Advertisement
Thematic Review Series Thematic Review Series: Seeing 2020: Lipids and Lipid-Soluble Molecules in the Eye| Volume 62, 100042, January 01, 2021

Bisretinoid phospholipid and vitamin A aldehyde: shining a light

Open AccessPublished:February 05, 2021DOI:https://doi.org/10.1194/jlr.TR120000742

      Abstract

      Vitamin A aldehyde covalently bound to opsin protein is embedded in a phospholipid-rich membrane that supports photon absorption and phototransduction in photoreceptor cell outer segments. Following absorption of a photon, the 11-cis-retinal chromophore of visual pigment in photoreceptor cells isomerizes to all-trans-retinal. To maintain photosensitivity 11-cis-retinal must be replaced. At the same time, however, all-trans-retinal has to be handled so as to prevent nonspecific aldehyde activity. Some molecules of retinaldehyde upon release from opsin are efficiently reduced to retinol. Other molecules are released into the lipid phase of the disc membrane where they form a conjugate [N-retinylidene-PE (NRPE)] through a Schiff base linkage with PE. The reversible formation of NRPE serves as a transient sink for retinaldehyde that is intended to return retinaldehyde to the visual cycle. However, if instead of hydrolyzing to PE and retinaldehyde, NRPE reacts with a second molecule of retinaldehyde, a synthetic pathway is initiated that leads to the formation of multiple species of unwanted bisretinoid fluorophores. We report on recently identified members of the bisretinoid family, some of which differ with respect to the acyl chains associated with the glycerol backbone. We discuss processing of the lipid moieties of these fluorophores in lysosomes of retinal pigment epithelial cells, their fluorescence characters, and new findings related to light- and iron-associated oxidation of bisretinoids.

      Supplementary key words

      Abbreviations:

      A2-DHP-PE (A2-dihydropyridine-PE), A2-GPE (A2-glycerophosphoethanolamine), all-trans-retinal dimer-E (all-trans-retinal dimer-ethanolamine), A2PE (phosphatidyl-pyridinium bisretinoid), GPE (glycerophosphoethanolamine), NRPE (N-retinylidene-PE), PLA2 (phospholipase A2), PLD (phospholipase D), RDH (NADPH-dependent retinol dehydrogenase), RPE (retinal pigment epithelium), λmax (absorbance maximum)

      Formation of bisretinoid fluorophores by reaction of vitamin a aldehyde with lipid

      After absorption of a photon of light, the 11-cis-retinaldehye chromophore of visual pigment isomerizes to all-trans-retinaldehyde, thereby initiating a series of conformational rearrangements leading to the phototransduction cascade (Fig. 1). Because it bears a reactive aldehyde, all-trans-retinaldehyde must be reduced to the less reactive alcohol (all-trans-retinol) by NADPH-dependent retinol dehydrogenases (RDH8, RDH11, and RDH12) in the photoreceptor cell (
      • Lamb T.D.
      • Pugh E.N.
      Dark adaptation and the retinoid cycle of vision.
      ,
      • Rattner A.
      • Smallwood P.M.
      • Nathans J.
      Identification and characterization of all-trans- retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol.
      ,
      • Maeda A.
      • Golczak M.
      • Maeda T.
      • Palczewski K.
      Limited roles of Rdh8, Rdh12, and Abca4 in all-trans-retinal clearance in mouse retina.
      ,
      • Chrispell J.D.
      • Feathers K.L.
      • Kane M.A.
      • Kim C.Y.
      • Brooks M.
      • Khanna R.
      • Kurth I.
      • Huebner C.A.
      • Gal A.
      • Mears A.J.
      • et al.
      Rdh12 activity and effects on retinoid processing in the murine retina.
      ,
      • Molday R.S.
      ATP-binding cassette transporter ABCA4: molecular properties and role in vision and macular degeneration.
      ,
      • Travis G.H.
      • Golczak M.
      • Moise A.R.
      • Palczewski K.
      Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents.
      ). To facilitate this deactivation, some molecules of retinaldehyde react with the primary amine of PE in the outer segment disc membrane, thereby forming the adduct NRPE via a Schiff base linkage (C=C–N) (
      • Anderson R.E.
      • Maude M.B.
      Phospholipids of bovine outer segments.
      ). This is a mechanism by which NRPE serves to sequester reactive all-trans-retinal. NRPE is also the ligand that binds photoreceptor-specific ABCA4 in outer segments (
      • Papermaster D.S.
      • Schneider B.G.
      • Zorn M.A.
      • Kraehenbuhl J.P.
      Immunocytochemical localization of a large intrinsic membrane protein to the incisures and margins of frog rod outer segment disks.
      ,
      • Illing M.
      • Molday L.L.
      • Molday R.S.
      The 220-kDa rim protein of retinal rod outer segments is a member of the ABC transporter superfamily.
      ,
      • Sun H.
      • Molday R.S.
      • Nathans J.
      Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease.
      ,
      • Sun H.
      • Nathans J.
      Stargardt’s ABCR is localized to the disc membrane of retinal rod outer segments.
      ,
      • Quazi F.
      • Molday R.S.
      ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.
      ,
      • Molday L.L.
      • Rabin A.R.
      • Molday R.S.
      ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy.
      ,
      • Ahn J.
      • Wong J.T.
      • Molday R.S.
      The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy.
      ). The function of ABCA4 is to transport NRPE across the lipid bilayer to the cytoplasmic face of the disc membrane where NRPE hydrolyzes and all-trans-retinaldehyde is released and reduced to all-trans-retinol by RDHs (
      • Rattner A.
      • Smallwood P.M.
      • Nathans J.
      Identification and characterization of all-trans- retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol.
      ,
      • Maeda A.
      • Golczak M.
      • Maeda T.
      • Palczewski K.
      Limited roles of Rdh8, Rdh12, and Abca4 in all-trans-retinal clearance in mouse retina.
      ,
      • Chrispell J.D.
      • Feathers K.L.
      • Kane M.A.
      • Kim C.Y.
      • Brooks M.
      • Khanna R.
      • Kurth I.
      • Huebner C.A.
      • Gal A.
      • Mears A.J.
      • et al.
      Rdh12 activity and effects on retinoid processing in the murine retina.
      ). The 11-cis isomer of NRPE (N-11-cis-retinylidene-PE) can also be flipped by ABCA4 from the luminal to the cytoplasmic leaflet of disk membranes; this activity is presumed to prevent excess levels of 11-cis-retinal (
      • Quazi F.
      • Molday R.S.
      ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.
      ).
      Figure thumbnail gr1
      Fig. 1Retinoid cycle of retina. The visual chromophore 11-cis-retinal forms a covalent Schiff base with lysine 296 (Lys 296) of opsin. Vision is initiated when a photon is captured by 11-cis-retinal; as a result, the chromophore is isomerized to all-trans-retinal. With all-trans-retinal still covalently bound to opsin, the activated pigment transitions to the metarhodopsin II conformation, the Schiff base is hydrolyzed, and all-trans-retinal is reduced to all-trans-retinol by RDHs. Alternatively, some all-trans-retinal reacts with PE in the lipid bilayer of photoreceptor outer segments to form NRPE, which is transported by ABCA4 and then hydrolyzes to release PE and all-trans-retinal. The latter is subsequently reduced to all-trans-retinol. Within the RPE cell, all-trans-retinol is esterified by the enzyme lecithin retinol acyl transferase (LRAT) and is isomerized from the all-trans configuration to the 11-cis-retinol by RPE65. The alcohol is then oxidized by 11-cis-retinol dehydrogenase (11cRDH) to 11-cis-retinal. The bisretinoid synthesis pathway (orange) is initiated when NRPE, rather than hydrolyzing to all-trans-retinal and PE, reacts with a second molecule of retinaldehyde. A multi-step pathway leads to formation of the intermediate dihydropyridinium-A2PE. Automatic oxidation of dihydropyridinium-A2PE with loss of two hydrogens generates A2PE, the immediate precursor of A2E and loss of one hydrogen generates A2-DHP-PE; phosphate hydrolysis of the latter produces A2-DHP-E. A2E, lysoA2PE, and A2-GPE are produced from A2PE. Via an alternative pathway, all-trans-retinal dimer forms from the condensation of two all-trans-retinals. Reaction of the all-trans-retinal dimer with PE with formation of a protonated Schiff base linkage generates all-trans-retinal dimer-PE (atRALdi-PE), and phosphate hydrolysis of the latter yields all-trans-retinal dimer-ethanolamine (atRALdi-E).
      If not efficiently reduced, NRPE also reacts nonenzymatically and irreversibly with a second retinaldehyde molecule to form toxic di-retinal fluorophores (bisretinoids) within the lipid bilayers of the photoreceptor outer segment (Fig. 1). To rid outer segments of these randomly produced light absorbing molecules, the photoreceptor cell undergoes a process of outer segment membrane renewal during which bisretinoids are transferred to retinal pigment epithelium (RPE) and become the lipofuscin of these cells (
      • Sparrow J.R.
      • Wu Y.
      • Kim C.Y.
      • Zhou J.
      Phospholipid meets all-trans-retinal: the making of RPE bisretinoids.
      ,
      • Katz M.L.
      • Drea C.M.
      • Eldred G.E.
      • Hess H.H.
      • Robison Jr., W.G.
      Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium.
      ,
      • Katz M.L.
      • Eldred G.E.
      • Robison Jr., W.G.
      Lipofuscin autofluorescence: evidence for vitamin A involvement in the retina.
      ,
      • Sparrow J.R.
      RPE lipofuscin: formation, properties and relevance to retinal degeneration.
      ). Deficiency in ABCA4 due to gene mutation in humans and mice leads to elevated levels of bisretinoid fluorophores (
      • Maeda A.
      • Golczak M.
      • Maeda T.
      • Palczewski K.
      Limited roles of Rdh8, Rdh12, and Abca4 in all-trans-retinal clearance in mouse retina.
      ,
      • Weng J.
      • Mata N.L.
      • Azarian S.M.
      • Tzekov R.T.
      • Birch D.G.
      • Travis G.H.
      Insights into the function of Rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in abcr knockout mice.
      ,
      • Kim S.R.
      • Fishkin N.
      • Kong J.
      • Nakanishi K.
      • Allikmets R.
      • Sparrow J.R.
      The Rpe65 Leu450Met variant is associated with reduced levels of the RPE lipofuscin fluorophores A2E and iso-A2E.
      ,
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Palczewski K.
      Retinopathy in mice induced by disrupted all-trans-retinal clearance.
      ).
      Incubation of bovine outer segments with all-trans-retinal mimics bisretinoid formation in vivo, and by optimizing the detection of these bisretinoid species, their formation by all-trans-retinal reactivity was confirmed (
      • Sparrow J.R.
      • Gregory-Roberts E.
      • Yamamoto K.
      • Blonska A.
      • Ghosh S.K.
      • Ueda K.
      • Zhou J.
      The bisretinoids of retinal pigment epithelium.
      ). Indeed, adjusting the ratio of all-trans-retinal to PE in synthetic mixtures to determine how the two precursors affect bisretinoid production showed that the yield of bisretinoid saturates when the ratio of equivalents of all-trans-retinal to PE (egg-PE as starting material) is 4:2; no further increase occurred with eight equivalents of all-trans-retinal (8:2 ratio) (
      • Sparrow J.R.
      • Gregory-Roberts E.
      • Yamamoto K.
      • Blonska A.
      • Ghosh S.K.
      • Ueda K.
      • Zhou J.
      The bisretinoids of retinal pigment epithelium.
      ). Conversely, increasing the concentration of PE from an equivalent ratio of 4:1 through 4:8 (all-trans-retinal to PE) was associated with a steady increase in bisretinoid product. These findings indicated that the concentration of PE in the environment of the outer segment membrane has a marked effect on the rate of bisretinoid synthesis. Formation of NRPE, the first step in the synthetic pathway, is likely the most facile reaction. In the Abca4−/− mouse, a mouse model exhibiting accelerated bisretinoid formation, the level of PE in the outer segment membrane was reported to be increased (
      • Weng J.
      • Mata N.L.
      • Azarian S.M.
      • Tzekov R.T.
      • Birch D.G.
      • Travis G.H.
      Insights into the function of Rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in abcr knockout mice.
      ). It has been suggested that a higher level of PE in Abca4−/− mice may facilitate sequestering of all-trans-retinal as NRPE, thereby accelerating recovery of the rod photoresponse after a 30–40% bleach (
      • Pawar A.S.
      • Qtaishat N.M.
      • Little D.M.
      • Pepperberg D.R.
      Recovery of rod photoresponses in ABCR-deficient mice.
      ).
      The unsaturated fatty acid 22:6n-3 (DHA) is abundant in the outer segments of photoreceptor cells, and DHA content has been shown to have several effects on retina (
      • Tanito M.
      • Brush R.S.
      • Elliott M.H.
      • Wicker L.D.
      • Henry K.R.
      • Anderson R.E.
      High levels of retinal membrane docosahexaenoic acid increase susceptibility to stress-induced degeneration.
      ), including susceptibility to light damage. When 22:6n-3-containing PE was used as precursor in the synthetic reaction mixture versus egg-PE (a mixture of fatty acid moieties), phosphatidyl-pyridinium bisretinoid (A2PE) production was found to be greater with the latter. Then again, if free DHA was added to the egg-PE/all-trans-retinal reaction mixture, the yield of A2PE was increased. Under deprotonating conditions associated with the addition of base (triethylamine) to the reactants, the generation of A2PE was increased. Altogether, not only is the phospholipid PE the reactant that combines with all-trans-retinal to form bisretinoid, the lipid composition of outer segments may also impact the extent to which the reaction may be favored.

      Structural features of bisretinoids

      Several members of the family of bisretinoids of RPE lipofuscin have been isolated and structurally characterized. All of these fluorophores form by reaction of retinaldehyde in a 2:1 ratio (A2) with glycerol-based phospholipids linked to an ethanolamine by means of a phosphate ester. The bisretinoids of retina consist of a complex mixture of fluorophores that have been identified by chromatography and MS. They have been detected in human and mouse eyes and they have been characterized structurally (
      • Sparrow J.R.
      • Gregory-Roberts E.
      • Yamamoto K.
      • Blonska A.
      • Ghosh S.K.
      • Ueda K.
      • Zhou J.
      The bisretinoids of retinal pigment epithelium.
      ) (Fig. 2). Chromatographic quantitation in murine eyes has revealed an age-related accumulation of each of these known bisretinoids (
      • Sparrow J.R.
      • Gregory-Roberts E.
      • Yamamoto K.
      • Blonska A.
      • Ghosh S.K.
      • Ueda K.
      • Zhou J.
      The bisretinoids of retinal pigment epithelium.
      ,
      • Kim S.R.
      • Jang Y.P.
      • Jockusch S.
      • Fishkin N.E.
      • Turro N.J.
      • Sparrow J.R.
      The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
      ,
      • Yamamoto K.
      • Yoon K.D.
      • Ueda K.
      • Hashimoto M.
      • Sparrow J.R.
      A novel bisretinoid of retina is an adduct on glycerophosphoethanolamine.
      ,
      • Wu Y.
      • Fishkin N.E.
      • Pande A.
      • Pande J.
      • Sparrow J.R.
      Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease.
      ). Based on noninvasive quantitative fundus autofluorescence in human subjects, these fluorophores also increase with age in human subjects (
      • Greenberg J.P.
      • Duncker T.
      • Woods R.L.
      • Smith R.T.
      • Sparrow J.R.
      • Delori F.C.
      Quantitative fundus autofluorescence in healthy eyes.
      ). Bisretinoids are present at elevated levels in mice with a null mutation in the Abca4 transporter, the gene causative for recessive Stargardt macular degeneration. A signature feature of bisretinoids is that they have two absorbance peaks, one in the UV range and the other in the visible spectrum (Fig. 2). These fluorophores include the pyridinium-containing molecules A2-glycerophosphoethanolamine (A2-GPE) (
      • Yamamoto K.
      • Yoon K.D.
      • Ueda K.
      • Hashimoto M.
      • Sparrow J.R.
      A novel bisretinoid of retina is an adduct on glycerophosphoethanolamine.
      ), A2E, and isomers of A2E (
      • Maeda A.
      • Golczak M.
      • Maeda T.
      • Palczewski K.
      Limited roles of Rdh8, Rdh12, and Abca4 in all-trans-retinal clearance in mouse retina.
      ,
      • Weng J.
      • Mata N.L.
      • Azarian S.M.
      • Tzekov R.T.
      • Birch D.G.
      • Travis G.H.
      Insights into the function of Rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in abcr knockout mice.
      ,
      • Kim S.R.
      • Fishkin N.
      • Kong J.
      • Nakanishi K.
      • Allikmets R.
      • Sparrow J.R.
      The Rpe65 Leu450Met variant is associated with reduced levels of the RPE lipofuscin fluorophores A2E and iso-A2E.
      ,
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Palczewski K.
      Retinopathy in mice induced by disrupted all-trans-retinal clearance.
      ,
      • Sakai N.
      • Decatur J.
      • Nakanishi K.
      • Eldred G.E.
      Ocular age pigment “A2E”: an unprecedented pyridinium bisretinoid.
      ,
      • Ren R.F.
      • Sakai N.
      • Nakanishi K.
      Total synthesis of the ocular age pigment A2E: a convergent pathway.
      ,
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      • Dillon J.
      • Sparrow J.R.
      Isolation and one- step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium.
      ,
      • Ablonczy Z.
      • Smith N.
      • Anderson D.M.
      • Grey A.C.
      • Spraggins J.
      • Koutalos Y.
      • Schey K.L.
      • Crouch R.K.
      The utilization of fluorescence to identify the components of lipofuscin by imaging mass spectrometry.
      ,
      • Ablonczy Z.
      • Higbee D.
      • Anderson D.M.
      • Dahrouj M.
      • Grey A.C.
      • Gutierrez D.
      • Koutalos Y.
      • Schey K.L.
      • Hanneken A.
      • Crouch R.K.
      Lack of correlation between the spatial distribution of A2E and lipofuscin fluorescence in the human retinal pigment epithelium.
      ,
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane.
      ); dimers of all-trans-retinal having a cyclohexadiene head group (all-trans-retinal dimer) (
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Palczewski K.
      Retinopathy in mice induced by disrupted all-trans-retinal clearance.
      ,
      • Kim S.R.
      • Jang Y.P.
      • Jockusch S.
      • Fishkin N.E.
      • Turro N.J.
      • Sparrow J.R.
      The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
      ,
      • Fishkin N.E.
      • Sparrow J.R.
      • Allikmets R.
      • Nakanishi K.
      Isolation and characterization of a retinal pigment epithelial cell fluorophore: an all-trans-retinal dimer conjugate.
      ), the associated protonated Schiff base conjugate (
      • Kim S.R.
      • Jang Y.P.
      • Jockusch S.
      • Fishkin N.E.
      • Turro N.J.
      • Sparrow J.R.
      The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
      ,
      • Fishkin N.E.
      • Sparrow J.R.
      • Allikmets R.
      • Nakanishi K.
      Isolation and characterization of a retinal pigment epithelial cell fluorophore: an all-trans-retinal dimer conjugate.
      ), and the uncharged A2-dihydropyridine-PE (A2-DHP-PE) (
      • Wu Y.
      • Fishkin N.E.
      • Pande A.
      • Pande J.
      • Sparrow J.R.
      Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease.
      ). Notably, all of these compounds are fluorescent molecules that exhibit absorbances in both the UV and visible range, a signature feature of bisretinoids.
      Figure thumbnail gr2
      Fig. 2Structures and UV-visible absorbance maxima (shown in nm) of the fluorophores that constitute the bisretinoids of retinal lipofuscin. Absorbance maxima of these bischromophores can be assigned to the shorter and longer side-arms of the molecules. For all-trans-retinal dimer-PE and all-trans-retinal dimer-E, the absorbance generated from the long-arm exhibits a bathochromic shift (red-shift) due to protonation of the imine functional group (–CH=N–). R, R1, and R2 are fatty acids with various carbon numbers and multiple double bonds.
      The first bisretinoid to be identified was A2E (
      • Sakai N.
      • Decatur J.
      • Nakanishi K.
      • Eldred G.E.
      Ocular age pigment “A2E”: an unprecedented pyridinium bisretinoid.
      ,
      • Ren R.F.
      • Sakai N.
      • Nakanishi K.
      Total synthesis of the ocular age pigment A2E: a convergent pathway.
      ,
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      • Dillon J.
      • Sparrow J.R.
      Isolation and one- step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium.
      ) (Fig. 2, compound 4); A2 in the nomenclature indicates its formation from two vitamin A-aldehyde molecules. The pyridinium bisretinoid (
      • Sun H.
      • Molday R.S.
      • Nathans J.
      Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease.
      ,
      • Sun H.
      • Nathans J.
      Stargardt’s ABCR is localized to the disc membrane of retinal rod outer segments.
      ,
      • Quazi F.
      • Molday R.S.
      ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.
      ,
      • Katz M.L.
      • Eldred G.E.
      • Robison Jr., W.G.
      Lipofuscin autofluorescence: evidence for vitamin A involvement in the retina.
      ,
      • Sparrow J.R.
      RPE lipofuscin: formation, properties and relevance to retinal degeneration.
      ) structure of A2E (C42H58ON; molecular weight, 592) was confirmed by extensive nuclear magnetic resonance studies and by total synthesis (
      • Sakai N.
      • Decatur J.
      • Nakanishi K.
      • Eldred G.E.
      Ocular age pigment “A2E”: an unprecedented pyridinium bisretinoid.
      ,
      • Ren R.F.
      • Sakai N.
      • Nakanishi K.
      Total synthesis of the ocular age pigment A2E: a convergent pathway.
      ,
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      • Dillon J.
      • Sparrow J.R.
      Isolation and one- step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium.
      ). A2E formation begins in photoreceptor outer segments when all-trans-retinal, instead of being reduced to all-trans-retinol, reacts nonenzymatically with PE in a 2:1 ratio thus forming the precursor A2PE (Fig. 2, compound 1). A2E is released when A2PE is acted upon by phospholipase D (PLD) in RPE lysosomes. The double bonds along the side arms of A2E are all in the trans (E) position. In addition, Z-isomers of A2E that have double bonds at the C13-C14 (isoA2E) (Fig. 2, compound 5), C9-C10, and C11-C12 positions are also all detectable in human and mouse RPE (
      • Kim S.R.
      • Jang Y.P.
      • Jockusch S.
      • Fishkin N.E.
      • Turro N.J.
      • Sparrow J.R.
      The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
      ,
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      • Dillon J.
      • Sparrow J.R.
      Isolation and one- step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium.
      ,
      • Ben-Shabat S.
      • Parish C.A.
      • Vollmer H.R.
      • Itagaki Y.
      • Fishkin N.
      • Nakanishi K.
      • Sparrow J.R.
      Biosynthetic studies of A2E, a major fluorophore of retinal pigment epithelial lipofuscin.
      ). These pigments exhibit absorbances in both the UV and visible regions of the spectrum [A2E: absorbance maximum (λmax), 440 and 340 nm; iso-A2E: λmax, 430 and 340 nm]. The polar head of A2E consists of an aromatic ring carrying a permanent positive charge conferred by a quaternary amine nitrogen. The nitrogen does not undergo deprotonation (
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      • Dillon J.
      • Sparrow J.R.
      Isolation and one- step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium.
      ,
      • Sparrow J.R.
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture.
      ), and the charge on the pyridinium nitrogen is neutralized by a counter ion. The aromatic ring also exhibits excellent stability.
      Another bisretinoid that is characterized by a central pyridinium ring housing a quaternary amine nitrogen (Fig. 2, compounds 3) exists as an A2-adduct on glycerophosphoethanolamine (GPE) (A2-GPE) (C45H65O6NP; molecular weight, 746) (
      • Yamamoto K.
      • Zhou J.
      • Hunter J.J.
      • Williams D.R.
      • Sparrow J.R.
      Toward an understanding of bisretinoid autofluorescence bleaching and recovery.
      ). Direct bisretinoid adduct formation on GPE would indicate that in addition to A2-adducts on PE, GPE is accessible for reaction. GPE is the ethanolamine ester of glycerophosphoric acid. In human retina, levels of GPE are 22% of PE (
      • Fliesler S.J.
      • Anderson R.E.
      Chemistry and metabolism of lipids in the vertebrate retina.
      ). The significance of this relatively high content is not known. GPE, a key product of PE catabolism, is generated as a result of phospholipase A2 (PLA2)-mediated cleavage of the acyl chains at both the sn-1 and sn-2 position of the glycerol backbone; further hydrolysis can yield ethanolamine and glycerophosphate. Alternatively, GPE could also be produced from plasmalogens (
      • Kim H.J.
      • Sparrow J.R.
      Novel bisretinoids of human retina are lyso alkyl ether glycerophosphoethanolamine-bearing A2PE species.
      ), a class of glycerophospholipid with a vinyl-ether moiety at the sn-1-position of the glycerol backbone; plasmalogens are highly susceptible to oxidation. The bisretinoid A2-DHP-PE has a noncharged dihydropyridine ring at its core (Fig. 2, compound 9); this structure was confirmed by HPLC-ESI-tandem MS with corroboration by Fourier transform infrared spectroscopy and modeling using density functional theory (
      • Wu Y.
      • Fishkin N.E.
      • Pande A.
      • Pande J.
      • Sparrow J.R.
      Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease.
      ). As with the other bisretinoids, this lipofuscin pigment is a fluorescent compound with absorbance maxima at 490 and 330 nm. A2-DHP-PE was identified in human, mouse, and bovine eyes, and we found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and PE. The stability of A2-DHP-PE was evinced by its detection in mouse eyecups and in human and bovine RPE, and with results demonstrating that A2-DHP-PE accumulates with age (
      • Wu Y.
      • Fishkin N.E.
      • Pande A.
      • Pande J.
      • Sparrow J.R.
      Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease.
      ). Another bisretinoid fluorophore of RPE lipofuscin also absorbs in the short-wavelength region of the visible spectrum (
      • Molday L.L.
      • Rabin A.R.
      • Molday R.S.
      ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy.
      ,
      • Ahn J.
      • Wong J.T.
      • Molday R.S.
      The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy.
      ,
      • Kim S.R.
      • Fishkin N.
      • Kong J.
      • Nakanishi K.
      • Allikmets R.
      • Sparrow J.R.
      The Rpe65 Leu450Met variant is associated with reduced levels of the RPE lipofuscin fluorophores A2E and iso-A2E.
      ). This pigment, all-trans-retinal dimer (λmax, 430 and 290 nm) (Fig. 2, compound 8), forms from the condensation of two all-trans-retinals and is present in RPE lipofuscin as Schiff base conjugates with either PE or ethanolamine [all-trans-retinal dimer-PE and all-trans-retinal dimer-ethanolamine (all-trans-retinal dimer-E), respectively] or as unconjugated all-trans-retinal dimer. The pigments all-trans-retinal dimer-PE (Fig. 2, compound 6) and all-trans-retinal dimer-E (Fig. 2, compound 7) absorb in the visible range at about 510 nm; the “red” shift relative to all-trans-retinal dimer is attributable to protonation of the Schiff base linkage. The protonation state of the Schiff base linkage in all-trans-retinal dimer-PE and all-trans-retinal dimer-E is pH dependent (
      • Kim S.R.
      • Jang Y.P.
      • Jockusch S.
      • Fishkin N.E.
      • Turro N.J.
      • Sparrow J.R.
      The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
      ).
      The compound all-trans-retinal dimer contains a cyclohexadiene ring from which two polyene arms extend: seven double-bond conjugations on the long arm and four on the short arm. All-trans-retinal dimer-E and all-trans-retinal dimer-PE are dimers of all-trans-retinal attached to PE via an imine function group (–C=N) with a protonation state that is pH dependent (
      • Fishkin N.E.
      • Sparrow J.R.
      • Allikmets R.
      • Nakanishi K.
      Isolation and characterization of a retinal pigment epithelial cell fluorophore: an all-trans-retinal dimer conjugate.
      ) (Fig. 2, compounds 6 and 7).
      The most recently discovered member of the bisretinoid family is alkyl-ether-lysoA2PE (1-alkyl ether-2-lysoA2PE) (1-octadecyl-2-lyso-sn-glyceroA2PE) presenting with a single alkyl chain at the sn-1 position (Fig. 2, compound 2) (
      • Kim H.J.
      • Sparrow J.R.
      Novel bisretinoids of human retina are lyso alkyl ether glycerophosphoethanolamine-bearing A2PE species.
      ). This bisretinoid forms by reaction of two vitamin A aldehydes with the ethanolamine head-group of a glycerophospholipid having an ether bond at the sn-1 position rather than the more common ester linkage. This structural assignment was based on molecular mass (m/z 998), UV-visible absorbance maxima (340 and 440 nm), and a retention time corroborated by biomimetic synthesis using all-trans-retinal and GPE analogs as starting materials. UPLC profiles of retinal extracts acquired from human donor eyes revealed that alkyl-ether-lysoA2PE was detectable in RPE but not neural retina. The structure of alkenyl-ether-lysoA2PE, an analog of alkyl-ether-lysoA2PE, including the saturated hydrocarbon long chain and vinyl ether linkage at sn-1 of glycerol phosphate moieties, is more complex than A2E. Specifically, due to the negative charge on the glycerol phosphate moiety, alkenyl-ether-lysoA2PE is likely to resist the tight packing required to form micelles with SDS in an aqueous milieu, while A2E with to its cationic polar head group would readily associate with SDS. On the other hand, alkenyl-ether-lysoA2PE may be able to aggregate in association with the hydrophobic retinaldehyde side-arms in aqueous conditions, thus conferring greater photooxidation under blue light radiation.

      Processing of the lipid moiety in lysosomes

      Bisretinoid fluorophores are deposited in RPE as a result of two processes: random reactions of vitamin A aldehyde in photoreceptor cell outer segments and phagocytosis of shed photoreceptor outer segment discs by RPE. These fluorophores accumulate with age in the lysosomal compartment of RPE cells and constitute the lipofuscin of retina.
      The lysosomal enzyme PLD catalyzes the cleavage of the phosphodiester bond of glycerophospholipids thereby generating phosphatidic acid and a free ethanolamine (Fig. 3) (
      • Kobayashi M.
      • Kanfer J.N.
      Solubilization and purification of rat tissue phospholipase D.
      ,
      • Salvador G.A.
      • Giusto N.M.
      Characterization of phospholipase D activity in bovine photoreceptor membranes.
      ,
      • Sciorra V.A.
      • Hammond S.M.
      • Morris A.J.
      Potent direct inhibition of mammalian phospholipase D isoenzymes by calphostin-c.
      ). In the case of diacyl A2PE species, PLD-mediated enzymatic hydrolysis (
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane.
      ,
      • Ben-Shabat S.
      • Parish C.A.
      • Vollmer H.R.
      • Itagaki Y.
      • Fishkin N.
      • Nakanishi K.
      • Sparrow J.R.
      Biosynthetic studies of A2E, a major fluorophore of retinal pigment epithelial lipofuscin.
      ,
      • Ben-Shabat S.
      • Itagaki Y.
      • Jockusch S.
      • Sparrow J.R.
      • Turro N.J.
      • Nakanishi K.
      Formation of a nona-oxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement.
      ,
      • Sparrow J.R.
      • Kim S.R.
      • Cuervo A.M.
      • Bandhyopadhyayand U.
      A2E, a pigment of RPE lipofuscin is generated from the precursor A2PE by a lysosomal enzyme activity.
      ) releases phosphatidic acid and A2E (pyridinium compound). Evidence for this cleavage event was obtained when A2PE was incubated in the presence of PLD and A2E appeared in chromatographic profiles (
      • Ben-Shabat S.
      • Itagaki Y.
      • Jockusch S.
      • Sparrow J.R.
      • Turro N.J.
      • Nakanishi K.
      Formation of a nona-oxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement.
      ). Hydrolytic activity in lysosomes isolated from liver and RPE also released A2E from A2PE (
      • Sparrow J.R.
      • Kim S.R.
      • Cuervo A.M.
      • Bandhyopadhyayand U.
      A2E, a pigment of RPE lipofuscin is generated from the precursor A2PE by a lysosomal enzyme activity.
      ). Additionally, this activity was inhibited by the PLD inhibitor calphostin C and by a protease inhibitor cocktail. After phosphate cleavage of A2PE, no further degradation of the molecule occurred (
      • Yamamoto K.
      • Yoon K.D.
      • Ueda K.
      • Hashimoto M.
      • Sparrow J.R.
      A novel bisretinoid of retina is an adduct on glycerophosphoethanolamine.
      ,
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane.
      ). The enzymatic processing in lysosomes appeared to be efficient: A2E was always a substantial peak in RPE extracts (Fig. 4), and A2PE was present at relatively low levels (
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane.
      ). In in vitro assays, A2E was also shown to be released from A2-GPE by PLD-mediated activity; however, the abundance of A2-GPE in RPE extracts indicated that this reaction is not favored. Because A2-GPE does not contain fatty acid chains at either the sn-1 or the sn-2 ester linkage, PLD-mediated hydrolysis at phosphodiester moieties of glycerophospholipid constituents of A2PE is evidently not affected by fatty acid chains attached to the glycerol backbone (Fig. 3). On the other hand, the relative abundance of A2-DHP-PE in mouse eyecups and human and bovine RPE indicated that A2-DHP-PE is more refractory to cleavage. Consequently, this bisretinoid retains the phospholipid-derived tail. Another bisretinoid in RPE that retains the phospholipid moiety is all-trans-retinal dimer-PE. A2PE, A2-DHP-PE, and all-trans-retinal dimer-PE are not single molecular species. Rather, each consists of a series of bisretinoids having fatty acids of varying lengths and numbers of double bonds, for instance 22:6 (DHA) and 18:0 (stearic acid) attached at the sn-1 and sn-2 through ester or ether linkages (Fig. 5).
      Figure thumbnail gr3
      Fig. 3A: Schematic summarizing bisretinoid production in photoreceptor outer segments with processing in RPE. RAL, retinaldehyde. B. Structure of A2PE, A2-GPE, and A2E with UV-visible absorbance (nanometers). The structure includes an ether-linked (–O–) saturated alkyl chain at the sn-1 position; a vinyl ether (-O-C=C-) at the sn-1 position (plasmalogen) with alkyl groups; and an ester (–O–C=O–) linkage at the sn-1 position with alkyl groups. Positions on the GPE that are subject to hydrolysis by PLA2 and PLD are indicated (pink arrows). Cleavage by PLA2 yields lysoA2PE. Cleavage by PLD yields A2E. Carbon numbers on the glycerol backbone are indicated by 1, 2, and 3. Absorbance peaks at 440 and 340 nm can be assigned to the long and short arms of the molecule, respectively.
      Figure thumbnail gr4
      Fig. 4UPLC profile of an extract of human RPE [donor age 74, 1 eye (A, B)]. The chromatogram represents analysis of six eyes. A: Chromatogram with monitoring at 430 nm absorbance. Top insets: UV-visible absorbance spectra of isoA2E, A2E, A2-GPE, A2-DHP-PE, atRALdi-PE, and lysoA2PE species. B: Fluorescence monitoring at an excitation of 430 nm and emission of 600 nm.
      Figure thumbnail gr5
      Fig. 5UPLC profiles derived from chloroform extracts of bovine rod outer segments (ROS) (four eyes per sample) incubated with 2 mM atRAL (all-trans-retinal) in 2% DMSO in DBPS, in the dark at 37°C for 3 days. Chromatographic separation by UPLC-PDA-MS with monitoring at 430 nm (A) and total ion chromatogram (TIC) (B). Insets: UV-visible absorbance spectra of peaks ∼1–5 from A. Peaks ∼1–5 exhibited corresponded ion at m/z 1,296, 1,295, 1,281, 1,323, and 1,323, respectively. A2PE species detected in B exhibit m/z 1,323.0 consistent with stearic acid (C18:0) and DHA (C22:6) attached by ester linkages to GPE.
      Recently, we have added to our understanding of enzyme processing by showing that PLA2 can also process A2PE (1-alkyl ether-2-acyl-A2PE) (i.e., an ether bond at the sn-1 site and an ester bond at the sn-2 site) by mediating cleavage at the ester bond positioned at the sn-2 site so as to produce lysoA2PE presenting with a single alkyl chain at the sn-1 position (
      • Kim H.J.
      • Sparrow J.R.
      Novel bisretinoids of human retina are lyso alkyl ether glycerophosphoethanolamine-bearing A2PE species.
      ) (Fig. 3). The detection of the bisretinoid alkyl-ether-lysoA2PE not in neural retina but in human RPE (Fig. 4) indicated that the PLA2 activity likely resides in RPE. In addition, we showed that the plasmalogen-lysoA2PE can subsequently undergo hydrolysis under acidic conditions expected for RPE lysosomes. This process yielded A2-GPE and thereby disclosed a new mechanism contributing to the final processing of bisretinoid (Fig. 3).

      Why do bisretinoids accumulate in the lysosomal compartment of rpe cells?

      RPE lipofuscin differs from other forms of lipofuscin. Specifically, the bisretinoid composition of RPE lipofuscin distinguishes this material from lipofuscin forms that consist of cross-linked oxidatively modified proteins (
      • Brunk U.T.
      • Terman A.
      Lipofuscin: mechanisms of age-related accumulation and influence on cell function.
      ) or protein accumulations (i.e., subunit c of mitochondrial ATP synthase), as in neuronal ceroid lipofuscinosis, a lysosomal storage disease (
      • Johnson T.B.
      • Cain J.T.
      • White K.A.
      • Ramirez-Montealegre D.
      • Pearce D.A.
      • Weimer J.M.
      Therapeutic landscape for Batten disease: current treatments and future prospects.
      ). Other reported molecular constituents of RPE lipofuscin are adducts of 2-(ω-carboxyethyl)pyrrole (
      • Ng K.P.
      • Gugiu B.G.
      • Renganathan K.
      • Davies M.W.
      • Gu X.
      • Crabb J.S.
      • Kim S.R.
      • Rozanowska M.B.
      • Bonilha V.L.
      • Rayborn M.E.
      • et al.
      Retinal pigment epithelium lipofuscin proteomics.
      ), 4-hydroxynonenal, and malondialdehyde (
      • Schutt F.
      • Bergmann M.
      • Holz F.G.
      • Kopitz J.
      Proteins modified by malondialdehyde, 4- hydroxynonenal or advanced glycation end products in lipofuscin of human retinal pigment epithelium.
      ) that are derived from oxidative fragmentation of lipid. The oxidative processes that produce these 2-(ω-carboxyethyl)pyrrole-protein adducts could occur in photoreceptor cells before RPE phagocytosis of outer segment membrane. It is just as likely that these products of lipid oxidation are generated within the lysosomal bodies in which RPE lipofuscin is stored; there they would be generated by bisretinoid-initiated photooxidation. Otherwise the spectral properties of the blue-green emitting fluorescent products of lipid oxidation are markedly different (excitation maxima ∼350 nm, emission maxima ∼435 nm) (
      • Rein D.
      • Tappel A.L.
      Fluorescent lipid oxidation products and heme spectra index antioxidant efficacy in kidney tissue of hamsters.
      ) than spectra generated from RPE lipofuscin (
      • Eldred G.E.
      • Katz M.L.
      The lipid peroxidation theory of lipofuscinogenesis cannot yet be confirmed.
      ,
      • Eldred G.E.
      • Miller G.V.
      • Stark W.S.
      • Feeney-Burns L.
      Lipofuscin: resolution of discrepant fluorescence data.
      ). It has been suggested that bisretinoids such as A2E are amassed in lysosomes of RPE cells because they become trapped after protonation in the acidic environment of the lysosome. It is also assumed that the resulting alkalinization of the lysosome milieu inhibits lysosomal enzymes (
      • Liu J.
      • Lu W.
      • Reigada D.
      • Nguyen J.
      • Laties A.M.
      • Mitchell C.H.
      Restoration of lysosomal pH in RPE cells from cultured human and ABCA4(−/−) mice: pharmacologic approaches and functional recovery.
      ,
      • Eldred G.E.
      • Lasky M.R.
      Retinal age pigments generated by self-assembling lysosomotropic detergents.
      ). However, A2E is a quaternary pyridinium salt that does not deprotonate or reprotonate; the positive charge on the pyridinium nitrogen is neutralized by a counterion, probably chloride (
      • Sparrow J.R.
      • Gregory-Roberts E.
      • Yamamoto K.
      • Blonska A.
      • Ghosh S.K.
      • Ueda K.
      • Zhou J.
      The bisretinoids of retinal pigment epithelium.
      ,
      • Pawar A.S.
      • Qtaishat N.M.
      • Little D.M.
      • Pepperberg D.R.
      Recovery of rod photoresponses in ABCR-deficient mice.
      ,
      • Kobayashi M.
      • Kanfer J.N.
      Solubilization and purification of rat tissue phospholipase D.
      ). The bisretinoid A2-DHP-PE presents with an uncharged dihydropyridine ring at its core; it does not undergo protonation and deprotonation (
      • Wu Y.
      • Fishkin N.E.
      • Pande A.
      • Pande J.
      • Sparrow J.R.
      Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease.
      ). Similarly, the bisretinoid, all-trans-retinal dimer also accumulates in lysosomal storage bodies; this fluorophore presents with a noncharged cyclohexadiene ring (
      • Kim S.R.
      • Jang Y.P.
      • Jockusch S.
      • Fishkin N.E.
      • Turro N.J.
      • Sparrow J.R.
      The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
      ,
      • Fishkin N.E.
      • Sparrow J.R.
      • Allikmets R.
      • Nakanishi K.
      Isolation and characterization of a retinal pigment epithelial cell fluorophore: an all-trans-retinal dimer conjugate.
      ).
      All-trans-retinal dimer can also form a conjugate with PE (all-trans-retinal dimer-PE) via a Schiff base linkage that exhibits pH-dependent protonation, and as with unprotonated unconjugated all-trans-retinal dimer, all trans-retinal dimer-PE accumulates in RPE lysosomes. Moreover, as we have shown here, the final step in the formation of A2E, A2-GPE, and lysoA2PE, all of which are amassed in RPE lysosomes, depends on the activity of at least two hydrolytic enzymes in lysosomes, PLD and PLA2 (Fig. 3). Considered together, these findings indicate that one cannot attribute bisretinoid accumulation to inhibition of lysosomal enzyme activity. Reduced activity of lysosomal degradative enzymes, if it were to occur, would result in a generalized increase in protein/peptide accumulation as in lysosomal storage diseases (
      • Brady R.O.
      Emerging strategies for the treatment of hereditary metabolic storage disorders.
      ), but this is not observed (
      • Ng K.P.
      • Gugiu B.G.
      • Renganathan K.
      • Davies M.W.
      • Gu X.
      • Crabb J.S.
      • Kim S.R.
      • Rozanowska M.B.
      • Bonilha V.L.
      • Rayborn M.E.
      • et al.
      Retinal pigment epithelium lipofuscin proteomics.
      ). Specifically, a proteomic study of purified lipofuscin granules revealed that the amino acid content was only 2% (w/w) (
      • Ng K.P.
      • Gugiu B.G.
      • Renganathan K.
      • Davies M.W.
      • Gu X.
      • Crabb J.S.
      • Kim S.R.
      • Rozanowska M.B.
      • Bonilha V.L.
      • Rayborn M.E.
      • et al.
      Retinal pigment epithelium lipofuscin proteomics.
      ). Other investigators have noted (
      • Eldred G.E.
      • Katz M.L.
      The lipid peroxidation theory of lipofuscinogenesis cannot yet be confirmed.
      ,
      • Eldred G.E.
      • Miller G.V.
      • Stark W.S.
      • Feeney-Burns L.
      Lipofuscin: resolution of discrepant fluorescence data.
      ,
      • Boulton M.E.
      Lipofuscin of the retinal pigment epithelium.
      ) that the presence of photoreceptor proteins in preparations enriched in lipofuscin-containing lysosomal organelles (
      • Schutt F.
      • Ueberle B.
      • Schnolzer M.
      • Holz F.G.
      • Kopitz J.
      Proteome analysis of lipofuscin in human retinal pigment epithelial cells.
      ,
      • Warburton S.
      • Southwick K.
      • Hardman R.M.
      • Secrest A.M.
      • Grow R.K.
      • Xin H.
      • Woolley A.T.
      • Burton G.F.
      • Thulin C.D.
      Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin.
      ) is attributable to contamination with unprocessed phagosomes. And finally, the view that RPE lipofuscin accumulates because of inhibition of lysosomal enzymes, cannot be reconciled with the accumulation of this material in all healthy eyes even at young ages (
      • Greenberg J.P.
      • Duncker T.
      • Woods R.L.
      • Smith R.T.
      • Sparrow J.R.
      • Delori F.C.
      Quantitative fundus autofluorescence in healthy eyes.
      ). Instead, it is likely that RPE lysosomal enzymes that would otherwise degrade the bisretinoid, do not recognize the structures that constitute this material. PLA2 may be of additional interest. Toxic sodium iodate, when internalized by ARPE-19 cells, induces increased expression of calcium independent PLA2 (
      • Kolko M.
      • Vohra R.
      • Westlund van der Burght B.
      • Poulsen K.
      • Nissen M.H.
      Calcium- independent phospholipase A(2), group VIA, is critical for RPE cell survival.
      ), while benign flecks retina is associated with the gene encoding PLA2 group V (
      • Garcia J.M.B.B.
      • Isaac D.L.C.
      • Sardeiro T.
      • Aquino E.
      • Avila M.
      Benign familial fleck retina: multimodal imaging including optical coherence tomography angiography.
      • Neriyanuri S.
      • Rao C.
      • Raman R.
      Structural and functional characterization of benign fleck retina using multimodal imaging.
      ).

      Fluorescence of bisretinoids

      Bisretinoids exhibit a central six-membered ring from which extends dual polyene arms terminating in β-ionine rings (Fig. 2). Each of the arms constitutes a distinct system of double bond conjugations with each arm serving as a retinaldehyde-derived chromophore, one arm absorbing in the UV range and the other in the visible region of the spectrum (
      • Sparrow J.R.
      • Gregory-Roberts E.
      • Yamamoto K.
      • Blonska A.
      • Ghosh S.K.
      • Ueda K.
      • Zhou J.
      The bisretinoids of retinal pigment epithelium.
      ,
      • Sparrow J.R.
      • Wu Y.
      • Nagasaki T.
      • Yoon K.D.
      • Yamamoto K.
      • Zhou J.
      Fundus autofluorescence and the bisretinoids of retina.
      ). The wavelengths at which bisretinoids absorb are determined by the lengths of the systems of alternating double and single bonds, including the double bonds in the β-ionone and central rings. Accordingly, the UV absorbance can be assigned to the short-arm while the long-arm generates the absorbance in the visible range. Absorbances are in the visible spectrum range from 430 to 510 nm. The phospholipid moiety, if retained, does not make a contribution to an absorbance above 250 nm.
      Although the excitation maxima of the various bisretinoid fluorophores varies, their fluorescence emission maxima are similar and generally peak between ∼600 and 620 nm. The retina exhibits an intrinsic autofluorescence that is excited by short-wavelength visible light and that has been monitored in human subjects by in vivo spectrofluorometry (
      • Delori F.C.
      Spectrophotometer for noninvasive measurement of intrinsic fluorescence and reflectance of the ocular fundus.
      ) and by fluorescence adaptive optics ophthalmoscopy (
      • Morgan J.I.
      • Hunter J.J.
      • Masella B.
      • Wolfe R.
      • Gray D.C.
      • Merigan W.H.
      • Delori F.C.
      • Williams D.R.
      Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium.
      ), and that in human subjects is imaged clinically by noninvasive confocal scanning laser ophthalmoscopy (
      • von Rückmann A.
      • Fitzke F.W.
      • Bird A.C.
      Distribution of fundus autofluorescence with a scanning laser ophthalmoscope.
      ,
      • von Rückmann A.
      • Fitzke F.W.
      • Bird A.C.
      In vivo fundus autofluorescence in macular dystrophies.
      ,
      • Delori F.C.
      • Dorey C.K.
      • Staurenghi G.
      • Arend O.
      • Goger D.G.
      • Weiter J.J.
      In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics.
      ). This autofluorescence has a broad excitation spectrum that peaks between 490 and 510 nm. The fluorescence emission is also broad and centered at approximately 600 nm (
      • Delori F.C.
      • Dorey C.K.
      • Staurenghi G.
      • Arend O.
      • Goger D.G.
      • Weiter J.J.
      In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics.
      ,
      • Delori F.C.
      • Goger D.G.
      • Dorey C.K.
      Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects.
      ). The spectral characteristics of short-wavelength fundus autofluorescence are consistent with those of RPE lipofuscin (
      • Eldred G.E.
      • Katz M.L.
      The lipid peroxidation theory of lipofuscinogenesis cannot yet be confirmed.
      ,
      • Eldred G.E.
      • Miller G.V.
      • Stark W.S.
      • Feeney-Burns L.
      Lipofuscin: resolution of discrepant fluorescence data.
      ,
      • Boulton M.
      • Docchio F.
      • Dayhaw-Barker P.
      • Ramponi R.
      • Cubeddu R.
      Age-related changes in the morphology, absorption and fluorescence of melanosomes and lipofuscin granules of the retinal pigment epithelium.
      ,
      • Feeney-Burns L.
      • Eldred G.E.
      The fate of the phagosome: conversion to ‘age pigment’ and impact in human retinal pigment epithelium.
      ) and principally with an origin from the bisretinoid fluorescent pigments that are the constituents (
      • Sparrow J.R.
      • Wu Y.
      • Nagasaki T.
      • Yoon K.D.
      • Yamamoto K.
      • Zhou J.
      Fundus autofluorescence and the bisretinoids of retina.
      ). Moreover, the emission spectra recorded from whole lipofuscin, bisretinoid, and short-wavelength fundus autofluorescence all exhibit red-shifts when excited by progressively longer wavelengths (
      • Sparrow J.R.
      • Wu Y.
      • Nagasaki T.
      • Yoon K.D.
      • Yamamoto K.
      • Zhou J.
      Fundus autofluorescence and the bisretinoids of retina.
      ).
      A significant increase in fluorescence emission is observed with limited photooxidation of A2E and all-trans-retinal dimer (discussed below) as compared with the parent compounds (
      • Kim S.R.
      • Jang Y.
      • Sparrow J.R.
      Photooxidation of RPE Lipofuscin bisretinoids enhanced fluorescence intensity.
      ). Moreover, this increase in fluorescence emission can occur without a change in visible-spectrum absorbance when the oxidation occurs on the short arm of the molecules (
      • Kim S.R.
      • Jang Y.
      • Sparrow J.R.
      Photooxidation of RPE Lipofuscin bisretinoids enhanced fluorescence intensity.
      ). With further oxidation on the long-arms, fluorescence bleaching is observed.

      Photooxidation and photodegradation of bisretinoid lipofuscin

      Because the cellular build-up of bisretinoid has adverse consequences for RPE cells, efforts have been made to understand the processes by which these fluorophores are damaging. While the bisretinoids of lipofuscin are unlikely to undergo lysosomal digestion, these fluorophores are nevertheless subject to photodegradation. Accordingly, whole lipofuscin mixtures (
      • Rózanowska M.
      • Jarvis-Evans J.
      • Korytowski W.
      • Boulton M.E.
      • Burke J.M.
      • Sarna T.
      Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species.
      ,
      • Gaillard E.R.
      • Atherton S.J.
      • Eldred G.
      • Dillon J.
      Photophysical studies on human retinal lipofuscin.
      ) and individual fluorophores such as A2-GPE (
      • Yamamoto K.
      • Yoon K.D.
      • Ueda K.
      • Hashimoto M.
      • Sparrow J.R.
      A novel bisretinoid of retina is an adduct on glycerophosphoethanolamine.
      ), A2E (
      • Ben-Shabat S.
      • Itagaki Y.
      • Jockusch S.
      • Sparrow J.R.
      • Turro N.J.
      • Nakanishi K.
      Formation of a nona-oxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement.
      ), and all-trans-retinal dimer (
      • Kim S.R.
      • Jang Y.P.
      • Jockusch S.
      • Fishkin N.E.
      • Turro N.J.
      • Sparrow J.R.
      The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
      ) have been shown to initiate photosensitization reactions that generate superoxide anion and singlet oxygen (
      • Kim S.R.
      • Jang Y.P.
      • Jockusch S.
      • Fishkin N.E.
      • Turro N.J.
      • Sparrow J.R.
      The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
      ,
      • Ben-Shabat S.
      • Itagaki Y.
      • Jockusch S.
      • Sparrow J.R.
      • Turro N.J.
      • Nakanishi K.
      Formation of a nona-oxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement.
      ,
      • Jang Y.P.
      • Matsuda H.
      • Itagaki Y.
      • Nakanishi K.
      • Sparrow J.R.
      Characterization of peroxy-A2E and furan-A2E photooxidation products and detection in human and mouse retinal pigment epithelial cells lipofuscin.
      ). Singlet oxygen, as a highly reactive form of oxygen, adds to conjugated double bonds along the side-arms of the bisretinoid molecules. In the case of A2E, this oxidation is evidenced in ESI/FAB-MS as mass increments of 16 (molecular weight of oxygen) starting from the molecular ion [M+] at m/z 592 peak attributable to A2E (
      • Ben-Shabat S.
      • Itagaki Y.
      • Jockusch S.
      • Sparrow J.R.
      • Turro N.J.
      • Nakanishi K.
      Formation of a nona-oxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement.
      ). Oxidized bisretinoids (e.g., mono- and bis-peroxy forms of A2E and those of all-trans-retinal dimer) have been detected in human and mouse retina (
      • Kim S.R.
      • Jang Y.P.
      • Jockusch S.
      • Fishkin N.E.
      • Turro N.J.
      • Sparrow J.R.
      The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
      ,
      • Jang Y.P.
      • Matsuda H.
      • Itagaki Y.
      • Nakanishi K.
      • Sparrow J.R.
      Characterization of peroxy-A2E and furan-A2E photooxidation products and detection in human and mouse retinal pigment epithelial cells lipofuscin.
      ). At these unstable oxygen-containing moieties, bisretinoids cleave and release a complex mixture of aldehyde-containing fragments and the dicarbonyls methylglyoxal and glyoxal (Fig. 6) (
      • Wu Y.
      • Yanase E.
      • Feng X.
      • Siegel M.M.
      • Sparrow J.R.
      Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration.
      ,
      • Yoon K.D.
      • Yamamoto K.
      • Ueda K.
      • Zhou J.
      • Sparrow J.R.
      A novel source of methylglyoxal and glyoxal in retina: implications for age-related macular degeneration.
      ,
      • Zhou J.
      • Cai B.
      • Jang Y.P.
      • Pachydaki S.
      • Schmidt A.M.
      • Sparrow J.R.
      Mechanisms for the induction of HNE- MDA- and AGE-adducts, RAGE and VEGF in retinal pigment epithelial cells.
      ) that ravage cellular and extracellular molecules and reflect a link between RPE bisretinoid lipofuscin and the formation of sub-RPE deposits. The photodegradation products released from bisretinoids cross-link protein and suppress matrix metalloproteinase activity (
      • Zhou J.
      • Ueda K.
      • Zhao J.
      • Sparrow J.R.
      Correlations between photodegradation of bisretinoid constituents of retina and dicarbonyl-adduct deposition.
      ) and inhibit the proteasome (
      • Fernandes A.F.
      • Zhou J.
      • Zhang X.
      • Bian Q.
      • Sparrow J.R.
      • Taylor A.
      • Pereira P.
      • Shang F.
      Oxidative inactivation of the proteasome in retinal pigment epithelial cells. A potential link between oxidative stress and up-regulation of interleukin-8.
      ). Given that photooxidation of A2E and all-trans-retinal dimer has also been shown to incite complement activation (
      • Zhou J.
      • Jang Y.P.
      • Kim S.R.
      • Sparrow J.R.
      Complement activation by photooxidation products of A2E, a lipofuscin constituent of the retinal pigment epithelium.
      ,
      • Zhou J.
      • Kim S.R.
      • Westlund B.S.
      • Sparrow J.R.
      Complement activation by bisretinoid constituents of RPE lipofuscin.
      ) together with evidence that these carbonyl-modified proteins are present in deposits (drusen) that accumulate on the basal side of RPE cells in vivo (
      • Handa J.T.
      • Verzijl N.
      • Matsunaga H.
      • Aotaki-Keen A.
      • Lutty G.A.
      • Koppele J.M.
      • Miyata T.
      • Hjelmeland L.M.
      Increase in advanced glycation end product pentosidine in Bruch’s membrane with age.
      ,
      • Crabb J.W.
      • Miyagi M.
      • Gu X.
      • Shadrach K.
      • West K.A.
      • Sakaguchi H.
      • Kamei M.
      • Hasan A.
      • Yan L.
      • Raybourn M.E.
      • et al.
      Drusen proteome analysis: an approach to the etiology of age-related macular degeneration.
      ), the photodegradation of bisretinoids is likely linked to AMD. These processes likely also contribute to Bruch’s membrane thickening observed in Abca4−/− mice (
      • Radu R.A.
      • Hu J.
      • Yuan Q.
      • Welch D.L.
      • Makshanoff J.
      • Lloyd M.
      • McMullen S.
      • Travis G.H.
      • Bok D.
      Complement system dysregulation and inflammation in the retinal pigment epithelium of a mouse model for Stargardt macular degeneration.
      ).
      Figure thumbnail gr6
      Fig. 6The dicarbonyls methylglyoxal and glyoxal are released from the photodegradation of RPE bisretinoids. Shown here is the photodegradation of A2E and all-trans-retinal dimer (atRALdi). Potential cleavage sites at carbon-carbon double bonds are exhibited (dashed lines). Each molecule of A2E and atRALdi could release methylglyoxal or glyoxal depending on the photodegradation patterns. Adapted from (
      • Sparrow J.R.
      • Gregory-Roberts E.
      • Yamamoto K.
      • Blonska A.
      • Ghosh S.K.
      • Ueda K.
      • Zhou J.
      The bisretinoids of retinal pigment epithelium.
      ).
      In studies of albino versus pigmented mice and mice raised in continuous darkness versus cyclic light, levels of bisretinoid were found to be lower in mice exposed to higher levels of intraocular light (albino mice and cyclic-light reared mice). Moreover, in mice treated with the antioxidant vitamin E, photodegradation of bisretinoid was suppressed resulting in increased bisretinoid and sparing of photoreceptor cells. Vitamin E intercepts bisretinoid oxidation by scavenging reactive oxygen species generated by bisretinoid photosensitization (
      • Wu Y.
      • Yanase E.
      • Feng X.
      • Siegel M.M.
      • Sparrow J.R.
      Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration.
      ). And finally, these photoreactive processes explain photoreceptor cell degeneration in Abca4−/− mice (
      • Wu L.
      • Nagasaki T.
      • Sparrow J.R.
      Photoreceptor cell degeneration in Abcr−/− mice.
      ,
      • Radu R.A.
      • Yuan Q.
      • Hu J.
      • Peng J.H.
      • Lloyd M.
      • Nusinowitz S.
      • Bok D.
      • Travis G.H.
      Accelerated accumulation of lipofuscin pigments in the RPE of a mouse model for ABCA4- mediated retinal dystrophies following vitamin A supplementation.
      ) and are a cause of the increased vulnerability of albino Abca4−/− mice to retinal light damage (
      • Wu L.
      • Ueda K.
      • Nagasaki T.
      • Sparrow J.R.
      Light damage in Abca4 and Rpe65rd12 mice.
      ).
      Intracellular iron can also promote the oxidation and degradation of bisretinoid by generating hydroxyl free radical via the Fenton reaction. Specifically, in mice treated with the iron chelator deferiprone, bisretinoid levels, measured by HPLC and noninvasively by quantitative fundus autofluorescence, were elevated (
      • Ueda K.
      • Kim H.J.
      • Zhao J.
      • Song Y.
      • Dunaief J.L.
      • Sparrow J.R.
      Iron promotes oxidative cell death caused by bisretinoids of retina.
      ). Loss of photoreceptor cell viability that is detected by outer nuclear layer thinning was also mitigated in deferiprone-treated albino Abca4−/− mice. Conversely, mice exhibiting increased iron in RPE cells due to deficiency in the iron export proteins hephaestin and ceruloplasmin, presented with reduced bisretinoid levels.

      Cones and rod photoreceptor cells

      Two types of photoreceptor cells populate the retina. Rod photoreceptors provide vision at low light levels and three types of cones enable color vision and vision in ambient lighting. Common to all of these light-sensitive cells is the visual chromophore 11-cis-retinal that binds to opsin molecules specific to each type of photoreceptor. Photon absorption triggers the isomerization of 11-cis-retinal to all-trans-retinal; continued light perception requires reconversion of the chromophore to the 11-cis isomer. The canonical visual cycle operating in RPE cells is responsible for regeneration and provision of the visual chromophore 11-cis-retinal to rods and cones in order to sustain photosensitivity. A supply of vitamin A is stored in RPE as esterified fatty acids. Subsequently the energy required to drive the double bond isomerization is derived from the hydrolysis of fatty acid retinyl esters. However, abundant evidence indicates that the efficiency with which the RPE visual cycle generates 11-cis-retinal is not sufficient to support cone-mediated vision in daylight. Various additional pathways have been suggested that include: light-dependent reverse isomerization of all-trans-retinal bound in the form of NRPE (
      • Kaylor J.J.
      • Xu T.
      • Ingram N.T.
      • Tsan A.
      • Hakobyan H.
      • Fain G.L.
      • Travis G.H.
      Blue light regenerates functional visual pigments in mammals through a retinyl-phospholipid intermediate.
      ); light-dependent pathways involving retinal G protein-coupled receptor (RGR) with all-trans-retinal as substrate (
      • Zhang J.
      • Choi E.H.
      • Tworak A.
      • Salom D.
      • Leinonen H.
      • Sander C.L.
      • Hoang T.V.
      • Handa J.T.
      • Blackshaw S.
      • Palczewska G.
      • et al.
      Photic generation of 11-cis-retinal in bovine retinal pigment epithelium.
      ); or light-independent processes that harness the retinoid isomerase isomerase sphingolipid delta(4)-desaturase 1 (DES1) (
      • Kaylor J.J.
      • Yuan Q.
      • Cook J.
      • Sarfare S.
      • Makshanoff J.
      • Miu A.
      • Kim A.
      • Kim P.
      • Habib S.
      • Roybal C.N.
      • et al.
      Identification of DES1 as a vitamin A isomerase in Muller glial cells of the retina.
      ,
      • Kiser P.D.
      • Kolesnikov A.V.
      • Kiser J.Z.
      • Dong Z.
      • Chaurasia B.
      • Wang L.
      • Summers S.A.
      • Hoang T.
      • Blackshaw S.
      • Peachey N.S.
      • et al.
      Conditional deletion of Des1 in the mouse retina does not impair the visual cycle in cones.
      ). An intraretinal cone-specific visual cycle would likely involve Muller glial cells (
      • Kolesnikov A.V.
      • Tang P.H.
      • Parker R.O.
      • Crouch R.K.
      • Kefalov V.J.
      The mammalian cone visual cycle promotes rapid M/L-cone pigment regeneration independently of the interphotoreceptor retinoid-binding protein.
      ).
      Questions have arisen as to whether the high visual chromophore demands of cone photoreceptor cells confer greater susceptibility to bisretinoid formation. While bisretinoid fluorophores are the source of short-wavelength fundus autofluorescence, the signal from foveal and perifoveal cones in humans is attenuated by macular pigment and by the increased optical density of melanin. Also complicating this issue is the possibility of greater photocleavage and loss of bisretinoid in central cone-rich retina due to higher light exposures (
      • Sparrow J.R.
      • Duncker T.
      Fundus autofluorescence and RPE lipofuscin in age-related macular degeneration.
      ). Thus, the question of cone versus rod bisretinoid formation cannot be addressed by noninvasive quantitative fundus autofluorescence imaging (
      • Duncker T.
      • Tsang S.H.
      • Lee W.
      • Zernant J.
      • Allikmets R.
      • Delori F.C.
      • Sparrow J.R.
      Quantitative fundus autofluorescence distinguishes ABCA4-associated and non-ABCA4-associated bull’s-eye maculopathy.
      ) in human subjects.
      Nevertheless, some insight was provided by studies of mice deficient in the Nrl transcription factor (Nrl−/−) (
      • Conley S.M.
      • Cai X.
      • Makkia R.
      • Wu Y.
      • Sparrow J.R.
      • Naash M.I.
      Increased cone sensitivity to ABCA4 deficiency provides insight into macular vision loss in Stargardt’s dystrophy.
      ). In these mice, rods are largely replaced by cone-like photoreceptor cells. These cells express cone proteins and exhibit cone electrophysiological and structural features. With placement of Nrl−/− mice on a background of Abca4−/−, bisretinoid levels, as expected, were higher in Abca4−/− than in Abca4+/+ mice. In addition, analysis by HPLC revealed that the cone-dominant eye generated more bisretinoid than the rod-dominant eye. Indeed, when bisretinoid levels were normalized to 11-cis-retinal quantity, bisretinoid (per eye) was found to be 6.8-fold higher in cone dominant Abca4−/−/Nrl−/− mice than in the primarily rod retina in Abca4−/− mice.

      Bisretinoids and retinal disease

      The efficiency with which the retinoid cycle replenishes the 11-cis chromophore of cone and rod visual pigment determines all-trans-retinal flux and thus is tightly coupled to the formation of lipofuscin bisretinoids (
      • Kim S.R.
      • Fishkin N.
      • Kong J.
      • Nakanishi K.
      • Allikmets R.
      • Sparrow J.R.
      The Rpe65 Leu450Met variant is associated with reduced levels of the RPE lipofuscin fluorophores A2E and iso-A2E.
      ,
      • Radu R.A.
      • Mata N.L.
      • Nusinowitz S.
      • Liu X.
      • Sieving P.A.
      • Travis G.H.
      Treatment with isotretinoin inhibits lipofuscin and A2E accumulation in a mouse model of recessive Stargardt’s macular degeneration.
      ,
      • Radu R.A.
      • Han Y.
      • Bui T.V.
      • Nusinowitz S.
      • Bok D.
      • Lichter J.
      • Widder K.
      • Travis G.H.
      • Mata N.L.
      Reductions in serum vitamin A arrest accumulation of toxic retinal fluorophores: a potential therapy for treatment of lipofuscin-based retinal diseases.
      ,
      • Maiti P.
      • Kong J.
      • Kim S.R.
      • Sparrow J.R.
      • Allikmets R.
      • Rando R.R.
      Small molecule RPE65 antagonists limit the visual cycle and prevent lipofuscin formation.
      ,
      • Maeda A.
      • Maeda T.
      • Sun W.
      • Zhang H.
      • Baehr W.
      • Palczewski K.
      Redundant and unique roles of retinol dehydrogenases in the mouse retina.
      ). On the other hand, conditions that interfere with clearance of all-trans-retinal from the interior of outer segment discs result in accelerated formation of the bisretinoids. For instance, as a consequence of deficient ABCA4-facilitated removal of all-trans-retinal from the interior of outer segment discs (
      • Papermaster D.S.
      • Schneider B.G.
      • Zorn M.A.
      • Kraehenbuhl J.P.
      Immunocytochemical localization of a large intrinsic membrane protein to the incisures and margins of frog rod outer segment disks.
      ,
      • Sun H.
      • Nathans J.
      Stargardt’s ABCR is localized to the disc membrane of retinal rod outer segments.
      ,
      • Molday L.L.
      • Rabin A.R.
      • Molday R.S.
      ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy.
      ,
      • Sun H.
      • Nathans J.
      ABCR, the ATP-binding cassette transporter responsible for Stargardt macular dystrophy, is an efficient target of all-trans retinal-mediated photo-oxidative damage in vitro: implications for retinal disease.
      ), RPE lipofuscin is elevated in ABCA4-related disease and in Abca4−/− mice (
      • Weng J.
      • Mata N.L.
      • Azarian S.M.
      • Tzekov R.T.
      • Birch D.G.
      • Travis G.H.
      Insights into the function of Rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in abcr knockout mice.
      ,
      • Kim S.R.
      • Fishkin N.
      • Kong J.
      • Nakanishi K.
      • Allikmets R.
      • Sparrow J.R.
      The Rpe65 Leu450Met variant is associated with reduced levels of the RPE lipofuscin fluorophores A2E and iso-A2E.
      ,
      • Eagle R.C.
      • Lucier A.C.
      • Bernardino V.B.
      • Yanoff M.
      Retinal pigment epithelial abnormalities in fundus flavimaculatus.
      ,
      • Delori F.C.
      • Staurenghi G.
      • Arend O.
      • Dorey C.K.
      • Goger D.G.
      • Weiter J.J.
      In vivo measurement of lipofuscin in Stargardt’s disease–fundus flavimaculatus.
      ,
      • Lois N.
      • Holder G.E.
      • Bunce C.V.
      • Fitzke F.W.
      • Bird A.C.
      Phenotypic subtypes of Stargardt macular dystrophy-fundus flavimaculatus.
      ). Mutations in ABCA4 can present clinically as recessive Stargardt macular degeneration (
      • Allikmets R.
      • Singh N.
      • Sun H.
      • Shroyer N.F.
      • Hutchinson A.
      • Chidambaram A.
      • Gerrard B.
      • Baird L.
      • Stauffer D.
      • Peiffer A.
      • et al.
      A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy.
      ), as a form of cone-rod dystrophy, or as retinitis pigmentosa (
      • Shroyer N.F.
      • Lewis R.A.
      • Allikmets R.
      • Singh N.
      • Dean M.
      • Leppert M.
      • Lupski J.R.
      The rod photoreceptor ATP-binding cassette transporter gene, ABCR, and retinal disease: from monogenic to multifactorial.
      ). Knockout of the photoreceptor cell enzymes (all-trans-retinol dehydrogenases) responsible for detoxifying all-trans-retinal (by conversion to all-trans-retinol) also leads to enhanced formation of bisretinoid (
      • Chrispell J.D.
      • Feathers K.L.
      • Kane M.A.
      • Kim C.Y.
      • Brooks M.
      • Khanna R.
      • Kurth I.
      • Huebner C.A.
      • Gal A.
      • Mears A.J.
      • et al.
      Rdh12 activity and effects on retinoid processing in the murine retina.
      ,
      • Maeda A.
      • Maeda T.
      • Sun W.
      • Zhang H.
      • Baehr W.
      • Palczewski K.
      Redundant and unique roles of retinol dehydrogenases in the mouse retina.
      ). Dysfunctional photoreceptor cells may also generate bisretinoid at accelerated levels. For instance, in acute zonal occult outer retinopathy, short-wavelength autofluorescence is elevated at the border between diseased and nondiseased retina and, at this lesion border, SD-OCT imaging reveals a loss of photoreceptor cell integrity (
      • Boudreault K.A.
      • Schuerch K.
      • Zhao J.
      • Lee W.
      • Cabral T.
      • Yannuzzi L.A.
      • Tsang S.H.
      • Sparrow J.R.
      Quantitative autofluorescence intensities in acute zonal occult outer retinopathy vs healthy eyes.
      ). The hyperautofluorescent ring that marks the junction between functional and nonfunctional retina in many cases of retinitis pigmentosa, also signals accelerated bisretinoid formation (
      • Schuerch K.
      • Woods R.L.
      • Lee W.
      • Duncker T.
      • Delori F.C.
      • Allikmets R.
      • Tsang S.H.
      • Sparrow J.R.
      Quantifying fundus autofluorescence in patients with retinitis pigmentosa.
      ). And finally, in mice homozygous for a targeted deletion of the Mer receptor tyrosine kinase gene (merkd; Mertk−/−), it has been shown that the autofluorescence in the photoreceptor cell debris that accumulates in the subretinal space originates from bisretinoids that form in abundance in this model (
      • Zhao J.
      • Ueda K.
      • Riera M.
      • Kim H.J.
      • Sparrow J.R.
      Bisretinoids mediate light sensitivity resulting in photoreceptor cell degeneration in mice lacking the receptor tyrosine kinase Mer.
      ). Moreover, these photoreactive compounds are linked to the mechanisms by which light potentiates photoreceptor cell degeneration in these mice.

      Concluding remarks

      PE in the lipid bilayer of photoreceptor outer segments participates in the sequestration and reduction of vitamin A aldehyde by forming a reversible Schiff base linkage (NRPE) with this reactive molecule. The formation of NRPE likely serves to both retain vitamin A within the visual cycle and limit the reactivity of this acutely toxic aldehyde. Under some conditions, NRPE reacts irreversibly with a second molecule of vitamin A aldehyde, thus forming bisretinoid fluorophore. RPE phagocytosis serves to transfer bisretinoid-burdened outer segment discs to the RPE but as has been demonstrated in mice deficient in the receptor tyrosine kinase Mer (
      • Zhao J.
      • Ueda K.
      • Riera M.
      • Kim H.J.
      • Sparrow J.R.
      Bisretinoids mediate light sensitivity resulting in photoreceptor cell degeneration in mice lacking the receptor tyrosine kinase Mer.
      ), phagocytosis is not required for the formation of these fluorophores. When known isomers (e.g., cis isomers of A2E) and photooxidized forms of bisretinoids are included together with biosynthetic intermediates such as A2PE (Fig. 2, compound 1) and dihydropyridinium-A2PE, at least 27 bisretinoid fluorophores can be identified chromatographically and by MS. The accumulation of bisretinoid by RPE is well-known to have adverse consequences for the cells and is implicated in disease processes.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Author Contribution

      H.J.K. and J.R.S. wrote, reviewed, and revised the paper.

      Funding and additional information

      This research was supported by National Institutes of Health Grants EY012951, EY024091, and EY028131 (J.R.S.) and unrestricted funds from Research to Prevent Blindness to the Department of Ophthalmology, Columbia University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      References

        • Niu S.L.
        • Mitchell D.C.
        Effect of packing density on rhodopsin stability and function in polyunsaturated membranes.
        Biophys. J. 2005; 89: 1833-1840
        • Tyminski P.N.
        • Latimer L.H.
        • O’Brien D.F.
        Reconstitution of rhodopsin and the cGMP cascade in polymerized bilayer membranes.
        Biochemistry. 1988; 27: 2696-2705
        • Fliesler S.J.
        • Anderson R.E.
        Chemistry and metabolism of lipids in the vertebrate retina.
        Prog. Lipid Res. 1983; 22: 79-131
        • Sparrow J.R.
        • Wu Y.
        • Kim C.Y.
        • Zhou J.
        Phospholipid meets all-trans-retinal: the making of RPE bisretinoids.
        J. Lipid Res. 2010; 51: 247-261
        • Lamb T.D.
        • Pugh E.N.
        Dark adaptation and the retinoid cycle of vision.
        Prog. Retin. Eye Res. 2004; 23: 307-380
        • Rattner A.
        • Smallwood P.M.
        • Nathans J.
        Identification and characterization of all-trans- retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol.
        J. Biol. Chem. 2000; 275: 11034-11043
        • Maeda A.
        • Golczak M.
        • Maeda T.
        • Palczewski K.
        Limited roles of Rdh8, Rdh12, and Abca4 in all-trans-retinal clearance in mouse retina.
        Invest. Ophthalmol. Vis. Sci. 2009; 50: 5435-5443
        • Chrispell J.D.
        • Feathers K.L.
        • Kane M.A.
        • Kim C.Y.
        • Brooks M.
        • Khanna R.
        • Kurth I.
        • Huebner C.A.
        • Gal A.
        • Mears A.J.
        • et al.
        Rdh12 activity and effects on retinoid processing in the murine retina.
        J. Biol. Chem. 2009; 284: 21468-21477
        • Molday R.S.
        ATP-binding cassette transporter ABCA4: molecular properties and role in vision and macular degeneration.
        J. Bioenerg. Biomembr. 2007; 39: 507-517
        • Travis G.H.
        • Golczak M.
        • Moise A.R.
        • Palczewski K.
        Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents.
        Annu. Rev. Pharmacol. Toxicol. 2007; 47: 469-512
        • Anderson R.E.
        • Maude M.B.
        Phospholipids of bovine outer segments.
        Biochemistry. 1970; 9: 3624-3628
        • Papermaster D.S.
        • Schneider B.G.
        • Zorn M.A.
        • Kraehenbuhl J.P.
        Immunocytochemical localization of a large intrinsic membrane protein to the incisures and margins of frog rod outer segment disks.
        J. Cell Biol. 1978; 78: 415-425
        • Illing M.
        • Molday L.L.
        • Molday R.S.
        The 220-kDa rim protein of retinal rod outer segments is a member of the ABC transporter superfamily.
        J. Biol. Chem. 1997; 272: 10303-10310
        • Sun H.
        • Molday R.S.
        • Nathans J.
        Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease.
        J. Biol. Chem. 1999; 274: 8269-8281
        • Sun H.
        • Nathans J.
        Stargardt’s ABCR is localized to the disc membrane of retinal rod outer segments.
        Nat. Genet. 1997; 17: 15-16
        • Quazi 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. USA. 2014; 111: 5024-5029
        • Molday L.L.
        • Rabin A.R.
        • Molday R.S.
        ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy.
        Nat. Genet. 2000; 25: 257-258
        • Ahn J.
        • Wong J.T.
        • Molday R.S.
        The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy.
        J. Biol. Chem. 2000; 275: 20399-20405
        • Katz M.L.
        • Drea C.M.
        • Eldred G.E.
        • Hess H.H.
        • Robison Jr., W.G.
        Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium.
        Exp. Eye Res. 1986; 43: 561-573
        • Katz M.L.
        • Eldred G.E.
        • Robison Jr., W.G.
        Lipofuscin autofluorescence: evidence for vitamin A involvement in the retina.
        Mech. Ageing Dev. 1987; 39: 81-90
        • Sparrow J.R.
        RPE lipofuscin: formation, properties and relevance to retinal degeneration.
        in: Tombran-Tink J. Barnstable C.J. Retinal Degenerations: Biology, Diagnostics and Therapeutics. Humana Press, Totowa, NJ2007: 213-236
        • Weng J.
        • Mata N.L.
        • Azarian S.M.
        • Tzekov R.T.
        • Birch D.G.
        • Travis G.H.
        Insights into the function of Rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in abcr knockout mice.
        Cell. 1999; 98: 13-23
        • Kim S.R.
        • Fishkin N.
        • Kong J.
        • Nakanishi K.
        • Allikmets R.
        • Sparrow J.R.
        The Rpe65 Leu450Met variant is associated with reduced levels of the RPE lipofuscin fluorophores A2E and iso-A2E.
        Proc. Natl. Acad. Sci. USA. 2004; 101: 11668-11672
        • Maeda A.
        • Maeda T.
        • Golczak M.
        • Palczewski K.
        Retinopathy in mice induced by disrupted all-trans-retinal clearance.
        J. Biol. Chem. 2008; 283: 26684-26693
        • Sparrow J.R.
        • Gregory-Roberts E.
        • Yamamoto K.
        • Blonska A.
        • Ghosh S.K.
        • Ueda K.
        • Zhou J.
        The bisretinoids of retinal pigment epithelium.
        Prog. Retin. Eye Res. 2012; 31: 121-135
        • Pawar A.S.
        • Qtaishat N.M.
        • Little D.M.
        • Pepperberg D.R.
        Recovery of rod photoresponses in ABCR-deficient mice.
        Invest. Ophthalmol. Vis. Sci. 2008; 49: 2743-2755
        • Tanito M.
        • Brush R.S.
        • Elliott M.H.
        • Wicker L.D.
        • Henry K.R.
        • Anderson R.E.
        High levels of retinal membrane docosahexaenoic acid increase susceptibility to stress-induced degeneration.
        J. Lipid Res. 2009; 50: 807-819
        • Kim S.R.
        • Jang Y.P.
        • Jockusch S.
        • Fishkin N.E.
        • Turro N.J.
        • Sparrow J.R.
        The all- trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.
        Proc. Natl. Acad. Sci. USA. 2007; 104: 19273-19278
        • Yamamoto K.
        • Yoon K.D.
        • Ueda K.
        • Hashimoto M.
        • Sparrow J.R.
        A novel bisretinoid of retina is an adduct on glycerophosphoethanolamine.
        Invest. Ophthalmol. Vis. Sci. 2011; 52: 9084-9090
        • Wu Y.
        • Fishkin N.E.
        • Pande A.
        • Pande J.
        • Sparrow J.R.
        Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease.
        J. Biol. Chem. 2009; 284: 20155-20166
        • Greenberg J.P.
        • Duncker T.
        • Woods R.L.
        • Smith R.T.
        • Sparrow J.R.
        • Delori F.C.
        Quantitative fundus autofluorescence in healthy eyes.
        Invest. Ophthalmol. Vis. Sci. 2013; 54: 5684-5693
        • Sakai N.
        • Decatur J.
        • Nakanishi K.
        • Eldred G.E.
        Ocular age pigment “A2E”: an unprecedented pyridinium bisretinoid.
        J. Am. Chem. Soc. 1996; 118: 1559-1560
        • Ren R.F.
        • Sakai N.
        • Nakanishi K.
        Total synthesis of the ocular age pigment A2E: a convergent pathway.
        J. Am. Chem. Soc. 1997; 119: 3619-3620
        • Parish C.A.
        • Hashimoto M.
        • Nakanishi K.
        • Dillon J.
        • Sparrow J.R.
        Isolation and one- step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium.
        Proc. Natl. Acad. Sci. USA. 1998; 95: 14609-14613
        • Ablonczy Z.
        • Smith N.
        • Anderson D.M.
        • Grey A.C.
        • Spraggins J.
        • Koutalos Y.
        • Schey K.L.
        • Crouch R.K.
        The utilization of fluorescence to identify the components of lipofuscin by imaging mass spectrometry.
        Proteomics. 2014; 14: 936-944
        • Ablonczy Z.
        • Higbee D.
        • Anderson D.M.
        • Dahrouj M.
        • Grey A.C.
        • Gutierrez D.
        • Koutalos Y.
        • Schey K.L.
        • Hanneken A.
        • Crouch R.K.
        Lack of correlation between the spatial distribution of A2E and lipofuscin fluorescence in the human retinal pigment epithelium.
        Invest. Ophthalmol. Vis. Sci. 2013; 54: 5535-5542
        • Liu J.
        • Itagaki Y.
        • Ben-Shabat S.
        • Nakanishi K.
        • Sparrow J.R.
        The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane.
        J. Biol. Chem. 2000; 275: 29354-29360
        • Fishkin N.E.
        • Sparrow J.R.
        • Allikmets R.
        • Nakanishi K.
        Isolation and characterization of a retinal pigment epithelial cell fluorophore: an all-trans-retinal dimer conjugate.
        Proc. Natl. Acad. Sci. USA. 2005; 102: 7091-7096
        • Ben-Shabat S.
        • Parish C.A.
        • Vollmer H.R.
        • Itagaki Y.
        • Fishkin N.
        • Nakanishi K.
        • Sparrow J.R.
        Biosynthetic studies of A2E, a major fluorophore of retinal pigment epithelial lipofuscin.
        J. Biol. Chem. 2002; 277: 7183-7190
        • Sparrow J.R.
        • Parish C.A.
        • Hashimoto M.
        • Nakanishi K.
        A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture.
        Invest. Ophthalmol. Vis. Sci. 1999; 40: 2988-2995
        • Yamamoto K.
        • Zhou J.
        • Hunter J.J.
        • Williams D.R.
        • Sparrow J.R.
        Toward an understanding of bisretinoid autofluorescence bleaching and recovery.
        Invest. Ophthalmol. Vis. Sci. 2012; 53: 3536-3544
        • Kim H.J.
        • Sparrow J.R.
        Novel bisretinoids of human retina are lyso alkyl ether glycerophosphoethanolamine-bearing A2PE species.
        J. Lipid Res. 2018; 59: 1620-1629
        • Kobayashi M.
        • Kanfer J.N.
        Solubilization and purification of rat tissue phospholipase D.
        Methods Enzymol. 1991; 197: 575-583
        • Salvador G.A.
        • Giusto N.M.
        Characterization of phospholipase D activity in bovine photoreceptor membranes.
        Lipids. 1998; 33: 853-860
        • Sciorra V.A.
        • Hammond S.M.
        • Morris A.J.
        Potent direct inhibition of mammalian phospholipase D isoenzymes by calphostin-c.
        Biochemistry. 2001; 40: 2640-2646
        • Ben-Shabat S.
        • Itagaki Y.
        • Jockusch S.
        • Sparrow J.R.
        • Turro N.J.
        • Nakanishi K.
        Formation of a nona-oxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement.
        Angew. Chem. Int. Ed. Engl. 2002; 41: 814-817
        • Sparrow J.R.
        • Kim S.R.
        • Cuervo A.M.
        • Bandhyopadhyayand U.
        A2E, a pigment of RPE lipofuscin is generated from the precursor A2PE by a lysosomal enzyme activity.
        Adv. Exp. Med. Biol. 2008; 613: 393-398
        • Brunk U.T.
        • Terman A.
        Lipofuscin: mechanisms of age-related accumulation and influence on cell function.
        Free Radic. Biol. Med. 2002; 33: 611-619
        • Johnson T.B.
        • Cain J.T.
        • White K.A.
        • Ramirez-Montealegre D.
        • Pearce D.A.
        • Weimer J.M.
        Therapeutic landscape for Batten disease: current treatments and future prospects.
        Nat. Rev. Neurol. 2019; 15: 161-178
        • Ng K.P.
        • Gugiu B.G.
        • Renganathan K.
        • Davies M.W.
        • Gu X.
        • Crabb J.S.
        • Kim S.R.
        • Rozanowska M.B.
        • Bonilha V.L.
        • Rayborn M.E.
        • et al.
        Retinal pigment epithelium lipofuscin proteomics.
        Mol. Cell. Proteomics. 2008; 7: 1397-1405
        • Schutt F.
        • Bergmann M.
        • Holz F.G.
        • Kopitz J.
        Proteins modified by malondialdehyde, 4- hydroxynonenal or advanced glycation end products in lipofuscin of human retinal pigment epithelium.
        Invest. Ophthalmol. Vis. Sci. 2003; 44: 3663-3668
        • Rein D.
        • Tappel A.L.
        Fluorescent lipid oxidation products and heme spectra index antioxidant efficacy in kidney tissue of hamsters.
        Free Radic. Biol. Med. 1998; 24: 1278-1284
        • Eldred G.E.
        • Katz M.L.
        The lipid peroxidation theory of lipofuscinogenesis cannot yet be confirmed.
        Free Radic. Biol. Med. 1991; 10: 445-447
        • Eldred G.E.
        • Miller G.V.
        • Stark W.S.
        • Feeney-Burns L.
        Lipofuscin: resolution of discrepant fluorescence data.
        Science. 1982; 216: 757-759
        • Liu J.
        • Lu W.
        • Reigada D.
        • Nguyen J.
        • Laties A.M.
        • Mitchell C.H.
        Restoration of lysosomal pH in RPE cells from cultured human and ABCA4(−/−) mice: pharmacologic approaches and functional recovery.
        Invest. Ophthalmol. Vis. Sci. 2008; 49: 772-780
        • Eldred G.E.
        • Lasky M.R.
        Retinal age pigments generated by self-assembling lysosomotropic detergents.
        Nature. 1993; 361: 724-726
        • Brady R.O.
        Emerging strategies for the treatment of hereditary metabolic storage disorders.
        Rejuvenation Res. 2006; 9: 237-244
        • Boulton M.E.
        Lipofuscin of the retinal pigment epithelium.
        in: Lois N. Forrester J.V. Fundus Autofluorescence. Wolters Klumer/Lippincott Williams and Wilkins, Philadelphia. PA2009: 14-26
        • Schutt F.
        • Ueberle B.
        • Schnolzer M.
        • Holz F.G.
        • Kopitz J.
        Proteome analysis of lipofuscin in human retinal pigment epithelial cells.
        FEBS Lett. 2002; 528: 217-221
        • Warburton S.
        • Southwick K.
        • Hardman R.M.
        • Secrest A.M.
        • Grow R.K.
        • Xin H.
        • Woolley A.T.
        • Burton G.F.
        • Thulin C.D.
        Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin.
        Mol. Vis. 2005; 11: 1122-1134
        • Kolko M.
        • Vohra R.
        • Westlund van der Burght B.
        • Poulsen K.
        • Nissen M.H.
        Calcium- independent phospholipase A(2), group VIA, is critical for RPE cell survival.
        Mol. Vis. 2014; 20: 511-521
        • Garcia J.M.B.B.
        • Isaac D.L.C.
        • Sardeiro T.
        • Aquino E.
        • Avila M.
        Benign familial fleck retina: multimodal imaging including optical coherence tomography angiography.
        Arq. Bras. Oftalmol. 2017; 80: 321-323
        • Neriyanuri S.
        • Rao C.
        • Raman R.
        Structural and functional characterization of benign fleck retina using multimodal imaging.
        Retin. Cases Brief Rep. 2017; 11: 186-190
        • Sparrow J.R.
        • Wu Y.
        • Nagasaki T.
        • Yoon K.D.
        • Yamamoto K.
        • Zhou J.
        Fundus autofluorescence and the bisretinoids of retina.
        Photochem. Photobiol. Sci. 2010; 9: 1480-1489
        • Delori F.C.
        Spectrophotometer for noninvasive measurement of intrinsic fluorescence and reflectance of the ocular fundus.
        Appl. Opt. 1994; 33: 7439-7452
        • Morgan J.I.
        • Hunter J.J.
        • Masella B.
        • Wolfe R.
        • Gray D.C.
        • Merigan W.H.
        • Delori F.C.
        • Williams D.R.
        Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium.
        Invest. Ophthalmol. Vis. Sci. 2008; 49: 3715-3729
        • von Rückmann A.
        • Fitzke F.W.
        • Bird A.C.
        Distribution of fundus autofluorescence with a scanning laser ophthalmoscope.
        Br. J. Ophthalmol. 1995; 79: 407-412
        • von Rückmann A.
        • Fitzke F.W.
        • Bird A.C.
        In vivo fundus autofluorescence in macular dystrophies.
        Arch. Ophthalmol. 1997; 115: 609-615
        • Delori F.C.
        • Dorey C.K.
        • Staurenghi G.
        • Arend O.
        • Goger D.G.
        • Weiter J.J.
        In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics.
        Invest. Ophthalmol. Vis. Sci. 1995; 36: 718-729
        • Delori F.C.
        • Goger D.G.
        • Dorey C.K.
        Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects.
        Invest. Ophthalmol. Vis. Sci. 2001; 42: 1855-1866
        • Boulton M.
        • Docchio F.
        • Dayhaw-Barker P.
        • Ramponi R.
        • Cubeddu R.
        Age-related changes in the morphology, absorption and fluorescence of melanosomes and lipofuscin granules of the retinal pigment epithelium.
        Vision Res. 1990; 30: 1291-1303
        • Feeney-Burns L.
        • Eldred G.E.
        The fate of the phagosome: conversion to ‘age pigment’ and impact in human retinal pigment epithelium.
        Trans. Ophthalmol. Soc. U. K. 1983; 103: 416-421
        • Kim S.R.
        • Jang Y.
        • Sparrow J.R.
        Photooxidation of RPE Lipofuscin bisretinoids enhanced fluorescence intensity.
        Vision Res. 2010; 50: 729-736
        • Rózanowska M.
        • Jarvis-Evans J.
        • Korytowski W.
        • Boulton M.E.
        • Burke J.M.
        • Sarna T.
        Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species.
        J. Biol. Chem. 1995; 270: 18825-18830
        • Gaillard E.R.
        • Atherton S.J.
        • Eldred G.
        • Dillon J.
        Photophysical studies on human retinal lipofuscin.
        Photochem. Photobiol. 1995; 61: 448-453
        • Jang Y.P.
        • Matsuda H.
        • Itagaki Y.
        • Nakanishi K.
        • Sparrow J.R.
        Characterization of peroxy-A2E and furan-A2E photooxidation products and detection in human and mouse retinal pigment epithelial cells lipofuscin.
        J. Biol. Chem. 2005; 280: 39732-39739
        • Wu Y.
        • Yanase E.
        • Feng X.
        • Siegel M.M.
        • Sparrow J.R.
        Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration.
        Proc. Natl. Acad. Sci. USA. 2010; 107: 7275-7280
        • Yoon K.D.
        • Yamamoto K.
        • Ueda K.
        • Zhou J.
        • Sparrow J.R.
        A novel source of methylglyoxal and glyoxal in retina: implications for age-related macular degeneration.
        PLoS One. 2012; 7: e41309
        • Zhou J.
        • Cai B.
        • Jang Y.P.
        • Pachydaki S.
        • Schmidt A.M.
        • Sparrow J.R.
        Mechanisms for the induction of HNE- MDA- and AGE-adducts, RAGE and VEGF in retinal pigment epithelial cells.
        Exp. Eye Res. 2005; 80: 567-580
        • Zhou J.
        • Ueda K.
        • Zhao J.
        • Sparrow J.R.
        Correlations between photodegradation of bisretinoid constituents of retina and dicarbonyl-adduct deposition.
        J. Biol. Chem. 2015; 290: 27215-27227
        • Fernandes A.F.
        • Zhou J.
        • Zhang X.
        • Bian Q.
        • Sparrow J.R.
        • Taylor A.
        • Pereira P.
        • Shang F.
        Oxidative inactivation of the proteasome in retinal pigment epithelial cells. A potential link between oxidative stress and up-regulation of interleukin-8.
        J. Biol. Chem. 2008; 283: 20745-20753
        • Zhou J.
        • Jang Y.P.
        • Kim S.R.
        • Sparrow J.R.
        Complement activation by photooxidation products of A2E, a lipofuscin constituent of the retinal pigment epithelium.
        Proc. Natl. Acad. Sci. USA. 2006; 103: 16182-16187
        • Zhou J.
        • Kim S.R.
        • Westlund B.S.
        • Sparrow J.R.
        Complement activation by bisretinoid constituents of RPE lipofuscin.
        Invest. Ophthalmol. Vis. Sci. 2009; 50: 1392-1399
        • Handa J.T.
        • Verzijl N.
        • Matsunaga H.
        • Aotaki-Keen A.
        • Lutty G.A.
        • Koppele J.M.
        • Miyata T.
        • Hjelmeland L.M.
        Increase in advanced glycation end product pentosidine in Bruch’s membrane with age.
        Invest. Ophthalmol. Vis. Sci. 1999; 40: 775-779
        • Crabb J.W.
        • Miyagi M.
        • Gu X.
        • Shadrach K.
        • West K.A.
        • Sakaguchi H.
        • Kamei M.
        • Hasan A.
        • Yan L.
        • Raybourn M.E.
        • et al.
        Drusen proteome analysis: an approach to the etiology of age-related macular degeneration.
        Proc. Natl. Acad. Sci. USA. 2002; 99: 14682-14687
        • Radu R.A.
        • Hu J.
        • Yuan Q.
        • Welch D.L.
        • Makshanoff J.
        • Lloyd M.
        • McMullen S.
        • Travis G.H.
        • Bok D.
        Complement system dysregulation and inflammation in the retinal pigment epithelium of a mouse model for Stargardt macular degeneration.
        J. Biol. Chem. 2011; 286: 18593-18601
        • Wu L.
        • Nagasaki T.
        • Sparrow J.R.
        Photoreceptor cell degeneration in Abcr−/− mice.
        Adv. Exp. Med. Biol. 2010; 664: 533-539
        • Radu R.A.
        • Yuan Q.
        • Hu J.
        • Peng J.H.
        • Lloyd M.
        • Nusinowitz S.
        • Bok D.
        • Travis G.H.
        Accelerated accumulation of lipofuscin pigments in the RPE of a mouse model for ABCA4- mediated retinal dystrophies following vitamin A supplementation.
        Invest. Ophthalmol. Vis. Sci. 2008; 49: 3821-3829
        • Wu L.
        • Ueda K.
        • Nagasaki T.
        • Sparrow J.R.
        Light damage in Abca4 and Rpe65rd12 mice.
        Invest. Ophthalmol. Vis. Sci. 2014; 55: 1910-1918
        • Ueda K.
        • Kim H.J.
        • Zhao J.
        • Song Y.
        • Dunaief J.L.
        • Sparrow J.R.
        Iron promotes oxidative cell death caused by bisretinoids of retina.
        Proc. Natl. Acad. Sci. USA. 2018; 115: 4963-4968
        • Kaylor J.J.
        • Xu T.
        • Ingram N.T.
        • Tsan A.
        • Hakobyan H.
        • Fain G.L.
        • Travis G.H.
        Blue light regenerates functional visual pigments in mammals through a retinyl-phospholipid intermediate.
        Nat. Commun. 2017; 8: 16
        • Zhang J.
        • Choi E.H.
        • Tworak A.
        • Salom D.
        • Leinonen H.
        • Sander C.L.
        • Hoang T.V.
        • Handa J.T.
        • Blackshaw S.
        • Palczewska G.
        • et al.
        Photic generation of 11-cis-retinal in bovine retinal pigment epithelium.
        J. Biol. Chem. 2019; 294: 19137-19154
        • Kaylor J.J.
        • Yuan Q.
        • Cook J.
        • Sarfare S.
        • Makshanoff J.
        • Miu A.
        • Kim A.
        • Kim P.
        • Habib S.
        • Roybal C.N.
        • et al.
        Identification of DES1 as a vitamin A isomerase in Muller glial cells of the retina.
        Nat. Chem. Biol. 2013; 9: 30-36
        • Kiser P.D.
        • Kolesnikov A.V.
        • Kiser J.Z.
        • Dong Z.
        • Chaurasia B.
        • Wang L.
        • Summers S.A.
        • Hoang T.
        • Blackshaw S.
        • Peachey N.S.
        • et al.
        Conditional deletion of Des1 in the mouse retina does not impair the visual cycle in cones.
        FASEB J. 2019; 33: 5782-5792
        • Kolesnikov A.V.
        • Tang P.H.
        • Parker R.O.
        • Crouch R.K.
        • Kefalov V.J.
        The mammalian cone visual cycle promotes rapid M/L-cone pigment regeneration independently of the interphotoreceptor retinoid-binding protein.
        J. Neurosci. 2011; 31: 7900-7909
        • Sparrow J.R.
        • Duncker T.
        Fundus autofluorescence and RPE lipofuscin in age-related macular degeneration.
        J. Clin. Med. 2014; 3: 1302-1321
        • Duncker T.
        • Tsang S.H.
        • Lee W.
        • Zernant J.
        • Allikmets R.
        • Delori F.C.
        • Sparrow J.R.
        Quantitative fundus autofluorescence distinguishes ABCA4-associated and non-ABCA4-associated bull’s-eye maculopathy.
        Ophthalmology. 2015; 122: 345-355
        • Conley S.M.
        • Cai X.
        • Makkia R.
        • Wu Y.
        • Sparrow J.R.
        • Naash M.I.
        Increased cone sensitivity to ABCA4 deficiency provides insight into macular vision loss in Stargardt’s dystrophy.
        Biochim. Biophys. Acta. 2012; 1822: 1169-1179
        • Radu R.A.
        • Mata N.L.
        • Nusinowitz S.
        • Liu X.
        • Sieving P.A.
        • Travis G.H.
        Treatment with isotretinoin inhibits lipofuscin and A2E accumulation in a mouse model of recessive Stargardt’s macular degeneration.
        Proc. Natl. Acad. Sci. USA. 2003; 100: 4742-4747
        • Radu R.A.
        • Han Y.
        • Bui T.V.
        • Nusinowitz S.
        • Bok D.
        • Lichter J.
        • Widder K.
        • Travis G.H.
        • Mata N.L.
        Reductions in serum vitamin A arrest accumulation of toxic retinal fluorophores: a potential therapy for treatment of lipofuscin-based retinal diseases.
        Invest. Ophthalmol. Vis. Sci. 2005; 46: 4393-4401
        • Maiti P.
        • Kong J.
        • Kim S.R.
        • Sparrow J.R.
        • Allikmets R.
        • Rando R.R.
        Small molecule RPE65 antagonists limit the visual cycle and prevent lipofuscin formation.
        Biochemistry. 2006; 45: 852-860
        • Maeda A.
        • Maeda T.
        • Sun W.
        • Zhang H.
        • Baehr W.
        • Palczewski K.
        Redundant and unique roles of retinol dehydrogenases in the mouse retina.
        Proc. Natl. Acad. Sci. USA. 2007; 104: 19565-19570
        • Sun H.
        • Nathans J.
        ABCR, the ATP-binding cassette transporter responsible for Stargardt macular dystrophy, is an efficient target of all-trans retinal-mediated photo-oxidative damage in vitro: implications for retinal disease.
        J. Biol. Chem. 2001; 276: 11766-11774
        • Eagle R.C.
        • Lucier A.C.
        • Bernardino V.B.
        • Yanoff M.
        Retinal pigment epithelial abnormalities in fundus flavimaculatus.
        Ophthalmology. 1980; 87: 1189-1200
        • Delori F.C.
        • Staurenghi G.
        • Arend O.
        • Dorey C.K.
        • Goger D.G.
        • Weiter J.J.
        In vivo measurement of lipofuscin in Stargardt’s disease–fundus flavimaculatus.
        Invest. Ophthalmol. Vis. Sci. 1995; 36: 2327-2331
        • Lois N.
        • Holder G.E.
        • Bunce C.V.
        • Fitzke F.W.
        • Bird A.C.
        Phenotypic subtypes of Stargardt macular dystrophy-fundus flavimaculatus.
        Arch. Ophthalmol. 2001; 119: 359-369
        • Allikmets R.
        • Singh N.
        • Sun H.
        • Shroyer N.F.
        • Hutchinson A.
        • Chidambaram A.
        • Gerrard B.
        • Baird L.
        • Stauffer D.
        • Peiffer A.
        • et al.
        A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy.
        Nat. Genet. 1997; 15: 236-246
        • Shroyer N.F.
        • Lewis R.A.
        • Allikmets R.
        • Singh N.
        • Dean M.
        • Leppert M.
        • Lupski J.R.
        The rod photoreceptor ATP-binding cassette transporter gene, ABCR, and retinal disease: from monogenic to multifactorial.
        Vision Res. 1999; 39: 2537-2544
        • Boudreault K.A.
        • Schuerch K.
        • Zhao J.
        • Lee W.
        • Cabral T.
        • Yannuzzi L.A.
        • Tsang S.H.
        • Sparrow J.R.
        Quantitative autofluorescence intensities in acute zonal occult outer retinopathy vs healthy eyes.
        JAMA Ophthalmol. 2017; 135: 1330-1338
        • Schuerch K.
        • Woods R.L.
        • Lee W.
        • Duncker T.
        • Delori F.C.
        • Allikmets R.
        • Tsang S.H.
        • Sparrow J.R.
        Quantifying fundus autofluorescence in patients with retinitis pigmentosa.
        Invest. Ophthalmol. Vis. Sci. 2017; 58: 1843-1855
        • Zhao J.
        • Ueda K.
        • Riera M.
        • Kim H.J.
        • Sparrow J.R.
        Bisretinoids mediate light sensitivity resulting in photoreceptor cell degeneration in mice lacking the receptor tyrosine kinase Mer.
        J. Biol. Chem. 2018; 293: 19400-19410

      Linked Article

      • The emerging roles of the macular pigment carotenoids throughout the lifespan and in prenatal supplementation
        Journal of Lipid ResearchVol. 62
        • Preview
          Since the publication of the Age-Related Eye Disease Study 2 (AREDS2) in 2013, the macular pigment carotenoids lutein (L) and zeaxanthin (Z) have become well known to both the eye care community and the public. It is a fascinating aspect of evolution that primates have repurposed photoprotective pigments and binding proteins from plants and insects to protect and enhance visual acuity. Moreover, utilization of these plant-derived nutrients has been widely embraced for preventing vision loss from age-related macular degeneration.
        • Full-Text
        • PDF
        Open Access
      • Lipid metabolism dysregulation in diabetic retinopathy
        Journal of Lipid ResearchVol. 62
        • Preview
          Lipid metabolic abnormalities have emerged as potential risk factors for the development and progression of diabetic complications, including diabetic retinopathy (DR). This review article provides an overview of the results of clinical trials evaluating the potential benefits of lipid-lowering drugs, such as fibrates, omega-3 fatty acids, and statins, for the prevention and treatment of DR. Although several clinical trials demonstrated that treatment with fibrates leads to improvement of DR, there is a dissociation between the protective effects of fibrates in the retina, and the intended blood lipid classes, including plasma triglycerides, total cholesterol, or HDL:LDL cholesterol ratio.
        • Full-Text
        • PDF
        Open Access
      • Fatty acid oxidation and photoreceptor metabolic needs
        Journal of Lipid ResearchVol. 62
        • Preview
          Photoreceptors have high energy demands and a high density of mitochondria that produce ATP through oxidative phosphorylation (OXPHOS) of fuel substrates. Although glucose is the major fuel for CNS brain neurons, in photoreceptors (also CNS), most glucose is not metabolized through OXPHOS but is instead metabolized into lactate by aerobic glycolysis. The major fuel sources for photoreceptor mitochondria remained unclear for almost six decades. Similar to other tissues (like heart and skeletal muscle) with high metabolic rates, photoreceptors were recently found to metabolize fatty acids (palmitate) through OXPHOS.
        • Full-Text
        • PDF
        Open Access
      • Signaling roles of phosphoinositides in the retina
        Journal of Lipid ResearchVol. 62
        • Preview
          The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (also known as phosphatidylinositol phosphates or PIPs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of phosphoinositide kinases and phosphoinositide phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule, phosphatidylinositol.
        • Full-Text
        • PDF
        Open Access
      • Introduction to the Thematic Review Series: Seeing 2020: lipids and lipid-soluble molecules in the eye
        Journal of Lipid ResearchVol. 62
        • Preview
          In 2010, this journal published a series of review articles in a Thematic Issue entitled “Lipids and Lipid Metabolism in the Eye.” Over the ensuing decade, a number of significant advances have been made that are pertinent to this broad topic, which prompted us to launch a follow-up Thematic Issue to present updates on several of the topics reviewed in that prior issue as well as to expand into new areas that previously had not been addressed. In addition to considering the conventional classes of lipids (e.g., glycerophospholipids, sphingolipids, fatty acids, and sterols), we also wanted to address some key lipid-soluble molecules (e.g., retinoids, bisretinoids, and carotenoids) that play important physiological roles in ocular tissues.
        • Full-Text
        • PDF
        Open Access
      • Docosanoid signaling modulates corneal nerve regeneration: effect on tear secretion, wound healing, and neuropathic pain
        Journal of Lipid ResearchVol. 62
        • Preview
          The cornea is densely innervated, mainly by sensory nerves of the ophthalmic branch of the trigeminal ganglia (TG). These nerves are important to maintain corneal homeostasis, and nerve damage can lead to a decrease in wound healing, an increase in corneal ulceration and dry eye disease (DED), and neuropathic pain. Pathologies, such as diabetes, aging, viral and bacterial infection, as well as prolonged use of contact lenses and surgeries to correct vision can produce nerve damage. There are no effective therapies to alleviate DED (a multifunctional disease) and several clinical trials using ω-3 supplementation show unclear and sometimes negative results.
        • Full-Text
        • PDF
        Open Access
      • Lipid conformational order and the etiology of cataract and dry eye
        Journal of Lipid ResearchVol. 62
        • Preview
          Lens and tear film lipids are as unique as the systems they reside in. The major lipid of the human lens is dihydrosphingomylein, found in quantity only in the lens. The lens contains a cholesterol to phospholipid molar ratio as high as 10:1, more than anywhere else in the body. Lens lipids contribute to maintaining lens clarity, and alterations in lens lipid composition due to age are likely to contribute to cataract. Lens lipid composition reflects adaptations to the unique characteristics of the lens: no turnover of lens lipids or proteins; the lowest amount of oxygen of any tissue; and contains almost no intracellular organelles.
        • Full-Text
        • PDF
        Open Access
      • Retinoids in the visual cycle: role of the retinal G protein-coupled receptor
        Journal of Lipid ResearchVol. 62
        • Preview
          Driven by the energy of a photon, the visual pigments in rod and cone photoreceptor cells isomerize 11-cis-retinal to the all-trans configuration. This photochemical reaction initiates the signal transduction pathway that eventually leads to the transmission of a visual signal to the brain and leaves the opsins insensitive to further light stimulation. For the eye to restore light sensitivity, opsins require recharging with 11-cis-retinal. This trans-cis back conversion is achieved through a series of enzymatic reactions composing the retinoid (visual) cycle.
        • Full-Text
        • PDF
        Open Access
      • Very long chain fatty acid-containing lipids: a decade of novel insights from the study of ELOVL4
        Journal of Lipid ResearchVol. 62
        • Preview
          Lipids play essential roles in maintaining cell structure and function by modulating membrane fluidity and cell signaling. The fatty acid elongase-4 (ELOVL4) protein, expressed in retina, brain, Meibomian glands, skin, testes and sperm, is an essential enzyme that mediates tissue-specific biosynthesis of both VLC-PUFA and VLC-saturated fatty acids (VLC-SFA). These fatty acids play critical roles in maintaining retina and brain function, neuroprotection, skin permeability barrier maintenance, and sperm function, among other important cellular processes.
        • Full-Text
        • PDF
        Open Access
      • Sphingolipids as critical players in retinal physiology and pathology
        Journal of Lipid ResearchVol. 62
        • Preview
          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.
        • Full-Text
        • PDF
        Open Access