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• W2620565698 abstract "Docosahexaenoic acid (DHA) has essential roles in photoreceptor cells in the retina and is therefore crucial to healthy vision. Although the influence of dietary DHA on visual acuity is well known and the retina has an abundance of DHA-containing phospholipids (PL-DHA), the mechanisms associated with DHA's effects on visual function are unknown. We previously identified lysophosphatidic acid acyltransferase 3 (LPAAT3) as a PL-DHA biosynthetic enzyme. Here, using comprehensive phospholipid analyses and imaging mass spectroscopy, we found that LPAAT3 is expressed in the inner segment of photoreceptor cells and that PL-DHA disappears from the outer segment in the LPAAT3-knock-out mice. Dynamic light-scattering analysis of liposomes and molecular dynamics simulations revealed that the physical characteristics of DHA reduced membrane-bending rigidity. Following loss of PL-DHA, LPAAT3-knock-out mice exhibited abnormalities in the retinal layers, such as incomplete elongation of the outer segment and decreased thickness of the outer nuclear layers and impaired visual function, as well as disordered disc morphology in photoreceptor cells. Our results indicate that PL-DHA contributes to visual function by maintaining the disc shape in photoreceptor cells and that this is a function of DHA in the retina. This study thus provides the reason why DHA is required for visual acuity and may help inform approaches for overcoming retinal disorders associated with DHA deficiency or dysfunction. Docosahexaenoic acid (DHA) has essential roles in photoreceptor cells in the retina and is therefore crucial to healthy vision. Although the influence of dietary DHA on visual acuity is well known and the retina has an abundance of DHA-containing phospholipids (PL-DHA), the mechanisms associated with DHA's effects on visual function are unknown. We previously identified lysophosphatidic acid acyltransferase 3 (LPAAT3) as a PL-DHA biosynthetic enzyme. Here, using comprehensive phospholipid analyses and imaging mass spectroscopy, we found that LPAAT3 is expressed in the inner segment of photoreceptor cells and that PL-DHA disappears from the outer segment in the LPAAT3-knock-out mice. Dynamic light-scattering analysis of liposomes and molecular dynamics simulations revealed that the physical characteristics of DHA reduced membrane-bending rigidity. Following loss of PL-DHA, LPAAT3-knock-out mice exhibited abnormalities in the retinal layers, such as incomplete elongation of the outer segment and decreased thickness of the outer nuclear layers and impaired visual function, as well as disordered disc morphology in photoreceptor cells. Our results indicate that PL-DHA contributes to visual function by maintaining the disc shape in photoreceptor cells and that this is a function of DHA in the retina. This study thus provides the reason why DHA is required for visual acuity and may help inform approaches for overcoming retinal disorders associated with DHA deficiency or dysfunction. Docosahexaenoic acid (DHA) 2The abbreviations used are: DHAdocosahexaenoic acidERGelectroretinogramPEphosphatidylethanolamineRPEretinal pigment epithelialLPClysophosphatidylcholinePAphosphatidic acidPCphosphatidylcholinePL-DHADHA-containing phospholipidOSouter segmentPLphospholipidDLSdynamic light scatteringMDmolecular dynamicsPSphosphatidylserinePGphosphatidylglycerolPIphosphatidylinositolAAarachidonic acidDOPCdi-oleoyl-PCDAPCdi-arachidonoyl-PCDDPCdi-DHA-PC (DDPC)DOPEdi-oleoyl-PEDDPEdi-DHA-PCONLouter nuclear layerINLinner nuclear layerISinner segmentTEMtransmission electron microscopyDPPCdipalmitoyl-PCANOVAanalysis of variancecdcandela. plays essential roles in photoreceptor cells in acquisition of visual function. Dietary DHA modulates the maturation and survival of photoreceptor cells (1Benolken R.M. Anderson R.E. Wheeler T.G. Membrane fatty acids associated with the electrical response in visual excitation.Science. 1973; 182: 1253-1254Crossref PubMed Scopus (250) Google Scholar, 2Jastrzebska B. Debinski A. Filipek S. Palczewski K. Role of membrane integrity on G protein-coupled receptors: rhodopsin stability and function.Prog. Lipid Res. 2011; 50: 267-277Crossref PubMed Scopus (54) Google Scholar, 3Jeffrey B.G. Neuringer M. Age-related decline in rod phototransduction sensitivity in rhesus monkeys fed an n-3 fatty acid-deficient diet.Invest. Ophthalmol. Vis. Sci. 2009; 50: 4360-4367Crossref PubMed Scopus (17) Google Scholar), and animals grown with polyunsaturated fatty acid-free diets develop abnormal electroretinograms (ERG) with decreased retinal DHA contents (4Neuringer M. Connor W.E. Van Petten C. Barstad L. Dietary ω-3 fatty acid deficiency and visual loss in infant rhesus monkeys.J. Clin. Invest. 1984; 73: 272-276Crossref PubMed Scopus (506) Google Scholar) suggesting that dietary DHA is essential for visual function. DHA is a dominant fatty acid of retinal phospholipids and affects rhodopsin content at discs, as well as photoresponses (2Jastrzebska B. Debinski A. Filipek S. Palczewski K. Role of membrane integrity on G protein-coupled receptors: rhodopsin stability and function.Prog. Lipid Res. 2011; 50: 267-277Crossref PubMed Scopus (54) Google Scholar, 5Antonny B. Vanni S. Shindou H. Ferreira T. From zero to six double bonds: phospholipid unsaturation and organelle function.Trends Cell Biol. 2015; 25: 427-436Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Many studies report the beneficial effects of DHA on visual functions (5Antonny B. Vanni S. Shindou H. Ferreira T. From zero to six double bonds: phospholipid unsaturation and organelle function.Trends Cell Biol. 2015; 25: 427-436Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 6Birch D.G. Birch E.E. Hoffman D.R. Uauy R.D. Retinal development in very-low-birth-weight infants fed diets differing in ω-3 fatty acids.Invest. Ophthalmol. Vis. Sci. 1992; 33: 2365-2376PubMed Google Scholar, 7Birch E.E. Birch D.G. Hoffman D.R. Uauy R. Dietary essential fatty acid supply and visual acuity development.Invest. Ophthalmol. Vis. Sci. 1992; 33: 3242-3253PubMed Google Scholar, 8Quazi F. Molday R.S. ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 5024-5029Crossref PubMed Scopus (83) Google Scholar, 9Rice D.S. Calandria J.M. Gordon W.C. Jun B. Zhou Y. Gelfman C.M. Li S. Jin M. Knott E.J. Chang B. Abuin A. Issa T. Potter D. Platt K.A. Bazan N.G. Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival.Nat. Commun. 2015; 6: 6228Crossref PubMed Scopus (80) Google Scholar, 10Wong B.H. Chan J.P. Cazenave-Gassiot A. Poh R.W. Foo J.C. Galam D.L. Ghosh S. Nguyen L.N. Barathi V.A. Yeo S.W. Luu C.D. Wenk M.R. Silver D.L. Mfsd2a is a transporter for the essential ω-3 fatty acid docosahexaenoic acid (DHA) in eye and is important for photoreceptor cell development.J. Biol. Chem. 2016; 291: 10501-10514Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar); however, the molecular mechanisms and direct roles of DHA in the retina remain unknown. docosahexaenoic acid electroretinogram phosphatidylethanolamine retinal pigment epithelial lysophosphatidylcholine phosphatidic acid phosphatidylcholine DHA-containing phospholipid outer segment phospholipid dynamic light scattering molecular dynamics phosphatidylserine phosphatidylglycerol phosphatidylinositol arachidonic acid di-oleoyl-PC di-arachidonoyl-PC di-DHA-PC (DDPC) di-oleoyl-PE di-DHA-PC outer nuclear layer inner nuclear layer inner segment transmission electron microscopy dipalmitoyl-PC analysis of variance candela. All-trans- and 11-cis-retinal are flipped from the lumen to the cytoplasmic leaflet of the disc membrane via ATP-binding cassette transporter ABCA4, which is associated with Stargardt macular degeneration (11Quazi F. Lenevich S. Molday R.S. ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer.Nat. Commun. 2012; 3: 925Crossref PubMed Scopus (174) Google Scholar). Although essential for vision, the clearance of 11-cis- and all-trans-retinal from the photoreceptor disc membrane is important due to their highly reactive aldehyde groups. 11-cis-Retinal with phosphatidylethanolamine (PE) is isomerized to the all-trans form, which is then reduced by retinol dehydrogenase 8 for entry into the visual cycle of retinal pigment epithelial (RPE) cells (8Quazi F. Molday R.S. ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 5024-5029Crossref PubMed Scopus (83) Google Scholar, 12Chen C. Thompson D.A. Koutalos Y. Reduction of all-trans-retinal in vertebrate rod photoreceptors requires the combined action of RDH8 and RDH12.J. Biol. Chem. 2012; 287: 24662-24670Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). During this step, PE-containing DHA enhances isomerization (8Quazi F. Molday R.S. ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 5024-5029Crossref PubMed Scopus (83) Google Scholar); however, the mechanism of how DHA affects this reaction, such as a possible binding site for DHA, is unknown. Although in vitro analyses using rhodopsin reconstituted into liposomes revealed that replacing C16:0-C18:1 with C18:1-C22:6 (DHA) phospholipids increased rhodopsin activation (5Antonny B. Vanni S. Shindou H. Ferreira T. From zero to six double bonds: phospholipid unsaturation and organelle function.Trends Cell Biol. 2015; 25: 427-436Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Mitchell D.C. Niu S.L. Litman B.J. Optimization of receptor-G protein coupling by bilayer lipid composition I: kinetics of rhodopsin-transducin binding.J. Biol. Chem. 2001; 276: 42801-42806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 14Niu S.L. Mitchell D.C. Lim S.Y. Wen Z.M. Kim H.Y. Salem Jr., N. Litman B.J. Reduced G protein-coupled signaling efficiency in retinal rod outer segments in response to n-3 fatty acid deficiency.J. Biol. Chem. 2004; 279: 31098-31104Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), the reason DHA has this effect remains unclear. Currently, the elucidation of molecular mechanisms associated with DHA is needed for understanding of visual functions. Recently, we partially identified the molecular mechanism associated with DHA incorporation into phospholipids (15Harayama T. Eto M. Shindou H. Kita Y. Otsubo E. Hishikawa D. Ishii S. Sakimura K. Mishina M. Shimizu T. Lysophospholipid acyltransferases mediate phosphatidylcholine diversification to achieve the physical properties required in vivo.Cell metabolism. 2014; 20: 295-305Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). DHA is first activated to DHA-CoA, which is esterified to lysophosphatidic acid to form phosphatidic acid (PA) by lysophosphatidic acid acyltransferase 3 (LPAAT3) (16Yuki K. Shindou H. Hishikawa D. Shimizu T. Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.J. Lipid Res. 2009; 50: 860-869Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 17Koeberle A. Shindou H. Harayama T. Shimizu T. Role of lysophosphatidic acid acyltransferase 3 for the supply of highly polyunsaturated fatty acids in TM4 Sertoli cells.FASEB J. 2010; 24: 4929-4938Crossref PubMed Scopus (33) Google Scholar). The DHA-containing PA is converted into other phospholipids, such as phosphatidylcholine (PC) and PE (18Shindou H. Shimizu T. Acyl-CoA:lysophospholipid acyltransferases.J. Biol. Chem. 2009; 284: 1-5Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). LPAAT3 is expressed in DHA-rich tissues, such as the testis (16Yuki K. Shindou H. Hishikawa D. Shimizu T. Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.J. Lipid Res. 2009; 50: 860-869Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 19Koeberle A. Shindou H. Harayama T. Yuki K. Shimizu T. Polyunsaturated fatty acids are incorporated into maturating male mouse germ cells by lysophosphatidic acid acyltransferase 3.FASEB J. 2012; 26: 169-180Crossref PubMed Scopus (46) Google Scholar). Here, we investigated the relationship between LPAAT3 and retinas, which contain high amounts of DHA-containing phospholipids (PL-DHA). We observed that LPAAT3 was highly expressed in the retina and that LPAAT3 deficiency dramatically lowered PL-DHA levels in the outer segment (OS) of photoreceptors and impaired visual functions. Additionally, disc morphology and/or organization in the OS of LPAAT3-knock-out (LPAAT3-KO) mice was disrupted. Cellular membrane with PL-DHA was more flexible than those with phospholipid-containing arachidonic acid (PL-AA) and other fatty acids according to analyses using dynamic light scattering (DLS) and molecular dynamics (MD) simulations. Our findings demonstrate DHA involvement in maintaining disc properties in the retina and provide insight into the roles of DHA in visual function. In the accompanying paper (56Iizuka-Hishikawa Y. Hishikawa D. Sasaki J. Takubo K. Goto M. Nagata K. Nakanishi H. Shindou H. Okamura T. Ito C. Toshimori K. Sasaki T. Shimizu T. Lysophosphatidic acid acyltransferase 3 tunes the membrane status of germ cells by incorporating docosahexaenoic acid during spermatogenesis.J. Biol. Chem. 2017; 292: 12065-12076Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), we reported that DHA is critical also for sperm maturation. We have previously reported the age-dependent up-regulation of mouse LPAAT3 in the testis (16Yuki K. Shindou H. Hishikawa D. Shimizu T. Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.J. Lipid Res. 2009; 50: 860-869Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 19Koeberle A. Shindou H. Harayama T. Yuki K. Shimizu T. Polyunsaturated fatty acids are incorporated into maturating male mouse germ cells by lysophosphatidic acid acyltransferase 3.FASEB J. 2012; 26: 169-180Crossref PubMed Scopus (46) Google Scholar). In this study, LPAAT3 levels were mainly evaluated in DHA-abundant mouse tissues. Quantitative-PCR analysis showed that LPAAT3 mRNA levels were higher in the retina, followed by the testis, in 8-week-old mice (Fig. 1A). LPAAT3 protein levels also increased in an age-dependent manner in the retina (wild-type (WT) in Fig. 1B). To examine the role of LPAAT3 in the retina, we constructed LPAAT3-KO mice as described in detail in the accompanying paper (56Iizuka-Hishikawa Y. Hishikawa D. Sasaki J. Takubo K. Goto M. Nagata K. Nakanishi H. Shindou H. Okamura T. Ito C. Toshimori K. Sasaki T. Shimizu T. Lysophosphatidic acid acyltransferase 3 tunes the membrane status of germ cells by incorporating docosahexaenoic acid during spermatogenesis.J. Biol. Chem. 2017; 292: 12065-12076Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and confirmed the absence of LPAAT3 protein in the retina (Fig. 1B). Comprehensive phospholipid analysis of the retina was performed using liquid chromatography-mass spectrometry (LC-MS) to detect PA, PC, PE, phosphatidylserine (PS) (Fig. 2, A–D), and PC with long chain fatty acids (total carbon number >54) (Fig. 2E), as well as phosphatidylglycerol (PG), phosphatidylinositol (PI), oxidized PC, LPA, lysophosphatidylcholine (LPC), lyso-PE, lyso-PG, lyso-PS, and lyso-PI (supplemental Fig. S1, A–I). In the retina of LPAAT3-KO mice, PL-DHA levels were almost abolished, whereas the PL-AA levels increased compared with WT mice. Oxidized PC generated from PC-DHA also decreased in LPAAT3-KO retinas (supplemental Fig. S1C). We also performed MS/MS analyses to investigate the acyl-chain composition of the representative PC species (PC38:4, PC38:6, and PC40:6). Each PC subspecies had an almost single acyl-chain composition (supplemental Fig. S1J). From the results, PC38:4, PC38:6, and PC40:6 were identified as PC18:0/20:4, PC16:0/22:6, and PC18:0/22:6 molecular species, respectively.Figure 2Attenuation of PL-DHA in LPAAT3-KO retinas. PA (A), PC (B), PE (C), PS (D), and PC with very long chain fatty acids (E) were measured by comprehensive phospholipid analysis and indicated by the area ratio (y axis, 100% indicates sum of the detected signals for each phospholipid). The x axis indicates the summation of fatty acid information at the sn-1 and sn-2 positions (number of carbon and double bonds, i.e. 34:0) in the phospholipids. 38:6, 40:6, and 44:12 in all phospholipids and very long chain in PC may contain DHA, which were suppressed in LPAAT3-KO mice. By contrast, 36:4, 38:4, and 40:4 may contain AA and were increased in LPAAT3-KO mice. 34:1 and 36:1 thought to have oleic acid were increased. These data were obtained from p11 to 8-week-old mice; WT (black) and LPAAT3-KO (magenta). Results are expressed as the mean ± S.E. of four independent experiments. F, PC localization of 8-week-old mice (WT, upper; KO, lower) was observed by imaging mass microscope. PC32:0, PC34:1, PC38:4, and PC40:6 are supposed to have C16:0 (palmitic acid), C18:1 (oleic acid), C20:4 (AA), and C22:6 (DHA), respectively. The signals of PC40:6 were disappeared in the OS of LPAAT3-KO retina. Imaging data of phospholipids were merged with captured light images. Scale bar, 70 μm. Two independent experiments were performed with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To identify the cell type in which PL-DHA was depleted in LPAAT3-KO retinas, we next examined PL-DHA localization in the retina by using an imaging mass microscope, iMscope TRIO (Shimadzu Corp., Kyoto, Japan). In addition to PC containing DHA and AA, representative PCs are shown in Fig. 2F. PC32:0, PC34:1, PC38:4, and PC40:6 are supposed to have C16:0 (palmitic acid), C18:1 (oleic acid), C20:4 (arachidonic acid, AA), and C22:6 (DHA), respectively. PC40:6 (supposed to have DHA, PC-DHA) was detected in the OS of WT retinas but disappeared in the OS of LPAAT3-KO retinas (Fig. 2F). By contrast, PC38:4 (PC-AA) levels increased in LPAAT3-KO retinas (Fig. 2F). These results were consistent with more quantitative analyses by LC-MS data (Fig. 2B). The levels of PC32:0 and PC34:1 were similar between WT and LPAAT3-KO retinas (Fig. 2F). From these results, abundant PL-DHA was located at the OS of the retina, but it almost disappeared during LPAAT3 deficiency. Our observations of disrupted PL-DHA levels in LPAAT3-KO mice encouraged us to examine the physical properties of lipid bilayers composed of several PCs, including PC-DHA. Thus, we studied the size of liposomes with known phospholipid contents. Liposomes composed of di-oleoyl-PC (DOPC), di-arachidonoyl-PC (DAPC), or di-DHA-PC (DDPC) (Fig. 3A) were constructed and analyzed by DLS using a Zetasizer NanoZSP (Malvern Instruments Ltd., UK). Although each liposome size was adjusted by passage through a 100-nm filter, the z-average diameters (light-intensity weighted hydrodynamic diameters) of DAPC or DDPC liposomes were smaller than that of DOPC liposomes (Fig. 3B). After additional passage through a 50-nm filter, DDPC liposome size decreased significantly relative to those of DAPC and DOPC (Fig. 3B). These results indicate that differences in fatty acid physical characteristics directly affected each liposome size, i.e. the physical property of membranes containing DDPC differed from those containing DAPC or DOPC. Liposomes consisting of PC with mixed acyl chains having stearic acid at the sn-1 position and oleic acid, AA, or DHA at the sn-2 position did not show any differences (data not shown); the differences in diameter, if they existed, might have been less than the sensitivity limit of the assay. We then calculated membrane elasticity by MD simulations using GROMACS 5.1 simulation packages (20Pall S. Abraham M.J. Kutzner C. Hess B. Lindahl E. Tackling exascale software challenges in molecular dynamics simulations with gromacs.In Solving software challenges for exascale. 2014; : 3-27Google Scholar, 21Abraham M.J. Murtola T. Schulz R. Páll S. Smith J.C. Hess B. Lindahl E. Gromacs: High performance molecular simulations through multi-level parallelism from laptops to supercomputers.SoftwareX. 2015; 1: 19-25Crossref Scopus (9737) Google Scholar). Each membrane area expansion modulus (KA) was estimated (supplemental Fig. S2) to calculate the membrane bending rigidity (κ) for three different membranes composed of either DOPC, DAPC, or DDPC. The membrane rigidity of DDPC was lower than the rigidity of DOPC and DAPC at 30 °C, indicating that DDPC is more flexible than the others (Fig. 3C). These differences in κ might be attributed to the number of double bonds in the three phospholipid species. PE liposomes using di-oleoyl-PE (DOPE), di-arachidonoyl-PE (DAPE), or di-DHA-PC (DDPE) showed a similar trend (Fig. 3C). Although the effect of double bonds on bending rigidity was decreased at 50 and 70 °C (supplemental Fig. S2), DDPC showed the lowest κ at all three temperatures. LPAAT3-KO retinas indicated decreases and increases of PL-DHA and PL-AA levels, respectively, with each exhibiting distinct physical properties. To examine whether the elimination of PL-DHA would affect retinal structure, we next performed histological analyses of LPAAT3-KO retinas. The thicknesses of the outer nuclear layer (ONL), inner nuclear layer (INL), inner segment (IS), and OS were comparable between WT and LPAAT3-KO retinas at ∼2 weeks following birth (Fig. 4, A–D), indicating that early retinal development was not significantly affected by PL-DHA deficiency. However, 3–8-week-old mice exhibited fully elongated OS with IS in WT retinas but not in LPAAT3-KO retinas (Fig. 4, A and B). This developmental benchmark in OS coincided with increased LPAAT3 expression in the retinas (Fig. 1B). Furthermore, the ONL in 6- and 8-week-old LPAAT3-KO retinas were thinner than those in WT retinas (Fig. 4, A, C, and E), whereas the INL thickness was similar between WT and LPAAT3-KO retinas (Fig. 4, A, D, and F). Next, the expression patterns of LPAAT3 and markers for rod photoreceptor cells (rhodopsin and recoverin) and cone photoreceptor cells (M-opsin) were detected by immunohistochemistry. We observed that LPAAT3 was localized in the IS indicating that PL-DHA was biosynthesized by LPAAT3 in the endoplasmic reticulum and/or other organelles in photoreceptor cells (Fig. 4G). Rhodopsin was primarily localized to the OS of rod photoreceptor cells in WT and LPAAT3-KO retinas; however, in LPAAT3-KO retinas, rhodopsin expression may have shown a slightly disturbed pattern in ONL. By contrast, recoverin was mainly expressed in the IS of WT retinas but appeared diffusely in both the IS and OS of LPAAT3-KO retinas (Fig. 4G). Furthermore, M-opsin was aligned in the OS of WT retinas but exhibited fragmented signals in LPAAT3-KO retinas (Fig. 4G). Thus, the structures of the IS and OS were disorganized, and rhodopsin was partially stacked at ONL in LPAAT3-KO retinas. Next, we examined whether the damage of retinal structure was affected by phototoxicity. ABCA4 clears 11-cis- and all-trans-retinal from the photoreceptor disc membrane to protect against phototoxicity (11Quazi F. Lenevich S. Molday R.S. ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer.Nat. Commun. 2012; 3: 925Crossref PubMed Scopus (174) Google Scholar). During this step, 11-cis-retinal combined with PE-DHA is effectively isomerized to an all-trans isomer by retinol dehydrogenase 8 and enters the visual cycle (8Quazi F. Molday R.S. ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 5024-5029Crossref PubMed Scopus (83) Google Scholar). We also investigated the effect of phototoxicity on retinal degeneration in LPAAT3-KO mice. Mice were housed under dark conditions for a 2-week period when they were 1–3 weeks old, and then their retinal layers were analyzed. The OS/IS and ONL of LPAAT3-KO retinas were lower than those of WT mice (Fig. 5) with similar results obtained under normal light/dark cycle conditions (Fig. 4, A–F). These results demonstrate that retinal degeneration of LPAAT3-KO mice was not caused by phototoxicity. To examine the effect of PL-DHA attenuation and OS damage on visual function, 8-week-old LPAAT3-KO mice were assessed by ERGs. A dark-adapted ERG revealed 80 and 50% decreases in the a- and b-wave amplitudes, respectively, in LPAAT3-KO retinas at light intensities of 4.5 cd·s/m2 (Fig. 6, A–C). Similarly, light-adapted ERG showed 80 and 70% decreases in the a- and b-waves, respectively, in LPAAT3-KO retinas at light intensities of 22.3 cd·s/m2 (Fig. 6, D–F). Therefore, these results indicate that loss of PL-DHA as a consequence of LPAAT3 deficiency resulted in visual dysfunction. We further examined the precise structure of the IS and OS of photoreceptor cells in LPAAT3-KO retinas by transmission electron microscopy (TEM). The retinas of 3-week-old mice were investigated, because the OS length was shortened significantly in LPAAT3-KO retinas according to the retinal sections in this stage (Fig. 4, A and B). At low magnification, the OS was well organized, and the disc exhibited normal formation in WT retinas (Fig. 7, A–C) but not in LPAAT3-KO retinas (Fig. 7, D–H). Highly disorganized but membranous disc-like structures were detected at the basal side of the OS in LPAAT3-KO retinas (Fig. 7E). However, RPE cells exhibiting normal structures may not affect OS abnormality in LPAAT3-KO retinas (Fig. 7D). Close examination of the IS-OS junction in LPAAT3-KO retinas revealed no drastic morphological changes in the IS and the connecting cilium (Fig. 7, F–H). Given that discs showed abnormal morphology compared with WT (Fig. 7C), but were at least formed and located at the OS in LPAAT3-KO retinas, membrane evagination and/or fusion at the base of the OS (22Ding J.D. Salinas R.Y. Arshavsky V.Y. Discs of mammalian rod photoreceptors form through the membrane evagination mechanism.J. Cell Biol. 2015; 211: 495-502Crossref PubMed Scopus (67) Google Scholar) might still be processed normally (WT, Fig. 7C; KO, Fig. 7, F and G). However, abnormal discs parallel to the axoneme, which is a hallmark of abnormal OS morphogenesis (23Gilliam J.C. Chang J.T. Sandoval I.M. Zhang Y. Li T. Pittler S.J. Chiu W. Wensel T.G. Three-dimensional architecture of the rod sensory cilium and its disruption in retinal neurodegeneration.Cell. 2012; 151: 1029-1041Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), were also observed in LPAAT3 KO retinas (Fig. 7, F and H). These results indicate that PL-DHA might be more essential for disc organization and/or maintenance of morphology than for new disc formation at the basal OS. Combined with DLS and MD simulation data, our results reveal that the different physical properties of phospholipids induced the distinct disc characteristics between WT and LPAAT3-KO retinas. Based on their structural flexibilities, PL-DHA but not PL-AA may be essential for disc organization/morphology (Fig. 8).Figure 8Model figures of DHA roles. Model figures of proposed DHA roles in photoreceptor cells. Low levels of PL-DHA in LPAAT3-KO-induced abnormal disc morphology/organization, which was not rescued by increased PL-AA levels.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This study provides a molecular mechanism associated with how DHA promotes visual functions. Attenuation of PL-DHA in retina as a consequence of LPAAT3 deficiency induced visual dysfunction due to the disruption of disc morphology in photoreceptor cells. DLS analysis revealed distinct physical properties of DDPC liposomes as compared with those of DAPC liposomes according to the increased number of double bonds in fatty acids. These results from DLS analysis were supported by MD simulations. This study proposes a molecular mechanism of DHA functions in photoreceptor cells formation (Fig. 8). In LPAAT3-KO retinas, although PL-DHA levels were strongly suppressed, PL-AA levels were increased (Fig. 2 and supplemental Fig. S1). We do not have a clear answer regarding the mechanism by which PL-AA increased; therefore, fatty acid metabolism in the LPAAT3-KO retinas requires future investigation. However, increased PL-AA did not compensate for lack of PL-DHA in disc maintenance and/or organization. One possible reason might be explained from the results of DLS analysis and MD simulations (Fig. 3, B and C). These results consistently indicate that the limit sizes of liposomes were reduced coinciding decreases with phase transition temperature, which decreases with increasing double bond number in fatty acids (24Zhigaltsev I.V. Tam Y.K. Leung A.K. Cullis P.R. Production of limit size nanoliposomal systems with potential utility as ultra-small drug delivery agents.J. Liposome Res. 2016; 26: 96-102PubMed Google Scholar). The high flexibility resulting from fatty acid desaturation in phospholipids might contribute to the maintenance of precise disc conformations (Fig. 8). Recently, polyunsaturation of fatty acids was reported to promote fluidity on both two-dimensional (x-y plane) and three-" @default.
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• W2620565698 date "2017-07-01" @default.
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• W2620565698 title "Docosahexaenoic acid preserves visual function by maintaining correct disc morphology in retinal photoreceptor cells" @default.
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