Sphingosine 1-Phosphate Receptors in Cerebral Ischemia.

Sphingosine 1-phosphate (S1P) is an important lipid biomolecule that exerts pleiotropic cellular actions as it binds to and activates its five G-protein-coupled receptors, S1P1-5. Through these receptors, S1P can mediate diverse biological activities in both healthy and diseased conditions. S1P is produced by S1P-producing enzymes, sphingosine kinases (SphK1 and SphK2), and is abundantly present in different organs, including the brain. The medically important roles of receptor-mediated S1P signaling are well characterized in multiple sclerosis because FTY720 (Gilenya™, Novartis), a non-selective S1P receptor modulator, is currently used as a treatment for this disease. In cerebral ischemia, its role is also notable because of FTY720's efficacy in both rodent models and human patients with cerebral ischemia. In particular, some of the S1P receptors, including S1P1, S1P2, and S1P3, have been identified as pathogenic players in cerebral ischemia. Other than these receptors, S1P itself and S1P-producing enzymes have been shown to play certain roles in cerebral ischemia. This review aims to compile the current updates and overviews about the roles of S1P signaling, along with a focus on S1P receptors in cerebral ischemia, based on recent studies that used in vivo rodent models of cerebral ischemia.

Preclinical and Clinical Evidence for the Involvement of Sphingosine 1-Phosphate Signaling in the Pathophysiology of Vascular Cognitive Impairment.

Sphingosine 1-phosphates (S1Ps) are bioactive lipids that mediate a diverse range of effects through the activation of cognate receptors, S1P1-S1P5. Scrutiny of S1P-regulated pathways over the past three decades has identified important and occasionally counteracting functions in the brain and cerebrovascular system. For example, while S1P1 and S1P3 mediate proinflammatory effects on glial cells and directly promote endothelial cell barrier integrity, S1P2 is anti-inflammatory but disrupts barrier integrity. Cumulatively, there is significant preclinical evidence implicating critical roles for this pathway in regulating processes that drive cerebrovascular disease and vascular dementia, both being part of the continuum of vascular cognitive impairment (VCI). This is supported by clinical studies that have identified correlations between alterations of S1P and cognitive deficits. We review studies which proposed and evaluated potential mechanisms by which such alterations contribute to pathological S1P signaling that leads to VCI-associated chronic neuroinflammation and neurodegeneration. Notably, S1P receptors have divergent but overlapping expression patterns and demonstrate complex interactions. Therefore, the net effect produced by S1P represents the cumulative contributions of S1P receptors acting additively, synergistically, or antagonistically on the neural, vascular, and immune cells of the brain. Ultimately, an optimized therapeutic strategy that targets S1P signaling will have to consider these complex interactions.

The Role of Sphingolipids and Specialized Pro-Resolving Mediators in Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia worldwide giving rise to devastating forms of cognitive decline, which impacts patients' lives and that of their proxies. Pathologically, AD is characterized by extracellular amyloid deposition, neurofibrillary tangles and chronic neuroinflammation. To date, there is no cure that prevents progression of AD. In this review, we elaborate on how bioactive lipids, including sphingolipids (SL) and specialized pro-resolving lipid mediators (SPM), affect ongoing neuroinflammatory processes during AD and how we may exploit them for the development of new biomarker panels and/or therapies. In particular, we here describe how SPM and SL metabolism, ranging from ω-3/6 polyunsaturated fatty acids and their metabolites to ceramides and sphingosine-1-phosphate, initiates pro- and anti-inflammatory signaling cascades in the central nervous system (CNS) and what changes occur therein during AD pathology. Finally, we discuss novel therapeutic approaches to resolve chronic neuroinflammation in AD by modulating the SPM and SL pathways.

[Basic mechanisms of action of fingolimod in relation to multiple sclerosis]. / Mecanismos basicos de accion del fingolimod en relacion con la esclerosis multiple.

INTRODUCTION: Fingolimod has recently been approved for the therapy of relapsing multiple sclerosis. This drug binds to different sphingosine-1-phosphate receptors. AIM: To analyze basic mechanisms of action that can account for the efficacy of this drug in multiple sclerosis. DEVELOPMENT: Fingolimod acts as an inverse agonist on sphingosine-1-phosphate receptors, inducing degradation of receptors. On lymphoid circulation, this effect causes retention in lymph nodes of naive and central memory T cells, including Th17 T lymphocytes, bearing CCR7 and CD62L receptors. As a result, the level of circulating T cells is markedly decreased. B ell circulation is impaired and complex effects on other immune cells are also induced. Fingolimod enters the central nervous system and binds to receptors on glial cells and neurons. In experimental autoimmune encephalomyelitis, the therapeutic efficacy of fingolimod is not only associated with a reduced entry of inflammatory cells into the nervous system, but also with a direct effect mostly on astroglial cells. CONCLUSIONS: In multiple sclerosis patients, the available evidence indicates that fingolimod efficacy is directly associated with impairment of circulation of several T cell subsets and possibly B cells. Animal studies raise the possibility that an additional effect on glial cells might also contribute to the clinical efficacy.

The reduction of allograft arteriosclerosis in intestinal transplant is associated with sphingosine kinase 1/sphingosine-1-phosphate signaling after fish oil treatment.

BACKGROUND: Transplant arteriosclerosis is a major cause of late intestinal allograft dysfunction. However, little is known about the immunologic and molecular mechanisms underlying it, and no effective treatment is available. This study aimed to investigate the role of sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) in transplant arteriosclerosis and find out whether fish oil (FO) attenuates allograft arteriosclerosis through S1P signaling. METHODS: A rat model with orthotopic intestinal transplantation was conducted in this study. Animals received daily FO supplementation after intestinal transplant. The allogeneic recipients by phosphate-buffered saline or corn oil treatment served as controls. The allograft arteriosclerosis was characterized, and the expression of SPHK1 and S1P receptors (S1P1, S1P2, and S1P3) was determined on day 190 posttransplant. RESULTS: The allogeneic controls presented transplant vasculopathy in mesenteric vessels, including intimal thickening, fibrosis, and leukocyte infiltration. The transplant arteriosclerosis was markedly reduced in FO-fed animals. The pression of SPHK1 and its activity were significantly augmented, and the expression of S1P1 and S1P3 messenger RNA was up-regulated in the allogeneic controls. FO supplementation suppressed the activation of SPHK1 and led to a decrease in the expression of S1P1 and S1P3 in these tissues in transplant arteriosclerosis. CONCLUSIONS: These results demonstrate that the activation of SPHK1/S1P signaling plays a possible role in the pathogenesis of transplant arteriosclerosis. The reduction of allograft arteriosclerosis by FO may be associated with down-regulation of SPHK1/S1P signaling. Understanding the role of FO for SPHK1/S1P may help us to identify considerable therapeutic targets for transplant arteriosclerosis.

Sphingosine-1-phosphate is a key regulator of proliferation and differentiation in retina photoreceptors.

PURPOSE: Identifying the cues required for the survival and development of photoreceptors is essential for treating retinal neurodegeneration. The authors previously established that glial-derived neurotrophic factor (GDNF) stimulates proliferation and that docosahexaenoic acid (DHA) promotes photoreceptor survival and differentiation. Later findings that ceramide triggers photoreceptor apoptosis suggested sphingolipids might also control photoreceptor development. The present study investigated whether sphingosine-1-phophate (S1P), which promotes survival and differentiation in several cell types, regulates photoreceptor proliferation and differentiation and whether it is a mediator in GDNF and DHA effects. METHODS: Rat retina neuronal cultures were supplemented at day 0 or 1 with S1P, GDNF, or DHA and were treated with DL-threo-dihydrosphingosine to inhibit S1P synthesis or with brefeldin A (BFA) to block intracellular trafficking. Proliferation was quantified to determine bromodeoxyuridine uptake and number of mitotic figures. Opsin, peripherin, and sphingosine kinase (SphK), the enzyme required for S1P synthesis, were quantified by immunocytochemistry and Western blot analysis. RESULTS: S1P increased the proliferation of photoreceptor progenitors. It also stimulated the formation of apical processes, enhanced opsin and peripherin expression, and promoted their localization in these processes; DHA had similar effects. BFA prevented S1P and DHA enhancement of apical process formation without affecting opsin expression. GDNF and DHA enhanced SphK expression in photoreceptors, while inhibiting S1P synthesis blocked GDNF mitogenic effects and DHA effects on differentiation. CONCLUSIONS: The authors propose S1P as a key regulator in photoreceptor development. GDNF and DHA might upregulate SphK levels to promote S1P synthesis, which would initially promote proliferation and then advance photoreceptor differentiation.

Antagonism of sphingosine 1-phosphate receptor-2 enhances migration of neural progenitor cells toward an area of brain.

BACKGROUND AND PURPOSE: We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P(1)R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR. METHODS: S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P(2)R antagonist JTE-013. RESULTS: The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P(1)R and S1P(2)R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P(1)R. However, an S1P(1)R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P(2)R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia. CONCLUSIONS: Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P(2)R function further enhances the migration of NPCs toward a brain infarction.

Sphingosine-1-phosphate and endothelin-1 induce the expression of rgs16 protein in cardiac myocytes by transcriptional activation of the rgs16 gene.

The expression of the negative Regulator of G protein signaling 16 (RGS16) is rapidly induced in cardiomyocytes by various stimuli. To identify the promoter of the mouse RGS16 gene, a 1.8-kb deoxyribonucleic acid fragment 5' of the RGS16-coding region was subcloned into a firefly-luciferase reporter vector and four overlapping fragments were analyzed. The luciferase production was quantified in neonatal rat cardiac myocytes (NRCM). A 0.6-kb fragment that induced a tenfold increase in luciferase activity contained the minimal promoter sequence. Its activity was twofold stimulated by fetal calf serum, endothelin-1 (ET-1), and sphingosine 1-phosphate (S1P), which stimuli also elevated the level of RGS16 protein. Stimulation of NRCM with ET-1 induced activation of the monomeric GTPases RhoA and Rac1, whereas S1P and the selective S1P1 receptor agonist SEW2871 only induced a pronounced activation of Rac1. In accordance, the treatment with the Rho-, Rac-, and Cdc42-inactivating Clostridium difficile Toxin B (TcdB) 10463 inhibited ET-1 and S1P-induced transcriptional activation. The ET-1-induced activation was insensitive to pertussis toxin but selectively suppressed by the RhoA-C-specific C2I-C3 ADP-ribosyl transferase and the ET(B) receptor antagonist BQ788. The S1P-induced activation was specifically inhibited by pertussis toxin and the Rac-inactivating TcdB 1470. All stimulated transcriptional activity was abolished by the negative transcription factor Yin Yang 1 (YY1), which binds to a consensus sequence within the minimal promoter. Taken together, our data show that most likely ET(B)- and S1P1-receptors induce RGS16 protein expression in cardiac myocytes by increasing the transcriptional activity of the rgs16 gene. This activation is mediated by heterotrimeric G proteins, Rho GTPases, and is under negative control of the transcription factor YY1.

Sphingosine 1-phosphate protects mouse extensor digitorum longus skeletal muscle during fatigue.

Sphingomyelin derivatives exert various second messenger actions in numerous tissues. Sphingosine (SPH) and sphingosine 1-phosphate (S1P) are two major sphingomyelin derivatives present at high levels in blood. The aim of the present work was to investigate whether S1P and SPH exert relevant actions in mouse skeletal muscle contractility and fatigue. Exogenous S1P and SPH administration caused a significant reduction of tension decline during fatigue of extensor digitorum longus muscle. Final tension after the fatiguing protocol was 40% higher than in untreated muscle. Interestingly, N,N-dimethylsphingosine, an inhibitor of SPH kinase (SK), abolished the effect of supplemented SPH but not that of S1P, suggesting that SPH acts through its conversion to S1P. Moreover, SPH was not effective in Ca(2+)-free solutions, in agreement with the hypothesis that SPH action is dependent on its conversion to S1P by the Ca(2+)-requiring enzyme SK. In contrast to SPH, S1P produced its positive effects on fatigue in Ca(2+)-free conditions, indicating that S1P action does not require Ca(2+) entry and most likely is receptor mediated. The effects of S1P could be ascribed in part to its ability to prevent the reduction (-20 mV) of action potential amplitude caused by fatigue. In conclusion, these results indicate that extracellular S1P has protective effects during the development of muscle fatigue and that the extracellular conversion of SPH to S1P may represent a rheostat mechanism to protect skeletal muscle from possible cytotoxic actions of SPH.

Modulation of protein kinase C by endogenous sphingosine: inhibition of phorbol dibutyrate binding in Niemann-Pick C fibroblasts.

The abnormal and variable increase in levels of free sphingoid bases recently described in fibroblasts from Niemann-Pick C patients allowed us to investigate the modulation of protein kinase C in vivo by endogenous sphingosine. The specific binding of [20-3H]phorbol 12, 13-dibutyrate to the regulatory domain of membrane-bound protein kinase C was significantly decreased in fibroblasts from patients compared with controls. A pronounced difference between the two groups (P<0.0001) was demonstrated in low-density lipoprotein-supplemented medium, i.e. under conditions known to disclose abnormal mobilization of unesterified cholesterol in Niemann-Pick C fibroblasts. Furthermore the degree of impairment of [3H]phorbol 12,13-dibutyrate binding was highly correlated (r=0.95) with the sphingosine levels measured in fibroblasts from those patients. Scatchard analysis of the binding data indicated that Niemann-Pick C and control fibroblasts contained almost the same number of binding sites per cell. A 8-34-fold increase in Kd was measured in Niemann-Pick C fibroblasts with at least a 5-fold increase in sphingosine levels. Removal, by cell fractionation, of membrane-bound protein kinase C from the bulk of sphingosine induced a normalization of Kd values. The overall results suggest that protein kinase C inhibition is directly related to sphingosine accumulation.