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.

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.

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.

Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling.

Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2-/- thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2-/- animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.

[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.

Lysophosphatidic acid enhances antimycobacterial activity both in vitro and ex vivo.

Lysophosphatidic acid (LPA) is a polar lipid metabolite which is involved in a wide range of biological processes, including cell proliferation and migration, wound healing, and increase of endothelial permeability. The present study reports evidences showing that LPA is able to enhance the antimicrobial activity of human macrophages and of bronchoalveolar lavage cells from tuberculosis patients leading to intracellular growth control of Mycobacterium tuberculosis. Such antimicrobial activity is mediated by the activation of phospholipase D which in turn induces acidification of M. tuberculosis containing phagosomes and is associated with the enhanced expression of Cathepsin D. These results suggest the possible protective role of this lysophospholipid in the activation of innate antimycobacterial response.

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.

In vivo modification of the UDP-glucuronosyltransferase functional state in rat liver following hypophysectomy and partial or complete hormonal restoration.

The effects of growth hormone on the uridine diphosphate glucuronosyltransferase functional state, biophysical membrane parameters (order parameters and rotational correlation frequency) and the composition in phospholipids were studied in male rat hepatic microsomes. Sham-operated and hypophysectomized animals were injected with two different dosages of growth hormone, mimicking either the male or female growth hormone secretion pattern. Half the animals received thyroxine and cortisol in concentrations chosen to compensate for the lack of thyroid hormones and glucocorticoids in hypophysectomized rats. Growth hormone treatment resulted in a decrease in the latency (that gives a quantification of uridine diphosphate glucuronosyltransferase functional state) of the glucuronidation activities towards various substrates (testosterone, androsterone, bilirubin and 4-nitrophenol). This decrease with growth hormone treatment was particularly evident in hypophysectomized animals that had received cortisol and thyroxine supplementation treatment. These modifications were strongly correlated with modifications in the microsomal membrane lysophospholipid content and to a lower extent with microsomal membrane fatty acid composition. The cytosolic phospholipase A(2)-dependent increase in the lysophospholipid content in the endoplasmic reticulum is probably a major determinant in the regulation of the functional state of glucuronoyltransferases in response to high dosage growth hormone treatment.

The influence of lysophosphatidic acid on the functions of human dendritic cells.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator which is generated by secretory phospholipase A(2). In this study, we studied the biological activity of LPA on human dendritic cells (DCs), which are specialized APCs characterized by their ability to migrate into target sites and secondary lymphoid organs to process Ags and activate naive T cells. We show that immature and mature DCs express the mRNA for different LPA receptors such as endothelial differentiation gene (EDG)-2, EDG-4, and EDG-7. In immature DCs, LPA stimulated pertussis toxin-sensitive Ca(2+) increase, actin polymerization, and chemotaxis. During the maturation process, DCs lost their ability to respond toward LPA with Ca(2+) transients, actin polymerization, and chemotaxis. However, LPA inhibited in a pertussis toxin-insensitive manner the secretion of IL-12 and TNFalpha as well as enhanced secretion of IL-10 from mature DCs. Moreover, LPA did not affect the endocytic or phagocytic capacities and the surface phenotype of DCs, although it increased the allostimulatory function of mature DC and inhibited their capacity to induce Th1 differentiation. In summary, our study implicates that LPA might regulate the trafficking, cytokine production, and T cell-activating functions of DCs.

Identification of the serum factor required for in vitro activation of macrophages. Role of vitamin D3-binding protein (group specific component, Gc) in lysophospholipid activation of mouse peritoneal macrophages.

In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.