The all-trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model

Departments of *Ophthalmology,
^‡Chemistry, and
^§Pathology and Cell Biology, Columbia University, New York, NY 10032

Edited by John E. Dowling, Harvard University, Cambridge, MA, and approved October 17, 2007 (received for review September 14, 2007)

Abstract

The bis-retinoid pigments that accumulate in retinal pigment epithelial cells as lipofuscin are associated with inherited and age-related retinal disease. In addition to A2E and related cis isomers, we previously showed that condensation of two molecules of all-trans-retinal leads to the formation of a protonated Schiff base conjugate, all-trans-retinal dimer-phosphatidylethanolamine. Here we report the characterization of the related pigments, all-trans-retinal dimer-ethanolamine and unconjugated all-trans-retinal dimer, in human and mouse retinal pigment epithelium. In eyecups of Abcr ^−/− mice, a model of recessive Stargardt macular degeneration, all-trans-retinal dimer-phosphatidylethanolamine was increased relative to wild type and was more abundant than A2E. Total pigment of the all-trans-retinal dimer series (sum of all-trans-retinal dimer-phosphatidylethanolamine, all-trans-retinal dimer-ethanolamine, and all-trans-retinal dimer) increased with age in Abcr ^−/− mice and was modulated by amino acid variants in Rpe65. In in vitro assays, enzyme-mediated hydrolysis of all-trans-retinal dimer-phosphatidylethanolamine generated all-trans-retinal dimer-ethanolamine, and protonation/deprotonation of the Schiff base nitrogen of all-trans-retinal dimer-ethanolamine was pH-dependent. Unconjugated all-trans-retinal dimer was a more efficient generator of singlet oxygen than A2E, and the all-trans-retinal dimer series was more reactive with singlet oxygen than was A2E. By analyzing chromatographic properties and UV-visible spectra together with mass spectrometry, mono- and bis-oxygenated all-trans-retinal dimer photoproducts were detected in Abcr ^−/− mice. The latter findings are significant to an understanding of the adverse effects of retinal pigment epithelial cell lipofuscin.

Footnotes

^¶To whom correspondence should be addressed at:
Department of Ophthalmology, Columbia University, 630 West 168th Street, New York, NY 10032.
E-mail: jrs88{at}columbia.edu
Author contributions: S.R.K. and Y.P.J. contributed equally to this work; S.R.K., Y.P.J., S.J., and J.R.S. designed research; S.R.K., Y.P.J., S.J., and J.R.S. performed research; N.E.F., N.J.T., and J.R.S. contributed new reagents/analytic tools; S.R.K., Y.P.J., S.J., and J.R.S. analyzed data; and S.R.K., Y.P.J., S.J., and J.R.S. wrote the paper.
↵ ^†Present address: College of Pharmacy, Kyung Hee University, Hoegi-dong, Dongdaemun-gu, Seoul 130-701, South Korea.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0708714104/DC1.