Retinal sphingolipids and their very-long-chain fatty acid-containing species.

Sphingolipids are a family of membrane lipids that play important roles in the regulation of the fluidity and subdomain structure of the lipid bilayer—especially, lipid rafts.1–4 They play crucial functional roles in membrane raft formation, receptor function, membrane conductance, cell–cell interactions, and internalization of pathogens.2,5,6 Many of them, such as ceramide (CER), ceramide-1-phosphate (C1P), sphingosine (SP), and sphingosine-1-phosphate (S1P), are bioactive molecules that have been implicated in the regulation of cell growth, apoptosis, angiogenesis, vesicular trafficking, and a multitude of specific cell actions and responses.6,7 Aberrant sphingolipid metabolism is associated with inflammation, tumorigenesis, diabetes, and neurodegenerative disorders.8–12 Sphingolipid metabolic defects that cause severe congenital or childhood-onset neurodegenerative diseases, such as Tay-Sachs disease, Fabry's disease, and Niemann-Pick disease, are often associated with blindness, indicating the importance of sphingolipid metabolism in retinal cells.13–15 Patients with Farber's (acid ceramidase), Gaucher's (glucosylceramidase), Krabbe's (galactosylceramidase), and Niemann-Pick (sphingomyelinase) diseases lose vision due to retinal neuronal cell death.16 The processes of apoptosis, neuronal dedifferentiation, and neovascularization are integral to major retinal diseases including retinitis pigmentosa (RP), Stargardt's disease, Leber's congenital amaurosis (LCA), diabetic retinopathy, and age-related macular degeneration (AMD).17–20 Recent evidence suggests a strong correlation between sphingolipid signaling and survival and homeostasis of photoreceptor and retinal pigment epithelial (RPE) cells: (1) CERs are mediators of retinal photoreceptor apoptosis, especially in oxidative stress–induced apoptosis21; (2) reducing the level of free CER through genetic manipulation rescues Drosophila photoreceptor cells from lethal mutations in the phototransduction genes22; (3) aberrant sphingolipid metabolism is reported in diabetic retinopathy16; (4) S1P signaling is involved in pathologic angiogenesis and choroidal neovascularization in the mouse retina23,24; and (5) mutations in the ceramide kinase–like (CERKL) gene, a gene predicted to be involved in CER metabolism, are involved in nonsyndromic retinal degeneration in human (RP26).25–27 These results clearly support the functional role of sphingolipid signaling in retinal physiology and pathophysiology. However, very little is known about their metabolism, abundance, and composition in the retina. As the first step toward understanding their role(s) in the retina, we determined the abundance and molecular composition of retinal sphingolipids. We have recently shown that the human Stargardt 3 (STGD3) retinal dystrophy protein ELOVL4 (elongation of very-long-chain fatty acid 4) is involved in biosynthesis of fatty acids with chain lengths >26 carbons.28 These very-long-chain saturated, mono- and polyunsaturated fatty acids are present in tissues that express ELOVL4, such as retina, skin, and testis, but the specific role that they play is not well understood. Homozygous Elovl4 knockout and knockin mice die after birth as a result of a peculiar skin barrier defect29–31 resulting from a deficiency in a particular sphingolipid (ω-O-acylceramide) that contains very-long-chain fatty acids. As in the skin of these mice, there is a possibility that sphingolipids containing very-long-chain fatty acids are involved in ELOVL4-mediated retinal disease in humans. We, therefore, extended our studies to analyze, for the first time, retinal sphingolipids containing very-long-chain fatty acids.