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Thematic Review Series Thematic Review Series: Seeing 2020: Lipids and Lipid-Soluble Molecules in the Eye| Volume 62, 100037, January 01, 2021

Sphingolipids as critical players in retinal physiology and pathology

  • Author Footnotes
    ‡ These authors contributed equally to this work.
    M. Victoria Simon
    Footnotes
    ‡ These authors contributed equally to this work.
    Affiliations
    Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Departamento De Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS), Argentine National Research Council (CONICET), Bahía Blanca, Argentina
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
    Sandip K. Basu
    Footnotes
    ‡ These authors contributed equally to this work.
    Affiliations
    Departments of Ophthalmology and Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA
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  • Bano Qaladize
    Affiliations
    Departments of Ophthalmology and Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA
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  • Richard Grambergs
    Affiliations
    Departments of Ophthalmology and Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA
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  • Nora P. Rotstein
    Correspondence
    For correspondence: Nora P. Rotstein; Nawajes Mandal
    Affiliations
    Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Departamento De Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS), Argentine National Research Council (CONICET), Bahía Blanca, Argentina
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  • Nawajes Mandal
    Correspondence
    For correspondence: Nora P. Rotstein; Nawajes Mandal
    Affiliations
    Departments of Ophthalmology and Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
Open AccessPublished:February 05, 2021DOI:https://doi.org/10.1194/jlr.TR120000972

      Abstract

      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.

      Supplementary key words

      Abbreviations:

      ADIPOR1 (adiponectin receptor 1), AH (aqueous humor), AMD (age-related macular degeneration), ASAH (N-acyl-sphingosine amidohydrolase), aSMase (acid SMase), BEST1 (bestrophin-1), BDNF (brain-derived neurotrophic factor), CDase (ceramidase), Cer (ceramide), CerK (ceramide kinase), CERKL (ceramide kinase-like), CerS (ceramide synthase), CFH (complement factor H), C1P (ceramide 1-phosphate), DHCer (dihydroceramide), DR (diabetic retinopathy), EAU (experimental autoimmune uveoretinitis), GA (geographic atrophy), GalCer (galactosylceramide), GCS (glucosylceramide synthase), GlcCer (glucosylceramide), hBest1 (human bestrophin-1), HexCer (hexosylceramide), IOP (intraocular pressure), LacCer (lactosylceramide), Mac Tel (macular telangiectasia), NGF (nerve growth factor), nSMase (neutral sphingomyelinase), OAG (open-angle glaucoma), PARP-1 (poly-ADP ribose polymerase 1), PDR (proliferative diabetic retinopathy), PKC (protein kinase C), POAG (primary open-angle glaucoma), RP (retinitis pigmentosa), RPE (retinal pigment epithelium), SMS (SM synthase), Sph (sphingosine), SphK (sphingosine kinase), S1PR (S1P receptor), SPT (serine palmitoyl transferase), VEGF (vascular endothelial growth factor), VMD (vitelliform macular dystrophy)

      Why sphingolipids?

      The notion that lipids are part of cellular signaling networks in addition to their canonical roles as energy reserves or structural membrane components was first proposed in the 1950s and is now widely accepted. However, outside the lipid community, their involvement is still overshadowed by their nonlipidic counterparts. Sphingolipids, one of the three main classes of membrane lipids, are among the latest incorporations to the club of recognized bioactive lipids. They owe their name to the initial enigma regarding their functions, which reminded J. L. Thudichum, who first isolated them from brain tissue during the late 19th century, of the riddle posed by the Sphinx in Greek mythology. After almost a century, this riddle was thought to be resolved when they were classified only as stable membrane structural components. Groundbreaking findings in the mid-1980s and early 1990s revealed novel roles for sphingosine (Sph) and ceramide (Cer) as signaling molecules involved in the induction of cell death and the inhibition of proliferation (
      • Dressler K.A.
      • Mathias S.
      • Kolesnick R.N.
      Tumor necrosis factor-alpha activates the sphingomyelin signal transduction pathway in a cell-free system.
      ,
      • Hannun Y.A.
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      Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets.
      ,
      • Kolesnick R.N.
      1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells.
      ,
      • Obeid L.M.
      • Linardic C.M.
      • Karolak L.A.
      • Hannun Y.A.
      Programmed cell death induced by ceramide.
      ). Later work established that their phosphorylated derivatives, S1P and ceramide 1-phosphate (C1P), promote survival, proliferation, and differentiation (
      • Cuvillier O.
      • Pirianov G.
      • Kleuser B.
      • Vanek P.G.
      • Cosot O.A.
      • Gutkind J.S.
      • Spiegel S.
      Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.
      ,
      • Gomez-Muñoz A.
      • Duffy P.A.
      • Martin A.
      • O’Brien L.
      • Byun H.S.
      • Bittman R.
      • Brindley D.N.
      Short-chain ceramide-1-phosphates are novel stimulators of DNA synthesis and cell division: antagonism by cell-permeable ceramides.
      ,
      • Gomez-Muñoz A.
      • Frago L.M.
      • Alvarez L.
      • Varela-Nieto I.
      Stimulation of DNA synthesis by natural ceramide 1-phosphate.
      ,
      • Zhang H.
      • Desai N.N.
      • Olivera A.
      • Seki T.
      • Brooker G.
      • Spiegel S.
      Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation.
      ,
      • Olivera A.
      • Spiegel S.
      Sphingosine-1-phosphate as second messenger in cell proliferation induced by PDGF and FCS mitogens.
      ), thus increasing the repertoire of bioactive sphingolipids. Accumulated evidence has not only expanded this family of bioactive sphingolipids to include glucosylceramide (GlcCer), lactosylceramide (LacCer), and some gangliosides, such as GM1, but has also established sphingolipids as amazingly versatile signaling molecules regulating multiple physiological and pathological processes.
      In the last two decades, work from several groups had uncovered key roles for sphingolipids as signaling molecules in the retina. Sphingolipids are now known to modulate the functionality of the multiple cell types present in the retina and, through dysregulation of their metabolism and populations, contribute to multiple retinal pathologies (
      • Mondal K.
      • Mandal N.
      Role of bioactive sphingolipids in inflammation and eye diseases.
      ,
      • Rotstein N.P.
      • Miranda G.E.
      • Abrahan C.E.
      • German O.L.
      Regulating survival and development in the retina: key roles for simple sphingolipids.
      ,
      • Simón M.V.
      • Prado Spalm F.H.
      • Vera M.S.
      • Rotstein N.P.
      Sphingolipids as emerging mediators in retina degeneration.
      ). In this review, we first introduce the roles and metabolism of simple sphingolipids, with particular focus on Cer and S1P, and then integrate the roles played by these lipids in several retinal pathologies.

      The complexity of sphingolipid structure and metabolic pathways

      The amazing diversity of sphingolipid structures is the basis for their extraordinary functional versatility. Structurally, sphingolipids are amphipathic molecules that share a hydrophobic region, a sphingoid long-chain (18–20 carbon) base, that constitutes the building block of mammalian sphingolipids. Addition of a fatty acid through an amide bond to carbon 2 of the Sph backbone gives rise to Cer (Fig. 1). Modifications of the sphingoid backbone can generate a large variety of structures. On the other hand, sphingolipid hydrophilic regions can vary widely; the addition of a single phosphate to Sph and Cer generates S1P and C1P, respectively, while attachment of diverse headgroups at the C-1 position of Cer gives rise to more complex sphingolipids, such as SM and glycosphingolipids. The variability and potential combination of these moieties give rise to an astonishing assortment of sphingolipid molecular species. Over 60 sphingoid bases have been reported, varying in their chain length (usually 18–20 carbons), the number of double bonds (often zero to one, but up to two), and number of hydroxyl groups (two to four) (
      • Chen Y.
      • Liu Y.
      • Sullards M.C.
      • Merrill A.H.
      An introduction to sphingolipid metabolism and analysis by new technologies.
      ), with Sph being the most common. Similarly, over 20 fatty acids are found in Cers, differing in their chain lengths (generally 14–36 carbon atoms), unsaturation (typically saturated but occasionally highly unsaturated), and hydroxylation. Finally, the existence of hundreds of headgroups that can be attached to Cer has established the amount of sphingolipid molecular species in the order of tens of thousands (
      • Merrill A.H.
      • Hannun Y.A.
      • Bell R.M.
      Introduction: sphingolipids and their metabolites in cell regulation.
      ).
      Figure thumbnail gr1
      Fig. 1Chemical structures of sphingolipids. The Sph backbone (black) is shared by all sphingolipids. Sph is amide-linked to a fatty acid moiety (brown), forming Cer. Later additions of a phosphate (blue) or hexose residues (orange) give rise to several sphingolipid molecules.
      To fully unravel the sphingolipid puzzle, it is necessary to consider their bewildering diversity in the context of their myriad metabolic pathways. The complexity and high interconnection of these pathways are the basis for the crucial roles in controlling numerous cellular functions. The biosynthesis and catabolism of sphingolipids involves multiple metabolic intermediates, many of which have biological functions of their own. The constant flux of cellular sphingolipid levels is the key to their ability to modulate multiple cellular processes. Sphingolipid concentrations vary among the different cell types, with SM being the most abundant. In most cells, their relative ratios can be illustrated as SM (30,000):Cer (3,000):Sph (100):S1P (1). Thus, minor changes in SM levels translate into significant variations in Cer and S1P concentrations, which result in a specific cellular response (
      • Hannun Y.A.
      • Obeid L.M.
      Principles of bioactive lipid signalling: lessons from sphingolipids.
      ).
      Cer is the undisputed hub of the highly interconnected sphingolipid metabolic network and controls key cellular responses such as growth arrest, senescence, and cell death (
      • Hannun Y.A.
      • Obeid L.M.
      Principles of bioactive lipid signalling: lessons from sphingolipids.
      ). Three different pathways lead to Cer formation: de novo synthesis, hydrolysis of SM, and recycling of Sph and complex sphingolipids (Fig. 2). It is noteworthy that these three pathways are activated by different cellular cues and contribute differentially to Cer signaling capacity. The de novo pathway of Cer synthesis takes place in the ER and starts with the condensation of l-serine and palmitoyl-CoA, catalyzed by l-serine palmitoyl transferase (SPT), to form 3-ketosphinganine, which is then reduced to sphinganine (Fig. 2). Next, Cer synthases (CerSs) catalyze the N-acylation of sphinganine, giving rise to dihydroceramides (DHCers). In mammals, the CerS family is formed by six isoforms (CerS1–6), which differentially utilize fatty acylCoAs differing in chain length from 14 to 34 carbons. DHCer desaturases then reduce DHCer to yield a diversity of Cer species (
      • Mullen T.D.
      • Hannun Y.A.
      • Obeid L.M.
      Ceramide synthases at the centre of sphingolipid metabolism and biology.
      ). The de novo pathway is activated upon different environmental events to induce stress responses and cell death (
      • Bose R.
      • Verheij M.
      • Haimovitz-Friedman A.
      • Scotto K.
      • Fuks Z.
      • Kolesnick R.
      Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals.
      ,
      • Jenkins G.M.
      • Ashley Cowart L.
      • Signorelli P.
      • Pettus B.J.
      • Chalfant C.E.
      • Hannun Y.A.
      Acute activation of de novo sphingolipid biosynthesis upon heat shock causes an accumulation of ceramide and subsequent dephosphorylation of SR proteins.
      ). Once generated, Cer can then be transferred to the Golgi, either through a Cer transporter (CERT) or through vesicular transport pathways, where it serves as a precursor for SM, C1P, or other glycosphingolipids. SM is synthesized through the addition of a phosphorylcholine to Cer, catalyzed by SM synthase (SMS) (
      • Tafesse F.G.
      • Ternes P.
      • Holthuis J.C.M.
      The multigenic sphingomyelin synthase family.
      ); the resulting SM is then conveyed to the plasma membrane through vesicular transport. In turn, the phosphorylation of Cer, catalyzed by Cer kinase (CerK), produces C1P (
      • Chalfant C.E.
      • Spiegel S.
      Sphingosine 1-phosphate and ceramide 1-phosphate: expanding roles in cell signaling.
      ,
      • Wijesinghe D.S.
      • Massiello A.
      • Subramanian P.
      • Szulc Z.
      • Bielawska A.
      • Chalfant C.E.
      Substrate specificity of human ceramide kinase.
      ).
      Figure thumbnail gr2
      Fig. 2The sphingolipid networks. A schematic view of the interconnected sphingolipid network, which has Cers (purple) forming its central hub. Cer can be synthesized through the de novo pathway (green), initiated by the condensation of l-serine and palmitoyl-CoA; through the SMase pathway (blue), from the degradation of SM catalyzed by different SMases; or through the Salvage pathway (yellow), from the Sph generated by the degradation of complex sphingolipids. Cer can then serve as a substrate for sphingomyelin synthesis by SMS; be phosphorylated by a CerK to generate C1P; or be deacylated by CDases to form Sph, which can in turn be phosphorylated by SphK to produce S1P. S1P can be dephosphorylated by S1P phosphatase (S1PP) to regenerate Sph or be irreversibly degraded by S1P lyase to render ethanolamine 1-phosphate and hexadecenal, an irreversible reaction that provides the only escape pathway from this intricate metabolic network. DES, dihydroceramide desaturase-1; LPP, lipid phosphate phosphatases.
      Glycosphingolipids are formed from Cer by sequential addition of sugar moieties; the addition of glucose or galactose to Cer gives rise to GlcCer and galactosylceramide (GalCer), respectively, which are two structural isomers collectively called hexosylceramides (HexCers) (
      • Hammad S.M.
      • Pierce J.S.
      • Soodavar F.
      • Smith K.J.
      • Al Gadban M.M.
      • Rembiesa B.
      • Klein R.L.
      • Hannun Y.A.
      • Bielawski J.
      • Bielawska A.
      Blood sphingolipidomics in healthy humans: impact of sample collection methodology.
      ). Their biosynthetic mechanism is similar: a glucosyltransferase (GlcCer synthase) transfers UDP-glucose to Cer, forming GlcCer, while a galactosyltransferase transfers UDP-galactose to Cer, generating GalCer (
      • Tettamanti G.
      Ganglioside/glycosphingolipid turnover: new concepts.
      ). They both serve as precursors for more complex sphingolipids, such as LacCer, which is the precursor for most gangliosides. The synthesis of these sialic acid-containing glycosphingolipids starts with the attachment of a sialic acid to LacCer, generating GM3, which serves as an essential core structure for the building of the complex oligosaccharide chains found in gangliosides. These sequential glycosylation reactions, catalyzed by different glycosyltransferases, take place mainly in the luminal surface of the Golgi and in trans-Golgi membranes (
      • Yu R.K.
      • Tsai Y.T.
      • Ariga T.
      • Yanagisawa M.
      Structures, biosynthesis, and functions of gangliosides–an overview.
      ,
      • Allende M.L.
      • Proia R.L.
      Sphingosine-1-phosphate receptors and the development of the vascular system.
      ,
      • Breiden B.
      • Sandhoff K.
      Ganglioside metabolism and its inherited diseases.
      ).
      Complex sphingolipids are distributed to the plasma membrane and different subcellular compartments and can, in turn, be catabolized to serve as sources of Cer in basal or signal-activated intracellular pathways. Earlier work identified signaling roles of Cer generated by a group of SM-hydrolyzing enzymes called SMases, which produce Cer via hydrolysis of the SM phosphodiester bond and consequent release of the phosphorylcholine headgroup. This mechanism of Cer generation is called the SMase pathway (Fig. 2). There are at least five different isoforms of SMase that differ in their cationic dependence, pH optimum, and subcellular localization, and are found in the plasma membrane, cytosol, mitochondria, and endo-lysosomal compartments (
      • Jenkins R.W.
      • Canals D.
      • Hannun Y.A.
      Roles and regulation of secretory and lysosomal acid sphingomyelinase.
      ,
      • Marchesini N.
      • Hannun Y.A.
      Acid and neutral sphingomyelinases: roles and mechanisms of regulation.
      ,
      • Wu B.X.
      • Clarke C.J.
      • Hannun Y.A.
      Mammalian Neutral Sphingomyelinases: Regulation and Roles in Cell Signaling Responses.
      ). Acid SMase (aSMase) catalyzes the hydrolysis of SM present in the endo-lysosomal compartments and the outer leaflet of the plasma membrane, as well as SM carried by lipoproteins (
      • Zeidan Y.H.
      • Hannun Y.A.
      The Acid Sphingomyelinase/Ceramide Pathway: Biomedical Significance and Mechanisms of Regulation.
      ). At least four neutral SMases (nSMases) have been identified, with nSMase1 facilitating hydrolysis of SM found in the ER/Golgi and nSMase2 within multilamellar bodies in cytosol, in the cytosolic leaflet of the plasma membrane, and in the nuclear envelope (
      • Airola M.V.
      • Hannun Y.A.
      Sphingolipid metabolism and neutral sphingomyelinases.
      ). SMases enable rapid increase in Cer levels in multiple cellular compartments, making them indispensable in both intracellular signaling and modifying membrane microdomains, such as rafts.
      The breakdown of complex sphingolipids by different hydrolases in the lysosomal and late endosomal compartments constitutes the third pathway for Cer generation, the so-called salvage pathway (Fig. 2) (
      • Kitatani K.
      • Idkowiak-Baldys J.
      • Hannun Y.A.
      The sphingolipid salvage pathway in ceramide metabolism and signaling.
      ). Cer cannot leave this compartment, but once hydrolyzed by ceramidases (CDases), the resulting Sph can be released and recycled in the ER where further reacylation by CerS regenerates Cer (
      • Kitatani K.
      • Idkowiak-Baldys J.
      • Hannun Y.A.
      The sphingolipid salvage pathway in ceramide metabolism and signaling.
      ,
      • Canals D.
      • Salamone S.
      • Hannun Y.A.
      Visualizing bioactive ceramides.
      ). Hence, CDases are crucial controllers of the interconversion of Cer and Sph. At least five CDases have been identified, with different optimal pH levels and localizations in cellular compartments such as lysosomes, ER, Golgi, and the plasma membrane (
      • Coant N.
      • Sakamoto W.
      • Mao C.
      • Hannun Y.A.
      Ceramidases, roles in sphingolipid metabolism and in health and disease.
      ). The reverse action of neutral CDase can participate in Cer formation from Sph and acyl-CoA in the mitochondria (
      • Novgorodov S.A.
      • Wu B.X.
      • Gudz T.I.
      • Bielawski J.
      • Ovchinnikova T.V.
      • Hannun Y.A.
      • Obeid L.M.
      Novel pathway of ceramide production in mitochondria: thioesterase and neutral ceramidase produce ceramide from sphingosine and acyl-CoA.
      ). Notably, mitochondria house most of the enzymes involved in sphingolipid metabolism; mitochondrial CerS, SMase, and neutral CDase give rise to a local Cer pool (
      • Bionda C.
      • Portoukalian J.
      • Schmitt D.
      • Rodriguez-Lafrasse C.
      • Ardail D.
      Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria?.
      ,
      • El Bawab S.
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      • Qian T.
      • Bielawska A.
      • Lemasters J.J.
      • Hannun Y.A.
      Molecular cloning and characterization of a human mitochondrial ceramidase.
      ,
      • Mullen T.D.
      • Obeid L.M.
      Ceramide and apoptosis: exploring the enigmatic connections between sphingolipid metabolism and programmed cell death.
      ,
      • Senkal C.E.
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      • Sinha D.
      • Jiang J.C.
      • Jazwinski S.M.
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      • Ogretmen B.
      Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas.
      ,
      • Wu B.X.
      • Rajagopalan V.
      • Roddy P.L.
      • Clarke C.J.
      • Hannun Y.A.
      Identification and characterization of murine mitochondria-associated neutral sphingomyelinase (MA-nSMase), the mammalian sphingomyelin phosphodiesterase 5.
      ), which regulates diverse cell death mechanisms, as discussed below.
      Sph can also be phosphorylated by two distinct Sph kinases (SphKs), to form S1P (
      • Hait N.C.
      • Oskeritzian C.A.
      • Paugh S.W.
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      • Spiegel S.
      Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases.
      ), which in turn can be dephosphorylated by S1P phosphatase to regenerate Sph (Fig. 2). Interestingly, S1P can also provide the sole escape from the intricate sphingolipid metabolic pathways; S1P can be irreversibly degraded to ethanolamine 1-phosphate and hexadecenal in a reaction catalyzed by S1P lyase, the only irreversible reaction in this pathway that does not render a sphingolipid metabolic intermediate (
      • Bandhuvula P.
      • Saba J.D.
      Sphingosine-1-phosphate lyase in immunity and cancer: silencing the siren.
      ).
      For further details on sphingolipid structure and metabolism, readers are referred to excellent reviews that extensively cover these issues (
      • Kitatani K.
      • Idkowiak-Baldys J.
      • Hannun Y.A.
      The sphingolipid salvage pathway in ceramide metabolism and signaling.
      ,
      • Canals D.
      • Salamone S.
      • Hannun Y.A.
      Visualizing bioactive ceramides.
      ,
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      • Obeid L.M.
      Many ceramides.
      ,
      • Lahiri S.
      • Futerman A.H.
      The metabolism and function of sphingolipids and glycosphingolipids.
      ).

      The sphingolipid rheostat: at the crossroads between cellular survival or death

      By the mid-1990s, it was clearly established that Cer and Sph regulated the induction of cell cycle arrest and cell death, whereas S1P modulated the opposite processes, enhancing proliferation and promoting cell survival and differentiation. Overwhelming evidence demonstrated a rapid and effective interconversion between these sphingolipids (Fig. 2), arising from the modulations in the activity and levels of the enzymes involved. These modulations, in turn, occur due to changes in multiple intracellular cues, resulting from the interaction of the cells with their environment. This led to the proposal that the levels of Cer and S1P provide an effective tool to monitor intracellular conditions and rapidly respond to changes in the environment, as fluctuations in their levels would activate numerous signaling pathways that control cell fate. This concept, later denominated “the sphingolipid rheostat” (
      • Cuvillier O.
      • Pirianov G.
      • Kleuser B.
      • Vanek P.G.
      • Cosot O.A.
      • Gutkind J.S.
      • Spiegel S.
      Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.
      ,
      • Gomez-Muñoz A.
      • Duffy P.A.
      • Martin A.
      • O’Brien L.
      • Byun H.S.
      • Bittman R.
      • Brindley D.N.
      Short-chain ceramide-1-phosphates are novel stimulators of DNA synthesis and cell division: antagonism by cell-permeable ceramides.
      ), has been supported by numerous reports, and it now provides the basis for understanding the crucial roles of sphingolipids as modulators of life or death in the cell.
      Later findings have extended our knowledge on the signaling pathways and molecular actors, such as C1P and Sph, participating in the sphingolipid metabolic cycle and have contributed to our understanding of their signaling capacity and extraordinary complexity. This amplified sphingolipid rheostat has been confirmed to be involved not only in normal cell physiology but also in numerous pathologies (
      • Hannun Y.A.
      • Obeid L.M.
      Principles of bioactive lipid signalling: lessons from sphingolipids.
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      Ceramide 1-phosphate/ceramide, a switch between life and death.
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      Revisiting the sphingolipid rheostat: Evolving concepts in cancer therapy.
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      Sphingosine kinase: biochemical and cellular regulation and role in disease.
      ,
      • Wang G.
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      Sphingolipids in neurodegeneration (with focus on ceramide and S1P).
      ,
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      • Kester M.
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      Sphingolipids: regulators of crosstalk between apoptosis and autophagy.
      ). Cumulative evidence supports its participation in diseases affecting the retina (
      • Mondal K.
      • Mandal N.
      Role of bioactive sphingolipids in inflammation and eye diseases.
      ,
      • Rotstein N.P.
      • Miranda G.E.
      • Abrahan C.E.
      • German O.L.
      Regulating survival and development in the retina: key roles for simple sphingolipids.
      ,
      • Simón M.V.
      • Prado Spalm F.H.
      • Vera M.S.
      • Rotstein N.P.
      Sphingolipids as emerging mediators in retina degeneration.
      ,
      • Grambergs R.
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      Inflammatory ocular diseases and sphingolipid signaling.
      ), thus providing new clues in the quest for innovative treatments for these pathologies.

      Main sphingolipids involved in retinal diseases

      Individually, sphingolipids are minor components of the retinal lipid pool; however, they collectively add up to 11–13 (mole) percent of lipids in rat and bovine retinas (
      • Brush R.S.
      • Tran J.-T.A.
      • Henry K.R.
      • McClellan M.E.
      • Elliott M.H.
      • Mandal M.N.A.
      Retinal sphingolipids and their very-long-chain fatty acid-containing species.
      ). SM is the most abundant (2.4–2.5% of total retinal lipids), and accounts for 80% of the sphingolipids analyzed in mouse retinas (
      • Garanto A.
      • Mandal N.A.
      • Egido-Gabás M.
      • Marfany G.
      • Fabriàs G.
      • Anderson R.E.
      • Casas J.
      • Gonzàlez-Duarte R.
      Specific sphingolipid content decrease in Cerkl knockdown mouse retinas.
      ). Cer is the second, amounting to around 11%. GlcCer and GalCer together represent around 4%, and Sph accounts for 0.45% of total sphingolipids (
      • Garanto A.
      • Mandal N.A.
      • Egido-Gabás M.
      • Marfany G.
      • Fabriàs G.
      • Anderson R.E.
      • Casas J.
      • Gonzàlez-Duarte R.
      Specific sphingolipid content decrease in Cerkl knockdown mouse retinas.
      ). Most of these sphingolipids have long and very long-chain saturated fatty acids, with 16:0 and 18:0 being the major ones. Notably, they just have 2–3% DHA (22:6 n-3) and completely lack very long-chain PUFAs over 24 carbons, contrasting with the high levels of PUFA usually found in retinal lipids (
      • Brush R.S.
      • Tran J.-T.A.
      • Henry K.R.
      • McClellan M.E.
      • Elliott M.H.
      • Mandal M.N.A.
      Retinal sphingolipids and their very-long-chain fatty acid-containing species.
      ,
      • Aveldaño M.I.
      • Sprecher H.
      Very long chain (C24 to C36) polyenoic fatty acids of the n-3 and n-6 series in dipolyunsaturated phosphatidylcholines from bovine retina.
      ).
      This sphingolipid profile and the enzymes involved in their metabolism are altered in retinal pathologies, as will be discussed later. We will first briefly describe the characteristics and functions of the sphingolipids most affected in these pathologies for a better understanding of their impact.

      Cer: the death orchestrator

      Cer has been a prime suspect in the cellular “crime scene” ever since its key roles in controlling cell death and growth arrest were uncovered over four decades ago. This bioactive sphingolipid is involved in senescence, inhibition of cell proliferation, inflammation, and in the induction of several pathways of cell death, including apoptosis, autophagy, and Parthanatos (
      • Hannun Y.A.
      • Obeid L.M.
      Principles of bioactive lipid signalling: lessons from sphingolipids.
      ,
      • Young M.M.
      • Kester M.
      • Wang H.-G.
      Sphingolipids: regulators of crosstalk between apoptosis and autophagy.
      ,
      • Ji L.
      • Zhang G.
      • Uematsu S.
      • Akahori Y.
      • Hirabayashi Y.
      Induction of apoptotic DNA fragmentation and cell death by natural ceramide.
      ,
      • Prado Spalm F.H.
      • Vera M.S.
      • Dibo M.J.
      • Simón M.V.
      • Politi L.E.
      • Rotstein N.P.
      Ceramide induces the death of retina photoreceptors through activation of Parthanatos.
      ). Cer-mediated cell death is frequently associated with mitochondrial dysfunction (
      • Law B.A.
      • Liao X.
      • Moore K.S.
      • Southard A.
      • Roddy P.
      • Ji R.
      • Szulc Z.
      • Bielawska A.
      • Schulze P.C.
      • Cowart L.A.
      Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.
      ,
      • Mizumura K.
      • Justice M.J.
      • Schweitzer K.S.
      • Krishnan S.
      • Bronova I.
      • Berdyshev E.V.
      • Hubbard W.C.
      • Pewzner-Jung Y.
      • Futerman A.H.
      • Choi A.M.K.
      • et al.
      Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.
      ,
      • Panda P.K.
      • Naik P.P.
      • Meher B.R.
      • Das D.N.
      • Mukhopadhyay S.
      • Praharaj P.P.
      • Maiti T.K.
      • Bhutia S.K.
      PUMA dependent mitophagy by Abrus agglutinin contributes to apoptosis through ceramide generation.
      ,
      • Stoica B.A.
      • Movsesyan V.A.
      • Lea Iv P.M.
      • Faden A.I.
      Ceramide-induced neuronal apoptosis is associated with dephosphorylation of Akt, BAD, FKHR, GSK-3β, and induction of the mitochondrial-dependent intrinsic caspase pathway.
      ).
      In understanding the complexity of Cer synthesis, it is crucial to note that its high hydrophobicity keeps Cer in the membrane in which it has been synthesized, except when carried away through a specific transport mechanism. Although Cer was initially considered a homogeneous sphingolipid class, a “many Cers” paradigm has evolved with the isolation of over 200 distinct mammalian Cers resulting from different combinations of enzymes localized to different cell compartments (
      • Hannun Y.A.
      • Obeid L.M.
      Many ceramides.
      ). This “compartment-specific” synthesis model provides a basis for understanding the different functions or mechanisms triggered by Cer in different cellular locations, highlighting the relevance of the specific enzymes involved in Cer-mediated actions. Cer fatty acyl chains are vital for these effects, supporting the critical role of CerSs, which have a distinct selectivity for specific fatty acids. For example, C16:0-Cer participates in the induction of cell death (
      • Hernández-Corbacho M.J.
      • Canals D.
      • Adada M.M.
      • Liu M.
      • Senkal C.E.
      • Yi J.K.
      • Mao C.
      • Luberto C.
      • Hannun Y.A.
      • Obeid L.M.
      Tumor necrosis factor-α (TNFα)-induced ceramide generation via ceramide synthases regulates loss of focal adhesion kinase (FAK) and programmed cell death.
      ,
      • Stiban J.
      • Perera M.
      Very long chain ceramides interfere with C16-ceramide-induced channel formation: A plausible mechanism for regulating the initiation of intrinsic apoptosis.
      ); low levels of CerS6 in colon cancer cells are sufficient to cause an ineffective C16-Cer response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induction of apoptosis, which is restored by expressing this enzyme (
      • White-Gilbertson S.
      • Mullen T.
      • Senkal C.
      • Lu P.
      • Ogretmen B.
      • Obeid L.
      • Voelkel-Johnson C.
      Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase-3 in colon cancer cells.
      ).
      The fact that its highly hydrophobic properties confine Cer to cell membranes does not diminish its ability to regulate cellular processes. In fact, it contributes to Cer’s diverse range of actions, which include modifying membrane properties, forming channels, and acting as an intracellular messenger. The biophysical properties of Cer are critical for its unique interactions with other membrane components and the modulation of membrane characteristics, leading to the reorganization of membranes and rafts. The ability of Cer to self-associate promotes the formation of small highly-ordered Cer-enriched microdomains, which can spontaneously fuse upon increases in Cer levels resulting from SMase activation, forming macrodomains that serve as signaling platforms by selective trapping of proteins. This allows for segregation, interaction, and oligomerization of proteins such as cytokines and death receptors, leading to the activation of signaling pathways such as those triggered by the proapoptotic protein Bax (
      • Ganesan V.
      • Perera M.N.
      • Colombini D.
      • Datskovskiy D.
      • Chadha K.
      • Colombini M.
      Ceramide and activated Bax act synergistically to permeabilize the mitochondrial outer membrane.
      ,
      • Grassmé H.
      • Cremesti A.
      • Kolesnick R.
      • Gulbins E.
      Ceramide-mediated clustering is required for CD95-DISC formation.
      ,
      • Grassmé H.
      • Riethmüller J.
      • Gulbins E.
      Biological aspects of ceramide-enriched membrane domains.
      ,
      • Grassmé H.
      • Schwarz H.
      • Gulbins E.
      Molecular mechanisms of ceramide-mediated CD95 clustering.
      ,
      • Silva L.
      • De Almeida R.F.M.
      • Fedorov A.
      • Matos A.P.A.
      • Prieto M.
      Ceramide-platform formation and -induced biophysical changes in a fluid phospholipid membrane.
      ). Moreover, Cer increase has been shown to displace cholesterol and caveolin from membrane domains (
      • Bieberich E.
      Sphingolipids and lipid rafts: novel concepts and methods of analysis.
      ), thus modifying their biophysical properties. Hence, Cer-enriched domains differ both structurally and functionally from traditional membrane rafts and caveolae.
      Cer is essential for the formation and/or secretion of exosomes by facilitating or inducing membrane curvature (
      • Carrer D.C.
      • Härtel S.
      • Mónaco H.L.
      • Maggio B.
      Ceramide modulates the lipid membrane organization at molecular and supramolecular levels.
      ,
      • Elsherbini A.
      • Bieberich E.
      Ceramide and exosomes: a novel target in cancer biology and therapy.
      ). Cer enrichment in exosomes has led to the proposal that it may participate in the transmission of “mobile rafts” from donor to recipient cells (
      • Elsherbini A.
      • Bieberich E.
      Ceramide and exosomes: a novel target in cancer biology and therapy.
      ). The increase in Cer levels in mitochondria is decisive for the induction of cell death. Extensive evidence supports that Cer can self-assemble to form channels composed of many Cer monomers, which are able to translocate proteins and have been associated with Cer’s ability to induce both apoptosis and necrosis. These channels have been shown to promote the release of cytochrome c from mitochondria (
      • Stiban J.
      • Perera M.
      Very long chain ceramides interfere with C16-ceramide-induced channel formation: A plausible mechanism for regulating the initiation of intrinsic apoptosis.
      ,
      • Colombini M.
      Ceramide channels.
      ,
      • Siskind L.J.
      • Kolesnick R.N.
      • Colombini M.
      Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins.
      ,
      • Yamane M.
      • Moriya S.
      • Kokuba H.
      Visualization of ceramide channels in lysosomes following endogenous palmitoyl-ceramide accumulation as an initial step in the induction of necrosis.
      ). Interaction between Cer and the Bcl-2 family of proteins is crucial for controlling mitochondrial outer membrane permeability, a central step in apoptosis signaling. Formation of Cer channels and release of cytochrome c are inhibited by Bcl-2 anti-apoptotic proteins, such as Bcl-XL and Bcl-2; in turn, Cer has been shown to promote Bax oligomerization and pore formation and/or to act synergistically with Bax and Bak, possibly by forming hybrid channels (
      • Hernández-Corbacho M.J.
      • Salama M.F.
      • Canals D.
      • Senkal C.E.
      • Obeid L.M.
      Sphingolipids in mitochondria.
      ). In addition, Cer inhibits the respiratory chain and stimulates ROS overproduction (
      • Garcia-Ruíz C.
      • Colell A.
      • Mari M.
      • Morales A.
      • Fernandez-Checa J.C.
      Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. Role of mitochondrial glutathione.
      ,
      • Gudz T.I.
      • Tserng K.Y.
      • Hoppel C.L.
      Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide.
      ).
      Cer also acts as a versatile second messenger. Its capacity to activate protein phosphatases of the PP1 and PP2A families grants Cer a role in controlling the cellular phospho-proteome, including the activity of protein kinase C (PKC), Akt, and ezrin (
      • Canals D.
      • Salamone S.
      • Hannun Y.A.
      Visualizing bioactive ceramides.
      ,
      • Chalfant C.E.
      • Kishikawa K.
      • Mumby M.C.
      • Kamibayashi C.
      • Bielawska A.
      • Hannun Y.A.
      Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid.
      ,
      • Galadari S.
      • Rahman A.
      • Pallichankandy S.
      • Thayyullathil F.
      Tumor suppressive functions of ceramide: evidence and mechanisms.
      ,
      • Wang G.
      • Silva J.
      • Krishnamurthy K.
      • Tran E.
      • Condie B.G.
      • Bieberich E.
      Direct binding to ceramide activates protein kinase Cζ before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells.
      ). PP2A can dephosphorylate and inactivate anti-apoptotic proteins such as Bcl-2, AKT, and c-Myc (
      • Mukhopadhyay A.
      • Saddoughi S.A.
      • Song P.
      • Sultan I.
      • Ponnusamy S.
      • Senkal C.E.
      • Snook C.F.
      • Arnold H.K.
      • Sears R.C.
      • Hanniui Y.A.
      • et al.
      Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling.
      ,
      • Saddoughi S.A.
      • Gencer S.
      • Peterson Y.K.
      • Ward K.E.
      • Mukhopadhyay A.
      • Oaks J.
      • Bielawski J.
      • Szulc Z.M.
      • Thomas R.J.
      • Selvam S.P.
      • et al.
      Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis.
      ). Cer targets depend on its site of generation; thus, lysosome-generated Cer triggers cathepsin B activation, whereas mitochondrial Cer induces Bax-dependent apoptosis (
      • Jain A.
      • Beutel O.
      • Ebell K.
      • Korneev S.
      • Holthuis J.C.M.
      Diverting CERT-mediated ceramide transport to mitochondria triggers Bax-dependent apoptosis.
      ,
      • Taniguchi M.
      • Ogiso H.
      • Takeuchi T.
      • Kitatani K.
      • Umehara H.
      • Okazaki T.
      Lysosomal ceramide generated by acid sphingomyelinase triggers cytosolic cathepsin B-mediated degradation of X-linked inhibitor of apoptosis protein in natural killer/T lymphoma cell apoptosis.
      ).
      The most investigated function of Cer is its role in the induction of cell death. A diverse array of cell stressors such as hypoxia, DNA damage, growth factor withdrawal, ionizing radiation, oxidative damage, or death factors increase the levels of Cer, which then triggers either the intrinsic or the extrinsic apoptotic pathways (
      • Galadari S.
      • Rahman A.
      • Pallichankandy S.
      • Thayyullathil F.
      Tumor suppressive functions of ceramide: evidence and mechanisms.
      ,
      • Pettus B.J.
      • Chalfant C.E.
      • Hannun Y.A.
      Ceramide in apoptosis: an overview and current perspectives.
      ,
      • Santana P.
      • Peña L.A.
      • Haimovitz-Friedman A.
      • Martin S.
      • Green D.
      • McLoughlin M.
      • Cordon-Cardo C.
      • Schuchman E.H.
      • Fuks Z.
      • Kolesnick R.
      Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.
      ). Cer also plays a key role in the regulation of both survival and lethal autophagy, acting at steps ranging from initiation to autophagosome formation (
      • Lavieu G.
      • Scarlatti F.
      • Sala G.
      • Carpentier S.
      • Levade T.
      • Ghidoni R.
      • Botti J.
      • Codogno P.
      Sphingolipids in macroautophagy.
      ). C18-Cer, generated by CerS1, induces selective mitochondrial autophagy, also known as mitophagy (
      • Sentelle R.D.
      • Senkal C.E.
      • Jiang W.
      • Ponnusamy S.
      • Gencer S.
      • Panneer Selvam S.
      • Ramshesh V.K.
      • Peterson Y.K.
      • Lemasters J.J.
      • Szulc Z.M.
      • et al.
      Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy.
      ). Although mitophagy can play a role in either survival or cell death, mitochondria-generated Cer triggers lethal mitophagy, particularly by binding to LC3II-containing autophagosomes (
      • Sentelle R.D.
      • Senkal C.E.
      • Jiang W.
      • Ponnusamy S.
      • Gencer S.
      • Panneer Selvam S.
      • Ramshesh V.K.
      • Peterson Y.K.
      • Lemasters J.J.
      • Szulc Z.M.
      • et al.
      Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy.
      ,
      • Jiang W.
      • Ogretmen B.
      Ceramide stress in survival versus lethal autophagy paradox: ceramide targets autophagosomes to mitochondria and induces lethal mitophagy.
      ). Cer has also been shown to activate necroptosis, which is triggered by high levels of C16:0-Cer (
      • Sawai H.
      • Ogiso H.
      • Okazaki T.
      Differential changes in sphingolipids between TNF-induced necroptosis and apoptosis in U937 cells and necroptosis-resistant sublines.
      ). Recent evidence also establishes that Cer induces Parthanatos, causing neuroblastoma and photoreceptor cell death (
      • Prado Spalm F.H.
      • Vera M.S.
      • Dibo M.J.
      • Simón M.V.
      • Politi L.E.
      • Rotstein N.P.
      Ceramide induces the death of retina photoreceptors through activation of Parthanatos.
      ,
      • Czubowicz K.
      • Strosznajder R.
      Ceramide in the molecular mechanisms of neuronal cell death. the role of sphingosine-1-phosphate.
      ).
      Cer is now known to participate in the progression of multiple pathologies, including inflammation, metabolic syndromes such as obesity and insulin resistance, vascular diseases such as ischemic injury and atherosclerosis, cancer, and neurological disorders (
      • Meikle P.J.
      • Summers S.A.
      Sphingolipids and phospholipids in insulin resistance and related metabolic disorders.
      ,
      • Turpin-Nolan S.M.
      • Hammerschmidt P.
      • Chen W.
      • Jais A.
      • Timper K.
      • Awazawa M.
      • Brodesser S.
      • Brüning J.C.
      CerS1-derived C18:0 ceramide in skeletal muscle promotes obesity-induced insulin resistance.
      ,
      • Zhao L.
      • Spassieva S.D.
      • Jucius T.J.
      • Shultz L.D.
      • Shick H.E.
      • Macklin W.B.
      • Hannun Y.A.
      • Obeid L.M.
      • Ackerman S.L.
      A deficiency of ceramide biosynthesis causes cerebellar Purkinje cell neurodegeneration and lipofuscin accumulation.
      ). Recent cardiovascular trials highlight a novel role for Cer as a biomarker of cardiovascular diseases, associating plasma Cer levels and distinct serum Cers with the risk of major cardiovascular events (
      • Havulinna A.S.
      • Sysi-Aho M.
      • Hilvo M.
      • Kauhanen D.
      • Hurme R.
      • Ekroos K.
      • Salomaa V.
      • Laaksonen R.
      Circulating ceramides predict cardiovascular outcomes in the population-based FINRISK 2002 cohort.
      ,
      • Wang D.D.
      • Toledo E.
      • Hruby A.
      • Rosner B.A.
      • Willett W.C.
      • Sun Q.
      • Razquin C.
      • Zheng Y.
      • Ruiz-Canela M.
      • Guasch-Ferre M.
      • et al.
      Plasma ceramides, Mediterranean diet, and incident cardiovascular disease in the PREDIMED trial (Prevencion con Dieta Mediterranea).
      ). Knowledge of its involvement and roles in these diseases is constantly expanding, and excellent collation on its pathophysiological impact can be found in recent reviews (
      • Canals D.
      • Salamone S.
      • Hannun Y.A.
      Visualizing bioactive ceramides.
      ,
      • Hannun Y.A.
      • Obeid L.M.
      Sphingolipids and their metabolism in physiology and disease.
      ,
      • Kurz J.
      • Parnham M.J.
      • Geisslinger G.
      • Schiffmann S.
      Ceramides as novel disease biomarkers.
      ).
      The observation that Cer accumulates in the retinas of patients with Farber disease, which primarily affects ganglion cells and is associated with visual dysfunction, suggests its involvement in retinal pathologies (
      • Zarbin M.A.
      • Green W.R.
      • Moser A.B.
      • Tiffany C.
      Increased levels of ceramide in the retina of a patient with Farber’s disease.
      ). The first direct evidence of this involvement came from the observation that transgenic expression of a neutral CDase prevents retinal degeneration in Drosophila phototransduction mutants by decreasing Cer levels (
      • Acharya U.
      • Patel S.
      • Koundakjian E.
      • Nagashima K.
      • Han X.
      • Acharya J.K.
      Modulating sphingolipid biosynthetic pathway rescues photoreceptor degeneration.
      ,
      • Dasgupta U.
      • Bamba T.
      • Chiantia S.
      • Karim P.
      • Tayoun A.N.A.
      • Yonamine I.
      • Rawat S.S.
      • Rao R.P.
      • Nagashima K.
      • Fukusaki E.
      • et al.
      Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction.
      ). Since then, extensive work has shown its contribution to retinal physiology and pathology, as we will analyze in this review.

      S1P: the good, the bad, and the ugly combined?

      The last thirty years have seen the emergence of another star in the world of bioactive lipids: S1P. S1P plays an incredibly diverse array of vital functions in virtually every cell of every organism, having both beneficial and deleterious roles. The basis for this dichotomic behavior lies in the ability of S1P to regulate several cellular processes such as proliferation, survival, differentiation, and cell movement, as well as more complex responses such as vascular development, inflammation, and immune cell trafficking (
      • Kono M.
      • Mi Y.
      • Liu Y.
      • Sasaki T.
      • Allende M.L.
      • Wu Y.P.
      • Yamashita T.
      • Proia R.L.
      The sphingosine-1-phosphate receptors S1P1, S1P2, and S1P3 function coordinately during embryonic angiogenesis.
      ,
      • Tabasinezhad M.
      • Samadi N.
      • Ghanbari P.
      • Mohseni M.
      • Saei A.A.
      • Sharifi S.
      • Saeedi N.
      • Pourhassan A.
      Sphingosin 1-phosphate contributes in tumor progression.
      ).
      As described, S1P is a molecular intermediate in the complex sphingolipid network that can easily interconvert with its precursor, Sph, and be further metabolized to Cer. Because S1P displays opposing cellular roles to both Sph and Cer, the balance of the relative levels of these sphingolipids constitutes the “sphingolipid rheostat”, which ultimately determines cell fate (
      • Cuvillier O.
      • Pirianov G.
      • Kleuser B.
      • Vanek P.G.
      • Cosot O.A.
      • Gutkind J.S.
      • Spiegel S.
      Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.
      ). S1P is synthesized through the phosphorylation of Sph by two SphKs, SphK1 and SphK2, which not only differ in their cellular localization but also generate S1P with distinct and at times opposing functions (
      • Maceyka M.
      • Sankala H.
      • Hait N.C.
      • Le Stunff H.
      • Liu H.
      • Toman R.
      • Collier C.
      • Zhang M.
      • Satin L.S.
      • Merrill A.H.
      • et al.
      SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism.
      ). SphK1 resides in the cytosol and is preferentially located near the plasma membrane. The S1P it produces acts as a second messenger or is secreted to become an extracellular ligand. SphK2 is localized in the nucleus and mitochondria, and the S1P it generates functions as a histone deacetylase inhibitor, thus regulating gene expression (
      • Hait N.C.
      • Allegood J.
      • Maceyka M.
      • Strub G.M.
      • Harikumar K.B.
      • Singh S.K.
      • Luo C.
      • Marmorstein R.
      • Kordula T.
      • Milstien S.
      • et al.
      Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate.
      ). High plasma levels of S1P have been proposed to depend mainly upon its release by vascular endothelial cells and red blood cells (
      • Hänel P.
      • Andréani P.
      • Gräler M.H.
      Erythrocytes store and release sphingosine 1-phosphate in blood.
      ,
      • Venkataraman K.
      • Lee Y.-M.
      • Michaud J.
      • Thangada S.
      • Ai Y.
      • Bonkovsky H.L.
      • Parikh N.S.
      • Habrukowich C.
      • Hla T.
      Vascular endothelium as a contributor of plasma sphingosine 1-phosphate.
      ,
      • Yanagida K.
      • Hla T.
      Vascular and immunobiology of the circulatory sphingosine 1-phosphate gradient.
      ). Circulating S1P is transported bound to plasma protein chaperones, mainly HDL and albumin, but also in smaller amounts by other lipoproteins (
      • Yanagida K.
      • Hla T.
      Vascular and immunobiology of the circulatory sphingosine 1-phosphate gradient.
      ). Multiple cell stimuli promote intracellular generation of S1P, which then acts as an extracellular ligand. Following export to the extracellular milieu by different cell transporters such as Spinster 2 (Spns2) (
      • Kawahara A.
      • Nishi T.
      • Hisano Y.
      • Fukui H.
      • Yamaguchi A.
      • Mochizuki N.
      The sphingolipid transporter Spns2 functions in migration of zebrafish myocardial precursors.
      ), ABCA1 (
      • Sato K.
      • Malchinkhuu E.
      • Horiuchi Y.
      • Mogi C.
      • Tomura H.
      • Tosaka M.
      • Yoshimoto Y.
      • Kuwabara A.
      • Okajima F.
      Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes.
      ), ABCC1 (
      • Mitra P.
      • Oskeritzian C.A.
      • Payne S.G.
      • Beaven M.A.
      • Milstien S.
      • Spiegel S.
      Role of ABCC1 in export of sphingosine-1-phosphate from mast cells.
      ), and ABCG2 (
      • Takabe K.
      • Kim R.H.
      • Allegood J.C.
      • Mitra P.
      • Ramachandran S.
      • Nagahashi M.
      • Harikumar K.B.
      • Hait N.C.
      • Milstien S.
      • Spiegel S.
      Estradiol induces export of sphingosine 1-phosphate from breast cancer cells via ABCC1 and ABCG2.
      ), S1P then binds to and activates a family of five S1P receptors, termed S1PR1–5, in an autocrine/paracrine fashion termed “inside-out” signaling (
      • Takabe K.
      • Paugh S.W.
      • Milstien S.
      • Spiegel S.
      “Inside-out” signaling of sphingosine-1-phosphate: therapeutic targets.
      ). These receptors belong to the superfamily of G protein-coupled membrane receptors that are ubiquitously expressed and activate different G proteins to regulate multiple downstream effectors including PI3K, adenylate cyclase, protein kinase-C, phospholipase C, and intracellular calcium (
      • Hla T.
      • Lee M.-J.
      • Ancellin N.
      • Paik J.H.
      • Kluk M.J.
      Lysophospholipids–receptor revelations.
      ,
      • Spiegel S.
      • Milstien S.
      Sphingosine 1-phosphate, a key cell signaling molecule.
      ). To add further complexity to its signaling pathways, S1P has been proposed to upregulate the transcription of SphK1, activating an “outside-in” S1P/SphK1 signaling axis (
      • Huang K.
      • Huang J.
      • Chen C.
      • Hao J.
      • Wang S.
      • Huang J.
      • Liu P.
      • Huang H.
      AP-1 regulates sphingosine kinase 1 expression in a positive feedback manner in glomerular mesangial cells exposed to high glucose.
      ). These intricate signaling networks allow S1P to trigger a myriad of cellular responses resulting from diverse combinations of cellular localization, receptors, and downstream signaling cascades activated by S1P. Therefore, it is not surprising that S1P activation of S1PRs is not only involved in many pathophysiological processes by regulating proliferation, differentiation, cell migration, cellular barrier integrity, angiogenesis, and immunity, but also contributes to disease processes such as inflammation, atherosclerosis, fibrosis, and neoplasia (
      • Knapp M.
      Cardioprotective role of sphingosine-1-phosphate.
      ,
      • Maceyka M.
      • Harikumar K.B.
      • Milstien S.
      • Spiegel S.
      Sphingosine-1-phosphate signaling and its role in disease.
      ,
      • Takuwa Y.
      • Ikeda H.
      • Okamoto Y.
      • Takuwa N.
      • Yoshioka K.
      Sphingosine-1-phosphate as a mediator involved in development of fibrotic diseases.
      ). For instance, S1P activation of S1PR1 is critical for the progression of autoimmune diseases (
      • Maceyka M.
      • Harikumar K.B.
      • Milstien S.
      • Spiegel S.
      Sphingosine-1-phosphate signaling and its role in disease.
      ).
      In the retina, S1P has both beneficial and detrimental properties. On the one hand, S1P promotes normal retinal morphogenesis (
      • Bian G.
      • Yu G.
      • Liu L.
      • Fang C.
      • Chen K.
      • Ren P.
      • Zhang Q.
      • Liu F.
      • Zhang K.
      • Xue Q.
      • et al.
      Sphingosine 1-phosphate stimulates eyelid closure in the developing rat by stimulating EGFR signaling.
      ,
      • Fang C.
      • Bian G.
      • Ren P.
      • Xiang J.
      • Song J.
      • Yu C.
      • Zhang Q.
      • Liu L.
      • Chen K.
      • Liu F.
      • et al.
      S1p transporter spns2 regulates proper postnatal retinal morphogenesis.
      ) and facilitates signaling in the inner retinal cells (
      • Crousillac S.
      • Colonna J.
      • McMains E.
      • Dewey J.S.
      • Gleason E.
      Sphingosine-1-phosphate elicits receptor-dependent calcium signaling in retinal amacrine cells.
      ). S1P signaling through S1PR1–3 is essential for the adequate development of retinal vasculature; the coordinate signaling of retinal endothelial S1P and vascular endothelial growth factor (VEGF) results in the formation of the trophic factor gradient essential for the growth and maturation of retinal vasculature (
      • Yanagida K.
      • Hla T.
      Vascular and immunobiology of the circulatory sphingosine 1-phosphate gradient.
      ,
      • Carmeliet P.
      • Jain R.K.
      Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases.
      ,
      • Yanagida K.
      • Engelbrecht E.
      • Niaudet C.
      • Jung B.
      • Gaengel K.
      • Holton K.
      • Swendeman S.
      • Liu C.H.
      • Levesque M.V.
      • Kuo A.
      • et al.
      Sphingosine 1-phosphate receptor signaling establishes AP-1 gradients to allow for retinal endothelial cell specialization.
      ). S1P induces the proliferation and later differentiation of retinal progenitors into photoreceptors (
      • Miranda G.E.
      • Abrahan C.E.
      • Politi L.E.
      • Rotstein N.P.
      Sphingosine-1-phosphate is a key regulator of proliferation and differentiation in retina photoreceptors.
      ) and mediates photoreceptor survival upon oxidative damage (
      • Rotstein N.P.
      • Miranda G.E.
      • Abrahan C.E.
      • German O.L.
      Regulating survival and development in the retina: key roles for simple sphingolipids.
      ,
      • Miranda G.E.
      • Abrahan C.E.
      • Politi L.E.
      • Rotstein N.P.
      Sphingosine-1-phosphate is a key regulator of proliferation and differentiation in retina photoreceptors.
      ,
      • Fabiani C.
      • Zulueta A.
      • Bonezzi F.
      • Casas J.
      • Ghidoni R.
      • Signorelli P.
      • Caretti A.
      2-Acetyl-5-tetrahydroxybutyl imidazole (THI) protects 661W cells against oxidative stress.
      ); although this supports a role for S1P during retina development, this remains to be confirmed. Moreover, several photoreceptor trophic factors such as glial-derived neurotrophic factor, DHA (
      • Abrahan C.E.
      • Miranda G.E.
      • Agnolazza D.L.
      • Politi L.E.
      • Rotstein N.P.
      Synthesis of sphingosine is essential for oxidative stress-induced apoptosis of photoreceptors.
      ), and nerve growth factor (NGF) (
      • Toman R.E.
      • Payne S.G.
      • Watterson K.R.
      • Maceyka M.
      • Lee N.H.
      • Milstien S.
      • Bigbee J.W.
      • Spiegel S.
      Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension.
      ) stimulate the S1P/SphK1 axis to enhance the levels of S1P and thus elicit their beneficial roles. On the other hand, S1P triggers threatening processes in cells with crucial support functions in the retina, i.e., the retinal pigment epithelium (RPE) and Müller glial cells. They include secretion of pro-inflammatory cytokines, proliferation, trans-differentiation, and migration (
      • Qiao Y.
      • Hu R.
      • Wang Q.
      • Qi J.
      • Yang Y.
      • Kijlstra A.
      • Yang P.
      Sphingosine 1-phosphate elicits proinflammatory responses in ARPE-19 cells.
      ,
      • Simón M.V.
      • Prado Spalm F.H.
      • Politi L.E.
      • Rotstein N.P.
      Sphingosine-1-phosphate is a crucial signal for migration of retina Müller glial cells.
      ,
      • Swaney J.S.
      • Moreno K.M.
      • Gentile A.M.
      • Sabbadini R.A.
      • Stoller G.L.
      Sphingosine-1-phosphate (S1P) is a novel fibrotic mediator in the eye.
      ,
      • Terao R.
      • Honjo M.
      • Aihara M.
      Apolipoprotein M inhibits angiogenic and inflammatory response by sphingosine 1-phosphate on retinal pigment epithelium cells.
      ), all of which alter the retinal structure and may contribute to visual dysfunction. We will later discuss the role of S1P, among other sphingolipids, in the development of retinal pathologies.

      C1P: a complementing performer

      First identified in the brain in the late eighties, C1P is now an established bioactive sphingolipid involved in numerous cellular processes such as cell proliferation, survival, and growth, and chemotaxis. To date, CerK is the only enzyme known to catalyze C1P synthesis in mammals (
      • Bajjalieh S.M.
      • Martin T.F.
      • Floor E.
      Synaptic vesicle ceramide kinase. A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles.
      ). This enzyme is most abundant in the Golgi, though it is also expressed in the cytosol, plasma membrane, nucleus, and perinuclear membranes (
      • Presa N.
      • Gomez-Larrauri A.
      • Dominguez-Herrera A.
      • Trueba M.
      • Gomez-Muñoz A.
      Novel signaling aspects of ceramide 1-phosphate.
      ). C1P is present both intra- and extracellularly. Once synthesized, it is transported through a specific Cer phosphate transfer protein (CPTP) to the plasma membrane (
      • Simanshu D.K.
      • Kamlekar R.K.
      • Wijesinghe D.S.
      • Zou X.
      • Zhai X.
      • Mishra S.K.
      • Molotkovsky J.G.
      • Malinina L.
      • Hinchcliffe E.H.
      • Chalfant C.E.
      • et al.
      Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.
      ). Although it is not highly permeable, C1P can cross the cell membrane to be released to the extracellular milieu, where it is found in concentrations as high as 20 μM (
      • Hammad S.M.
      • Pierce J.S.
      • Soodavar F.
      • Smith K.J.
      • Al Gadban M.M.
      • Rembiesa B.
      • Klein R.L.
      • Hannun Y.A.
      • Bielawski J.
      • Bielawska A.
      Blood sphingolipidomics in healthy humans: impact of sample collection methodology.
      ,
      • Gomez-Muñoz A.
      The role of ceramide 1-phosphate in tumor cell survival and dissemination.
      ,
      • Mietla J.A.
      • Wijesinghe D.S.
      • Hoeferlin L.A.
      • Shultz M.D.
      • Natarajan R.
      • Fowler A.A.
      • Chalfant C.E.
      • Chalfant C.E.
      Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase.
      ). Existence of a C1P-specific transporter and its secretion in vesicles have been reported (
      • Simanshu D.K.
      • Kamlekar R.K.
      • Wijesinghe D.S.
      • Zou X.
      • Zhai X.
      • Mishra S.K.
      • Molotkovsky J.G.
      • Malinina L.
      • Hinchcliffe E.H.
      • Chalfant C.E.
      • et al.
      Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.
      ,
      • Kuc N.
      • Doermann A.
      • Shirey C.
      • Lee D.D.
      • Lowe C.W.
      • Awasthi N.
      • Schwarz R.E.
      • Stahelin R.V.
      • Schwarz M.A.
      Pancreatic ductal adenocarcinoma cell secreted extracellular vesicles containing ceramide-1-phosphate promote pancreatic cancer stem cell motility.
      ). C1P is a second messenger as well as an extracellular ligand, activating multiple signaling pathways including PI3K, ERK/MAPK, Jun N-terminal kinase (JNK), cytosolic phospholipase A2, NF-κB, and glycogen synthase kinase 3 (GSK3) (
      • Gangoiti P.
      • Granado M.H.
      • Arana L.
      • Ouro A.
      • Gomez-Muñoz A.
      Activation of protein kinase C-α is essential for stimulation of cell proliferation by ceramide 1-phosphate.
      ). As an extracellular ligand, C1P interacts with a G protein-coupled receptor, which is not yet fully characterized, although it is known to differ from S1PRs (
      • Granado M.H.
      • Gangoiti P.
      • Ouro A.
      • Arana L.
      • González M.
      • Trueba M.
      • Gómez-Muñoz A.
      Ceramide 1-phosphate (C1P) promotes cell migration. Involvement of a specific C1P receptor.
      ).
      Recent findings indicate that C1P promotes cell migration (
      • Arana L.
      • Gangoiti P.
      • Ouro A.
      • Rivera I.-G.
      • Ordoñez M.
      • Trueba M.
      • Lankalapalli R.S.
      • Bittman R.
      • Gomez-Muñoz A.
      Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate.
      ,
      • Arana L.
      • Ordoñez M.
      • Ouro A.
      • Rivera I.G.
      • Gangoiti P.
      • Trueba M.
      • Gomez-Muñoz A.
      Ceramide 1-phosphate induces macrophage chemoattractant protein-1 release: involvement in ceramide 1-phosphate-stimulated cell migration.
      ), proliferation (
      • Gangoiti P.
      • Granado M.H.
      • Wang S.W.
      • Kong J.Y.
      • Steinbrecher U.P.
      • Gómez-Muñoz A.
      Ceramide 1-phosphate stimulates macrophage proliferation through activation of the PI3-kinase/PKB, JNK and ERK1/2 pathways.
      ,
      • Mitra P.
      • Maceyka M.
      • Payne S.G.
      • Lamour N.
      • Milstien S.
      • Chalfant C.E.
      • Spiegel S.
      Ceramide kinase regulates growth and survival of A549 human lung adenocarcinoma cells.
      ,
      • Ouro A.
      • Arana L.
      • Riazy M.
      • Zhang P.
      • Gomez-Larrauri A.
      • Steinbrecher U.
      • Duronio V.
      • Gomez-Muñoz A.
      Vascular endothelial growth factor mediates ceramide 1-phosphate-stimulated macrophage proliferation.
      ), and survival, as C1P is also known to be antiapoptotic (
      • Granado M.H.
      • Gangoiti P.
      • Ouro A.
      • Arana L.
      • González M.
      • Trueba M.
      • Gómez-Muñoz A.
      Ceramide 1-phosphate (C1P) promotes cell migration. Involvement of a specific C1P receptor.
      ,
      • Gómez-Muñoz A.
      • Kong J.Y.
      • Salh B.
      • Steinbrecher U.P.
      Ceramide-1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages.
      ). These actions make C1P a relevant signal transducer for cancer progression (
      • Mitra P.
      • Maceyka M.
      • Payne S.G.
      • Lamour N.
      • Milstien S.
      • Chalfant C.E.
      • Spiegel S.
      Ceramide kinase regulates growth and survival of A549 human lung adenocarcinoma cells.
      ,
      • Rivera I.-G.
      • Ordoñez M.
      • Presa N.
      • Gangoiti P.
      • Gomez-Larrauri A.
      • Trueba M.
      • Fox T.
      • Kester M.
      • Gomez-Muñoz A.
      Ceramide 1-phosphate regulates cell migration and invasion of human pancreatic cancer cells.
      ). C1P can act as a pro- or anti-inflammatory signal, depending on the cell type (
      • Presa N.
      • Gomez-Larrauri A.
      • Dominguez-Herrera A.
      • Trueba M.
      • Gomez-Muñoz A.
      Novel signaling aspects of ceramide 1-phosphate.
      ,
      • Berwick M.L.
      • Dudley B.A.
      • Maus K.
      • Chalfant C.E.
      The role of ceramide 1-phosphate in inflammation, cellular proliferation, and wound healing.
      ,
      • Presa N.
      • Gomez-Larrauri A.
      • Rivera I.-G.
      • Ordoñez M.
      • Trueba M.
      • Gomez-Muñoz A.
      Regulation of cell migration and inflammation by ceramide 1-phosphate.
      ), and also has neuroprotective effects in the nervous system (
      • Aleshin S.
      • Reiser G.
      Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) protects against ceramide-induced cellular toxicity in rat brain astrocytes and neurons by activation of ceramide kinase.
      ,
      • Tabuchi K.
      • Hara A.
      Impact of sphingolipid mediators on the determination of cochlear survival in ototoxicity.
      ) contributing to neurotransmitter release (
      • Jeon H.J.
      • Lee D.H.
      • Kang M.S.
      • Lee M.-O.
      • Jung K.M.
      • Jung S.Y.
      • Kim D.K.
      Dopamine release in PC12 cells is mediated by Ca(2+)-dependent production of ceramide via sphingomyelin pathway.
      ).
      In the retina, C1P functions are still elusive. CerK is highly expressed in the retina (
      • Mandal N.A.
      • Tran J.T.
      • Saadi A.
      • Rahman A.K.
      • Huynh T.P.
      • Klein W.H.
      • Cho J.H.
      Expression and localization of CERKL in the mammalian retina, its response to light-stress, and relationship with NeuroD1 gene.
      ), and it is present in the RPE cells (
      • Zhu D.
      • Sreekumar P.G.
      • Hinton D.R.
      • Kannan R.
      Expression and regulation of enzymes in the ceramide metabolic pathway in human retinal pigment epithelial cells and their relevance to retinal degeneration.
      ). CerK is critical for controlling C1P levels in this tissue, as it is markedly reduced in Cerk−/− mouse retinas (
      • Graf C.
      • Niwa S.
      • Müller M.
      • Kinzel B.
      • Bornancin F.
      Wild-type levels of ceramide and ceramide-1-phosphate in the retina of ceramide kinase-like-deficient mice.
      ). C1P promotes the proliferation of photoreceptor progenitors and their differentiation as photoreceptors in vitro (
      • Miranda G.E.
      • Abrahan C.E.
      • Agnolazza D.L.
      • Politi L.E.
      • Rotstein N.P.
      Ceramide-1-phosphate, a new mediator of development and survival in retina photoreceptors.
      ). C1P also promotes photoreceptor survival through the preservation of their mitochondrial potential (
      • Miranda G.E.
      • Abrahan C.E.
      • Agnolazza D.L.
      • Politi L.E.
      • Rotstein N.P.
      Ceramide-1-phosphate, a new mediator of development and survival in retina photoreceptors.
      ) and probably also by preventing the accumulation of Cer, a mechanism already observed in macrophages (
      • Granado M.H.
      • Gangoiti P.
      • Ouro A.
      • Arana L.
      • González M.
      • Trueba M.
      • Gómez-Muñoz A.
      Ceramide 1-phosphate (C1P) promotes cell migration. Involvement of a specific C1P receptor.
      ,
      • Gómez-Muñoz A.
      • Kong J.Y.
      • Salh B.
      • Steinbrecher U.P.
      Ceramide-1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages.
      ). We will discuss the role of C1P in the development of retinal pathologies in a later part of this review.

      Other sphingolipid players in retinal pathologies

      Sph

      Along with Cer, Sph is an endogenous mediator of apoptosis and its addition inhibits proliferation and/or induces apoptosis in many cell types in vitro (
      • Cuvillier O.
      Sphingosine in apoptosis signaling.
      ). Different apoptotic inducers, such as oxidative stress, chemotherapy, environmental stress, and tumor necrosis factor α (TNF-α), rapidly increase the levels of both Cer and Sph, which then induce cell cycle arrest, senescence, or apoptosis (
      • Hannun Y.A.
      • Obeid L.M.
      Sphingolipids and their metabolism in physiology and disease.
      ,
      • Cuvillier O.
      Sphingosine in apoptosis signaling.
      ,
      • Cuvillier O.
      • Edsall L.
      • Spiegel S.
      Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells.
      ,
      • Ogretmen B.
      • Hannun Y.A.
      Biologically active sphingolipids in cancer pathogenesis and treatment.
      ,
      • Ohta H.
      • Sweeney E.A.
      • Masamune A.
      • Yatomi Y.
      • Hakomori S.
      • Igarashi Y.
      Induction of apoptosis by sphingosine in human leukemic HL-60 cells: a possible endogenous modulator of apoptotic DNA fragmentation occurring during phorbol ester-induced differentiation.
      ). Usually, Cer upsurge precedes that of Sph, implying that Sph accumulation results mainly from the deacylation of Cer, catalyzed by CDases (
      • Cuvillier O.
      • Edsall L.
      • Spiegel S.
      Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells.
      ,
      • Ohta H.
      • Sweeney E.A.
      • Masamune A.
      • Yatomi Y.
      • Hakomori S.
      • Igarashi Y.
      Induction of apoptosis by sphingosine in human leukemic HL-60 cells: a possible endogenous modulator of apoptotic DNA fragmentation occurring during phorbol ester-induced differentiation.
      ). The fact that Sph can be either rapidly recycled to regenerate Cer or phosphorylated by SphKs to render S1P (Fig. 2) has complicated ascertaining Sph’s effects. However, apoptosis in thymocytes and 3T3/A31 is drastically reduced by inhibiting Sph synthesis (
      • Lépine S.
      • Lakatos B.
      • Courageot M.-P.
      • Le Stunff H.
      • Sulpice J.-C.
      • Giraud F.
      Sphingosine contributes to glucocorticoid-induced apoptosis of thymocytes independently of the mitochondrial pathway.
      ,
      • Suzuki E.
      • Handa K.
      • Toledo M.S.
      • Hakomori S.
      Sphingosine-dependent apoptosis: a unified concept based on multiple mechanisms operating in concert.
      ). Sph itself induces apoptosis in cells under conditions where Cer is unable to do so and when Sph conversion to Cer is blocked (
      • Cuvillier O.
      • Edsall L.
      • Spiegel S.
      Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells.
      ,
      • Sweeney E.A.
      • Sakakura C.
      • Shirahama T.
      • Masamune A.
      • Ohta H.
      • Hakomori S.
      • Igarashi Y.
      Sphingosine and its methylated derivative N,N-dimethylsphingosine (DMS) induce apoptosis in a variety of human cancer cell lines.
      ). These findings have contributed in establishing Sph as a bona fide second messenger, whose increase is triggered by diverse apoptotic stimuli to induce cell death.
      Sph modulates the functions of several signaling molecules to promote cell death. In addition to PKC, Sph activates protein kinase A (
      • Ma Y.
      • Pitson S.
      • Hercus T.
      • Murphy J.
      • Lopez A.
      • Woodcock J.
      Sphingosine activates protein kinase A type II by a novel cAMP-independent mechanism.
      ) and inhibits calmodulin-dependent kinases (
      • Olivera A.
      • Spiegel S.
      Sphingosine-1-phosphate as second messenger in cell proliferation induced by PDGF and FCS mitogens.
      ). Sph induces apoptosis through the generation of ROS and downregulation of Bcl-2, with the consequent activation of the mitochondrial pathway, cytochrome c release, and caspase-3 activation (
      • Abrahan C.E.
      • Miranda G.E.
      • Agnolazza D.L.
      • Politi L.E.
      • Rotstein N.P.
      Synthesis of sphingosine is essential for oxidative stress-induced apoptosis of photoreceptors.
      ,
      • Cuvillier O.
      • Edsall L.
      • Spiegel S.
      Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells.
      ,
      • Sweeney E.A.
      • Sakakura C.
      • Shirahama T.
      • Masamune A.
      • Ohta H.
      • Hakomori S.
      • Igarashi Y.
      Sphingosine and its methylated derivative N,N-dimethylsphingosine (DMS) induce apoptosis in a variety of human cancer cell lines.
      ). This also involves downregulation of the pro-survival signaling through Akt signaling together with increased phosphorylation of 14-3-3 protein and its consequent inability to sequester BAD/Bax (
      • Suzuki E.
      • Handa K.
      • Toledo M.S.
      • Hakomori S.
      Sphingosine-dependent apoptosis: a unified concept based on multiple mechanisms operating in concert.
      ). Mitochondrial dysfunction is apparently instrumental in Sph-induced cell death, because preventing it by overexpressing Bcl-xL impedes cell death, even upon increased levels of Sph (
      • Cuvillier O.
      • Nava V.E.
      • Murthy S.K.
      • Edsall L.C.
      • Levade T.
      • Milstien S.
      • Spiegel S.
      Sphingosine generation, cytochrome c release, and activation of caspase-7 in doxorubicin-induced apoptosis of MCF7 breast adenocarcinoma cells.
      ). Mitochondrial accumulation of Sph impairs the electron transport chain and has been proposed to be critical for brain injury after trauma (
      • Novgorodov S.A.
      • Riley C.L.
      • Yu J.
      • Borg K.T.
      • Hannun Y.A.
      • Proia R.L.
      • Kindy M.S.
      • Gudz T.I.
      Essential roles of neutral ceramidase and sphingosine in mitochondrial dysfunction due to traumatic brain injury.
      ).
      In the retina, both enhanced endogenous synthesis and exogenous addition of Sph promote the death of photoreceptors and amacrine neurons (
      • Abrahan C.E.
      • Miranda G.E.
      • Agnolazza D.L.
      • Politi L.E.
      • Rotstein N.P.
      Synthesis of sphingosine is essential for oxidative stress-induced apoptosis of photoreceptors.
      ). Oxidative stress increases the synthesis of Sph, leading to photoreceptor death, and this death is prevented by inhibition of alkaline CDase. Sph promotes ROS formation, mitochondrial permeabilization, and cytochrome c release leading to photoreceptor apoptosis. Notably, DHA protects photoreceptors by increasing SphK1 expression and translocation to the plasma membrane, suggesting that the increased generation of S1P and/or the consequent decrease in Sph levels prevent their death (
      • Abrahan C.E.
      • Miranda G.E.
      • Agnolazza D.L.
      • Politi L.E.
      • Rotstein N.P.
      Synthesis of sphingosine is essential for oxidative stress-induced apoptosis of photoreceptors.
      ). In contrast, overexpression of acid CDase in a human RPE cell line, ARPE-19, increases Sph levels and protects these cells from oxidative damage with no visible accumulation of S1P (
      • Sugano E.
      • Edwards G.
      • Saha S.
      • Wilmott L.A.
      • Grambergs R.C.
      • Mondal K.
      • Qi H.
      • Stiles M.
      • Tomita H.
      • Mandal N.
      Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress.
      ). Although further research is required to establish Sph’s effects in different retinal cell types, the existing data have cemented Sph and Cer as crucial mediators in the onset of photoreceptor death and support the hypothesis that modulation of the sphingolipid pathways may provide powerful tools for treating neurodegenerative diseases of the retina.

      GlcCer and LacCer

      The complex sphingolipid metabolic routes provide alternative pathways to prevent the increase in Cer by converting it to glycosyl Cers and thus avoid the effects of its accumulation. Many different molecules regulate the expression and activity of GlcCer synthases (GCSs), which catalyze GlcCer synthesis from Cer (
      • Bleicher R.J.
      • Cabot M.C.
      Glucosylceramide synthase and apoptosis.
      ). GlcCer is found in multiple animal tissues, such as spleen, skin, erythrocytes, and the nervous system, and has often ambiguous roles in mammalian cells. It is essential for preserving the water permeability barrier of skin, and its levels in tissues are affected in skin disorders, diabetes, cardiovascular diseases, and cancer (
      • Messner M.C.
      • Cabot M.C.
      Glucosylceramide in humans.
      ). Its formation serves as an escape route preventing Cer accretion and the consequent induction of cell death and has been associated to drug resistance in several cancers (
      • Liu Y.-Y.
      • Yu J.Y.
      • Yin D.
      • Patwardhan G.A.
      • Gupta V.
      • Hirabayashi Y.
      • Holleran W.M.
      • Giuliano A.E.
      • Jazwinski S.M.
      • Gouaze-Andersson V.
      • et al.
      A role for ceramide in driving cancer cell resistance to doxorubicin.
      ). GCS expression is linked to poor prognosis in certain cancer patients (
      • Kim J.W.
      • Park Y.
      • Roh J.L.
      • Cho K.J.
      • Choi S.H.
      • Nam S.Y.
      • Kim S.Y.
      Prognostic value of glucosylceramide synthase and P-glycoprotein expression in oral cavity cancer.
      ), whereas its inhibition attenuates resistance to chemotherapy in different tumor cells (
      • Roh J.L.
      • Kim E.H.
      • Park J.Y.
      • Kim J.W.
      Inhibition of glucosylceramide synthase sensitizes head and neck cancer to cisplatin.
      ,
      • Stefanovic M.
      • Tutusaus A.
      • Martinez-Nieto G.A.
      • Bárcena C.
      • De Gregorio E.
      • Moutinho C.
      • Barbero-Camps E.
      • Villanueva A.
      • Colell A.
      • Marí M.
      • et al.
      Targeting glucosylceramide synthase upregulation reverts sorafenib resistance in experimental hepatocellular carcinoma.
      ). GlcCer is involved in cell proliferation, differentiation, oncogenic transformation, and tumor metastasis (
      • Bleicher R.J.
      • Cabot M.C.
      Glucosylceramide synthase and apoptosis.
      ,
      • Deguchi H.
      • Fernández J.A.
      • Pabinger I.
      • Heit J.A.
      • Griffin J.H.
      Plasma glucosylceramide deficiency as potential risk factor for venous thrombosis and modulator of anticoagulant protein C pathway.
      ). It has been shown to inhibit LPS-induced inflammation in macrophages by blocking nuclear translocation of NF-κB (
      • Yeom M.
      • Park J.
      • Lim C.
      • Sur B.
      • Lee B.
      • Han J.J.
      • Choi H.D.
      • Lee H.
      • Hahm D.H.
      Glucosylceramide attenuates the inflammatory mediator expression in lipopolysaccharide-stimulated RAW264.7 cells.
      ), and also have immunostimulatory functions, acting as a ligand for lectin receptors sensing damaged cells (
      • Nagata M.
      • Izumi Y.
      • Ishikawa E.
      • Kiyotake R.
      • Doi R.
      • Iwai S.
      • Omahdi Z.
      • Yamaji T.
      • Miyamoto T.
      • Bamba T.
      • et al.
      Intracellular metabolite β-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity.
      ).
      In the eye, GlcCer increases in the retinas of diabetic rats and preventing this increase augments insulin sensitivity and is neuroprotective, linking GlcCer accumulation to the pathogenesis of diabetic retinopathy (DR) (
      • Fox T.E.
      • Han X.
      • Kelly S.
      • Merrill A.H.
      • Martin R.E.
      • Anderson R.E.
      • Gardner T.W.
      • Kester M.
      Diabetes alters sphingolipid metabolism in the retina: a potential mechanism of cell death in diabetic retinopathy.
      ). GlcCer also accumulates in retinas of patients with Gaucher’s disease, resulting in visual loss (
      • Seidova S.F.
      • Kotliar K.
      • Foerger F.
      • Klopfer M.
      • Lanzl I.
      Functional retinal changes in Gaucher disease.
      ). Interestingly, inhibiting GlcCer synthesis in photoreceptors abrogates the protective effect of DHA upon oxidative stress and Cer increase (
      • German O.L.
      • Insua M.F.
      • Gentili C.
      • Rotstein N.P.
      • Politi L.E.
      Docosahexaenoic acid prevents apoptosis of retina photoreceptors by activating the ERK/MAPK pathway.
      ). Hence, GlcCer might be either protective or deleterious in the retina in a context- and concentration-dependent manner.
      In the Golgi, GlcCer can be converted by LacCer synthases to LacCer, which has a pivotal role in the synthesis of most major glycosphingolipids. Cellular functions of LacCer are still ill-defined. It has been proposed that several molecules, such as growth factors, pro-inflammatory cytokines, and modified LDL increase LacCer levels, activating multiple pathways that contribute to cell proliferation, adhesion, migration, angiogenesis, and apoptosis (
      • Chatterjee S.
      • Alsaeedi N.
      Lactosylceramide synthase as a therapeutic target to mitigate multiple human diseases in animal models.
      ,
      • Chatterjee S.
      • Pandey A.
      The Yin and Yang of lactosylceramide metabolism: Implications in cell function.
      ). LacCer is thought to mediate the attachment of many pathogens and may participate in the innate response to them, especially on nonimmune cells (
      • Hahn P.Y.
      • Evans S.E.
      • Kottom T.J.
      • Standing J.E.
      • Pagano R.E.
      • Limper A.H.
      Pneumocystis carinii cell wall β-glucan induces release of macrophage inflammatory protein-2 from alveolar epithelial cells via a -mediated mechanism.
      ,
      • Jimenez-Lucho V.
      • Ginsburg V.
      • Krivan H.C.
      Cryptococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipid lactosylceramide (GAlβ1-4Glcβ1-1Cer), a possible adhesion receptor for yeasts.
      ). It is enriched in the plasma membrane of neutrophils, promoting their migration and phagocytosis, and mediating innate immune functions (
      • Iwabuchi K.
      • Masuda H.
      • Kaga N.
      • Nakayama H.
      • Matsumoto R.
      • Iwahara C.
      • Yoshizaki F.
      • Tamaki Y.
      • Kobayashi T.
      • Hayakawa T.
      • et al.
      Properties and functions of lactosylceramide from mouse neutrophils.
      ). An accumulation of LacCer has been linked to pathogenic alterations in diseases affecting different organs. In diabetic mice, an increased Cer flux leads to elevated levels of LacCer in cardiac tissue and contributes to mitochondrial dysfunction (
      • Novgorodov S.A.
      • Riley C.L.
      • Yu J.
      • Keffler J.A.
      • Clarke C.J.
      • Van Laer A.O.
      • Baicu C.F.
      • Zile M.R.
      • Gudz T.I.
      Lactosylceramide contributes to mitochondrial dysfunction in diabetes.
      ). Furthermore, oxidative stress leads to LacCer accumulation in retinal endothelial cells, suggesting its possible role in inflammatory eye diseases (
      • Mondal K.
      • Mandal N.
      Role of bioactive sphingolipids in inflammation and eye diseases.
      ).

      Sphingolipids in retinal pathogenesis

      During the last decade, evidence has been acquired that supports the relevance and association of sphingolipids in multiple retinal diseases (Table 1). In the next part of the review we will focus on the involvement of sphingolipids like Cer, Sph, S1P, C1P, and glycosylceramides (HexCer and LacCer) in multiple retinal pathologies.
      Table 1Association of bioactive sphingolipids with different retinal diseases/pathologies

      Age-related macular degeneration: watching sphingolipids at work?

      Age-related macular degeneration (AMD) is a degenerative disease of the macula that accounts for approximately half of all legal blindness in industrialized countries (
      • Owen C.G.
      • Fletcher A.E.
      • Donoghue M.
      • Rudnicka A.R.
      How big is the burden of visual loss caused by age related macular degeneration in the United Kingdom?.
      ). Among the two subtypes, nonexudative or atrophic AMD (also called dry AMD) is a broad designation, encompassing all forms that do not result in neovascularization. This includes early and intermediate forms of AMD, as well as the advanced form of dry AMD known as geographic atrophy (GA). Atrophic AMD has a relatively poorly understood etiology and no effective treatment. It involves the formation of drusen between the RPE and the Bruch’s membrane, leading to slow but increasing RPE and photoreceptor degeneration and progressive GA (
      • Zając-Pytrus H.M.
      • Pilecka A.
      • Turno-Krecicka A.
      • Adamiec-Mroczek J.
      • Misiuk-Hojlo M.
      The dry form of age-related macular degeneration (AMD): the current concepts of pathogenesis and prospects for treatment.
      ). On the other hand, in exudative or neovascular AMD (also known as wet AMD), vision loss is due to abnormal choroidal neovascularization. It is characterized by overproduction of VEGF in the RPE, responsible for breakdown of the blood-retinal barrier and choroidal/subretinal neovascularization (
      • Nowak J.Z.
      Age-related macular degeneration (AMD): pathogenesis and therapy.
      ). The proliferation of abnormal blood vessels in the retina, which are more fragile than typical blood vessels, leads to hemorrhage, causing macular scarring and edema, which is the major cause of vision loss in exudative AMD (
      • Campochiaro P.A.
      Ocular neovascularization.
      ). However, degeneration of the RPE cells and subsequent photoreceptor death leading to loss of central vision is the hallmark of both forms of AMD. Several studies have proposed a connection between inflammatory mechanisms and AMD pathology (
      • Kauppinen A.
      • Paterno J.J.
      • Blasiak J.
      • Salminen A.
      • Kaarniranta K.
      Inflammation and its role in age-related macular degeneration.
      ,
      • Knickelbein J.E.
      • Chan C.-C.
      • Sen H.N.
      • Ferris F.L.
      • Nussenblatt R.B.
      Inflammatory mechanisms of age-related macular degeneration.
      ,
      • Parmeggiani F.
      • Romano M.R.
      • Costagliola C.
      • Semeraro F.
      • Incorvaia C.
      • D’Angelo S.
      • Perri P.
      • De Palma P.
      • De Nadai K.
      • Sebastiani A.
      Mechanism of inflammation in age-related macular degeneration.
      ). Subretinal drusen contain a variety of potentially harmful constituents such as lipids, RPE-derived cellular debris, oxidation byproducts, and inflammatory factors including complement components and immunoglobulins (
      • Johnson L.V.
      • Ozaki S.
      • Staples M.K.
      • Erickson P.A.
      • Anderson D.H.
      A potential role for immune complex pathogenesis in drusen formation.
      ,
      • Hageman G.S.
      • Luthert P.J.
      • Victor Chong N.H.
      • Johnson L.V.
      • Anderson D.H.
      • Mullins R.F.
      An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration.
      ,
      • Hageman G.S.
      • Mullins R.F.
      • Russell S.R.
      • Johnson L.V.
      • Anderson D.H.
      Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.
      ,
      • Anderson D.H.
      • Mullins R.F.
      • Hageman G.S.
      • Johnson L.V.
      A role for local inflammation in the formation of drusen in the aging eye.
      ). Complement factor H (CFH), a major inhibitor of the complement pathway, is synthesized by RPE cells and accumulates within drusen; the variant harboring a point mutation Y402H in the CFH gene has been identified as a major risk factor for the development of AMD (
      • Hageman G.S.
      • Anderson D.H.
      • Johnson L.V.
      • Hancox L.S.
      • Taiber A.J.
      • Hardisty L.I.
      • Hageman J.L.
      • Stockman H.A.
      • Borchardt J.D.
      • Gehrs K.M.
      • et al.
      A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.
      ,
      • Gangnon R.E.
      • Lee K.E.
      • Klein B.E.
      • Iyengar S.K.
      • Sivakumaran T.A.
      • Klein R.
      Effect of the Y402H variant in the complement factor H gene on the incidence and progression of age-related macular degeneration: results from multistate models applied to the Beaver Dam Eye Study.
      ,
      • Zareparsi S.
      • Branham K.E.H.
      • Li M.
      • Shah S.
      • Klein R.J.
      • Ott J.
      • Hoh J.
      • Abecasis G.R.
      • Swaroop A.
      Strong association of the Y402H variant in complement factor H at 1q32 with susceptibility to age-related macular degeneration.
      ,
      • Haines J.L.
      • Hauser M.A.
      • Schmidt S.
      • Scott W.K.
      • Olson L.M.
      • Gallins P.
      • Spencer K.L.
      • Kwan S.Y.
      • Noureddine M.
      • Gilbert J.R.
      • et al.
      Complement factor H variant increases the risk of age-related macular degeneration.
      ). Further associations have been identified between AMD and several complement pathway-associated genes: complement factor B, complement factor H-related 1 and 3, and complement components 2 and 3 (
      • Anderson D.H.
      • Radeke M.J.
      • Gallo N.B.
      • Chapin E.A.
      • Johnson P.T.
      • Curletti C.R.
      • Hancox L.S.
      • Hu J.
      • Ebright J.N.
      • Malek G.
      • et al.
      The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited.
      ). Interestingly, a recent study showed that the Y402H variant in the CFH gene influences the association of high serum Cer levels with GA, and high levels of HexCer in the serum of patients with choroidal neovascularization and GA (
      • Pujol-Lereis L.M.
      • Liebisch G.
      • Schick T.
      • Lin Y.
      • Grassmann F.
      • Uchida K.
      • Zipfel P.F.
      • Fauser S.
      • Skerka C.
      • Weber B.H.F.
      Evaluation of serum sphingolipids and the influence of genetic risk factors in age-related macular degeneration.
      ).
      Increasing evidence supports altered sphingolipid levels contributing to AMD pathology (
      • Zhu D.
      • Sreekumar P.G.
      • Hinton D.R.
      • Kannan R.
      Expression and regulation of enzymes in the ceramide metabolic pathway in human retinal pigment epithelial cells and their relevance to retinal degeneration.
      ). Degeneration and death of photoreceptor and RPE cells is the ultimate cause of blindness in AMD, and Cer-induced inflammation and apoptosis have been linked to degeneration of both cell types in different models of AMD and other ocular degenerative diseases (
      • Zhu D.
      • Sreekumar P.G.
      • Hinton D.R.
      • Kannan R.
      Expression and regulation of enzymes in the ceramide metabolic pathway in human retinal pigment epithelial cells and their relevance to retinal degeneration.
      ). Chen et al. (
      • Chen H.
      • Tran J.T.
      • Eckerd A.
      • Huynh T.P.
      • Elliott M.H.
      • Brush R.S.
      • Mandal N.A.
      Inhibition of de novo ceramide biosynthesis by FTY720 protects rat retina from light-induced degeneration.
      ) have shown that the increase in Cer levels by de novo biosynthesis mediates photoreceptor apoptosis in a rat model of light-induced retinal degeneration, a pathology with significant overlap with human atrophic AMD, whereas inhibiting Cer production protects the retina against light stress. Elevated Cer levels trigger photoreceptor death in different in vitro models of retinal degeneration. Oxidative stress increases de novo synthesis of Cer in cultured rat retinal neurons and induces photoreceptor death by affecting mitochondrial function, whereas lowering Cer levels by inhibiting its synthesis or promoting its glycosylation to GlcCer prevents photoreceptor death (
      • German O.L.
      • Miranda G.E.
      • Abrahan C.E.
      • Rotstein N.P.
      Ceramide is a mediator of apoptosis in retina photoreceptors.
      ). Oxidative stress also induces apoptosis of the 661W photoreceptor-like cell line through the activation of aSMase and subsequent Cer increase, which activates the mitochondrial pathway of apoptosis, the caspase cascade, and also the calpain- and cathepsin-mediated death pathways. Again, inhibiting aSMase-dependent Cer synthesis prevented cell death (
      • Sanvicens N.
      • Cotter T.G.
      Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells.
      ). Cer has recently been shown to induce cell death in cultured photoreceptors through the Parthanatos death pathway, involving activation of poly-ADP ribose polymerase 1 (PARP-1) and calpains (
      • Prado Spalm F.H.
      • Vera M.S.
      • Dibo M.J.
      • Simón M.V.
      • Politi L.E.
      • Rotstein N.P.
      Ceramide induces the death of retina photoreceptors through activation of Parthanatos.
      ). As a whole, these studies clearly establish Cer as a master controller of the cell death decision in photoreceptor cells independent of its biosynthetic pathway. The increase in Cer levels triggers cell death through a diversity of pathways, suggesting that the biosynthetic pathway and the cell death routines may be context- and cell type-dependent.
      Cer has also been shown to be a crucial player in the induction of RPE cell death. Cer addition to human cultured RPE cells increases the levels of ROS, promoting mitochondrial permeabilization and caspase-3 activation, leading to RPE cell apoptosis (
      • Kannan R.
      • Jin M.
      • Gamulescu M.A.
      • Hinton D.R.
      Ceramide-induced apoptosis: role of catalase and hepatocyte growth factor.
      ). Oxidative stress has been shown to induce Cer synthesis and promote apoptosis of human cultured RPE cells (
      • Barak A.
      • Morse L.S.
      • Goldkorn T.
      Ceramide: a potential mediator of apoptosis in human retinal pigment epithelial cells.
      ) and also induces cell death in ARPE-19 cells, increasing Cer and HexCer levels. Conversely, overexpression of acid CDase diminishes Cer levels by hydrolyzing it to Sph, and partially decreases cell death, probably by transforming at least part of the generated Sph into S1P (
      • Sugano E.
      • Edwards G.
      • Saha S.
      • Wilmott L.A.
      • Grambergs R.C.
      • Mondal K.
      • Qi H.
      • Stiles M.
      • Tomita H.
      • Mandal N.
      Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress.
      ). Conversely, overexpression of nSMase, which increases Cer generation, promotes ARPE-19 cell death (
      • Zhu D.
      • Sreekumar P.G.
      • Hinton D.R.
      • Kannan R.
      Expression and regulation of enzymes in the ceramide metabolic pathway in human retinal pigment epithelial cells and their relevance to retinal degeneration.
      ). Cer has been implicated in AMD-related RPE degeneration, wherein activation of aSMase results in RPE autophagy dysfunction, complement regulatory protein recycling, endosome biogenesis, and complement activation (
      • Tan L.X.
      • Toops K.A.
      • Lakkaraju A.
      Protective responses to sublytic complement in the retinal pigment epithelium.
      ,
      • Toops K.A.
      • Tan L.X.
      • Jiang Z.
      • Radu R.A.
      • Lakkaraju A.
      Cholesterol-mediated activation of acid sphingomyelinase disrupts autophagy in the retinal pigment epithelium.
      ,
      • Kaur G.
      • Tan L.X.
      • Rathnasamy G.
      • La Cunza N.
      • Germer C.J.
      • Toops K.A.
      • Fernandes M.
      • Blenkinsop T.A.
      • Lakkaraju A.
      Aberrant early endosome biogenesis mediates complement activation in the retinal pigment epithelium in models of macular degeneration.
      ,
      • Natoli R.
      • Fernando N.
      • Jiao H.
      • Racic T.
      • Madigan M.
      • Barnett N.L.
      • Chu-Tan J.A.
      • Valter K.
      • Provis J.
      • Rutar M.
      Retinal macrophages synthesize C3 and activate complement in AMD and in models of focal retinal degeneration.
      ). These data highlight the involvement of Cer in the degeneration and death of photoreceptors and RPE cells; because these are critical events for AMD onset and progression, Cer may have a role in triggering this disease and controlling its metabolism may provide a therapeutic strategy for this disease.
      Other sphingolipids may also be involved in AMD progression. Sph has also been implicated in photoreceptor death; its addition induces photoreceptor apoptosis, increasing ROS production and promoting cytochrome c release from mitochondria (
      • Abrahan C.E.
      • Miranda G.E.
      • Agnolazza D.L.
      • Politi L.E.
      • Rotstein N.P.
      Synthesis of sphingosine is essential for oxidative stress-induced apoptosis of photoreceptors.
      ). The pro-inflammatory state of RPE together with its release of proangiogenic factors is known to contribute to AMD development. Mounting evidence supports a role for S1P, a well-known mediator of inflammation and neovascularization, in these processes. Recent work has shown that S1P promotes the secretion of inflammatory cytokines by ARPE-19 cells (

      Deleted in proof.

      ). In addition, S1PR2 deficient mice show marked downregulation of laser-induced choroidal neovascularization (
      • Skoura A.
      • Sanchez T.
      • Claffey K.
      • Mandala S.M.
      • Proia R.L.
      • Hla T.
      Essential role of sphingosine 1-phosphate receptor 2 in pathological angiogenesis of the mouse retina.
      ), a hallmark of wet AMD. This neovascularization is also significantly reduced when S1P action is blocked with sonepcizumab, a humanized monoclonal antibody against S1P (
      • Xie B.
      • Shen J.
      • Dong A.
      • Rashid A.
      • Stoller G.
      • Campochiaro P.A.
      Blockade of sphingosine-1-phosphate reduces macrophage influx and retinal and choroidal neovascularization.
      ). Puzzlingly, S1P has been shown to prevent neuronal death in different models of retinal injuries. S1P also promotes differentiation and survival of cultured photoreceptors (
      • Zhang H.
      • Desai N.N.
      • Olivera A.
      • Seki T.
      • Brooker G.
      • Spiegel S.
      Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation.
      ). The expression of SphK1, S1PR2, and S1PR3 rapidly increases in a rat model of light-induced retinal degeneration, suggesting a function for S1P signaling in light stress responses in the retina (
      • Porter H.
      • Qi H.
      • Prabhu N.
      • Grambergs R.
      • McRae J.
      • Hopiavuori B.
      • Mandal N.
      Characterizing sphingosine kinases and sphingosine 1-phosphate receptors in the mammalian eye and retina.
      ). Due to the multiple processes it modulates, S1P may have opposing functions in the development of AMD, on the one hand promoting survival of photoreceptors and on the other hand contributing to the progression of inflammation and neovascularization. Further research is needed for establishing the functions of S1P and uncovering the signaling mechanisms it triggers.
      In conclusion, these findings establish that sphingolipids play important roles in central features contributing to AMD pathology by regulating retinal cell death, inflammation, and neovascularization and may therefore be involved in its onset and/or progression. Controlling their metabolism and the intracellular pathways they activate may provide novel targets and therapeutic strategies for treating this devastating disease.

      Retinal inflammation and uveitis: are sphingolipids critical regulators?

      Uveitis is an autoimmune eye disease characterized by inflammation of the uvea, specifically in the middle layer of the eye consisting of the anterior uvea (iris and ciliary body) and the posterior uvea (choroid) (
      • Mondal K.
      • Mandal N.
      Role of bioactive sphingolipids in inflammation and eye diseases.
      ). Common symptoms of anterior uveitis include pain, erythema, and photophobia, while intermediate and posterior uveitis results in visual deficits (
      • Grambergs R.
      • Mondal K.
      • Mandal N.
      Inflammatory ocular diseases and sphingolipid signaling.
      ) leading to loss of vision of approximately 30,000 people annually in the United States (
      • Acharya N.R.
      • Tham V.M.
      • Esterberg E.
      • Borkar D.S.
      • Parker J.V.
      • Vinoya A.C.
      • Uchida A.
      Incidence and prevalence of uveitis: results from the Pacific Ocular Inflammation Study.
      ). The inflammation resulting in uveitis can arise from a number of diseases ranging from a viral infection to ocular trauma and systemic disease (
      • Rathinam S.R.
      • Babu M.
      Algorithmic approach in the diagnosis of uveitis.
      ). It can cause severe damage to the retina, optic nerve, and vitreous, often leading to complications such as macular edema, development of cataracts, and glaucoma (
      • Ness T.
      • Boehringer D.
      • Heinzelmann S.
      Intermediate uveitis: pattern of etiology, complications, treatment and outcome in a tertiary academic center.
      ). The inflammation associated with uveitis is due to infiltration of both innate and adaptive immune cells (
      • Horai R.
      • Silver P.B.
      • Chen J.
      • Agarwal R.K.
      • Chong W.P.
      • Jittayasothorn Y.
      • Mattapallil M.J.
      • Nguyen S.
      • Natarajan K.
      • Villasmil R.
      • et al.
      Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen.
      ). The characteristic inflammatory reaction involves CD4+ T-cells activated against retinal cells, as has been shown in an animal model of experimental autoimmune uveoretinitis (EAU) (
      • Caspi R.R.
      • Roberge F.G.
      • Chan C.C.
      • Wiggert B.
      • Chader G.J.
      • Rozenszajn L.A.
      • Lando Z.
      • Nussenblatt R.B.
      A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens.
      ). Th17 and Th1 T-cells also play a significant role in the inflammatory mechanism of uveitis. The helper T-cells recruit different effector immune cells, including neutrophils and monocytes, responsible for tissue destruction, with pro-inflammatory cytokines playing a major role (
      • Perez V.L.
      • Caspi R.R.
      Immune mechanisms in inflammatory and degenerative eye disease.
      ).
      Uveitis can arise from inflammation in the eye itself or it can be a manifestation of diseases affecting multiple organs like systemic sarcoidosis (
      • Caspi R.R.
      A look at autoimmunity and inflammation in the eye.
      ), where about 70% of the cases result in anterior granulomatous uveitis (
      • Herbort C.P.
      • Rao N.A.
      • Mochizuki M.
      Scientific Committee of First International Workshop on Ocular Sarcoidosis Members
      International criteria for the diagnosis of ocular sarcoidosis: results of the first International Workshop on Ocular Sarcoidosis (IWOS).
      ). Uveitis can also be a complication of the autoimmune disease multiple sclerosis, affecting between 1% and 10% of patients with this disease (
      • Messenger W.
      • Hildebrandt L.
      • Mackensen F.
      What is the relationship between MS and uveitis?.
      ). Multiple sclerosis is characterized by immune-mediated demyelination and inflammation of the CNS, and both the innate and adaptive immune systems are known to be involved in its development, recruiting microglia, activated macrophages, and both B and T lymphocytes (
      • Thompson A.J.
      • Baranzini S.E.
      • Geurts J.
      • Hemmer B.
      • Ciccarelli O.
      Multiple sclerosis.
      ). The cause of uveitis in patients with multiple sclerosis is unknown, but myelin basic protein and myelin oligodendrocyte glycoprotein have been shown to promote autoimmune uveitis in animal models (
      • Shao H.
      • Sun S.L.
      • Kaplan H.J.
      • Sun D.
      Induction of autoimmune encephalomyelitis and uveitis in B6 and (B6 x SJL) mice by peptides derived from myelin/oligodendrocyte glycoprotein.
      ). An autoimmune reaction resulting from sensitization of the immune system to antigens expressed in the CNS has been proposed as a trigger. Because nerve and ocular tissues derive from the same embryonic cells, multiple sclerosis and uveitis may share some etiologic factors (
      • Olsen T.G.
      • Frederiksen J.
      The association between multiple sclerosis and uveitis.
      ).
      Recent evidence suggests a role of sphingolipids in autoimmune eye diseases such as uveitis. Fingolimod (FTY720), a Food and Drug Administration-approved therapeutic drug for multiple sclerosis, has been found to be effective in a rat model of experimental autoimmune uveitis (
      • Commodaro A.G.
      • Peron J.P.
      • Lopes C.T.
      • Arslanian C.
      • Belfort Jr., R.
      • Rizzo L.V.
      • Bueno V.
      Evaluation of experimental autoimmune uveitis in mice treated with FTY720.
      ). FTY720 is a structural analog of Sph and has different targets in the complex sphingolipid metabolic network. FTY720 phosphorylation by SphK2 results in its active form, FTY720-phosphate, which mimics S1P and is a functional antagonist of almost all S1PRs, with the exception of S1PR2 (
      • Brinkmann V.
      • Davis M.D.
      • Heise C.E.
      • Albert R.
      • Cottens S.
      • Hof R.
      • Bruns C.
      • Prieschl E.
      • Baumruker T.
      • Hiestand P.
      • et al.
      The immune modulator FTY720 targets sphingosine 1-phosphate receptors.
      ). FTY720-phosphate binds to S1PR1, preventing its activation by S1P, and promotes its internalization and degradation, thus blocking the egress of lymphocytes from the lymph nodes (
      • Brinkmann V.
      • Billich A.
      • Baumruker T.
      • Heining P.
      • Schmouder R.
      • Francis G.
      • Aradhye S.
      • Burtin P.
      Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.
      ). FTY720 also blocks de novo Cer synthesis by inhibiting CerSs (
      • Chen H.
      • Chan A.Y.
      • Stone D.U.
      • Mandal N.A.
      Beyond the cherry-red spot: Ocular manifestations of sphingolipid-mediated neurodegenerative and inflammatory disorders.
      ). This grants FTY720 the ability to modulate both Cer synthesis and S1P signaling, thus affecting the “sphingolipid rheostat” and, consequently, sphingolipid signaling (
      • Spiegel S.
      • Milstien S.
      Sphingosine-1-phosphate: an enigmatic signalling lipid.
      ). In patients with Vogt-Koyanagi-Harada uveitis, T cell clones from aqueous humor (AH) or peripheral blood mononuclear cells produce high levels of pro-inflammatory cytokines IL-6, IL-8, and IFN-γ; treatment with FTY720 suppresses T cell production of granulocyte monocyte colony stimulating factor (
      • Sakaguchi M.
      • Sugita S.
      • Sagawa K.
      • Itoh K.
      • Mochizuki M.
      Cytokine production by T cells infiltrating in the eye of uveitis patients.
      ). FTY720 has been shown to suppress both the incidence and intensity of inflammation in a dose-dependent manner in an animal model of EAU (
      • Kurose S.
      • Ikeda E.
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      ). FTY720’s effects may result from its antagonizing S1P signaling through S1PR1, suggesting that S1P is involved in promoting inflammation and migration in EAU. A similar effect has been reported in clinical cases of uveitis (
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