Isolation and characterization of a retinal pigment epithelial cell fluorophore: An all-trans -retinal dimer conjugate

Abstract

Several lines of investigation suggest that the nondegradable fluorophores that accumulate as lipofuscin in retinal pigment epithelium (RPE) cells contribute to the etiology of macular degeneration. Despite evidence that much of this fluorescent material may originate as inadvertent products of the retinoid cycle, the enzymatic pathway by which the 11-cis-retinal chromophore of rhodopsin is generated, the only fluorophores of the RPE to be characterized as yet have been A2E and its isomers. Here, we report the isolation and structural characterization of an additional RPE lipofuscin fluorophore that originates as a condensation product of two molecules of all-trans-retinal (ATR) dimer and forms a protonated Schiff base conjugate with phosphatidylethanolamine (PE), the latter conjugate (ATR dimer-PE) having UV-visible absorbance maxima at 285 and 506 nm. ATR dimer was found to form natively in bleached rod outer segments in vitro and when rod outer segments were incubated with ATR. HPLC analysis of eyecups that included RPE and isolated neural retina from Abcr ؊͞؊ mice and RPE isolated from human donor eyes revealed the presence of a pigment with the same UV-visible absorbance and retention time as synthetic ATR dimer-PE conjugate. Evidence that ATR dimer undergoes a photooxidation process involving the addition of oxygens at double bonds as well as an aromatic demethylation also may indicate a role for this molecule, or its derivatives, in the photoreactivity of RPE lipofuscin.W hereas the lipofuscin that is amassed by most nonproliferating cells is derived from autophagy (1), lipofuscin fluorophores of the retinal pigment epithelium (RPE) originate, in large part, from photoreceptor cells (2), with Ͼ90% of the fluorescent material being generated from conjugates formed by retinoids of the visual cycle (3, 4). RPE lipofuscin fluorophores accumulate with age (5, 6), the greatest accretion is in RPE cells underlying the central retina (6), and the retinoid-derived fluorophores are particularly abundant in Stargardt's disease (7, 8), a macular degeneration of juvenile onset. Indeed, several lines of evidence indicate that the lipofuscin fluorophores that accumulate in RPE contribute to the death of these cells in some forms of macular degeneration (5, 9-13). The loss of RPE is a critical event because it is thought that it leads to bystander-like degeneration of photoreceptors that culminates in visual impairment.As yet, the only RPE lipofuscin fluorophores that have been characterized are A2E, its 13-(Z)-double-bond isomer iso-A2E, and other minor Z-isomers of A2E (14-19). These compounds are generated by hydrolysis of the phosphate ester of A2-PE, the phosphatidyl-pyridinium bisretinoid precursor that forms in rod outer segments (ROS). The synthesis of A2-PE occurs when molecules of all-trans-retinal (ATR), instead of undergoing reduction to all-trans-retinol, form conjugates with phosphatidylethanolamine (PE) through a series of random͞nonenzyme-mediated reactions (16,19). This pyridinium bisretinoid pathway is ...

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

“…Ϫ/Ϫ Mouse Eyecups-The Abca4 null mutant mouse serves as a model of autosomal recessive Stargardt disease and has been shown to accumulate the bisretinoid compounds of RPE lipofuscin in abundance (8,10,17,18,28,29). Accordingly, we analyzed chloroform/methanol extracts of posterior eyecups of Abca4 Ϫ/Ϫ mice (age, 5 months) by reverse phase HPLC while monitoring at 490 nm.…”

“…Although the double bonds along the side arms of A2E are all in the trans (E) position, Z-isomers of A2E have double bonds at the C-13/14 (isoA2E), C-9/9Ј-10/10Ј, and C-11/11Ј-12/12Ј positions, and all are detectable in human and mouse RPE (16). These pigments exhibit absorbances in both the UV and visible regions of the spectrum (A2E: max , 439 and 338 nm; iso-A2E: max , 428 and 337 nm).Another bisretinoid compound of RPE lipofuscin also absorbs in the short wavelength region of the visible spectrum (17,18,23). This pigment, all-trans-retinal dimer (atRAL dimer; max , 432 and 290 nm) forms from the condensation of two atRAL and is present in RPE lipofuscin as Schiff base conjugates with either PE or ethanolamine (atRAL dimer-PE and atRAL dimer-E, respectively) or as unconjugated atRAL dimer.…”

“…Another bisretinoid compound of RPE lipofuscin also absorbs in the short wavelength region of the visible spectrum (17,18,23). This pigment, all-trans-retinal dimer (atRAL dimer; max , 432 and 290 nm) forms from the condensation of two atRAL and is present in RPE lipofuscin as Schiff base conjugates with either PE or ethanolamine (atRAL dimer-PE and atRAL dimer-E, respectively) or as unconjugated atRAL dimer.…”

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.…”

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

“…Synthesis-A2E and all-trans-retinal dimer were synthesized using starting materials and conditions as described previously; purification was by HPLC (30,31).…”