Limited roles of Rdh8, Rdh12, and Abca4 in all-trans-retinal clearance in mouse retina

Abstract

Purpose: Although the retinoid cycle is essential for vision, all-trans-retinal and the side products of this cycle are toxic. Delayed clearance of all-trans-retinal causes accumulation of its condensation products, A2E, and all-trans-retinal dimer (RALdi), both associated with human macular degeneration. The protective roles were examined of the all-trans-RDHs, Rdh8 and Rdh12, and the ATP-binding cassette transporter Abca4, retinoid cycle enzymes involved in all-trans-retinal clearance.

Methods: Mice genetically engineered to lack Rdh8, Rdh12, and Abca4, either singly or in various combinations, were investigated because all-trans-retinal clearance is achieved by all-trans-RDHs and Abca4. Knockout mice were evaluated by spectral-domain optical coherence tomography (SD-OCT), electroretinography, retinal morphology, and visual retinoid profiling with HPLC and MS. ARPE19 cells were examined to evaluate A2E and RALdi oxidation and toxicity induced by exposure to UV and blue light.

Results: Rdh8(-/-)Abca4(-/-) and Rdh8(-/-)Rdh12(-/-)Abca4(-/-) mice displayed slowly progressive, severe retinal degeneration under room light conditions. Intense light-induced acute retinal degeneration was detected by SD-OCT in Rdh8(-/-)Rdh12(-/-)Abca4(-/-) mice. Amounts of A2E in the RPE correlated with diminished all-trans-retinal clearance, and the highest A2E amounts were found in Rdh8(-/-)Rdh12(-/-)Abca4(-/-) mice. However oxidized A2E was not found in any of these mice, and A2E oxidation was not induced by blue light and UV illumination of A2E-loaded ARPE19 cells. Of interest, addition of all-trans-retinal did activate retinoic acid receptors in cultured cells.

Conclusions: Rdh8, Rdh12, and Abca4 all protect the retina and reduce A2E production by facilitating all-trans-retinal clearance. Delayed all-trans-retinal clearance contributes more than A2E oxidation to light-induced cellular toxicity.

Figure 1

Characterization of Rdh8^−/− Rdh12^−/− Abca4^−/− mice. (A) Retinal histology of 6-week-old Rdh8^−/− Rdh12^−/− Abca4^−/− mice (right) and WT mice (left). Representative images are shown (n > 10). RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. (B) Rates of dark-adaptation were assessed in 6-week-old Rdh8^−/−Rdh12^−/−Abca4^−/− and WT mice by monitoring ERG a-wave amplitudes after 3 minutes of exposure to light in a Ganzfeld chamber (500 cd · m⁻²). Bars, SD (n > 6). (C) Amounts of all-trans-retinal in eyes of 6-week-old Rdh8^−/−Rdh12^−/− Abca4^−/− and WT mice exposed for 3 minutes to light in a Ganzfeld chamber (500 cd · m⁻²). 0, immediately after light exposure; 1, 1-hour dark adaptation after light exposure; and 2, 2-hour dark adaptation after light exposure. Bars, SD (n > 3) Horizontal bar: the amount of all-trans-retinal found in dark-adapted WT mice. Numbers are the percentages of all-trans-retinal cleared relative to the WT dark-adapted level set at 100%. (D) Kinetics of 11-cis-retinal formation (top), all-trans-retinal disappearance (middle), and all-trans-retinyl ester accumulation (bottom) in 6-week-old Rdh8^−/−Rdh12^−/−Abca4^−/− and WT mice. Retinoids were quantified by HPLC in eye samples collected at different time points after a flash that bleached ∼40% of visual pigments. Bars, SD (n > 3). At 6 weeks of age Rdh8^−/−Rdh12^−/−Abca4^−/− mice exhibited mild retinal degeneration with a ∼25% loss of rhodopsin.

Figure 2

Retinal changes after intense light illumination in Rdh8^−/− Rdh12^−/−Abca4^−/− and WT mice. Normal-appearing retinas of 4-week-old Rdh8^−/−Rdh12^−/−Abca4^−/− and WT mice were exposed to bright light (10,000 lux) for 30 minutes and then at 0 and 24 hours and 3 and 7 days after being kept in the dark were examined by ultrahigh-resolution SD-OCT. Representative SD-OCT images (n = 4) of the same eye are shown (top). Cryosections of retinas were prepared 7 days after illumination, and nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI) (bottom). Representative images of the same eye imaged by SD-OCT are shown (n = 4). Bars, 20 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ELM, external limiting membrane; IS, inner segment; OS, outer segment; RPE, retinal pigment epithelium; PR, photoreceptor.

Figure 3

All-trans-retinal reduction in 6-week-old mice with either a single- or a double-knockout combination of Rdh8, Rdh12, and/or Abca4. Kinetics of all-trans-retinal disappearance in 6-week-old single-knockout (A) and double-knockout (B) mice lacking functional Rdh8, Rdh12, and Abca4 genes. Retinoids were quantified by HPLC in eyes collected at different time points after a flash that bleached ∼40% of visual pigments. Bars, SD (n > 3).

Figure 4

A2E quantities in the eyes of mice with single, double, and triple knockouts of Rdh8, Rdh12, and Abca4. A2E amounts in the whole eye were quantified by HPLC in 3- and 6-month-old mice with single, double, and triple knockouts of Rdh8, Rdh12, and Abca4. Mice were reared under 12-hour light (∼10 lux)/12-hour dark conditions. Bars, SD (n > 3). All knockout mice showed significant age-dependent increases in A2E compared with WT animals (P < 0.01).

Figure 5

Effect of UV light exposure on A2E and RALdi. A2E and RALdi in 75% aqueous acetonitrile were exposed to UV light (365 nm) for 1, 5, or 15 minutes, and the amounts of A2E and RALdi were analyzed by HPLC. A2E eluted slightly after 5 minutes and remained stable after exposure to UV light, whereas amounts of RALdi eluting at 20 minutes had virtually disappeared by 1 minute after exposure. Representative chromatograms are shown (n > 3)

Figure 6

A2E and RALdi disappearance after exposure to blue light. A2E and RALdi in 75% aqueous acetonitrile were exposed to blue light (440 nm) at room temperature for 1 minute or 5 minutes, and amounts of A2E and RALdi were determined by HPLC at 460 and 430 nm, respectively. Representative chromatograms are shown (n > 3) Amounts of A2E or RALdi after blue light illumination were compared to those in identical nonilluminated samples of A2E or RALdi (A), and indicated as percentages of the original substrate (B). Bars, SD (n > 3) The amount of A2E remained unchanged, whereas RALdi decreased markedly after exposure.

Figure 7

A2E oxidation by ARPE19 cells after exposure to blue light. (A) A2E-laden ARPE19 cells were produced by incubating them for 16 hours at 37°C in medium with 30 μM A2E in 2% DMSO followed by 3 days' incubation in media without A2E. Cells were exposed to blue light (440 nm) for 5 minutes and placed in the dark for 16 hours at 37°C. After collection of cells by centrifugation, A2E content was analyzed by HPLC. Representative chromatograms are shown (n > 5). (B) A2E in the ARPE19 cell culture medium (100 μL) was exposed to blue light (440 nm) for 5 minutes and then quantified by HPLC. Representative chromatograms are shown (n > 5). (C) Quantification of A2E in A2E-laden ARPE19 cells and medium with and without exposure to blue (440 nm) or UV (365 nm) light for 5 minutes. Bars, SD (n > 5). Exposure to blue and UV light decreased the amounts of A2E in the medium but not in the ARPE19 cells. (D) Oxidized products of A2E in medium after exposure to blue light detected by MS. Top: Representative chromatogram of an A2E peak showing several iso-forms (stereoisomers; asterisk) of A2E (gray trace). Representative chromatogram of a light-induced oxidized A2E peak (black trace). These peaks corresponded to masses of 592.6 (bottom left; A2E, gray line) and 608.5 (bottom right; oxidized A2E [A2E-oxi], black line).

Figure 8

Oxidation of RALdi in 75% aqueous acetonitrile under room light. RALdi in 75% aqueous acetonitrile was kept under room light (<100 lux) for 16 hours at 25°C, and samples were analyzed by MS. Top: A HPLC chromatogram at 290 nm. Bottom: oxidation products of RALdi appeared as multiple peaks with increasing masses. The mass of RALdi is 551.3.

Figure 9

Retinoid activation of RAR. RAR activation was assessed by the RAR element reporter cell line F9-RARE-lacZ (SIL15-RA) assay. All-trans-retinoic acid activated this receptor at concentrations of 10⁻⁹ M and higher, whereas all-trans-retinal activated it at 10⁻⁷ M. Decreased activity of all-trans-retinal at 10⁻⁶ M was caused by its cytotoxicity. Bars, SD (n > 5)