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. 2010 Jan 1;48(1):16-25.
doi: 10.1016/j.freeradbiomed.2009.08.005. Epub 2009 Aug 14.

Retinol Dehydrogenase 12 Detoxifies 4-hydroxynonenal in Photoreceptor Cells

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Free PMC article

Retinol Dehydrogenase 12 Detoxifies 4-hydroxynonenal in Photoreceptor Cells

Lea D Marchette et al. Free Radic Biol Med. .
Free PMC article

Abstract

Mutations of the photoreceptor retinol dehydrogenase 12 (RDH12) gene cause the early onset retinal dystrophy Leber congenital amaurosis (LCA) by mechanisms not completely resolved. Determining the physiological role of RDH12 in photoreceptors is the focus of this study. Previous studies showed that RDH12, and the closely related retinol dehydrogenase RDH11, can enzymatically reduce toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE), in vitro. To explore the significance of this activity, we investigated the ability of RDH11 and RDH12 to protect stably transfected HEK-293 cells against the toxicity of 4-HNE. Both enzymes protected against 4-HNE modification of proteins and 4-HNE-induced apoptosis in HEK-293 cells. In the retina, exposure to bright light induced lipid peroxidation, 4-HNE production, and 4-HNE modification of proteins in photoreceptor inner segments, where RDH11 and RDH12 are located. In mouse retina, RDH12-but not RDH11-protected against adduct formation, suggesting that 4-HNE is a physiological substrate of RDH12. RDH12-but not RDH11-also protected against light-induced apoptosis of photoreceptors. We conclude that in mouse retina RDH12 reduces 4-HNE to a nontoxic alcohol, protecting cellular macromolecules against oxidative modification and protecting photoreceptors from light-induced apoptosis. This activity is of particular significance to the understanding of the molecular mechanisms of RDH12-induced LCA.

Figures

Figure 1
Figure 1
Mouse RDH11 and RDH12 protect HEK-293 cells against 4-HNE-induced apoptosis. Cells were transfected with the expression plasmid pTarget, containing or not Flag-tagged RDH11 or RDH12. Stable cell lines were established and maintained by growing the cells in complete DMEM medium containing G418 (1mg/ml). A, Equal aliquots (50 μg) of cell homogenates were analyzed by Western-blot using anti-Flag antibody (1:1000 dilution) to compare expression levels of RDH11 and RDH12. B, Cells were treated over night (20 h) with indicated concentrations of 4-HNE in complete DMEM containing G418. Pictures of cell dishes subjected to 0 or 100 μM of 4-HNE were taken the next day, after removing the media containing floating cells, and replacing it with PBS. Note complete absence of adhering cells in the mock-transfected clone, in contrast with RDH11- and RDH12-transfected clones in which a significant number of cells are still attached to the dish. C, Quantification of Annexin V positive cells corresponding to apoptotic cells, by flow cytometry. After 20 h treatment with 4-HNE, floating and attached cells are pooled, washed in PBS, and stained with Annexin V-PE before analysis by flow cytometry. Graph shows results of 6 independent experiments. Data points represent the mean and error bars denote SEM. RDH-expressing clones were compared with the control using the Student’s t test for significance. *=p<0.05; **=p<0.001; and ***=p<0.0001.
Figure 2
Figure 2
An inactivating mutation of RDH12 associated with LCA abolishes protection against 4-HNE-induced apoptosis. Cells were transfected with the expression plasmid pcDNA3.1/HIS, expressing human RDH12 variants R161 (wild-type), R161Q (common variant), and T49M (mutant inducing LCA). Stable cell lines were established and maintained by growing the cells in complete DMEM medium containing G418 (1mg/ml). A, Equal aliquots (50 μg) of microsomal fractions were analyzed by Western-blot using anti-hRDH12 antibody (1:1000 dilution) to compare expression levels. B, Cells were treated over night (20 h) with 100 μM of 4-HNE in complete DMEM. LDH activity, released by cells with damaged membranes, was measured in medium to quantify non-viable cells. Percent cell death was calculated for each cell line, according to the manufacturer’s instructions. Percent cell death in each stable cell line was then expressed in comparison to the non-transfected cell death, set at 100%. Graph shows results of 2 independent experiments, each having 3 replicates of the same conditions and error bars denote SD.
Figure 3
Figure 3
Mouse RDH11 and RDH12 protect against 4-HNE-protein adduct formation in HEK-293 cells. Confluent cells were treated overnight (20 h) with indicated concentrations of 4-HNE. A, Whole cell homogenates are prepared and equal aliquots (5 μg) of protein are analyzed by dot blot. M, mock; 11, RDH11; 12, RDH12. Protein loading is first verified by staining the membrane with Ponceau red (lower picture) and the membrane is then incubated with anti-HNE coupled with HRP at 1:1000 dilution. B, Graph shows quantification of adduct formation in 6 independent experiments. Data points represent the mean and error bars denote SEM. RDH-expressing clones were compared with the control using the Student’s t test for significance. *=p<0.05; **=p<0.001; and ***=p<0.0001.
Figure 4
Figure 4
RDH12 protects against formation of 4-HNE-protein adducts in mouse retinal microsomes. Microsomal fractions were prepared from wild-type and Rdh12 knockout retinas. A, Equal aliquots (10 μg) of microsomal proteins from wild-type or Rdh12 knockout retinas were incubated with indicated concentrations of 4-HNE in reaction buffer containing NADPH, for 2 h at room temperature. Reactions were transferred to the membrane by vacuum filtration, and unbound 4-HNE was washed 3 times with PBS. Protein loading is verified and adduct formation is quantified as described in Figure 3. Graph shows quantification of adduct formation in 3 independent experiments. B, The same experiment is repeated with wild-type retinal microsomes and 125 μM of 4-HNE, with and without NADPH. Graph shows quantification of adduct formation in 3 independent experiments. Data points represent the mean and error bars denote SEM. Results were compared using the Student’s t test for significance. *=p<0.05; **=p<0.001; and ***=p<0.0001.
Figure 5
Figure 5
Exposure of Balb/C mice to bright light induces adduct formation in the retina followed by photoreceptor apoptosis. Mice were raised in dim (10 lux) cyclic light until 8 to 12 weeks-old. They were then subjected to bright light (3,000 lux) exposure for indicated times, and transferred to the dark for indicated times. A, Immunohistochemistry of retinal paraffin sections using anti-HNE coupled with biotin at 1:100 dilution (brown signal), and anti-RDH12 at 1:200 dilution (green signal). Pictures were taken at 20 × magnification. RPE, retinal pigmented epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Note the co-localization of adducts and RDH12 in photoreceptors indicated by brackets. B, Immediately after exposure to bright light, mice were killed and retinas were dissected. Whole retinal homogenates were prepared and equal aliquots (10 μg) of retinal homogenates were analyzed by dot blot using anti-HNE coupled with HRP to quantify total 4-HNE-protein adducts. Six mice were used in each group, and the mean and SEM are plotted. C, TUNEL nuclear staining was observed in the ONL at 4 h of exposure to bright light followed by at least 12 h in the dark. Pictures were taken at 40 × magnification.
Figure 6
Figure 6
RDH12 protects against modification of proteins by 4-HNE in mouse retina. Pigmented wild-type and Rdh12 knockout mice (A) or Balb/C wild-type, Rdh11 knockout and Rdh12 knockout mice (B) were raised in dim cyclic light for 8 to 12 weeks. Pigmented wild-type and Rdh12 knockout mice were then subjected to constant bright light (3,000 lux) for 48 h. Whole retinal homogenates were prepared and equal aliquots (10 μg) of retinal homogenates were analyzed by dot blot using anti-HNE coupled with HRP to quantify total 4-HNE-protein adducts. Six mice were used in each group, and the mean and SEM are plotted. Results were compared using the Student’s t test for significance. *=p<0.05; **=p<0.001; and ***=p<0.0001.
Figure 7
Figure 7
RDH12 protects photoreceptors against light-induced apoptosis in Balb/C mice. Mice were raised in dim cyclic light for 8 to 12 weeks and light damage was induced in wild-type, Rdh11 knockout and Rdh12 knockout by exposure to 3,000 lux of light for 2 h. After light exposure, mice are returned in dim cyclic light for 7 days to allow the retina to clear all dead cells and return to a well-organized morphology. A, Representative sections of wild-type and Rdh12 knockout retinas. Pictures are taken in the inferior retina, 0.5 mm from the optic nerve head (ONH) as indicated by the boxed area on the low magnification of mouse retina. The layer of photoreceptor nuclei (ONL) is thinner in the knockout than in the wild-type after exposure to bright light, indicating more photoreceptor apoptosis in the knockout. The thickness of the outer nuclear layer (ONL) was measured in wild-type and Rdh11 knockout retinas (B) and in Rdh12 knockout retinas (C) at 0.24 mm intervals from the ONH to the inferior and superior ends of retina, as shown by arrows on the low magnification of mouse retina (A, lower panel). Ten mice were used in each group and the mean and SEM are plotted.
Figure 7
Figure 7
RDH12 protects photoreceptors against light-induced apoptosis in Balb/C mice. Mice were raised in dim cyclic light for 8 to 12 weeks and light damage was induced in wild-type, Rdh11 knockout and Rdh12 knockout by exposure to 3,000 lux of light for 2 h. After light exposure, mice are returned in dim cyclic light for 7 days to allow the retina to clear all dead cells and return to a well-organized morphology. A, Representative sections of wild-type and Rdh12 knockout retinas. Pictures are taken in the inferior retina, 0.5 mm from the optic nerve head (ONH) as indicated by the boxed area on the low magnification of mouse retina. The layer of photoreceptor nuclei (ONL) is thinner in the knockout than in the wild-type after exposure to bright light, indicating more photoreceptor apoptosis in the knockout. The thickness of the outer nuclear layer (ONL) was measured in wild-type and Rdh11 knockout retinas (B) and in Rdh12 knockout retinas (C) at 0.24 mm intervals from the ONH to the inferior and superior ends of retina, as shown by arrows on the low magnification of mouse retina (A, lower panel). Ten mice were used in each group and the mean and SEM are plotted.

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