Sphingolipid changes in mouse brain and plasma after mild traumatic brain injury at the acute phases.

BACKGROUND: Traumatic brain injury (TBI) causes neuroinflammation and can lead to long-term neurological dysfunction, even in cases of mild TBI (mTBI). Despite the substantial burden of this disease, the management of TBI is precluded by an incomplete understanding of its cellular mechanisms. Sphingolipids (SPL) and their metabolites have emerged as key orchestrators of biological processes related to tissue injury, neuroinflammation, and inflammation resolution. No study so far has investigated comprehensive sphingolipid profile changes immediately following TBI in animal models or human cases. In this study, sphingolipid metabolite composition was examined during the acute phases in brain tissue and plasma of mice following mTBI. METHODS: Wildtype mice were exposed to air-blast-mediated mTBI, with blast exposure set at 50-psi on the left cranium and 0-psi designated as Sham. Sphingolipid profile was analyzed in brain tissue and plasma during the acute phases of 1, 3, and 7 days post-TBI via liquid-chromatography-mass spectrometry. Simultaneously, gene expression of sphingolipid metabolic markers within brain tissue was analyzed using quantitative reverse transcription-polymerase chain reaction. Significance (P-values) was determined by non-parametric t-test (Mann-Whitney test) and by Tukey's correction for multiple comparisons. RESULTS: In post-TBI brain tissue, there was a significant elevation of 1) acid sphingomyelinase (aSMase) at 1- and 3-days, 2) neutral sphingomyelinase (nSMase) at 7-days, 3) ceramide-1-phosphate levels at 1 day, and 4) monohexosylceramide (MHC) and sphingosine at 7-days. Among individual species, the study found an increase in C18:0 and a decrease in C24:1 ceramides (Cer) at 1 day; an increase in C20:0 MHC at 3 days; decrease in MHC C18:0 and increase in MHC C24:1, sphingomyelins (SM) C18:0, and C24:0 at 7 days. Moreover, many sphingolipid metabolic genes were elevated at 1 day, followed by a reduction at 3 days and an absence at 7-days post-TBI. In post-TBI plasma, there was 1) a significant reduction in Cer and MHC C22:0, and an increase in MHC C16:0 at 1 day; 2) a very significant increase in long-chain Cer C24:1 accompanied by significant decreases in Cer C24:0 and C22:0 in MHC and SM at 3 days; and 3) a significant increase of C22:0 in all classes of SPL (Cer, MHC and SM) as well as a decrease in Cer C24:1, MHC C24:1 and MHC C24:0 at 7 days. CONCLUSIONS: Alterations in sphingolipid metabolite composition, particularly sphingomyelinases and short-chain ceramides, may contribute to the induction and regulation of neuroinflammatory events in the early stages of TBI, suggesting potential targets for novel diagnostic, prognostic, and therapeutic strategies in the future.

Sphingolipids as critical players in retinal physiology and pathology.

Sphingolipids have emerged as bioactive lipids involved in the regulation of many physiological and pathological processes. In the retina, they have been established to participate in numerous processes, such as neuronal survival and death, proliferation and migration of neuronal and vascular cells, inflammation, and neovascularization. Dysregulation of sphingolipids is therefore crucial in the onset and progression of retinal diseases. This review examines the involvement of sphingolipids in retinal physiology and diseases. Ceramide (Cer) has emerged as a common mediator of inflammation and death of neuronal and retinal pigment epithelium cells in animal models of retinopathies such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa. Sphingosine-1-phosphate (S1P) has opposite roles, preventing photoreceptor and ganglion cell degeneration but also promoting inflammation, fibrosis, and neovascularization in AMD, glaucoma, and pro-fibrotic disorders. Alterations in Cer, S1P, and ceramide 1-phosphate may also contribute to uveitis. Notably, use of inhibitors that either prevent Cer increase or modulate S1P signaling, such as Myriocin, desipramine, and Fingolimod (FTY720), preserves neuronal viability and retinal function. These findings underscore the relevance of alterations in the sphingolipid metabolic network in the etiology of multiple retinopathies and highlight the potential of modulating their metabolism for the design of novel therapeutic approaches.

Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress.

Over 11 million people in the United States alone have some form of age-related macular degeneration (AMD). Oxidative stress, cell death, and the degeneration of retinal pigment epithelial (RPE) cells contribute to AMD pathology. Recent evidence suggests that ceramide (Cer), a cellular sphingolipid mediator that acts as a second messenger to induce apoptosis, might play a role in RPE cell death. The lysosomal breakdown of Cer by acid ceramidase [N-acylsphingosine amidohydrolase (ASAH)1] into sphingosine (Sph) is the major source for Sph 1-phosphate production, which has an opposing role to Cer and provides cytoprotection. Here, we investigated the role of Cer in human RPE-derived ARPE19 cells under hydrogen peroxide-induced oxidative stress, and show that Cer and hexosyl-Cer levels increase in the oxidatively stressed ARPE19 cells, which can be prevented by overexpression of lysosomal ASAH1. This study demonstrates that oxidative stress generates sphingolipid death mediators in retinal cells and that induction of ASAH1 could rescue retinal cells from oxidative stress by hydrolyzing excess Cers.

Inflammatory Ocular Diseases and Sphingolipid Signaling.

Inflammation is a powerful immune countermeasure to tissue damage and infection. The inflammatory response is complex and requires the involvement of myriad signaling pathways and metabolic processes, all governed by a multitude of regulatory systems. Although inflammation is a vital defense against tissue injury and a necessary step in tissue healing, the mechanisms which modulate the initiation, intensity, and duration of this innate immune response can malfunction and result in inappropriate or out-of-control inflammation, even in the absence of an appropriate stimulus. Though the human eye exists in an immune-privileged microenvironment, it is not spared from this. The eye is neither devoid of immune cells nor is it fully sequestered from systemic immune responses, and is therefore fully capable of ruining itself through localized inflammatory dysfunction and systemic inflammatory disease (Taylor AW, Front Immunol 7:37, 2016; Zhou R, Caspi RR, Biol Rep 2, 2010). In fact, a wide range of ocular inflammatory diseases exist and are major causes of blindness in humans. Advances in the understanding of inflammatory processes have revealed new key pathways and molecular factors involved in the mechanisms of inflammation. Lipids and sphingolipids are increasingly being recognized as having important signaling roles in the pathophysiology of ocular inflammatory diseases. What follows below is a discussion of fundamental inflammatory processes, the place of sphingolipids as mediators of said processes, brief descriptions of major inflammatory ocular diseases, and new findings implicating sphingolipids in their pathogenesis.

Characterizing Sphingosine Kinases and Sphingosine 1-Phosphate Receptors in the Mammalian Eye and Retina.

Sphingosine 1-phosphate (S1P) signaling regulates numerous biological processes including neurogenesis, inflammation and neovascularization. However, little is known about the role of S1P signaling in the eye. In this study, we characterize two sphingosine kinases (SPHK1 and SPHK2), which phosphorylate sphingosine to S1P, and three S1P receptors (S1PR1, S1PR2 and S1PR3) in mouse and rat eyes. We evaluated sphingosine kinase and S1P receptor gene expression at the mRNA level in various rat tissues and rat retinas exposed to light-damage, whole mouse eyes, specific eye structures, and in developing retinas. Furthermore, we determined the localization of sphingosine kinases and S1P receptors in whole rat eyes by immunohistochemistry. Our results unveiled unique expression profiles for both sphingosine kinases and each receptor in ocular tissues. Furthermore, these kinases and S1P receptors are expressed in mammalian retinal cells and the expression of SPHK1, S1PR2 and S1PR3 increased immediately after light damage, which suggests a function in apoptosis and/or light stress responses in the eye. These findings have numerous implications for understanding the role of S1P signaling in the mechanisms of ocular diseases such as retinal inflammatory and degenerative diseases, neovascular eye diseases, glaucoma and corneal diseases.

Plasma Ceramides and Other Sphingolipids in Relation to Incident Prediabetes in a Longitudinal Biracial Cohort.

CONTEXT: Sphingolipids are linked to the pathogenesis of type 2 diabetes. OBJECTIVE: To test the hypothesis that plasma sphingolipid profiles predict incident prediabetes. DESIGN: A case-control study nested in the Pathobiology of Prediabetes in a Biracial Cohort study, a 5-year follow-up study. SETTING: Academic health center. PARTICIPANTS: Normoglycemic adults enrolled in the Pathobiology of Prediabetes in a Biracial Cohort study. Assessments included oral glucose tolerance test, insulin sensitivity, and insulin secretion. Participants with incident prediabetes were matched in age, sex, and ethnicity with nonprogressors. INTERVENTIONS: We assayed 58 sphingolipid species (ceramides, monohexosyl ceramides, sphingomyelins, and sphingosine) using liquid chromatography/tandem mass spectrometry in baseline plasma levels from participants and determined association with prediabetes risk. MAIN OUTCOME MEASURE: The primary outcome was progression from normoglycemia to prediabetes, defined as impaired fasting glucose or impaired glucose tolerance. RESULTS: The mean age of participants (N = 140; 50% Black, 50% female) was 48.1 ± 8.69 years, body mass index 30.1 ± 5.78 kg/m2, fasting plasma glucose 92.7 ± 5.84 mg/dL, and 2-hour plasma glucose 121 ± 23.3 mg/dL. Of the 58 sphingolipid species assayed, higher ratios of sphingomyelin C26:0/C26:1 (OR, 2.73 [95% CI, 1.172-4.408], P = .015) and ceramide C18:0/C18:1 (OR, 1.236 [95% CI, 1.042-1.466], P = .015) in baseline plasma specimens were significantly associated with progression to prediabetes during the 5-year follow-up period, after adjustments for age, race, sex, body mass index, fasting plasma glucose, 2-hour plasma glucose, insulin sensitivity, and insulin secretion. CONCLUSION: We conclude that the saturated-to-monounsaturated ratios of long-chain ceramide C18:0/C18:1 and very-long-chain sphingomyelin C26:0/C26:1 are potential biomarkers of prediabetes risk among individuals with parental history of type 2 diabetes.

The role of sphingosine-1-phosphate receptor 2 in mouse retina light responses.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) acts as a ligand for a family of G protein-coupled S1P receptors (S1PR1-5) to participate in a variety of signaling pathways. However, their specific roles in the neural retina remain unclear. We previously showed that S1P receptor subtype 2 (S1PR2) is expressed in murine retinas, primarily in photoreceptors and bipolar cells, and its expression is altered by retinal stress. This study aims to elucidate the role of S1PR2 in the mouse retina. We examined light responses by electroretinography (ERG), structural differences by optical coherence tomography (OCT), and protein levels by immunohistochemistry (IHC) in wild-type (WT) and S1PR2 knockout (KO) mice at various ages between 3 and 6 months. We found that a- and b-wave responses significantly increased at flash intensities between 400∼2000 and 4∼2,000 cd.s/m 2 respectively, in S1PR2 KO mice relative to those of WT controls at baseline. S1PR2 KO mice also exhibited significantly increased retinal nerve fiber layer (RNFL) and outer plexiform layer (OPL) thickness by OCT relative to the WT. Finally, in S1PR2 KO mice, we observed differential labeling of synaptic markers by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). These results suggest a specific involvement of S1PR2 in the structure and synaptic organization of the retina and a potential role in light-mediated functioning of the retina.

The Role of Sphingosine-1-Phosphate Receptor 2 in Mouse Retina Light Responses.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) acts as a ligand for a family of G protein-coupled S1P receptors (S1PR1-5) to participate in a variety of signaling pathways. However, their specific roles in the neural retina remain unclear. We previously showed that S1P receptor subtype 2 (S1PR2) is expressed in murine retinas, primarily in photoreceptors and bipolar cells, and its expression is altered by retinal stress. This study aims to elucidate the role of S1PR2 in the mouse retina. We examined light responses by electroretinography (ERG), structural differences by optical coherence tomography (OCT), and protein levels by immunohistochemistry (IHC) in wild-type (WT) and S1PR2 knockout (KO) mice at various ages between 3 and 6 months. We found that a- and b-wave responses significantly increased at flash intensities between 400~2000 and 4~2000 cd.s/m2, respectively, in S1PR2 KO mice relative to those of WT controls at baseline. S1PR2 KO mice also exhibited significantly increased retinal nerve fiber layer (RNFL) and outer plexiform layer (OPL) thickness by OCT relative to the WT. Finally, in S1PR2 KO mice, we observed differential labeling of synaptic markers by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). These results suggest a specific involvement of S1PR2 in the structure and synaptic organization of the retina and a potential role in light-mediated functioning of the retina.

The role of sphingolipids in meibomian gland dysfunction and ocular surface inflammation.

Inflammation occurs in response to tissue injury and invasion of microorganisms and is carried out by the innate and adaptive immune systems, which are regulated by numerous chemokines, cytokines, and lipid mediators. There are four major families of bioactive lipid mediators that play an integral role in inflammation - eicosanoids, sphingolipids (SPL), specialized pro-resolving mediators (SPM), and endocannabinoids. SPL have been historically recognized as important structural components of cellular membranes; their roles as bioactive lipids and inflammatory mediators are recent additions. Major SPL metabolites, including sphingomyelin, ceramide, ceramide 1-phosphate (C1P), sphingosine, sphingosine 1-phosphate (S1P), and their respective enzymes have been studied extensively, primarily in cell-culture and animal models, for their roles in cellular signaling and regulating inflammation and apoptosis. Less focus has been given to the involvement of SPL in eye diseases. As such, the aim of this review was to examine relationships between the SPL family and ocular surface diseases, focusing on their role in disease pathophysiology and discussing the potential of therapeutics that disrupt SPL pathways.

A Comprehensive Profiling of Cellular Sphingolipids in Mammalian Endothelial and Microglial Cells Cultured in Normal and High-Glucose Conditions.

Sphingolipids (SPLs) play a diverse role in maintaining cellular homeostasis. Dysregulated SPL metabolism is associated with pathological changes in stressed and diseased cells. This study investigates differences in SPL metabolism between cultured human primary retinal endothelial (HREC) and murine microglial cells (BV2) in normal conditions (normal glucose, NG, 5 mM) and under high-glucose (HG, 25 mM)-induced stress by sphingolipidomics, immunohistochemistry, biochemical, and molecular assays. Measurable differences were observed in SPL profiles between HREC and BV2 cells. High-glucose treatment caused a >2.5-fold increase in the levels of Lactosyl-ceramide (LacCer) in HREC, but in BV2 cells, it induced Hexosyl-Ceramides (HexCer) by threefold and a significant increase in Sphingosine-1-phosphate (S1P) compared to NG. Altered SPL profiles coincided with changes in transcript levels of inflammatory and vascular permeability mediators in HREC and inflammatory mediators in BV2 cells. Differences in SPL profiles and differential responses to HG stress between endothelial and microglial cells suggest that SPL metabolism and signaling differ in mammalian cell types and, therefore, their pathological association with those cell types.

Ablation of Sphingosine Kinase 1 Protects Cornea from Neovascularization in a Mouse Corneal Injury Model.

The purpose of this study was to investigate the role of sphingosine kinase 1 (SphK1), which generates sphingosine-1-phosphate (S1P), in corneal neovascularization (NV). Wild-type (WT) and Sphk1 knockout (Sphk1-/-) mice received corneal alkali-burn treatment to induce corneal NV by placing a 2 mm round piece of Whatman No. 1 filter paper soaked in 1N NaOH on the center of the cornea for 20 s. Corneal sphingolipid species were extracted and identified using liquid chromatography/mass spectrometry (LC/MS). The total number of tip cells and those positive for ethynyl deoxy uridine (EdU) were quantified. Immunocytochemistry was done to examine whether pericytes were present on newly forming blood vessels. Cytokine signaling and angiogenic markers were compared between the two groups using multiplex assays. Data were analyzed using appropriate statistical tests. Here, we show that ablation of SphK1 can significantly reduce NV invasion in the cornea following injury. Corneal sphingolipid analysis showed that total levels of ceramides, monohexosyl ceramides (HexCer), and sphingomyelin were significantly elevated in Sphk-/- corneas compared to WT corneas, with a comparable level of sphingosine among the two genotypes. The numbers of total and proliferating endothelial tip cells were also lower in the Sphk1-/- corneas following injury. This study underscores the role of S1P in post-injury corneal NV and raises further questions about the roles played by ceramide, HexCer, and sphingomyelin in regulating corneal NV. Further studies are needed to unravel the role played by bioactive sphingolipids in maintenance of corneal transparency and clear vision.

Role of ceramides in the pathogenesis of diabetes mellitus and its complications.

Diabetes mellitus (DM) is a systemic metabolic disease that affects 463 million adults worldwide and is a leading cause of cardiovascular disease, blindness, nephropathy, peripheral neuropathy, and lower-limb amputation. Lipids have long been recognized as contributors to the pathogenesis and pathophysiology of DM and its complications, but recent discoveries have highlighted ceramides, a class of bioactive sphingolipids with cell signaling and second messenger capabilities, as particularly important contributors to insulin resistance and the underlying mechanisms of DM complications. Besides their association with insulin resistance and pathophysiology of type 2 diabetes, evidence is emerging that certain species of ceramides are mediators of cellular mechanisms involved in the initiation and progression of microvascular and macrovascular complications of DM. Advances in our understanding of these associations provide unique opportunities for exploring ceramide species as potential novel therapeutic targets and biomarkers. This review discusses the links between ceramides and the pathogenesis of DM and diabetic complications and identifies opportunities for novel discoveries and applications.

Analysis of sphingolipid composition in human vitreous from control and diabetic individuals.

OBJECTIVE: Sphingolipids have a fundamental role in many cellular processes, and they have been implicated in insulin resistance and Diabetes Mellitus (DM) and its complications, including diabetic retinopathy (DR). Little is known about how bioactive sphingolipids relate to retinopathies in human DM. In this study, we analyzed the sphingolipid composition of type 2 diabetic (T2DM) and non-diabetic human vitreous samples. METHODS: We conducted an observational study on post-mortem human vitreous samples from non-diabetic (Controls; n = 4; age: 71.6 ±â€¯11.0 years, mean ±â€¯SD) and type 2 diabetic (T2DM; n = 9; age: 67.0 ±â€¯9.2 years) donors to identify changes in sphingolipid composition. Samples were analyzed by a triple quadrupole mass spectrometer and individual sphingolipid species were identified and quantified using established protocols. RESULTS: The total quantity (pmol/mg) of ceramide (Cer), lactosylceramide (Lac-Cer), and sphingomyelin (SM) were increased in type 2 diabetic vitreous samples. Among individual species, we found a general trend of increase in the longer chain species of ceramides, hexosylceramides (Hex-Cer), Lac-Cer, and SM. CONCLUSIONS: This study shows the presence of measurable levels of sphingolipids in human vitreous. The results indicate changes in sphingolipid composition in the vitreous due to type 2 diabetes, which could be connected to the disease pathologies of the retina, retinal vessels, vitreous and the surrounding tissues.

Sphingosine Kinase-1 Is Essential for Maintaining External/Outer Limiting Membrane and Associated Adherens Junctions in the Aging Retina.

Sphingosine-1-phosphate (S1P) produced by sphingosine kinases (SPHK1 and SPHK2) is a signaling molecule involved in cell proliferation and formation of cellular junctions. In this study, we characterized the retinas of Sphk1 knockout (KO) mice by electron microscopy and immunocytochemistry. We also tested cultured Müller glia for their response to S1P. We found that S1P plays an important role in retinal and retinal pigment epithelial (RPE) structural integrity in aging mice. Ultrastructural analysis of Sphk1 KO mouse retinas aged to 15 months or raised with moderate light stress revealed a degenerated outer limiting membrane (OLM). This membrane is formed by adherens junctions between neighboring Müller glia and photoreceptor cells. We also show that Sphk1 KO mice have reduced retinal function in mice raised with moderate light stress. In vitro assays revealed that exogenous S1P modulated cytoskeletal rearrangement and increased N-cadherin production in human Müller glia cells. Aged mice also had morphological degeneration of the RPE, as well as increased lipid storage vacuoles and undigested phagosomes reminiscent of RPE in age-related macular degeneration. These findings show that SPHK1 and S1P play a vital role in the structural maintenance of the mammalian retina and retinal pigmented epithelium by supporting the formation of adherens junctions.

Sphingosine Kinase 2 Phosphorylation of FTY720 is Unnecessary for Prevention of Light-Induced Retinal Damage.

Mammalian Sphingosine kinase 2 is the primary enzyme responsible for phosphorylating FTY720 to its active form, FTY720-P. Systemic FTY720 treatment confers significant protection to murine retinas from light- and disease-mediated photoreceptor cell death. It is not clear whether FTY720-P, FTY720, or both are responsible for this photoreceptor protection. We investigated Sphingosine kinase 2 knockout (Sphk2 KO) mouse retinas, tested their sensitivity to light, and measured what degree of protection from light-induced damage they receive from systemic FTY720 treatment. Sphk2 KO retinas were found to be similar to their wild-type counterparts in sensitivity to light damage. Additionally, FTY720 treatment protected Sphk2 KO retinas from light-induced damage despite significant retardation of FTY720 phosphorylation in Sphk2 KO mice. We conclude that FTY720 serves an active role in preventing photoreceptor cell death. Furthermore, we conclude that the phosphorylation of FTY720 is not necessary to provide this protective effect.