Title: Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors
Abstract: Long-chain PUFAs (LC-PUFAs; C20–C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C ≥28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods. Long-chain PUFAs (LC-PUFAs; C20–C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C ≥28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods. DHA (22:6n3) and arachidonic acid (AA; 20:4n6) are the major long-chain PUFAs (LC-PUFAs) in the retina and rod outer segment (ROS) membranes of all vertebrate species examined thus far (1.Fliesler S.J. Anderson R.E. Chemistry and metabolism of lipids in the vertebrate retina.Prog. Lipid Res. 1983; 22: 79-131Crossref PubMed Scopus (826) Google Scholar, 2.SanGiovanni J.P. Chew E.Y. The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina.Prog. Retin. Eye Res. 2005; 24: 87-138Crossref PubMed Scopus (615) Google Scholar). DHA is also highly enriched in some, but not all, invertebrate retinas (3.Anderson R.E. Benolken R.M. Kelleher P.A. Maude M.B. Wiegand R.D. Chemistry of photoreceptor membrane preparations from squid retinas.Biochim. Biophys. 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In 1987, Avendaño and Sprecher reported the presence in bovine ROSs of n3 and n6 PUFAs containing up to 38 carbons, esterified exclusively to the sn-1 position of phosphatidylcholine, with DHA on the sn-2 position (10.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.J. Biol. Chem. 1987; 262: 1180-1186Abstract Full Text PDF PubMed Google Scholar, 11.Aveldaño M.I. A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina.J. Biol. Chem. 1987; 262: 1172-1179Abstract Full Text PDF PubMed Google Scholar). No specific function or importance was assigned to these very-long-chain PUFAs (VLC-PUFAs; ≥C28). In 2001, three groups reported that mutations in exon 6 of a gene named elongation of very-long-chain fatty acids-4 (ELOVL4) are the cause of autosomal dominant Stargardt-like macular dystrophy (STGD3) (12.Bernstein P.S. Tammur J. Singh N. Hutchinson A. Dixon M. Pappas C.M. Zabriskie N.A. Zhang K. Petrukhin K. Leppert M. et al.Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.Invest. Ophthalmol. Vis. Sci. 2001; 42: 3331-3336PubMed Google Scholar, 13.Edwards A.O. Donoso L.A. Ritter III, R. A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.Invest. Ophthalmol. Vis. Sci. 2001; 42: 2652-2663PubMed Google Scholar, 14.Zhang X.M. Yang Z. Karan G. Hashimoto T. Baehr W. Yang X.J. Zhang K. Elovl4 mRNA distribution in the developing mouse retina and phylogenetic conservation of Elovl4 genes.Mol. Vis. 2003; 9: 301-307PubMed Google Scholar). The gene was so named because it shared sequence homology with a family of yeast enzymes named ELOVL, which catalyze the rate-limiting condensation reaction in the addition of 2-carbon units to existing fatty acids to produce even longer chain saturated fatty acids and PUFAs (15.Dittrich F. Zajonc D. Huhne K. Hoja U. Ekici A. Greiner E. Klein H. Hofmann J. Bessoule J.J. Sperling P. et al.Fatty acid elongation in yeast–biochemical characteristics of the enzyme system and isolation of elongation-defective mutants.Eur. J. Biochem. 1998; 252: 477-485Crossref PubMed Scopus (50) Google Scholar, 16.Leonard A.E. Pereira S.L. Sprecher H. Huang Y.S. Elongation of long-chain fatty acids.Prog. Lipid Res. 2004; 43: 36-54Crossref PubMed Scopus (429) Google Scholar). Our laboratory cloned and expressed ELOVL4 and identified its products as saturated fatty acids and PUFAs of 28 carbons or greater in length (17.Agbaga M.P. Brush R.S. Mandal M.N. Henry K. Elliott M.H. Anderson R.E. Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.Proc. Natl. Acad. Sci. USA. 2008; 105: 12843-12848Crossref PubMed Scopus (203) Google Scholar, 18.Yu M. Benham A. Logan S. Brush R.S. Mandal M.N. Anderson R.E. Agbaga M.P. ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3.J. Lipid Res. 2012; 53: 494-504Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We subsequently showed that the STGD3 mutant protein did not have enzymatic activity and exerted a dominant negative effect on the activity of the WT enzyme in vitro (19.Logan S. Agbaga M.P. Chan M.D. Brush R.S. Anderson R.E. Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity.J. Lipid Res. 2014; 55: 698-708Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 20.Logan S. Agbaga M.P. Chan M.D. Kabir N. Mandal N.A. Brush R.S. Anderson R.E. Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.Proc. Natl. Acad. Sci. USA. 2013; 110: 5446-5451Crossref PubMed Scopus (36) Google Scholar), consistent with reports that the mutant formed dimers with the WT protein (21.Grayson C. Molday R.S. Dominant negative mechanism underlies autosomal dominant Stargardt-like macular dystrophy linked to mutations in ELOVL4.J. Biol. Chem. 2005; 280: 32521-32530Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 22.Ambasudhan R. Wang X. Jablonski M.M. Thompson D.A. Lagali P.S. Wong P.W. Sieving P.A. Ayyagari R. Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein.Genomics. 2004; 83: 615-625Crossref PubMed Scopus (56) Google Scholar, 23.Karan G. Yang Z. Howes K. Zhao Y. Chen Y. Cameron D.J. Lin Y. Pearson E. Zhang K. Loss of ER retention and sequestration of the wild-type ELOVL4 by Stargardt disease dominant negative mutants.Mol. 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Because the mutation is in an enzyme that utilizes LC-PUFA precursors to make VLC-PUFAs, there is considerable interest in knowing the size and nature of the precursor pool in human cones and the VLC-PUFA products generated from them. Van Kuijk and Buck (26.van Kuijk F.J. Buck P. Fatty acid composition of the human macula and peripheral retina.Invest. Ophthalmol. Vis. Sci. 1992; 33: 3493-3496PubMed Google Scholar) were the first to compare the fatty acid compositions of peripheral and macular regions of the human retina, and found that the macula had less DHA than the periphery. Their analyses did not include VLC-PUFAs. More recently, Bernstein's group showed that while there were no age-related changes in DHA in total lipids from whole human retinas, there was a significant reduction in DHA in retinas from age-matched patients with age-related macular degeneration (AMD) (27.Liu A. Chang J. Lin Y. Shen Z. Bernstein P.S. Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.J. Lipid Res. 2010; 51: 3217-3229Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Interestingly, there was no change in AA in these same retinas. The Bernstein group also reported significantly lower VLC-PUFAs in the whole retina lipids compared with age-matched control values (27.Liu A. Chang J. Lin Y. Shen Z. Bernstein P.S. Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.J. Lipid Res. 2010; 51: 3217-3229Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In a later study, using GC-MS technology to analyze the lipids in 4 mm trephine retinal punches, the Bernstein group found that human macula lipids contained less VLC-PUFAs than peripheral lipids, although the levels of DHA were the same (28.Liu A. Terry R. Lin Y. Nelson K. Bernstein P.S. Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina.J. Chromatogr. A. 2013; 1307: 191-200Crossref PubMed Scopus (28) Google Scholar). An interesting finding from the Bernstein study was that mouse retinas contain almost 10 times the level of VLC-PUFAs and almost twice the level of DHA as do normal human retinas. Given the differences in LC-PUFAs and VLC-PUFAs reported for peripheral versus central normal human retinas (26.van Kuijk F.J. Buck P. Fatty acid composition of the human macula and peripheral retina.Invest. Ophthalmol. Vis. Sci. 1992; 33: 3493-3496PubMed Google Scholar, 27.Liu A. Chang J. Lin Y. Shen Z. Bernstein P.S. Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.J. 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The mosaic of nerve cells in the mammalian retina.Proc. R. Soc. Lond. B Biol. Sci. 1978; 200: 441-461Crossref PubMed Scopus (442) Google Scholar). We performed comprehensive glycerophospholipid analyses on these rod- and cone-dominant retinas to define and compare their lipid compositions, which may account for the differences in their function. Here, we show that outer segment (OS) membranes from cone-dominant retinas have much lower levels of PUFAs and VLC-PUFAs than do rod-dominant retinas. Nrl−/− mice were generously provided by Anand Swaroop, PhD (National Eye Institute, Bethesda, MD) and were housed and bred at the Dean McGee Eye Institute and the University of Oklahoma Animal Research Facility vivarium on 12 h light:12 h dark cyclic light. The Nrl−/− mice were bred with C57BL6/J mice purchased from Jackson Laboratories (Bar Harbor, ME) to generate Nrl+/− and WT C57BL6/J controls. Because the retinas of Nrl−/− mice undergo age-related retinal structural changes by about three months of age (29.Mears A.J. Kondo M. Swain P.K. Takada Y. Bush R.A. Saunders T.L. Sieving P.A. Swaroop A. Nrl is required for rod photoreceptor development.Nat. Genet. 2001; 29: 447-452Crossref PubMed Scopus (710) Google Scholar, 34.Stuck M.W. Conley S.M. Naash M.I. Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina.PLoS One. 2012; 7: e32484Crossref PubMed Scopus (35) Google Scholar), our studies were carried out in 1-month-old WT, Nrl+/−, and Nrl−/− animals. Sprague Dawley rats were raised at the Dean McGee Eye Institute vivarium and TLGSs (I. tridecemlineatus) were raised at the University of Wisconsin Oshkosh (Oshkosh, WI). Tree squirrels (S. niger) were obtained from local licensed trappers. Tree shrews were bred and raised at the Max Planck Florida Institute, Jupiter, FL, by Rebekah Corlew, PhD, who generously provided us with retinal tissues. All animals were handled and euthanized according to the guidelines outlined in the Statement for the Use of Animals in Ophthalmic and Vision Research, and the protocols were approved by the University of Oklahoma Health Sciences Center IACUC. Prior to preparation of OS membranes, animals were dark-adapted overnight. Retinas were dissected under dim red light, flash-frozen in liquid nitrogen, and stored at −80°C until they were used. Cone OSs (COSs) or ROSs, collectively referred to as photoreceptor OSs, were prepared by discontinuous sucrose gradient ultracentrifugation according to protocols previously published (35.Martin R.E. Elliott M.H. Brush R.S. Anderson R.E. Detailed characterization of the lipid composition of detergent-resistant membranes from photoreceptor rod outer segment membranes.Invest. Ophthalmol. Vis. 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An aliquot of membranes from each fraction was taken for protein determination by quantitative BCA assay in order to use equal amounts of membrane fractions for lipid and fatty acid analyses. For Western blotting, the membranes were lysed in a lysis buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, EDTA-free protease inhibitor tablets (Roche Applied Sciences, Indianapolis, IN), and 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO). After brief sonication on ice, the lysates were incubated with gentle rocking at 4°C for 1 h, and centrifuged at 27,000 g for 30 min at 4°C. Supernatants were collected and protein concentrations determined using the Pierce BCA assay (Thermo Fisher Scientific, Rockford, IL). Equal amounts of protein (5 μg) were separated by SDS/PAGE on 12% polyacrylamide gels, followed by either silver staining or electro-transfer to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk and incubated with rhodopsin antibodies 1D4 (1:3,000), a gift from Dr. James McGinnis (Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK). Membranes were stripped and reprobed with M- and S-opsin antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX) at 1:1,000, and then with monoclonal antibody against β-actin (Sigma-Aldrich). Immunoreaction of horseradish peroxidase-conjugated donkey anti-rabbit or goat anti-mouse IgG secondary antibodies (GE Healthcare Bio-Sciences, Pittsburgh, PA) was detected using Super-Signal West Dura extended duration substrate (Thermo Fisher Scientific). Total lipids were extracted from 100 μg protein of the three retinal membrane fractions, 100 μl of plasma, or whole retinas following the method of Bligh and Dyer (38.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42828) Google Scholar) with minor modifications (35.Martin R.E. Elliott M.H. Brush R.S. Anderson R.E. Detailed characterization of the lipid composition of detergent-resistant membranes from photoreceptor rod outer segment membranes.Invest. Ophthalmol. Vis. Sci. 2005; 46: 1147-1154Crossref PubMed Scopus (79) Google Scholar). The purified lipid extracts were stored under nitrogen until use. To these lipid extracts, 50 nmol each of 15:0, 17:0, and 23:0, and 4 nmol of 30:3n6 were added as internal standards. One milliliter of 16.6% concentrated HCl in methanol was then added. The tubes were sealed under N2 with Teflon-lined caps and heated at 100°C overnight. The tubes were cooled on ice and fatty acid methyl esters (FAMEs) were extracted and processed as previously described (17.Agbaga M.P. Brush R.S. Mandal M.N. Henry K. Elliott M.H. Anderson R.E. Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.Proc. Natl. Acad. Sci. USA. 2008; 105: 12843-12848Crossref PubMed Scopus (203) Google Scholar). All reagents for fatty acid extraction and derivatization were of the highest quality available from Sigma-Aldrich. FAMEs were identified using an Agilent Technologies 7890A gas chromatograph with a 5975C inert XL mass spectrometer detector (Agilent Technologies, Lexington, MA). The gas chromatograph-mass spectrometer was operated in the electron impact total ion and single ion monitoring modes. The injection volume was 1 μl and the inlet, held at 280°C, was set to pulsed splitless mode. An Agilent Technologies DB-23 column (60 m × 0.32 mm × 0.25 μm) was used with a helium carrier gas flow rate of 1.9 ml/min. The oven temperature began at 130°C for 1.0 min, was ramped to 170°C at 6.8°C/min, and was then ramped to 215°C at 2.9°C/min. After holding at 215°C for 11.4 min, the oven was ramped to 230°C at 42°C/min and held for 9.6 min. The oven was then ramped to 290°C at 10°C/min and held for 14.4 min. The mass spectrometer transfer line, ion source, and quadrupole temperatures were 290°C, 230°C, and 150°C, respectively. The PUFAs were identified by using the m/z 79.1, 108.1, and 150.1 in single ion monitoring mode and the full scan mass spectra in total ion mode. FAMEs were quantified using an Agilent Technologies 6890N gas chromatograph with flame ionization detector. Sample concentrations were determined by comparison to internal standards 15:0, 17:0, 23:0, and 30:3n6. The injection volume was 1 μl and the inlet, held at 280°C, was set to pulsed split mode (10:1 ratio). An Agilent Technologies DB-23 column (60 m × 0.32 mm × 0.25 μm) was used with a hydrogen carrier gas constant pressure of 13.1 psi. The oven temperature began at 130°C for 0.8 min, was ramped to 170°C at 8.2°C/min, and was then ramped to 215°C at 3.5°C/min. After holding at 215°C for 9.5 min, the oven was ramped to 230°C at 50°C/min, and was then held for 8 min. The oven was then ramped to 290°C at 12.0°C/min and was held for 12 min. The detector was held at 290°C. The method used for MS/MS analysis of retinal lipids has been described previously (39.Busik J.V. Reid G.E. Lydic T.A. Global analysis of retina lipids by complementary precursor ion and neutral loss mode tandem mass spectrometry.Methods Mol. Biol. 2009; 579: 33-70Crossref PubMed Scopus (42) Google Scholar) and was used with slight modification. Briefly, tissue was homogenized in 40% aqueous methanol and diluted 1:40 with 2-propanol/methanol/chloroform (4:2:1 v/v/v) containing 20 mM ammonium formate and 1.0 μmol each of glycerophosphatidylcholine (PC) 14:0/14:0 and glycerophosphatidylethanolamine (PE) 14:0/14:0, 0.33 μmol of glycerophosphatidylserine (PS) 14:0/14:0, and 12.5 nmol of d18:1/12:0 ceramide as internal standards. Samples were introduced into an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) operating at a resolution of 100,000 (at m/z 400) using a chip-based nano-ESI source (Advion NanoMate, Ithaca, NY) operating in infusion mode. PC, PE, and PS molecular species were determined by high-resolution MS and MS/MS using accurate mass measurements in positive ion mode (40.Lydic T.A. Busik J.V. Reid G.E. A monophasic extraction strategy for the simultaneous lipidome analysis of polar and nonpolar retina lipids.J. Lipid Res. 2014; 55: 1797-1809Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Quantification of lipid molecular species was performed using the Lipid Mass Spectrum Analysis (LIMSA) software's peak model fit algorithm (41.Haimi P. Chaithanya K. Kainu V. Hermansson M. Somerharju P. Instrument-independent software tools for the analysis of MS-MS and LC-MS lipidomics data.Methods Mol. Biol. 2009; 580: 285-294PubMed Google Scholar, 42.Haimi P. Uphoff A. Hermansson M. Somerharju P. Software tools for analysis of mass spectrometric lipidome data.Anal. Chem. 2006; 78: 8324-8331Crossref PubMed Scopus (167) Google Scholar, 43.Song H. Ladenson J. Turk J. Algorithms for automatic processing of data from mass spectrometric analyses of lipids.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2847-2854Crossref PubMed Scopus (28) Google Scholar). Statistical analyses were performed using GraphPad Prism 5 software. Results are expressed as the mean ± SD. All relative mole percentages of n3 and n6 LC-PUFAs and VLC-PUFAs were from total lipids extracted from sample homogenates equivalent to 100 µg or 2.0 mg of protein. For all figures, significant differences are indicated by P < 0.05, while n.s. indicates no significant difference at P > 0.05, using a multivariate ANOVA with Neuman-Keuls posthoc test or Bonferroni's multiple comparisons test. To determine the enrichment and purity of OS membrane preparations, equal amounts of protein of OS membranes from WT mouse (C57B6/J), Nrl+/− mouse, Nrl−/− mouse, rat, and TLGS retinas were resolved on SDS-PAGE, as described by Laemmli (44.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar), and either stained with silver nitrate or immunoblotted with anti-opsin, anti-rod transducin, anti-M-opsin, or anti-actin antibodies (Fig. 1A, B). Band I, representing enriched OS membranes from WT and Nrl+/− retinas, showed prominent staining in the regions generally accepted for rhodopsin and rhodopsin polymers. This staining was absent in the Nrl−/− OS membranes. Similar staining patterns were found for band II, which represented broken OS and inner segment membranes, although the amounts of rhodopsin were less. There was very little staining for rhodopsin in the pellet membranes. M-opsin was present in the OS membranes from the three groups of mice, showing that all three had the visual pigment found in mouse cones. Rhodopsin and rod transducin were found only in OS preparations from WT and Nrl+/− retinas, indicating that the Nrl−/− OS membranes did not contain these two prominent components of ROSs. OS preparations from TLGS retinas were also examined by Western blot (Fig. 1B). We did not detect significant rhodopsin labeling from the all-cone TLGS OS membranes compared with the rat ROS membranes, which was expected due to the cone-dominant nature of the TLGS retina. However, it is possible that the 1D4 antibody does not recognize TLGS rhodopsin. Rat ROSs also contained a small amount of M-opsin, which was present along with S-opsin in OS membranes from TLGS. We determined the fatty acid composition of whole retina lipids from rod-dominant WT and Nrl+/