Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt's macular degeneration

Abstract

Recessive Stargardt's macular degeneration is a blinding disease of children caused by mutations in the ABCA4 (ABCR) gene. Mice with a knockout mutation in abcr accumulate toxic lipofuscin pigments in ocular tissues, similar to affected humans. The major fluorophore of lipofuscin is the bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E). In the current study, we sought to define the effect of increasing light on A2E accumulation. We crossed the abcr(-/-) mutation onto an albino background. The retinoid profiles in albino mice indicated higher retinal illuminance than in pigmented mice exposed to similar ambient light. Unexpectedly, A2E levels were not higher in the albino mice. Also, A2E levels in abcr(-/-) mice reared under cyclic light at 30, 120, or 1,700 lux were similar. Thus, increased retinal illuminance was not correlated with higher A2E. A2E has been shown to undergo light-dependent oxidation to yield a series of A2E epoxides or oxiranes. These oxiranes react with DNA in vitro, suggesting a potential mechanism for A2E cytotoxicity. We analyzed ocular tissues from abcr(-/-) mice for A2E oxiranes by mass spectrometry. Unlike A2E, the oxiranes were more abundant in albino vs. pigmented abcr(-/-) mice, and in abcr(-/-) mice exposed to increasing ambient light. These observations suggest that both the biosynthesis of A2E and its conversion to oxiranes are accelerated by light. Finally, we showed that the formation of A2E oxiranes is strongly suppressed by treating the abcr(-/-) mice with Accutane (isotretinoin), an inhibitor of rhodopsin regeneration.

Fig. 1.

Retinoid pathways in the retina and RPE. (A) Visual cycle mediating rhodopsin regeneration. Absorption of a photon (hv) by a rhodopsin molecule in a rod outer segment disk induces photoisomerization of the 11cRAL chromophore, yielding activated metarhodopsin II. After several seconds, metarhodopsin II decays to yield apo-rhodopsin and free atRAL. ABCR functions to accelerate removal of atRAL from the interior of outer segment discs to the cytoplasmic space by flipping N-ret-PE (5). The atRAL is subsequently reduced to atROL or vitamin A by all-trans-retinol dehydrogenase. The atROL is released from the outer segment and taken up by an adjacent RPE cell where it is esterified by lecithin retinol acyl transferase (LRAT) to form an atRE. Chemical isomerization is effected by an isomerase that uses atREs as a substrate, in conjunction with Rpe65 (46). The resulting 11cROL is oxidized by 11cRDH to form 11cRAL chromophore. 11cRDH is inhibited by isotretinoin with a K_i of ≈0.1 μM (40, 47). 11cROL may also serve as a substrate for LRAT to form 11-cis-retinyl esters. The final step is recombination of 11cRAL with aporhodopsin in the outer segment to form a new molecule of light-sensitive rhodopsin. (B) Synthesis of A2E. After light exposure, newly released atRAL condenses reversibly with phosphatidylethanolamine to N-ret-PE (step 1). Rarely, a second molecule of atRAL will condense with N-ret-PE to form A2PE-H₂ (step 2). The wavelength of maximal absorption (λ_max) for A2PE-H₂ is 500 nm. Within the acidic and oxidizing environment of RPE phagolysosomes, A2PE-H₂ is oxidized to A2PE (λ_max = 430 nm) (step 3). Hydrolysis of the phosphate ester yields A2E (λ_max = 435 nm) and phosphatidic acid (step 4) (10). Double bonds along the polyene chains of A2E may react with singlet oxygen to form a series of one to nine (shown) oxiranes (step 5).

Fig. 3.

A2E and A2PE-H₂ in abcr^–/– eyes. (A) Representative chromatogram of a phospholipid extract from a pigmented abcr^–/– eyecup showing absorbance at 435 nm. (Inset) UV spectra acquired from the peaks labeled A2E and A2PE-H₂. Note the λ_max of A2E at 435 and A2PE-H₂ at 500 nm. (B) Quantitation of A2E (pmol per eye) and A2PE-H₂ (milli-absorbance unit per eye at 500 nm) in eyecups from 3-mo-old pigmented (dark blue bars) and albino (cyan bars) abcr^–/– mice raised under cyclic light at 30 lux. Error bars show standard deviations (n = 4).

Fig. 2.

Visual retinoids in pigmented and albino mice. (A) Representative HPLC chromatogram of a retinoid extract from the eyecup of a 2-mo-old pigmented wild-type mouse showing absorbance at 340 nm. Labeled peaks corresponding to atREs, atROL, and the syn- and anti-oximes of 11cRAL (11cRox) and atRAL (atRox) were confirmed by spectral analysis. (B) Chromatogram of authentic retinoid standards at 340-nm absorbance. (C) Quantitation of retinoids from eyecups of 2- to 3-mo-old wild-type pigmented (dark blue bars) and albino (cyan bars) mice, light adapted at 30 lux. Values for the indicated retinoids are shown in pmol per eye. (D) Quantitation of retinoids from eyecups of 2- to 3-mo-old pigmented and albino abcr^–/– mice light adapted at 30 lux. Error bars show standard deviations (n = 4).

Fig. 4.

Effects of light on A2E and A2PE-H₂. Quantitation of A2E (magenta bars) in pmol per eye and A2PE-H₂ (blue bars) in milli-absorbance unit per eye from 5-mo-old pigmented abcr^–/– mice raised under cyclic light at 30, 120, or 1,700 lux. Error bars show standard deviations (n = 3).

Fig. 5.

Identification of A2E oxiranes generated in vitro and in vivo by MS. (A) Full-scan MS in the range 580–750 m/z of oxiranes after chemical oxidation of in vitro synthesized A2E. Note A2E at 592 and the family of ions that increase by 16 mass units to nonaoxirane at 736. (B) Product-ion spectrum showing fragmentation of a 640-m/z parent ion in a phospholipid extract of 7-mo-old abcr^–/– eyecups. Note the formation of bisoxirane (624), monooxirane (608), and A2E (592).

Fig. 6.

A2E and oxiranes in abcr^–/– mice at different retinal illuminance. (A) Quantitation of A2E and total oxiranes in eyecups from 7-mo-old abcr^–/– pigmented (dark blue bars) and albino (cyan bars) mice raised under cyclic light at 30 lux determined by LC-MS (n = 4). (B) Quantitation of A2E and total oxiranes in 7-mo-old abcr^–/– pigmented mice raised under cyclic light at 30 (dark green bars) or 120 lux (light green bars). Values are expressed as ion intensities relative to a lysophosphatidylcholine internal standard. Error bars show standard deviations (n = 3). Note the similar levels of A2E and elevated oxiranes with increased illuminance in both experiments.

Fig. 7.

Effect of treatment with isotretinoin on A2E and oxiranes in abcr^–/– mice. Quantitation of A2E and total oxiranes in eyecups from pigmented 4-mo-old abcr^–/– mice raised under cyclic light at 30 lux and treated (red bars) or not treated (green bars) with isotretinoin for 2 mo. Error bars show standard deviations (n = 4). Note the reduction of both A2E and oxiranes in the treated mice.