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 microgliato-neuron signalling pathway.

Alzheimer's disease (AD) is characterized by extracellular amyloid-b (Ab) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Ab species, rather than insoluble fibrils, are the most toxic forms of Ab. Preventing soluble Ab 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 Ab species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Ab forms trafficked to MVs after Ab internalization into microglia. MV neurotoxicity was neutralized by the Ab-interacting protein PrP and anti-Ab antibodies, which prevented binding to neurons of neurotoxic soluble Ab 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. Cell Death and Differentiation (2014) 21, 582-593; doi:10.1038/cdd.2013.180; published online 13 December 2013Alzheimer's disease (AD) is the major cause of dementia in humans. Neuronal loss and cognitive decline occurring in AD patients are traditionally linked to the accumulation in the brain of extracellular plaques consisting of short amyloid-b (Ab) peptides of 39-42 amino acids, generated by amyloidogenic cleavage of the amyloid precursor protein. 1 Among Ab peptides, Ab 1-42 and pyroglutamate-modified Ab very rapidly aggregate and initiate the complex multistep process that leads to mature fibrils and plaque. 2,3 Although association of amyloid plaques with AD has long been assumed, Ab load does not correlate with neuronal loss 4,5 and high plaque burden does not necessarily lead to dementia in humans. 6,7 Accordingly, recent evidence clearly showed that the amyloid load reaches a plateau early after the onset of clinical symptoms in AD patients 8 and does not substantially increase in size during clinical progression. 9 These observations agree with the current view that small, soluble pre-fibrillar Ab species, rather than plaques formed by insoluble Ab fibrils, are the most toxic forms of Ab. 10 These cause synaptic dysfunction and spine loss, and correlate most closely with the severity of human AD. 5,8,11 Recent biochemical studies indicated that natural sphingolipids and gangliosides, whose metabolism has been shown to be altered in AD patients, 12 destabilize and rapidly resolubilize long Ab fibrils to neurotoxic species. 13 These studies also showed that phospholipids stabilize toxic oligomers...

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.Electronic supplementary materialThe online version of this article (10.1007/s00401-019-02049-1) contains supplementary material, which is available to authorized users.

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.

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.

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.

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 C 6 -ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B 1 , 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.Sphingolipids are a structurally diverse family of membrane lipids. Several lines of evidence have implicated metabolites of sphingolipids such as ceramide, sphingosine, and sphingosine 1-phosphate (S1P) 5 in diverse cellular processes (1, 2). Ceramide and sphingosine have been implicated in pathways involving stress responses, cell differentiation, apoptosis, and cell cycle arrest (1). Unlike ceramide and sphingosine, S1P promotes cell growth and survival and inhibits apoptosis (2, 3). Because of their inter-convertibility and opposing effects, the dynamic balance between S1P and ceramide/sphingosine has been proposed to be an important factor that determines cell fate (2). Accumulating evidence suggests that the S1P/ceramide balance is ultimately regulated by the relative activities of enzymes controlling the turnover of these sphingolipid metabolites. However, the molecular mechanisms involved in the regulation of intracellular levels of these sphingolipids are not yet fully understood. Cellular levels of S1P are kept low by tight spatio-temporal regulation of its synthesis and degradation. Sphingosine kinases (SphKs) catalyze the synthesis of S1P by phosphorylation of sphingosine. Two distinct SphK isoforms, SphK1 and SphK2, have been cloned and characterized in mammals (2). Diverse external stimuli, particularly growth and survival factors, stimulate SphK1, generating S1P that has been implicated in their mitogenic and anti-apoptotic effects (4, 5). In contrast to SphK1, rather than promoting growth and survival, overexpression of SphK2 suppressed growth and...

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