Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration

Abstract

Increased accumulation of lipofuscin in cells of the retinal pigment epithelium (RPE) is seen in several forms of macular degeneration, a common cause of blindness in humans. A major fluorophore of lipofuscin is the toxic bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E). Previously, we generated mice with a knockout mutation in the abcr gene. This gene encodes rim protein (RmP), an ATP-binding cassette transporter in rod outer segments. Mice lacking RmP accumulate A2E in RPE cells at a greatly increased rate over controls. Here, we identify three precursors of A2E in ocular tissues from abcr-/- mice and humans with ABCR-mediated recessive macular degenerations. Our results corroborate the scheme proposed by C. A. Parish, M. Hashimoto, K. Nakanishi, J. Dillon & J. Sparrow [Proc. Natl. Acad. Sci. USA (1998) 95, 14609-14613], for the biosynthesis of A2E: (i) condensation of all-trans-retinaldehyde (all-trans-RAL) with phosphatidylethanolamine to form a Schiff base; (ii) condensation of the amine product with a second all-trans-RAL to form a bis-retinoid; (iii) oxidation to yield a pyridinium salt; and (iv) hydrolysis of the phosphate ester to yield A2E. The latter two reactions probably occur within RPE phagolysosomes. As predicted by this model, formation of A2E was completely inhibited when abcr-/- mice were raised in total darkness. Also, once formed, A2E was not eliminated by the RPE. These data suggest that humans with retinal or macular degeneration caused by loss of RmP function may slow progression of their disease by limiting exposure to light. The precursors of A2E identified in this study may represent pharmacological targets for the treatment of ABCR-mediated macular degeneration.

Figure 1

HPLC analysis showing A2PE-H₂ in rod outer segment (ROS) and RPE. (A) Chromatogram of phospholipid extracts from 12-week-old abcr+/+ (light blue tracing) and abcr−/− (black tracing) ROS. Detection wavelength is 500 nm. Inset shows absorption spectrum of A2PE-H₂ peak fraction, indicated by red arrow. (B) Histogram showing levels of A2PE-H₂ in ROS from abcr−/− mice at the indicated ages in area units per eye. Error bars show standard deviations. (C) Chromatogram of phospholipid extracts from 12-week-old abcr+/+ (light blue tracing) and abcr−/− (black tracing) RPE. Detection wavelength is 500 nm. Inset shows absorption spectrum of the A2PE-H₂ peak fraction, indicated by red arrow. (D) Histogram showing the relative levels of A2PE-H₂ in RPE from abcr−/− mice at the indicated ages in area units per eye, ± standard deviations. Note that A2PE-H₂ is exclusively present in ROS and RPE from abcr−/− mice.

Figure 2

HPLC analysis showing the A2PE intermediate in the formation of A2E. (A) Chromatogram of A2PE-H₂ purified from 11-month abcr−/− outer segments. Detection wavelength is 500 nm. Inset shows spectrum of the A2PE-H₂ peak, labeled with the red arrow. (B) Chromatogram of A2PE-H₂ fraction from A after 5 min incubation in HCl. Detection wavelength is 430 nm. The A2PE-H₂ peak is labeled with the red arrow. Inset shows spectrum of the A2PE peak, labeled with the purple arrow. (C) Chromatogram of A2PE-H₂ fraction from A after overnight incubation in HCl. Detection wavelength is 430 nm. The phosphatidic acid peak is labeled with the black arrow. Inset shows spectrum for the A2E peak, labeled with the blue arrow. Note the disappearance of A2PE-H₂ and A2PE and the appearance of phosphatidic acid and A2E. (D) Mass spectrum of A2E fraction from C. Note the major molecular-ion species with a m/z ratio of 592.3. The additional labeled peaks were also present in a sample containing only solvent. (E) Chromatogram of phospholipid extract from 6-month-old abcr−/− RPE. Detection wavelength is 430 nm. Inset shows spectra for the A2PE peak, labeled with the purple arrow, and A2PE-H₂ peak, labeled with the red arrow.

Figure 3

HPLC analysis of phospholipid extracts from human retina and RPE showing phosphatidylethanolamine, A2PE-H₂, and A2E accumulation. A and D show analysis of retina and RPE, respectively, from a 71-year-old female with no retinal pathology as a representative control. B and E show analysis of retina and RPE, respectively, from a 62-year-old female with FFM. C and F show analysis of retina and RPE, respectively, from a 73-year-old male with STGD1. Chromatograms are shown at detection wavelengths of 500 (dark purple tracing) and 205 nm (green tracing) to display retinoid-conjugate and phospholipid absorption, respectively. For A–F, the 205-nm scale (Left) is in absorption units (AU), and the 500-nm scale (Right) is in milliabsorption units (mAU). Peaks corresponding to phosphatidylethanolamine (PE) in the retina samples are labeled with the black arrows. Insets show absorption spectra for the labeled A2PE-H₂ (red arrow) and A2E (blue arrow) peaks.

Figure 4

A2E in RPE from mice raised under different light conditions. Data expressed in pmol A2E per eye from mice of the indicated ages. (A) Wild-type (abcr+/+) mice raised under cyclic light (open circles) or in total darkness (filled circles). (B) abcr−/− mutant mice raised under cyclic light (open circles) or in total darkness (filled circles). (C) abcr−/− mutant mice raised for 12 weeks under cyclic light and transferred to total darkness until reaching the indicated ages. Error bars show standard deviations. * indicates a significant difference between data points by Student's t test (P < 0.05).

Figure 5

Hypothetical biosynthetic pathway for A2E in mice and humans lacking the RmP transporter. This model is based on the proposed scheme for A2E biogenesis by Parish et al. (24). In outer segments after a photobleach, phosphatidylethanolamine and all-trans-RAL are transiently in equilibrium with APE (reaction 1). After a (1, 5) sigmatropic rearrangement, the resulting secondary amine may condense with another molecule of all-trans-RAL. Subsequent (3, 3) sigmatropic rearrangement of the bis-retinoid product results in the formation of A2PE-H₂ (reaction 2), an irreversible step at neutral pH. Oxidation of A2PE-H₂ to A2PE (reaction 3) occurs within RPE phagolysosomes and is accompanied by a shift in the visible λ_max from 500 to 430 nm. This blue shift is expected, because the two tetraene side chains are oriented meta on the pyridinium ring, hence resonance delocalization occurs at a higher energy in the oxidized form because of loss of aromaticity. Finally, A2E is formed within RPE phagolysosomes on acid hydrolysis of the phosphate ester and release of phosphatidic acid (reaction 4).