Metformin Lysosomal Targeting: A Novel Aspect to Be Investigated for Metformin Repurposing in Neurodegenerative Diseases?

Metformin is a widely employed drug in type 2 diabetes. In addition to warranting good short- and long-term glycemic control, metformin displays many intriguing properties as protection against cardiovascular and neurodegenerative diseases, anti-tumorigenic and longevity promotion. In addition to being a low-cost drug, metformin is generally well tolerated. However, despite the enthusiastic drive to aliment these novel studies, many contradictory results suggest the importance of better elucidating the complexity of metformin action in different tissues/cells to establish its possible employment in neurodegenerative diseases. This review summarises recent data identifying lysosomal-dependent processes and lysosomal targets, such as endosomal Na+/H+ exchangers, presenilin enhancer 2 (PEN2), the lysosomal pathway leading to AMP-activated protein kinase (AMPK) activation, and the transcription factor EB (TFEB), modulated by metformin. Lysosomal dysfunctions resulting in autophagic and lysosomal acidification and biogenesis impairment appear to be hallmarks of many inherited and acquired neurodegenerative diseases. Lysosomes are not yet seen as a sort of cellular dump but are crucial in determining key signalling paths and processes involved in the clearance of aggregated proteins. Thus, the possibility of pharmacologically modulating them deserves great interest. Despite the potentiality of metformin in this context, many additional important issues, such as dosing, should be addressed in the future.

Sphingosine 1-Phosphate Stimulates ER to Golgi Ceramide Traffic to Promote Survival in T98G Glioma Cells.

Glioblastoma multiforme is the most common and fatal brain tumor among human cancers. Ceramide (Cer) and Sphingosine 1-phosphate (S1P) have emerged as bioeffector molecules that control several biological processes involved in both cancer development and resistance. Cer acts as a tumor suppressor, inhibiting cancer progression, promoting apoptosis, enhancing immunotherapy and sensitizing cells to chemotherapy. In contrast, S1P functions as an onco-promoter molecule, increasing proliferation, survival, invasiveness, and resistance to drug-induced apoptosis. The pro-survival PI3K/Akt pathway is a recognized downstream target of S1P, and we have previously demonstrated that in glioma cells it also improves Cer transport and metabolism towards complex sphingolipids in glioma cells. Here, we first examined the possibility that, in T98G glioma cells, S1P may regulate Cer metabolism through PI3K/Akt signaling. Our research showed that exogenous S1P increases the rate of vesicular trafficking of Cer from the endoplasmic reticulum (ER) to the Golgi apparatus through S1P receptor-mediated activation of the PI3K/Akt pathway. Interestingly, the effect of S1P results in cell protection against toxicity arising from Cer accumulation in the ER, highlighting the role of S1P as a survival factor to escape from the Cer-generating cell death response.

Ceramide Is Involved in Temozolomide Resistance in Human Glioblastoma U87MG Overexpressing EGFR.

Glioblastoma multiforme (GBM) is the most frequent and deadly brain tumor. Many sphingolipids are crucial players in the regulation of glioma cell growth as well as in the response to different chemotherapeutic drugs. In particular, ceramide (Cer) is a tumor suppressor lipid, able to induce antiproliferative and apoptotic responses in different types of tumors including GBM, most of which overexpress the epidermal growth factor receptor variant III (EGFRvIII). In this paper, we investigated whether Cer metabolism is altered in the U87MG human glioma cell line overexpressing EGFRvIII (EGFR+ cells) to elucidate their possible interplay in the mechanisms regulating GBM survival properties and the response to the alkylating agent temozolomide (TMZ). Notably, we demonstrated that a low dose of TMZ significantly increases Cer levels in U87MG cells but slightly in EGFR+ cells (sensitive and resistant to TMZ, respectively). Moreover, the inhibition of the synthesis of complex sphingolipids made EGFR+ cells sensitive to TMZ, thus involving Cer accumulation/removal in TMZ resistance of GBM cells. This suggests that the enhanced resistance of EGFR+ cells to TMZ is dependent on Cer metabolism. Altogether, our results indicate that EGFRvIII expression confers a TMZ-resistance phenotype to U87MG glioma cells by counteracting Cer increase.

Impaired Autophagy in Krabbe Disease: The Role of BCL2 and Beclin-1 Phosphorylation.

Autophagic impairment was identified in many lysosomal storage diseases and adult neurodegenerative diseases. It seems that this defect could be directly related to the appearance of a neurodegenerative phenotype and could contribute to worsen metabolite accumulation and lysosomal distress. Thus, autophagy is becoming a promising target for supportive therapies. Autophagy alterations were recently identified also in Krabbe disease. Krabbe disease is characterized by extensive demyelination and dysmyelination and it is due to the genetic loss of function of the lysosomal enzyme galactocerebrosidase (GALC). This enzyme leads to the accumulation of galactosylceramide, psychosine, and secondary substrates such as lactosylceramide. In this paper, we induced autophagy through starvation and examined the cellular response occurring in fibroblasts isolated from patients. We demonstrated that the inhibitory AKT-mediated phosphorylation of beclin-1 and the BCL2-beclin-1 complex concur to reduce autophagosomes formation in response to starvation. These events were not dependent on the accumulation of psychosine, which was previously identified as a possible player in autophagic impairment in Krabbe disease. We believe that these data could better elucidate the capability of response to autophagic stimuli in Krabbe disease, in order to identify possible molecules able to stimulate the process.

Ceramide and Sphingosine-1-Phosphate in Neurodegenerative Disorders and Their Potential Involvement in Therapy.

Neurodegenerative disorders (ND) are progressive diseases of the nervous system, often without resolutive therapy. They are characterized by a progressive impairment and loss of specific brain regions and neuronal populations. Cellular and animal model studies have identified several molecular mechanisms that play an important role in the pathogenesis of ND. Among them are alterations of lipids, in particular sphingolipids, that play a crucial role in neurodegeneration. Overall, during ND, ceramide-dependent pro-apoptotic signalling is promoted, whereas levels of the neuroprotective spingosine-1-phosphate are reduced. Moreover, ND are characterized by alterations of the metabolism of complex sphingolipids. The finding that altered sphingolipid metabolism has a role in ND suggests that its modulation might provide a useful strategy to identify targets for possible therapies. In this review, based on the current literature, we will discuss how bioactive sphingolipids (spingosine-1-phosphate and ceramide) are involved in some ND (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis) and their possible involvement in therapies.

The Challenge of Long COVID-19 Management: From Disease Molecular Hallmarks to the Proposal of Exercise as Therapy.

Long coronavirus disease 19 (COVID-19) is the designation given to a novel syndrome that develops within a few months after infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and that is presenting with increasing incidence because of the numerous cases of infection. Long COVID-19 is characterized by a sequela of clinical symptoms that concern different organs and tissues, from nervous, respiratory, gastrointestinal, and renal systems to skeletal muscle and cardiovascular apparatus. The main common molecular cause for all long COVID-19 facets appears to be related to immune dysregulations, the persistence of inflammatory status, epigenetic modifications, and alterations of neurotrophin release. The prevention and management of long COVID-19 are still inappropriate because many aspects need further clarification. Exercise is known to exert a deep action on molecular dysfunctions elicited by long COVID-19 depending on training intensity, duration, and continuity. Evidence suggests that it could improve the quality of life of long COVID-19 patients. This review explores the main clinical features and the known molecular mechanisms underlying long COVID-19 in the perspective of considering exercise as a co-medication in long COVID-19 management.

The role of Sphingolipids in myelination and myelin stability and their involvement in childhood and adult demyelinating disorders.

Multiple sclerosis (MS) represents the most common demyelinating disease affecting the central nervous system (CNS) in adults as well as in children. Furthermore, in children, in addition to acquired diseases such as MS, genetically inherited diseases significantly contribute to the incidence of demyelinating disorders. Some genetic defects lead to sphingolipid alterations that are able to elicit neurological symptoms. Sphingolipids are essential for brain development, and their aberrant functionality may thus contribute to demyelinating diseases such as MS. In particular, sphingolipidoses caused by deficits of sphingolipid-metabolizing enzymes, are often associated with demyelination. Sphingolipids are not only structural molecules but also bioactive molecules involved in the regulation of cellular events such as development of the nervous system, myelination and maintenance of myelin stability. Changes in the sphingolipid metabolism deeply affect plasma membrane organization. Thus, changes in myelin sphingolipid composition might crucially contribute to the phenotype of diseases characterized by demyelinalization. Here, we review key features of several sphingolipids such as ceramide/dihydroceramide, sphingosine/dihydrosphingosine, glucosylceramide and, galactosylceramide which act in myelin formation during rat brain development and in human brain demyelination during the pathogenesis of MS, suggesting that this knowledge could be useful in identifying targets for possible therapies.

The Role of Sphingolipids in Cancer Immunotherapy.

Immunotherapy is now considered an innovative and strong strategy to beat metastatic, drug-resistant, or relapsing tumours. It is based on the manipulation of several mechanisms involved in the complex interplay between cancer cells and immune system that culminates in a form of immune-tolerance of tumour cells, favouring their expansion. Current immunotherapies are devoted enforcing the immune response against cancer cells and are represented by approaches employing vaccines, monoclonal antibodies, interleukins, checkpoint inhibitors, and chimeric antigen receptor (CAR)-T cells. Despite the undoubted potency of these treatments in some malignancies, many issues are being investigated to amplify the potential of application and to avoid side effects. In this review, we discuss how sphingolipids are involved in interactions between cancer cells and the immune system and how knowledge in this topic could be employed to enhance the efficacy of different immunotherapy approaches. In particular, we explore the following aspects: how sphingolipids are pivotal components of plasma membranes and could modulate the functionality of surface receptors expressed also by immune cells and thus their functionality; how sphingolipids are related to the release of bioactive mediators, sphingosine 1-phosphate, and ceramide that could significantly affect lymphocyte egress and migration toward the tumour milieu, in addition regulating key pathways needed to activate immune cells; given the renowned capability of altering sphingolipid expression and metabolism shown by cancer cells, how it is possible to employ sphingolipids as antigen targets.

Extracellular Sphingosine-1-Phosphate Downstream of EGFR Increases Human Glioblastoma Cell Survival.

Sphingosine-1-phosphate (S1P) is a crucial mediator involved in the progression of different cancers, including glioblastoma multiforme (GBM), the most frequent and deadly human brain tumor, characterized by extensive invasiveness and rapid cell growth. Most of GBMs overexpress the epidermal growth factor receptor (EGFR), and we investigated the possible link between S1P and EGFR signaling pathways, focusing on its role in GBM survival, using the U87MG human cell line overexpressing EGFR (EGFR+). We previously demonstrated that EGFR+ cells have higher levels of extracellular S1P and increased sphingosine kinase-1 (SK1) activity than empty vector expressing cells. Notably, we demonstrated that EGFR+ cells are resistant to temozolomide (TMZ), the standard chemotherapeutic drug in GBM treatment, and the inhibition of SK1 or S1P receptors made EGFR+ cells sensitive to TMZ; moreover, exogenous S1P reverted this effect, thus involving extracellular S1P as a survival signal in TMZ resistance in GBM cells. In addition, both PI3K/AKT and MAPK inhibitors markedly reduced cell survival, suggesting that the enhanced resistance to TMZ of EGFR+ cells is dependent on the increased S1P secretion, downstream of the EGFR-ERK-SK1-S1P pathway. Altogether, our study provides evidence of a functional link between S1P and EGFR signaling pathways enhancing the survival properties of GBM cells.

Lipid rafts and neurodegeneration: structural and functional roles in physiologic aging and neurodegenerative diseases.

Lipid rafts are small, dynamic membrane areas characterized by the clustering of selected membrane lipids as the result of the spontaneous separation of glycolipids, sphingolipids, and cholesterol in a liquid-ordered phase. The exact dynamics underlying phase separation of membrane lipids in the complex biological membranes are still not fully understood. Nevertheless, alterations in the membrane lipid composition affect the lateral organization of molecules belonging to lipid rafts. Neural lipid rafts are found in brain cells, including neurons, astrocytes, and microglia, and are characterized by a high enrichment of specific lipids depending on the cell type. These lipid rafts seem to organize and determine the function of multiprotein complexes involved in several aspects of signal transduction, thus regulating the homeostasis of the brain. The progressive decline of brain performance along with physiological aging is at least in part associated with alterations in the composition and structure of neural lipid rafts. In addition, neurodegenerative conditions, such as lysosomal storage disorders, multiple sclerosis, and Parkinson's, Huntington's, and Alzheimer's diseases, are frequently characterized by dysregulated lipid metabolism, which in turn affects the structure of lipid rafts. Several events underlying the pathogenesis of these diseases appear to depend on the altered composition of lipid rafts. Thus, the structure and function of lipid rafts play a central role in the pathogenesis of many common neurodegenerative diseases.jlr;61/5/636/F1F1f1.

Detrimental and protective action of microglial extracellular vesicles on myelin lesions: astrocyte involvement in remyelination failure.

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.

A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

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 ß-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., Soldà, 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.

Human Remyelination Promoting Antibody Stimulates Astrocytes Proliferation Through Modulation of the Sphingolipid Rheostat in Primary Rat Mixed Glial Cultures.

Remyelination promoting human IgMs effectively increase the number of myelinated axons in animal models of multiple sclerosis. Hence, they ultimately stimulate myelin production by oligodendrocytes (OLs); however, their exact mechanism of action remains to be elucidated, and in particular, it remains unclear whether they are directly targeting OLs, or their action is mediated by effects on other cell types. We assessed the effect of remyelination promoting antibody rHIgM22 on the proliferative response and on the ceramide/sphingosine 1-phosphate rheostat in mixed glial cell cultures (MGCs). rHIgM22 treatment caused a time-dependent increase in PDGFαR protein in MGCs. Forty-eight hours of treatment with rHIgM22 induced a dose-dependent proliferative response (evaluated as total cell number and as EdU(+) cell number) in MGCs. When the proliferation response of MGCs to rHIgM22 was analyzed as a function of the cell types, the most significant proliferative response was associated with GLAST(+) cells, i.e., astrocytes. In many cell types, the balance between different sphingolipid mediators (the "sphingolipid rheostat"), in particular ceramide and sphingosine 1-phosphate, is critical in determining the cell fate. rHIgM22 treatment in MGCs induced a moderate but significant inhibition of total acidic sphingomyelinase activity (measured in vitro on cell lysates), the main enzyme responsible for the stimulus-mediated production of ceramide, when treatment was performed in serum containing medium, but no significant differences were observed when antibody treatment was performed in the absence of serum. Moreover, rHIgM22 treatment, either in the presence or in absence of serum, had no effects on ceramide levels. On the other hand, rHIgM22 treatment for 24 h induced increased production and release of sphingosine 1-phosphate in the extracellular milieu of MGC. Release of sphingosine 1-phosphate upon rHIgM22 treatment was strongly reduced by a selective inhibitor of PDGFαR. Increased sphingosine 1-phosphate production does not seem to be mediated by regulation of the biosynthetic enzymes, sphingosine kinase 1 and 2, since protein levels of these enzymes and phosphorylation of sphingosine kinase 1 were unchanged upon rHIgM22 treatment. Instead, we observed a significant reduction in the levels of sphingosine 1-phosphate lyase 1, one of the key catabolic enzymes. Remarkably, rHIgM22 treatment under the same experimental conditions did not induce changes in the production and/or release of sphingosine 1-phosphate in pure astrocyte cultures. Taken together, these data suggest that rHIgM22 indirectly influences the proliferation of astrocytes in MGCs, by affecting the ceramide/sphingosine 1-phosphate balance. The specific cell population directly targeted by rHIgM22 remains to be identified, however our study unveils another aspect of the complexity of rHIgM22-induced remyelinating effect.

Role of sphingolipids in the biogenesis and biological activity of extracellular vesicles.

Extracellular vesicles (EVs) are membrane vesicles released by both eukaryotic and prokaryotic cells; they not only serve physiological functions, such as disposal of cellular components, but also play pathophysiologic roles in inflammatory and degenerative diseases. Common molecular mechanisms for EV biogenesis are evident in different cell biological contexts across eukaryotic phyla, and inhibition of this biogenesis may provide an avenue for therapeutic research. The involvement of sphingolipids (SLs) and their enzymes on EV biogenesis and release has not received much attention in current research. Here, we review how SLs participate in EV biogenesis by shaping membrane curvature and how they contribute to EV action in target cells. First, we describe how acid and neutral SMases, by generating the constitutive SL, ceramide, facilitate biogenesis of EVs at the plasma membrane and inside the endocytic compartment. We then discuss the involvement of other SLs, such as sphingosine-1-phosphate and galactosyl-sphingosine, in EV formation and cargo sorting. Last, we look ahead at some biological effects of EVs mediated by changes in SL levels in recipient cells.

Abiraterone and Ionizing Radiation Alter the Sphingolipid Homeostasis in Prostate Cancer Cells.

Prostate cancer (PC) is one of the most common leading causes of cancer-related death in men. Currently, the main therapeutic approaches available for PC are based on the androgen deprivation and on radiotherapy. However, despite these treatments being initially effective in cancer remission, several patients undergo recurrence, developing a most aggressive and resistant PC.Emerging evidence showed that abiraterone acetate drug will reduce PC recurrence by a mechanism independent of the inhibition of Cytochrome P450 17α-hydroxylase/17,20-lyase. Here we describe the involvement in the abiraterone-mediated PC cell death of a particular class of bioactive lipids called sphingolipids (SL). Sphingolipids are components of plasma membrane (PM) that organize macromolecular complexes involved in the control of several signaling pathways including the tumor cell death induced by radiotherapy. Here, we show for the first time that both in androgen-sensitive and insensitive PC cells abiraterone and ionizing radiation induce a reorganization of the plasma membrane SL composition. This event is triggered by activation of the PM-associated glycohydrolases that induce the production of cytotoxic ceramide by the in situ hydrolyses of glycosphingolipids. Taken together our data open a new scenario on the SL involvement in the therapy of PC.

Sphingosine-1-Phosphate (S1P) Impacts Presynaptic Functions by Regulating Synapsin I Localization in the Presynaptic Compartment.

Growing evidence indicates that sphingosine-1-P (S1P) upregulates glutamate secretion in hippocampal neurons. However, the molecular mechanisms through which S1P enhances excitatory activity remain largely undefined. The aim of this study was to identify presynaptic targets of S1P action controlling exocytosis. Confocal analysis of rat hippocampal neurons showed that S1P applied at nanomolar concentration alters the distribution of Synapsin I (SynI), a presynaptic phosphoprotein that controls the availability of synaptic vesicles for exocytosis. S1P induced SynI relocation to extrasynaptic regions of mature neurons, as well as SynI dispersion from synaptic vesicle clusters present at axonal growth cones of developing neurons. S1P-induced SynI relocation occurred in a Ca(2+)-independent but ERK-dependent manner, likely through the activation of S1P3 receptors, as it was prevented by the S1P3 receptor selective antagonist CAY1044 and in neurons in which S1P3 receptor was silenced. Our recent evidence indicates that microvesicles (MVs) released by microglia enhance the metabolism of endogenous sphingolipids in neurons and stimulate excitatory transmission. We therefore investigated whether MVs affect SynI distribution and whether endogenous S1P could be involved in the process. Analysis of SynI immunoreactivity showed that exposure to microglial MVs induces SynI mobilization at presynaptic sites and growth cones, whereas the use of inhibitors of sphingolipid cascade identified S1P as the sphingolipid mediating SynI redistribution. Our data represent the first demonstration that S1P induces SynI mobilization from synapses, thereby indicating the phosphoprotein as a novel target through which S1P controls exocytosis. SIGNIFICANCE STATEMENT: Growing evidence indicates that the bioactive lipid sphingosine and its metabolite sphingosine-1-P (S1P) stimulate excitatory transmission. While it has been recently clarified that sphingosine influences directly the exocytotic machinery by activating the synaptic vesicle protein VAMP2 to form SNARE fusion complexes, the molecular mechanism by which S1P promotes neurotransmission remained largely undefined. In this study, we identify Synapsin I, a presynaptic phosphoprotein involved in the control of availability of synaptic vesicles for exocytosis, as the key target of S1P action. In addition, we provide evidence that S1P can be produced at mature axon terminals as well as at immature growth cones in response to microglia-derived signals, which may be important to stabilize nascent synapses and to restore or potentiate transmission.

Evidence for the Involvement of Lipid Rafts and Plasma Membrane Sphingolipid Hydrolases in Pseudomonas aeruginosa Infection of Cystic Fibrosis Bronchial Epithelial Cells.

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.

Microvesicles released from microglia stimulate synaptic activity via enhanced sphingolipid metabolism.

Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell-to-cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia-to-neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose-dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired-pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro, injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia-derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microglia-to-neuron signalling pathway.

Role of palmitate-induced sphingoid base-1-phosphate biosynthesis in INS-1 ß-cell survival.

Sphingoid base-1-phosphates represent a very low portion of the sphingolipid pool but are potent bioactive lipids in mammals. This study was undertaken to determine whether these lipids are produced in palmitate-treated pancreatic ß cells and what role they play in palmitate-induced ß cell apoptosis. Our lipidomic analysis revealed that palmitate at low and high glucose supplementation increased (dihydro)sphingosine-1-phosphate levels in INS-1 ß cells. This increase was associated with an increase in sphingosine kinase 1 (SphK1) mRNA and protein levels. Over-expression of SphK1 in INS-1 cells potentiated palmitate-induced accumulation of dihydrosphingosine-1-phosphate. N,N-dimethyl-sphingosine, a potent inhibitor of SphK, potentiated ß-cell apoptosis induced by palmitate whereas over-expression of SphK1 significantly reduced apoptosis induced by palmitate with high glucose. Endoplasmic reticulum (ER)-targeted SphK1 also partially inhibited apoptosis induced by palmitate. Inhibition of INS-1 apoptosis by over-expressed SphK1 was independent of sphingosine-1-phosphate receptors but was associated with a decreased formation of pro-apoptotic ceramides induced by gluco-lipotoxicity. Moreover, over-expression of SphK1 counteracted the defect in the ER-to-Golgi transport of proteins that contribute to the ceramide-dependent ER stress observed during gluco-lipotoxicity. In conclusion, our results suggest that activation of palmitate-induced SphK1-mediated sphingoid base-1-phosphate formation in the ER of ß cells plays a protective role against palmitate-induced ceramide-dependent apoptotic ß cell death.

Autocrine/paracrine sphingosine-1-phosphate fuels proliferative and stemness qualities of glioblastoma stem cells.

Accumulating reports suggest that human glioblastoma contains glioma stem-like cells (GSCs) which act as key determinants driving tumor growth, angiogenesis, and contributing to therapeutic resistance. The proliferative signals involved in GSC proliferation and progression remain unclear. Using GSC lines derived from human glioblastoma specimens with different proliferative index and stemness marker expression, we assessed the hypothesis that sphingosine-1-phosphate (S1P) affects the proliferative and stemness properties of GSCs. The results of metabolic studies demonstrated that GSCs rapidly consume newly synthesized ceramide, and export S1P in the extracellular environment, both processes being enhanced in the cells exhibiting high proliferative index and stemness markers. Extracellular S1P levels reached nM concentrations in response to increased extracellular sphingosine. In addition, the presence of EGF and bFGF potentiated the constitutive capacity of GSCs to rapidly secrete newly synthesized S1P, suggesting that cooperation between S1P and these growth factors is of central importance in the maintenance and proliferation of GSCs. We also report for the first time that S1P is able to act as a proliferative and pro-stemness autocrine factor for GSCs, promoting both their cell cycle progression and stemness phenotypic profile. These results suggest for the first time that the GSC population is critically modulated by microenvironmental S1P, this bioactive lipid acting as an autocrine signal to maintain a pro-stemness environment and favoring GSC proliferation, survival and stem properties.