceramide, increased expression and activity of GCS is present, and its inhibitors hinder resistance. Resistant cells exhibit cross-resistance, despite differing in marker expression, and cytotoxic mechanism. These findings suggest that GCS protects glioblastoma cells against autophagic and apoptotic death, and contributes to cell survival under chemotherapy.

Different lines of evidence indicate that both aberrant activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt survival pathway and down-regulation of the death mediator ceramide play a critical role in the aggressive behavior, apoptosis resistance, and adverse clinical outcome of glioblastoma multiforme. Furthermore, the inhibition of the PI3K/Akt pathway and the up-regulation of ceramide have been found functional to the activity of many cytotoxic treatments against glioma cell lines and glioblastomas as well. A reciprocal control between PI3K/Akt and ceramide signaling in glioma cell survival/death is suggested by data demonstrating a protective role of PI3K/Akt on ceramide-induced cell death in glial cells. In this study we investigated the role of the PI3K/Akt pathway in the regulation of the ceramide metabolism in C6 glioma cells, a cell line in which the PI3K/Akt pathway is constitutively activated. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids and microscopy studies with fluorescent ceramides demonstrated that the chemical inhibition of PI3K and the transfection with a dominant negative Akt strongly inhibited ceramide utilization for the biosynthesis of complex sphingolipids by controlling the endoplasmic reticulum (ER) to Golgi vesicular transport of ceramide. These findings constitute the first evidence for a PI3K/Akt-dependent regulation of vesicle-mediated movements of ceramide in the ER-Golgi district. Moreover, the findings also suggest the activation of the PI3K/Akt pathway as crucial to coordinate the biosynthesis of membrane complex sphingolipids with cell proliferation and growth and/or to maintain low ceramide levels, especially as concerns those treatments that promote ceramide biosynthesis in the ER.

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.

The mitogenic role of sphingosine-1-phosphate (S1P) and its involvement in basic fibroblast growth factor (bFGF)-induced proliferation were examined in primary cultures of cerebellar astrocytes. Exposure to bFGF resulted in a rapid increase of extracellular S1P formation, bFGF inducing astrocytes to release S1P, but not sphingosine kinase, in the extracellular milieu. The SK inhibitor N,N-dimethylsphingosine inhibited S1P release as well as bFGF-induced growth stimulation. S1P application in quiescent astrocytes caused a dose-dependent increase in DNA synthesis. This gliotrophic effect was induced by a brief exposure to low nanomolar S1P, mimicked by the S1P receptor agonist dihydro-S1P, and inhibited by pertussis toxin (PTX), an inactivator of G(i)/G(o)-proteins. S1P also induced activation of extracellular signal-regulated kinase that was inhibited again by PTX. Moreover, the S1P lyase inhibitor 4-deoxypyridoxine induced the cellular accumulation of S1P but did not affect DNA synthesis. These results support the view that S1P exerted a mitogenic effect on cerebellar astrocytes extracellularly, most likely through cell surface S1P receptors. In agreement, mRNAs for S1P1, S1P2, and S1P3 receptors are expressed in cerebellar astrocytes (Anelli et al., 2005. J Neurochem 92:1204-1215). Ceramide, a negative regulator of astrocyte proliferation and down-regulated by bFGF (Riboni et al., 2002. Cerebellum 1:129-135), efficiently inhibited S1P-induced proliferation. The S1P action appears to be part of an autocrine/paracrine cascade stimulated by bFGF and, together with ceramide down-regulation, essential for astrocytes to respond to bFGF. The results suggest that S1P and bFGF/S1P may play an important role in physiopathological glial proliferation, such as brain development, reactive gliosis and brain tumor formation.

Suppressors of cytokine signaling (SOCS) proteins are intracellular proteins that inhibit cytokine signaling in a variety of cell types. A number of viral infections have been associated with SOCS upregulation; however, not much is known about the mechanisms regulating SOCS expression during viral infection. In this study, we used two pathologically distinct intracerebral (i.c.) infection models to characterize temporal and spatial aspects of SOCS expression in the virus-infected central nervous system (CNS), and by employing various knockout mouse models, we sought to identify regulatory mechanisms that may underlie a virus induced upregulation of SOCS in the CNS. We found that i.c. infection with either lymphocytic choriomeningitis virus (LCMV) or yellow fever virus (YF) results in gradual upregulation of SOCS1/3 mRNA expression peaking at day 7 postinfection (p.i.). In the LCMV model, SOCS mRNA was expressed in brain resident cells, including astrocytes and some neurons, and for SOCS1 in particular this upregulation was almost entirely mediated by gamma interferon (IFN-γ) produced by infiltrating T cells. After infection with YF, we also found SOCS expression to be upregulated in brain resident cells with a peak on day 7 p.i., but in this model, the upregulation was only partially dependent on IFN-γ and T cells, indicating that at least one other mediator was involved in the upregulation of SOCS following YF infection. We conclude that virus-induced inflammation of the CNS is associated with upregulation of SOCS1/3 mRNA expression in brain resident cells and that at least two distinctive pathways can lead to this upregulation. IMPORTANCE In the present report, we have studied the induction of SOCS1 and SOCS3 expression in the context of virus-induced CNS infection. We found that both a noncytolytic and a cytolytic virus induce marked upregulation of SOCS1 and -3 expression. Notably, the kinetics of the observed upregulation follows that of activity within proinflammatory signaling pathways and, interestingly, type II interferon (IFN), which is also a key inducer of inflammatory mediators, seems to be essential in initiating this counterinflammatory response. Another key observation is that not only cells of the immune system but also CNS resident cells are actively involved in both the pro- and the counterinflammatory immune circuits; thus, for example, astrocytes upregulate both C-X-C-motif chemokine 10 (CXCL10) and SOCS when exposed to type II IFN in vivo .

Aiming to investigate the possible production of ceramide-1-phosphate from complex sphingolipid metabolism in neurons, we administered radiolabeled sphingolipids to cerebellar granule cells and inspected the formation of labeled ceramide-1-phosphate in different experimental conditions. We report that differentiated granule cells are capable to form Cer-1-P via ceramide derived from SM degradation at the plasma membrane level. Moreover we observed that ceramide-1-phosphate can be also produced from a metabolic pathway not involving SM degradation. In particular, we obtained evidence that ceramide, synthesized via the recycling of sphingosine produced from ganglioside catabolism, can also be the precursor of ceramide-1-phosphate. We also found that undifferentiated and differentiated granule cells display different capacities to phosphorylate Cer produced by the two different metabolic pathways. The results here obtained demonstrate that cerebellar neurons are able to metabolically produce ceramide-1-phosphate and support that this molecule may serve a potential role in sphingoid-mediated signaling in the nervous system.

The plasma membrane-associated sialidase NEU3 plays crucial roles in regulation of transmembrane signaling, and its aberrant up-regulation in various cancers contributes to malignancy. However, it remains uncertain how NEU3 is naturally activated and locates to plasma membranes, because of its Triton X-100 requirement for the sialidase activity in vitro and its often changing subcellular location. Among phospholipids examined, we demonstrate that phosphatidic acid (PA) elevates its sialidase activity 4 to 5 times at 50 μM in vitro at neutral pH and promotes translocation to the cell surface and cell migration through Ras-signaling in HeLa and COS-1 cells. NEU3 was found to interact selectively with PA as assessed by phospholipid array, liposome coprecipitation, and ELISA assays and to colocalize with phospholipase D (PLD) 1 in response to epidermal growth factor (EGF) or serum stimulation. Studies using tagged NEU3 fragments with point mutations identified PA- and calmodulin (CaM)-binding sites around the N terminus and confirmed its participation in translocation and catalytic activity. EGF induced PLD1 activation concomitantly with enhanced NEU3 translocation to the cell surface, as assessed by confocal microscopy. These results suggest that interactions of NEU3 with PA produced by PLD1 are important for regulation of transmembrane signaling, this aberrant acceleration probably promoting malignancy in cancers.

The aim of radiotherapy is to eradicate cancer cells with ionizing radiation; tumor cell death following irradiation can be induced by several signaling pathways, most of which are triggered as a consequence of DNA damage, the primary and major relevant cell response to radiation. Several lines of evidence demonstrated that ceramide, a crucial sensor and/or effector of different signalling pathways promoting cell cycle arrest, death and differentiation, is directly involved in the molecular mechanisms underlying cellular response to irradiation. Most of the studies strongly support a direct relationship between ceramide accumulation and radiation-induced cell death, mainly apoptosis; for this reason, defining the contribution of the multiple metabolic pathways leading to ceramide formation and the causes of its dysregulated metabolism represent the main goal in order to elucidate the ceramide-mediated signaling in radiotherapy. In this review, we summarize the current knowledge concerning the different routes leading to ceramide accumulation in radiation-induced cell response with particular regard to the role of the enzymes involved in both ceramide neogenesis and catabolism. Emphasis is placed on sphingolipid breakdown as mechanism of ceramide generation activated following cell irradiation; the functional relevance of this pathway, and the role of glycosphingolipid glycohydrolases as direct targets of ionizing radiation are also discussed. These new findings add a further attractive point of investigation to better define the complex interplay between sphingolipid metabolism and radiation therapy.

Gangliosides are a large group of complex lipids found predominantly on the outer layer of the plasma membranes of cells, and they are particularly concentrated in nerve endings. Their half-life in the nervous system is short, and their membrane composition and content are strictly connected to their metabolism. Their neobiosynthesis starts in the endoplasmic reticulum and is completed in the Golgi; catabolism occurs primarily in the lysosomes. However, the final content of gangliosides in the plasma membrane is affected by other cellular processes.

We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137–145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from eitherl-[3H]serine or [3H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.

The effects of different bioactive sphingoid molecules on NOS activity of differentiated cerebellar granule cells were investigated by measuring the conversion of [ 3 H]arginine to [ 3 H]citrulline. Cytosolic Ca 2+ -dependent NOS activity was strongly inhibited in a dose-dependent manner by sphingosine in concentrations of 1–40 μM. This inhibition seems to be peculiar to sphingosine in that ceramide, N -acetylsphingosine, sphingosine-1P, sphinganine and tetradecylamine have no effect on the cytosolic enzyme at the considered concentrations, suggesting that it is the bulk of the sphingosine hydrophilic portion that is critical for cytosolic NOS inhibition. This inhibition of cytosolic NOS is not reversed by increasing the arginine concentration, so a competitive mechanism can be excluded. Instead, increasing the concentrations of calmodulin led to loss of sphingosine inhibition, suggesting that sphingosine interferes with the calmodulin-dependent activation of the enzyme by a competitive mechanism. Sphingosine and related compounds had no effect on the particulate Ca 2+ -independent NOS activity. The data obtained suggest that sphingosine could be involved in the regulation of NO production in neurons.

The possible relationship between metabolism and biological effects of sphingosine was investigated in Neuro2a cells. [C3-3H]-sphingosine, administered at different doses (80 pmol-80 nmol/mg cell protein). Amounts up to hundredfold were rapidly taken up and metabolized, the intracellular content of sphingosine being processed within 2 h. At low doses, [3H]-sphingosine represented a minor portion of the cellular radiolabel, and N-acylated metabolites, particularly ceramide, prevailed over degradation products. Neuro2a cell differentiation took place in conjunction with ceramide increase. At increasing exogenous sphingosine/cell ratio, the acylation process became saturated while sphingosine degradation increased proportionally. From this point on [3H]-sphingosine accumulated and cell toxicity occurred. In conclusion, in Neuro2a cells the biological effects exerted by exogenous sphingosine are strictly connected to the exogenous sphingosine/cell ratio and to the capacity of the cell to metabolize sphingosine.

Glycosphingolipids are a large group of complex lipids particularly abundant in the outer layer of the neuronal plasma membranes. Qualitative and quantitative changes in glycosphingolipids have been reported along neuronal differentiation and aging. Their half-life is short in the nervous system and their membrane composition and content are the result of a complex network of metabolic pathways involving both the de novo synthesis in the Golgi apparatus and the lysosomal catabolism. In particular, most of the enzymes of glycosphingolipid biosynthesis and catabolism have been found also at the plasma membrane level. Their action could be responsible for the fine tuning of the plasma membrane glycosphingolipid composition allowing the formation of highly specialized membrane areas, such as the synapses and the axonal growth cones. While the correlation between the changes of GSL pattern and the modulation of the expression/activity of different glycosyltransferases during the neuronal differentiation has been widely discussed, the role of the glycohydrolytic enzymes in this process is still little explored. For this reason, in the present review, we focus on the main glycolipid catabolic enzymes β-hexosaminidases, sialidases, β-galactosidases, and β-glucocerebrosidases in the process of the neuronal differentiation.

Current studies indicate that ceramide is involved in the regulation of important cell functions, namely cell growth, differentiation, and apoptosis. In the present study, the possible role of ceramide in the differentiation of neuroblastoma Neuro2a cells was investigated. The following results were obtained. (a) Ceramide content of Neuro2a cells, induced to differentiate by retinoic acid (RA) treatment rapidly increased after addition of RA, was maintained at high levels in RA-differentiated cells and returned to the starting levels with removal of RA and reversal of differentiation; under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with [3H]sphingomyelin or [3H]sphingosine or L-[3H]serine, the metabolic formation of ceramide was markedly higher and more rapid in RA-differentiated than undifferentiated cells. (c) Inhibitors of ceramide biosynthesis (Fumonisin B1, beta-chloroalanine and L-cycloserine) diminished the extent of the differentiating effect of RA and concomitantly Cer content decreased. (d) The activity of neutral sphingomyelinase increased after addition of RA, maintained high levels in RA-differentiated cells, and returned to the initial levels with removal of RA. (e) Experimental conditions that cause an elevation of ceramide content (treatment with sphingosine or ceramide or C2-ceramide or bacterial sphingomyelinase) inhibited cell proliferation and stimulated neurite outgrowth; dihydro-analogues of sphingosine, ceramide, and C2-ceramide had no effect on differentiation. (f) treatment with Fumonisin B1 completely inhibited sphingosine-induced differentiation. These data suggest a specific bioregulatory function of ceramide in the control of Neuro2a cell growth and differentiation and pose the general hypothesis of a mediator role of ceramide in the differentiation of cells of neural origin.

The effect of BFA on the metabolic processing and intracellular traffic of gangliosides was studied in cerebellar granule cells, fed in culture for different periods with radiolabeled ganglioside GM1. The following results were obtained: (a) degradation of taken-up GM1 was markedly inhibited by BFA; this effect was rapid, reversible, and affected by reduced temperature, ATP depletion, and microtubule disruption: (b) direct glycosylation of internalized GM1 to GD1a was completely blocked by BFA; (c) the portion of GM1 that escaped BFA inhibition was degraded with formation of sphingosine, that was recycled for the biosynthesis of less glycosylated glycolipids (glucosyl-ceramide, GM3 and GD3); (d) in BFA-treated cells highly glycosylated gangliosides were undetectable, and the formation of sphingomyelin from liberated sphingosine was markedly reduced. These results suggest that, in the cells used: (a) the delivery of endocytosed gangliosides to lysosomes, (b) the flow of poorly glycosylated glycolipids from the Golgi stacks to the trans-Golgi network, and (c) the direct transport of part of endocytosed gangliosides to the late sites of glycosylation (possibly TGN) are mediated by BFA-sensitive vesicles. We propose that in cultured granule cells a BFA-sensitive mechanism regulates ganglioside traffic to and from the plasma membrane.

The glucose-dependent insulinotropic polypeptide receptor (GIPr) has been implicated in high fat diet-induced obesity and is proposed as an anti-obesity target despite an uncertainty regarding the mechanism of action. To independently investigate the contribution of the insulinotropic effects and the direct effects on adipose tissue, we generated transgenic mice with targeted expression of the human GIPr to white adipose tissue or beta-cells, respectively. These mice were then cross-bred with the GIPr knock-out strain. The central findings of the study are that mice with GIPr expression targeted to adipose tissue have a similar high fat diet -induced body weight gain as control mice, significantly greater than the weight gain in mice with a general ablation of the receptor. Surprisingly, this difference was due to an increase in total lean body mass rather than a gain in total fat mass that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass, but it does not support a direct and independent role for the adipocyte or beta-cell GIPr in promoting adipogenesis.

Cerebellar granule cells, differentiated in vitro, were parallelly fed with [Sph-3H]GM1 and [stearoyl-14C]GM1, under identical conditions (10−6 M ganglioside; pulse, from 1–4 h; chase, up to 24 h after 4 h pulse) and the salvage pathways of sphingosine and stearic acid were investigated. It was observed that both sphingosine and stearic acid, liberated during the intralysosomal degradation of ganglioside, are metabolically recycled, along distinct pathways. Sphingosine is used for the biosynthesis of a number of sphingolipids, particularly ceramide, glucosyl-ceramide, gangliosides and sphingomyelin; stearic acid is utilized for the biosynthesis of sphingolipids, and to a greater extent, glycero-phospholipids, especially those endogenously richer in stearic acid (phosphatidyl-ethanolamine and phosphatidyl-choline). No evidence was provided for a salvage pathway for ceramide.

The role of the ceramide moiety of gangliosides, together with the deriving aggregative properties of ganglioside in solution, in the process of ganglioside-cell interactions was studied. The natural GM1(stearoyl) and the synthetic GM1(acetyl), containing the stearoyl and acetyl groups as the acyl moiety, respectively, were used in binding experiments to rat cerebellar granule cells. Regardless of the cell culture conditions, such as the presence or absence of fetal calf serum, the association of GM1(acetyl) to the cells was much greater than that of GM1(stearoyl). GM1(acetyl) was present in the incubation medium as monomers. After incubation, a large part of the total GM1(acetyl) associated to cells, 76–93% depending on the experimental conditions, was removed by washing with protein solutions. The remaining associated ganglioside was not removed by repeating washing with protein solutions or trypsin treatments and was considered as a component of the membrane. The cell association of GM1(stearoyl), present in solution as monomers as well as micelles, could be classified as serum-labile, trypsin-labile and trypsin-stable. The trypsin-stable form of association, corresponding to the molecules stably inserted into the membrane, was proportionally higher, the proportions varying with increasing incubation time and decreasing ganglioside concentration. This form of association was particularly high when incubation was performed in the presence of fetal calf serum. Incubation experiments performed with a mixture of GM1(stearoyl) and GM1(acetyl) in a molar ratio which allowed their presence in the medium as monomers as well as mixed micelles, led to a ganglioside association suggesting that besides the aggregative properties of the molecule other ganglioside properties are involved in the ganglioside-cell interaction process.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GM1 in order to have it inserted into the plasma membrane and metabolized. Among the formed metabolites radioactive sphingosine and ceramide were identified. [3H]Ceramide started to be measurable after 10 min of incubation (pulse), and [3H]sphingosine after 15 min. Their concentrations increased with pulse time, and after a 1-hour pulse, with chase time. After a 1-hour pulse with 2 × 10−6 M [3H-Sph]GM1 followed by a 4-hour chase, the amount of [3H]sphingosine and [3H]ceramide formed were 0.04 and 0.4 pmol/106 cells, respectively. Particularly the ability to produce sphingosine was higher in differentiated than in undifferentiated cells. It is concluded that ganglioside turnover contributes to the maintenance of the intracellular levels of free sphingosine and ceramide.

As the majority of gliomas arise through malignant transformation of astrocytes, we aimed at investigating the interaction between malignant glioma cells and astrocytes in a co-culture experimental model. For this purpose we analyzed the expression of genes and proteins involved in tumor promotion and invasion, such as glial fibrillary acidic protein (GFAP), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), transforming growth factor-beta1 (TGF-beta1), secreted protein acidic and rich in cysteine (SPARC), and connexin 43 (CX43). Co-cultures of human neural stem cell-derived astrocytes and U87 MG astrocytoma cells were performed in a transwell system. Gene expression was evaluated by real-time RT-PCR, and protein analysis was performed by Western blotting, SDS-zymography, and immunofluorescence. GFAP tended to be up-regulated in astrocytes co-cultivated with U87, suggesting a reactive response induced by glioma cells. CX43 mRNA tended to be down- regulated in co-cultured astrocytes, as well as the non-phosphorylated isoform at the protein level. MMP-2 mRNA tended to be up-regulated, and MMP-2 protein levels were significantly increased in astrocytes co-cultivated with U87. TIMP-2 and SPARC mRNA decreased in astrocytes co-cultivated with U87, showing lower expression in glioma cells. By contrast, SPARC protein expression was strongly induced in supernatants of co-cultured astrocytes. TGF-beta1 was not modified. Our results suggest that U87 cells elicit phenotype modifications in the neighbouring resident astrocytes very likely mediated by soluble factors. Glioma/astrocyte interaction could possibly trigger an astrocyte phenotype modification consistent with a malignant transformation, and favouring a more permissive environment for glioma cells invasion.

The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelope that confers biochemical and morphological identity typical of the species. Purified chorions can be easily isolated from either oocytes or ovulated eggs. The aim of this work was to analyze the macromolecular composition of the various chorion components in Oncorhynchus mykiss (Salmonids). SDS-PAGE analysis of purified chorion showed a reproducible pattern of four major components (129, 62, 54, and 47 kD), representing about 80% of total chorion proteins. The 129 and 47 kD polypeptides were periodic-acid Schiff (PAS) and concanavalin A positive. After chemical and enzymatic deglycosylation treatments only the 129 and 47 kD components proved to be glycosylated and to belong to the "asparagine-linked" glycoprotein family. Furthermore, peptide mapping performed on isolated polypeptides showed comigrating fragments on SDS-PAGE. These results suggest that the four main chorion polypeptides might share common structural features.

HMGB1 (high mobility group box 1 protein) is a nuclear protein that can also act as an extracellular trigger of inflammation, proliferation and migration, mainly through RAGE (the receptor for advanced glycation end products); HMGB1-RAGE interactions have been found to be important in a number of cancers. We investigated whether HMGB1 is an autocrine factor in human glioma cells. Western blots showed HMGB1 and RAGE expression in human malignant glioma cell lines. HMGB1 induced a dose-dependent increase in cell proliferation, which was found to be RAGE-mediated and involved the MAPK/ERK pathway. Moreover, in a wounding model, it induced a significant increase in cell migration, and RAGE-dependent activation of Rac1 was crucial in giving the tumour cells a motile phenotype. The fact that blocking DNA replication with anti-mitotic agents did not reduce the distance migrated suggests the independence of the proliferative and migratory effects. We also found that glioma cells contain HMGB1 predominantly in the nucleus, and cannot secrete it constitutively or upon stimulation; however, necrotic glioma cells can release HMGB1 after it has translocated from the nucleus to cytosol. These findings provide the first evidence supporting the existence of HMGB1/RAGE signalling pathways in human glioblastoma cells, and suggest that HMGB1 may play an important role in the relationship between necrosis and malignancy in glioma tumours by acting as an autocrine factor that is capable of promoting the growth and migration of tumour cells.

Intracellular movements of ceramide are strongly limited by its hydrophobic nature, and the mechanisms involved in ceramide transport can represent a crucial aspect of sphingolipid metabolism and signaling. The recent identification of the ceramide specific carrier protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin biosynthesis. In this study we investigated the metabolic and functional role of CERT in C6 glioma cells. These cells were found to constitutively express CERT, the protein being mainly associated with the cytosolic fraction. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids demonstrated that the down regulation of CERT by RNAi technology resulted in a significant but not complete reduction of ceramide metabolism to sphingomyelin, without affecting its utilization for glycosphingolipid biosynthesis. Since nitric oxide is an inhibitor of ceramide ER-to-Golgi traffic and metabolism in C6 glioma cells, we evaluated the possibility that the CERT-mediated transport of ceramide might represent a target for nitric oxide. The data obtained demonstrate that CERT down regulation does not affect the inhibitory activity of nitric oxide on Cer metabolism, and the effects of nitric oxide and CERT silencing on ceramide utilization were additive. These results strongly suggest that a CERT-mediated and a CERT-independent, nitric oxide-sensitive Cer transport coexist in C6 glioma cells and can separately contribute to the control of sphingolipid metabolism and Cer levels in these cells.

S1P is involved in the regulation of multiple biological processes (cell survival, growth, migration and differentiation) both in neurons and glial cells. The study was aimed at investigating the possible effects of S1P on calcium signaling in cerebellar astrocytes and differentiated granule cells. In cerebellar astrocytes S1P is able to mediate calcium signaling mainly through Gi protein coupled receptors, whereas in differentiated neurons it failed to evoke any calcium signaling, despite acting both extracellularly and intracellularly. The data indicate strict cell specificity in S1P-evoked calcium response, which could be relevant to communication between neurons and glial cells in the cerebellum.

To evaluate the role of ceramide in glial growth, primary cultures of quiescent astrocytes from rat cerebellum were stimulated to proliferate by mitogenic doses of basic fibroblast growth factor (bFGF). Parallel to the bFGF mitogenic effect was a marked, and persistent, decrease in cellular ceramide levels. Both in vitro and in culture metabolic studies have led us to exclude both sphingomyelinase and ceramidase involvement in ceramide level variation. Instead, we found evidence of a functional connection between the decrease in ceramide levels and astrocyte proliferation. In fact, cell growth in bFGF-stimulated astrocytes was inhibited by exogenous ceramide and C2-ceramide, maximal inhibition being obtained at a ceramide concentration of 5–10 μM. Under the same conditions, the dihydroderivatives of ceramides were without effect. Following ceramide treatment, the phosphorylation of the MAP kinase isoforms ERK1/2, key components in bFGF-induced cell proliferation, was examined. The administration of antiproliferative doses of ceramide or C2-ceramide, but not of their dihydroderivatives, resulted in a significant inhibition of ERK1/2 activation. In conclusion, our data indicate that the prompt modulation of ceramide levels by bFGF is an early step associated with the signaling pathways responsible for the mitogenic activity of bFGF in astrocytes. GLIA 32:137–145, 2000. © 2000 Wiley-Liss, Inc.

The metabolic fate of exogenous [3H]sphingosine was investigated in five types of cultured cells: primary cultures of neurons and astrocytes, murine and human neuroblastoma cells and human skin fibroblasts. After administration of 40 nM [3-3H]sphingosine into a cell-conditioned medium containing fetal calf serum, all cell types rapidly and efficiently incorporated the long-chain base in a time-dependent fashion. In all cases, after a 120 min pulse, the amount of radioactivity taken up was in the range of the endogenous sphingosine content. However, unchanged [3H]sphingosine represented only a very minor portion of the label incorporated into cells throughout the pulse period (10–120 min), indicating rapid and efficient sphingosine metabolism in these cells. Most of the [3H]sphingosine taken up was metabolically processed, either by degradation (assessed as 3H2O release into the culture medium) or by N-acylation (mainly to radioactive ceramide, sphingomyelin, neutral glycolipids and gangliosides). [3H]Sphingosine 1-phosphate accounted for less than 2% of the total radioactivity incorporated in all cases. Throughout the pulse period and in all cell types, 3H-labelled organic metabolites largely prevailed over 3H2O, indicating that N-acylation is the major metabolic fate of sphingosine in these cells under apparently physiological conditions. These results are consistent with the notion that sphingosine has a rapid turnover in the cells studied, and indicate that regulation of the basal level of this bioactive molecule occurs mainly through N-acylation.

Many Golgi membrane-bound glycosyltransferases exist as intermolecular disulfide bonded species, some of which have been demonstrated to be homodimers. Evidence for homodimer formation has come primarily from radiation inactivation experiments. We utilized an alternative strategy to test for homodimer formation of the cloned beta 1,4 N-acetylgalactosaminyltransferase (GalNAcT) responsible for synthesis of the glycosphingolipids GM2, GD2, and GA2. We stably transfected CHO cells with myc epitopetagged GalNAcT, which localizes primarily to the Golgi, and a hemagglutinin (HA) epitope-tagged GalNAcT fusion protein in which the cytoplasmic domain of GalNAcT was replaced by an ER retention signal. We then sought evidence for dimer formation between the two forms of GalNAcT. Immunoprecipitation with anti-myc or anti-HA co-immunoprecipitated the HA-tagged form or the myc-tagged form, respectively, providing evidence for the physical association of the two forms of GalNAcT. As a result of this association, GalNAcT/myc increased in the ER as demonstrated by Western blots and immunofluorescence. The rapid formation of dimers provided further evidence for dimer formation occurring in the ER. In summary, these results demonstrate that GalNAcT forms homodimers as a result of intermolecular disulfide bond formation in the ER. Furthermore, this ER motif strategy is potentially useful for demonstrating homodimer formation of other Golgi enzymes.

The possible involvement of protein phosphatase in ceramide-mediated neural cell differentiation was investigated. Neuroblastoma Neuro2a cell differentiation induced by retinoic acid, or conditions causing an increase in cellular ceramide, was significantly inhibited by the serine/threonine phosphatase inhibitor okadaic acid, at concentrations as low as 2.5 nM. A crude cytosolic preparation from Neuro2a cells was found to have a cation-independent protein phosphatase activity that was stimulated by ceramide in a dose-dependent manner. Short- and long-chain ceramides, but not sphingosine and related dihydroderivatives, were active. Ceramide-activated protein phosphatase activity from Neuro2a cells was inhibited by 5 nM okadaic acid. The data indicate that a type 2A protein phosphatase is involved in ceramide-mediated differentiation of Neuro2a cells.

The involvement of ceramide in the differentiation of two neuroblastoma cell lines, Neuro2a and SH-SY5Y, and cerebellar granule cells in primary culture was investigated. The following results were obtained: (a) the cellular content of ceramide markedly increased with induced differentiation of Neuro2a cells (inducers: RA, FCS deprivation), SH-SY5Y cells (inducers: RA, PMA), and spontaneous differentiation of cerebellar granule cells; (b) all the investigated cells in the differentiated form displayed a higher ability to produce ceramide from exogenously administered [3H]Sph-SM and expressed a higher content of neutral sphingomyelinase and, in the case of cerebellar granule cells, also of acidic sphingomyelinase; (c) inhibition of ceramide biosynthesis by Fumonisin B1 blocked the process of differentiation in Neuro2a and cerebellar granule cells; and (d) treatments capable of enhancing ceramide level (administration of sphingosine or C2-Ceramide) induced differentiation in both Neuro2a and SH-SY5Y cells. The data obtained support the notion that ceramide plays a general biomodulatory role in neural cell differentiation.

Cerebellar granule cells in culture were subjected to a pulse (0.5–4 h)-chase (0–4 h) of 10 −6 M [ 3 H]ganglioside GM1 carrying the radioactive label at the level of NeuAc ([ 3 H-NeuAc]GM1), Sph ([ 3 H-Sph]GM1) or Gal ([ 3 H-GallGMl) and the formed [ 3 H]metabolites were determined. With all forms of [ 3 H]GM1, there was formation of [ 3 H]catabolites, including [ 3 H]H 2 O and [ 3 H]biosynthetic products obtained by recycling of [ 3 H]NeuAc, [ 3 H]Sph and [ 3 H]Gal released during intralysosomal ganglioside degradation (salvage processes). Much higher amounts of [ 3 MH 2 O were produced from [ 3 H-Gal]GM1 than [ 3 H-Sph]GM1 and [ 3 H-NeuAc]GM1; conversely, more products from salvage processes (polysialogangliosides GD1a, GDlb, GTlb, O-acetylated GT1b, protein-bound radioactivity) were obtained with [ 3 H-NeuAc]GM1; than the two other forms of [ 3 H]GM1, Liberated [ 3 H]NeuAc produced 10-fold less tritiated water and 10-fold higher salvage products than [ 3 H]Gal. Using [ 3 H-NeuAc]GM1; granule cells appeared to metabolize 7.7% of membrane-incorporated exogenous GM1 per hour with a high degree of NeuAc recycling and the calculated metabolic half-life was 6.5 h.

Exogenous sphingomyelin, radiolabelled at the sphingosine moiety, was administered to primary cultures of cerebellar granule cells and astrocytes for different pulse times (20 min–2 h) and the fate of the radioactivity was followed. Ceramide was the main metabolic product in both cells, whereas sphingosine, glucosyl-ceramide and gangliosides GM3 and GD3 were produced only in astrocytes. When endocytosis was prevented and the lysosomal apparatus inactivated, ceramide formation was reduced slightly in granule cells and almost completely blocked in astrocytes, with disappearance of sphingosine, glucosyl-ceramide, GM3 and GD3. These data indicate that (a) ceramide is rapidly produced in cerebellar granule cells and astrocytes, presumably at the level of the plasma membrane in the first cell type, and of the lysosomes in the second one; (b) sphingosine is produced in cerebellar astrocytes by lysosomal sphingomyelin degradation and is partly reused for glucosyl-ceramide and ganglioside biosynthesis.

Over the past decade, ceramide and sphingosine-1-phosphate have emerged as crucial mediators in many cells including those from the nervous system. In brain cells various stimuli, ranging from growth factors and differentiating agents, to oxidative stress and cytotoxic drugs, regulate one or more of the enzymes of sphingolipid metabolism. This results in the variation of cell sphingoids, which in turn act as mediators in regulating growth, differentiation and fate. Emerging data also indicate that sphingosine-1-phosphate can be released from brain cells and acts as intercellular mediator, after interaction with specific EDG receptors. The importance of sphingoid mediators in brain has been reinforced by recent findings implicating them in physiological processes as well as in the pathogenesis of brain diseases, including degenerative disorders and cancers. Evidence is accumulating that the manipulation of sphingolipid metabolism in brain cells will offer novel potential targets for therapeutic intervention in brain dysfunctions.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GD1a in order to have it inserted into the plasma membrane, internalized by endocytosis, and metabolized. The metabolites formed included GM1, product of GD1a desialosylation. No GM1 or other metabolites were present in the incubation medium, whereas with the lysosomal apparatus blocked by chloroquine, or GD1a endocytosis prevented at 4 degrees C, the only metabolite formed was GM1. These results suggest that GD1a desialosylation did not occur either extracellularly or intracellularly but likely, at the membrane level. Similar results were obtained with [3H-Gal]GD1b, whereas no degradation of [3H-NeuAc]GM1 took place in the presence of chloroquine or at 4 degrees C. In conclusion, cerebellar granule cells express in vivo a sialidase, presumably located on the cell surface, that affects GD1a and GD1b but not GM1.

We found that sparse and confluent C6 glioma cells differ both in GM3 content, which increases with cell density, and in endothelin-1 (ET-1)-induced phosphoinositide hydrolysis, which was markedly higher in the sparse cells than in the confluent. Also after manipulation of the cellular GM3 content through treatment with exogenous GM3 or with drugs known to affect GM3 metabolism, the ET-1 effect was inversely related to GM3 cellular levels. Cell treatment with an anti-GM3 mAb resulted in the enhancement of ET-1-induced phospholipase C activation and restored the capacity of GM3-treated cells to respond to ET-1. These findings suggest that the GM3 ganglioside represents a physiological modulator of ET-1 signaling in glial cells.

The possible relation between nitric oxide synthase (NOS) activity and neural differentiation was investigated using primary cultures of rat cerebellar granule cells differentiating in culture. NOS activity was measured in the cytosolic and particulate fractions obtained from cell homogenate. In the experimental conditions used the optimal pH for NOS activity was about 6.4, the activity being about 3-fold higher than at pH 7.4. Cerebellar granule cell differentiation was associated with marked increases in NOS activity. In undifferentiated cells the enzyme was almost evenly distributed between the cytosolic and particulate fractions, during differentiation there was a 12-fold increase in activity in the cytosolic enzyme and a 3-fold increase in the particulate one. This indicates a marked preferential enrichment of the cytosolic enzyme during differentiation. Cerebellar granule cells produced and released NO in the culture medium; NO formation being markedly higher in differentiated cells (7–12 DIC) than in undifferentiated (2–3 DIC) ones. These data demonstrate a relationship between NOS expression and NO production and the differentiation of cerebellar granule cells, supporting the notion that NO may play a role in this process.