Nrf2 signaling is impaired in the aging RPE given an oxidative insult

Abstract

Age-related macular degeneration (AMD) represents the leading cause of blindness in the elderly, yet no definitive therapy exists for early, dry disease. Several lines of evidence have implicated oxidative stress-induced damage to the retinal pigment epithelium (RPE) in the pathogenesis of AMD, suggesting that the aging RPE may exhibit increased susceptibility to cell damage induced by exogenous stressors. The transcription factor Nrf2 serves as the master regulator of a highly coordinated antioxidant response in virtually all cell types. We compared Nrf2 signaling in the RPE of young (2 months) and old (15 months) mice under unstressed and stressed (sodium iodate) conditions. The aging RPE expressed higher levels of the Nrf2 target genes NQO1, GCLM, and HO1 compared with the RPE of younger mice under unstressed conditions, suggesting an age-related increase in basal oxidative stress. Moreover, the RPE of older mice demonstrated impaired induction of the protective Nrf2 pathway following oxidative stress induced with sodium iodate. The RPE of old mice exposed to sodium iodate also exhibited higher levels of superoxide anion and malondialdehyde than young mice, suggesting inadequate protection against oxidative damage. Induction of Nrf2 signaling in response to sodium iodate was partially restored in the RPE of aging mice with genetic rescue, using conditional knockdown of the Nrf2 negative regulator Keap1 (Tam-Cre; Keap1loxP) compared to Keap1loxP mice. These data indicate that the aging RPE is vulnerable to oxidative damage due to impaired Nrf2 signaling, and that Nrf2 signaling is a promising target for novel pharmacologic or genetic therapeutic strategies.

Keywords: Nrf2; age-related macular degeneration; aging; oxidative stress; retinal pigment epithelium.

Figure 1. Pharmacologic induction of oxidative stress in the RPE of young and old mice

Oxidative stress was pharmacologically induced with NaIO3. The RPE of 2-month old mice treated with PBS (A) has similar DHE fluorescence (red), a marker of superoxide anion, as the RPE of 2-month-old mice given NaIO3 (B). The RPE of 15-month old mice treated with PBS (C) display similar DHE staining as their young counterparts treated also with PBS (A), indicating adequate neutralization of ROS in the RPE of aging mice at baseline, i.e. under unstressed conditions. The RPE of 15-month old mice treated with NaIO3 (D) have increased DHE labeling compared with their age-matched vehicle-treated controls (C) or with 2-month old mice treated with NaIO3 (B), which suggests inadequate neutralization of ROS. Bar=25um. RPE, retinal pigment epithelium; Ch, choroid. NaIO3 induces oxidative damage, as indicated by MDA immunolabeling. The RPE of 2-month old mice treated with PBS had no MDA immunolabeling (E) while the RPE of 2- month old mice treated with NaIO3 have mild MDA staining (F). This labeling pattern suggests that the RPE’s antioxidant response prevents oxidative damage after an oxidative stress stimulus in young mice. The RPE of 15-month old mice treated with PBS also have very little MDA labeling (G) while the RPE of 15-month old mice given NaIO3 (H) show the strongest MDA labeling among the experimental groups, suggesting inadequate neutralization of ROS that promotes oxidative damage. IgG control of 2-month (I) and 15- month (J) old mouse treated with NaIO3. Bar=50um. RPE, retinal pigment epithelium; Ch, choroid.

Figure 2. Increased basal Nrf2 activity in the RPE of aging mice

Two-month-old and 15-month-old C57Bl6J mice were treated with either NaIO3 or vehicle (PBS), and RNA was isolated from the RPE for quantitative RT-qPCR analyses of expression of Nrf2 target genes GCLM, HO1, and NQO1. (A) HO1 and NQO1 expression was approximately 11-fold and 10- fold higher, respectively, in aging RPE compared with their young counterparts. *p<0.05, n = 4 mice per group. (B) NaIO3 induced a 2-fold, 9-fold, and 10-fold increase in transcript levels of GCLM, HO1, and NQO1 respectively, in the RPE of young mice. NaIO3 treatment failed to stimulate an Nrf2 response in the RPE of older mice (15mo). *p<0.05; **p<0.005, n= 4 mice per group. (C) NaIO3 induced a 2.2-fold increase in Nrf2 mRNA in the RPE of young mice, but did not stimulate induction of Nrf2 mRNA in the RPE of older mice (15mo). *p<0.05; n= 4 mice per group. (D) After injection of tamoxifen, 2-month-old and 15-monthold Tam-Cre;Keap1loxP and Keap1loxP mice were treated with either PBS or NaIO3. Tamoxifen decreased Keap1 expression by 50%, as assayed by quantitative RT-qPCR in Tam-Cre;Keap1loxP mice compared with Keap1loxP mice which lack the tamoxifen-inducible Cre recombinase transgene. (E) NaIO3 treatment induced 2.5-fold higher levels of the Nrf2 target gene NQO1 in old Tam-Cre;Keap1loxP mice compared with age-matched Keap1loxP mice expressing wild type levels of Keap1. *p<0.05, **o<0.005. n= 6 mice per group.