Paola Giussani

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.

Sphingosine-1-phosphate is released by cerebellar astrocytes in response to bFGF and induces astrocyte proliferation through Gi-protein-coupled receptors.

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 Gi/Go‐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.

Microglia convert aggregated amyloid- β into neurotoxic forms through the shedding of microvesicles

Alzheimer’s disease (AD) is characterized by extracellular amyloid-β (Aβ) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aβ species, rather than insoluble fibrils, are the most toxic forms of Aβ. Preventing soluble Aβ formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aβ species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aβ forms trafficked to MVs after Aβ internalization into microglia. MV neurotoxicity was neutralized by the Aβ-interacting protein PrP and anti-Aβ antibodies, which prevented binding to neurons of neurotoxic soluble Aβ species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.

Recycling of sphingosine is regulated by the concerted actions of sphingosine-1-phosphate phosphohydrolase 1 and sphingosine kinase 2

In yeast, the long-chain sphingoid base phosphate phosphohydrolase Lcb3p is required for efficient ceramide synthesis from exogenous sphingoid bases. Similarly, in this study, we found that incorporation of exogenous sphingosine into ceramide in mammalian cells was regulated by the homologue of Lcb3p, sphingosine-1-phosphate phosphohydrolase 1 (SPP-1), an endoplasmic reticulum resident protein. Sphingosine incorporation into endogenous long-chain ceramides was increased by SPP-1 overexpression, whereas recycling of C6-ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B1, an inhibitor of ceramide synthase, but not by ISP-1, an inhibitor of serine palmitoyltransferase, the rate-limiting step in the de novo biosynthesis of ceramide. Mass spectrometry analysis revealed that SPP-1 expression increased the incorporation of sphingosine into all ceramide acyl chain species, particularly enhancing C16:0, C18:0, and C20:0 long-chain ceramides. The increased recycling of sphingosine into ceramide was accompanied by increased hexosylceramides and, to a lesser extent, sphingomyelins. Sphingosine kinase 2, but not sphingosine kinase 1, acted in concert with SPP-1 to regulate recycling of sphingosine into ceramide. Collectively, our results suggest that an evolutionarily conserved cycle of phosphorylation-dephosphorylation regulates recycling and salvage of sphingosine to ceramide and more complex sphingolipids.

HMGB1 as an autocrine stimulus in human T98G glioblastoma cells: role in cell growth and migration

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.

Sphingosine-1-Phosphate Phosphohydrolase Regulates Endoplasmic Reticulum-to-Golgi Trafficking of Ceramide‡

ABSTRACT Previous studies demonstrated that sphingosine-1-phosphate (S1P) phosphohydrolase 1 (SPP-1), which is located mainly in the endoplasmic reticulum (ER), regulates sphingolipid metabolism and apoptosis (H. Le Stunff et al., J. Cell Biol. 158:1039-1049, 2002). We show here that the treatment of SPP-1-overexpressing cells with S1P, but not with dihydro-S1P, increased all ceramide species, particularly the long-chain ceramides. This was not due to inhibition of ceramide metabolism to sphingomyelin or monohexosylceramides but rather to the inhibition of ER-to-Golgi trafficking, determined with the fluorescent ceramide analog N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (DMB-Cer). Fumonisin B1, an inhibitor of ceramide synthase, prevented S1P-induced elevation of all ceramide species and corrected the defect in ER transport of DMB-Cer, readily allowing its detection in the Golgi. In contrast, ceramide accumulation had no effect on either the trafficking or the metabolism of 6-([N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl)-sphingosine, which rapidly labels the Golgi even at 4°C. Protein trafficking from the ER to the Golgi, determined with vesicular stomatitis virus ts045 G protein fused to green fluorescent protein, was also inhibited in SPP-1-overexpressing cells in the presence of S1P but not in the presence of dihydro-S1P. Our results suggest that SPP-1 regulates ceramide levels in the ER and thus influences the anterograde membrane transport of both ceramide and proteins from the ER to the Golgi apparatus.

Sphingolipids: key regulators of apoptosis and pivotal players in cancer drug resistance.

Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8)αNeu5Ac(2-3)βGal(1-4)βGlc(1-1)Cer) or N-glycolyl GM3 (αNeu5Ac (2-3)βGal(1-4)βGlc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)βGal(1-4)βGlc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy.

Phosphatidylinositol 3-Kinase/AKT Pathway Regulates the Endoplasmic Reticulum to Golgi Traffic of Ceramide in Glioma Cells A LINK BETWEEN LIPID SIGNALING PATHWAYS INVOLVED IN THE CONTROL OF CELL SURVIVAL

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.

Cultured granule cells and astrocytes from cerebellum differ in metabolizing Sphingosine

Sphingosine metabolism was studied in primary cultures of differentiated cerebellar granule cells and astrocytes. After a 2‐h pulse with [C3‐3H]sphingosine at different doses (0.1‐200 nmol/mg of cell protein), both cell types efficiently incorporated the long chain base ; the percentage of cellular [3H]sphingosine over total label incorporation was extremely low at sphingosine doses of <10 nmol/mg of cell protein and increased at higher doses. Most of the [3H]sphingosine taken up underwent metabolic processing by N‐acylation, 1‐phosphorylation, and degradation (assessed as 3H2O released in the medium). The metabolic processing of exogenous sphingosine was extremely efficient in both cells, granule cells and astrocytes being able to metabolize, respectively, an amount of sphingosine up to 80‐ and 300‐fold the cellular content of this long chain base in 2 h. At the different doses, the prevailing metabolic route of sphingosine was different. At lower doses and in a wide dose range, the major metabolic fate of sphingosine was N‐acylation. With increasing doses, there was first increased sphingosine degradation and then increased levels of sphingosine‐1‐phosphate. The data demonstrate that, in neurons and astrocytes, the metabolic machinery devoted to sphingosine processing is different, astrocytes possessing an overall higher capacity to synthesize the bioactive compounds ceramide and sphingosine‐1‐phosphate.

Glucosylceramide Synthase Protects Glioblastoma Cells Against Autophagic and Apoptotic Death Induced by Temozolomide and Paclitaxel

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.