Novel Lipofuscin Bisretinoids Prominent in Human Retina and in a Model of Recessive Stargardt Disease

Abstract

Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve alltrans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-electrospray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridinephosphatidylethanolamine (A2-DHP-PE). The second pigment, A2-dihydropyridine-ethanolamine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at ϳ490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidylethanolamine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.Throughout the life of an individual, retinal pigment epithelial (RPE) 2 cells of the eye accumulate bisretinoid adducts that comprise the lipofuscin of these cells (1-3). The compounds form as a byproduct of light-mediated isomerization of the visual chromophore 11-cis-retinal. Accordingly, conditions that reduce the production of all-trans-retinal (atRAL) from 11-cisretinal photoisomerization, such as reduced serum vitamin A (4 -6), variants, or mutations in the visual cycle protein RPE65 (7-9) and inhibitors of RPE65 and 11-cis retinol dehydrogenase (10 -13), substantially reduce the formation of this material.Up to the present time, at least 17 constituents of RPE lipofuscin have been identified chromatographically and characterized structurally; added to these are biosynthetic intermediates such as N-retinylidene-phosphatidylethanolamine (NRPE), A2PE and dihydropyridinium-A2PE (see Fig. 1, A and B) (14 -19). The first RPE lipofuscin constituent to be described was A2E (see Fig. 1A, inset). The pyridinium bisretinoid (14 -16, 20, 21) structure of A2E (C 42 H 58 NO; molecular weight, 592) was confirmed by extensive nuclear magnetic resonance studies (14) and by total synthesis (22). A2E...

Enzymatic Degradation of A2E, a Retinal Pigment Epithelial Lipofuscin Bisretinoid

“…A human adult RPE cell line (ARPE-19; American Type Culture Collection, Manassas, VA) that is devoid of endogenous A2 1c was cultured in 35 mm cell culture dishes (Corning, NY) to confluence as previously reported 14,28 . The cells were allowed to accumulate synthesized A2E 1c for 3 weeks from a 10 µM concentration in culture media 9a that included 10% etal bovine serum (FBS; Invitrogen, Carlsbad, CA).…”

“…The cells were allowed to accumulate synthesized A2E 1c for 3 weeks from a 10 µM concentration in culture media 9a that included 10% etal bovine serum (FBS; Invitrogen, Carlsbad, CA). Incubation of ARPE-19 cells with A2E at higher concentrations perturbs cell membranes.…”

“…1 The nonenzymatic biosynthetic pathways leading to the formation of these bisretinoids have been elucidated, at least in part, and for all of these bisretinoids, biosynthesis begins in photoreceptor cells. 1a,2 When discarded photoreceptor outer segments are phagocytosed by RPE cells, the bisretinoid precursors are deposited into the lysosomal compartment of the cells. An enzyme activity that has been shown to be phospholipase-D and to be present in RPE cell lysosomes can remove phosphatidic acid from the bisretinoid precursors; as a result, A2E is released from A2PE and all- trans -retinal dimer-ethanolamine from all-trans-retinal dimer-phosphatidylethanolamine.…”

“…An enzyme activity that has been shown to be phospholipase-D and to be present in RPE cell lysosomes can remove phosphatidic acid from the bisretinoid precursors; as a result, A2E is released from A2PE and all- trans -retinal dimer-ethanolamine from all-trans-retinal dimer-phosphatidylethanolamine. 1b,2a,3 However, after this enzyme-mediated phosphate cleavage, little or no further enzyme degradation of the bisretinoid molecules occurs; thus, the pigment accumulates in the cell. We surmise that, since the structure of A2E is unprecedented, it may not be recognized by other lysosomal enzymes of the RPE and, thus, is largely indigestible.…”

Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration

“…Whereas these bisretinoids constitute a complex mixture, all appear to originate from reactions of all-trans-retinal and some have been identified, including A2E ( Fig. 1) and its isomers (8); A2-dihydropyridinephosphatidylethanolamine (A2-DHP-PE) and A2-dihydropyridine-ethanolamine (A2-DHP-E) (9); and all-trans-retinal dimer, all-trans-retinal dimer-phosphatidylethanolamine (all-transretinal dimer-PE), and all-trans-retinal dimer-ethanolamine (alltrans-retinal dimer-E) (10,11). Structural features common to all of these pigments are the alternating single and double carboncarbon bonds that originate from an aromatic head group, and extend along the two sidearms of the molecules and into the terminal β-ionone rings.…”