Relative Contributions of All-Trans and 11-Cis Retinal to Formation of Lipofuscin and A2E Accumulating in Mouse Retinal Pigment Epithelium

Abstract

Purpose: Bis-retinoids are a major component of lipofuscin that accumulates in the retinal pigment epithelium (RPE) in aging and age-related macular degeneration (AMD). Although bis-retinoids are known to originate from retinaldehydes required for the light response of photoreceptor cells, the relative contributions of the chromophore, 11-cis retinal, and photoisomerization product, all-trans retinal, are unknown. In photoreceptor outer segments, all-trans retinal, but not 11-cis retinal, is reduced by retinol dehydrogenase 8 (RDH8). Using Rdh8-/- mice, we evaluated the contribution of increased all-trans retinal to the formation and stability of RPE lipofuscin.

Methods: Rdh8-/- mice were reared in cyclic-light or darkness for up to 6 months, with selected light-reared cohorts switched to dark-rearing for the final 1 to 8 weeks. The bis-retinoid A2E was measured from chloroform-methanol extracts of RPE-choroid using HPLC-UV/VIS spectroscopy. Lipofuscin fluorescence was measured from whole flattened eyecups (excitation, 488 nm; emission, 565-725 nm).

Results: Cyclic-light-reared Rdh8-/- mice accumulated A2E and RPE lipofuscin approximately 1.5 times and approximately 2 times faster, respectively, than dark-reared mice. Moving Rdh8-/- mice from cyclic-light to darkness resulted in A2E levels less than expected to have accumulated before the move.

Conclusions: Our findings establish that elevated levels of all-trans retinal present in cyclic-light-reared Rdh8-/- mice, which remain low in wild-type mice, contribute only modestly to RPE lipofuscin formation and accumulation. Furthermore, decreases in A2E levels occurring after moving cyclic-light-reared Rdh8-/- mice to darkness are consistent with processing of A2E within the RPE and the existence of a mechanism that could be a therapeutic target for controlling A2E cytotoxicity.

Figure 1.

Lipofuscin accumulates in the RPE of Rdh8^−/− mice. (A) True color fluorescence micrograph (excitation: 450–490 nm) of a flat-mounted RPE from 3-month-old dark-reared Rdh8^−/− mouse. (B) Fluorescence emission spectra (excitation: 488 nm) of RPE lipofuscin granules (n = 101 for each) from 3-month-old cyclic-light- (CL, ○) and dark-reared (D, ●) Rdh8^−/− mice. Error bars represent SEM.

Figure 2.

Total RPE fluorescence increases faster with age in cyclic-light-reared compared to dark-reared Rdh8^−/− mice. (A) True color fluorescence micrograph (excitation: 450–490 nm) of a whole flat-mounted RPE of a 3-month-old cyclic-light-reared Rdh8^−/− mouse. (B) Total RPE fluorescence (excitation: 488 nm; emission: 565–725 nm; measured in bead units per megapixel) increases with age in cyclic-light- (CL, ○) and dark-reared (D, ●) Rdh8^−/− mice. Straight lines are regression lines with slopes of 20 BU/MP/month for cyclic-light-reared and 9.1 BU/MP/month for dark-reared mice. All experiments were conducted in triplicate. Error bars represent SEM. Asterisks denote statistical significance.

Figure 3.

The RPE levels of the bis-retinoid A2E increase faster with age in cyclic-light-reared compared to dark-reared Rdh8^−/− mice. (A) Chromatograms at 430 nm of RPE-choroid extracts from 6-month-old cyclic-light- (CL) and dark-reared (D) Rdh8^−/− mice. Trace absorbance has been normalized to the number of eyes used, so it represents amount per eye. (B) RPE levels of A2E increase with age in cyclic-light- (CL, ○) and dark-reared (D, ●) Rdh8^−/− mice. Straight lines are regression lines with slopes of 1.7 pmol/eye/month for cyclic-light-reared and 1.1 pmol/eye/month for dark-reared mice. All experiments were conducted in triplicate. Error bars represent SEM. Asterisks denote statistical significance.

Figure 4.

Moving Rdh8^−/− mice from cyclic light to darkness decreases the accumulation of A2E in the RPE. Animals were moved to dark for different lengths of time before becoming 6 months old. A2E levels were measured at 6 months. All experiments were conducted in triplicate. The straight line shows the expected levels of A2E accumulation at the time the animals were moved to darkness. The cyclic light (CL, ○) and dark (D, ●) data points are from the experiments in Figure 3B. Error bars represent SEM. Asterisks denote statistical significance.