Targeted deletion of the murine Lgr4 gene decreases lens epithelial cell resistance to oxidative stress and induces age-related cataract formation

Abstract

Oxidative stress contributes to the formation of cataracts. The leucine rich repeat containing G protein-coupled receptor 4 (LGR4, also known as GPR48), is important in many developmental processes. Since deletion of Lgr4 has previously been shown to lead to cataract formation in mice, we sought to determine the specific role that Lgr4 plays in the formation of cataracts. Initially, the lens opacities of Lgr4(-/-) mice at different ages without ocular anterior segment dysgenesis (ASD) were evaluated with slit-lamp biomicroscopy. Lenses from both Lgr4(-/-) and wild-type mice were subjected to oxidation induced protein denaturation to assess the ability of the lens to withstand oxidation. The expression of antioxidant enzymes was evaluated with real-time quantitative PCR. Phenotypically, Lgr4(-/-) mice showed earlier onset of lens opacification and higher incidence of cataract formation compared with wild-type mice of similar age. In addition, Lgr4(-/-) mice demonstrated increased sensitivity to environmental oxidative damage, as evidenced by altered protein expression. Real-time quantitative PCR showed that two prominent antioxidant defense enzymes, catalase (CAT) and superoxidase dismutase-1 (SOD1), were significantly decreased in the lens epithelial cells of Lgr4(-/-) mice. Our results suggest that the deletion of Lgr4 can lead to premature cataract formation, as well as progressive deterioration with aging. Oxidative stress and altered expression of several antioxidant defense enzymes contribute to the formation of cataracts.

Fig 1. Deletion of Lgr4 in mice causes different types of age-related cataract formation.

(A) Disruption of the mouse Lgr4 gene. Genotype analysis of Lgr4 ^+/+, Lgr4 ^−/−, and Lgr4 ^+/− mice. +/+, −/−, and +/− represent genomic DNAs isolated from Lgr4 ^+/+, Lgr4 ^−/−, and Lgr4 ^+/− mice, respectively. The 1 kb band is derived from the wild-type allele, and the 700 bp band is derived from the mutated allele. (B) Slit-lamp biomicroscopy examination of wild-type and Lgr4 knockout lenses. At 20 weeks of age, diverse morphologies and various degrees of lens opacification were observed in the anterior portion of the cortex in Lgr4 knockout mice, while most of the wild-type mice remained clear (a,b). Punctate anterior cortical cataract (c). Lamellar cortical cataract (d). The cataract region includes the Y-sutures and looks like a “fishbone” pattern (e). The whole lens becomes opaque (f). (C) Expression profile of Lgr4 in mouse lens. LacZ staining of heterozygous lenses showed strong Lgr4 expression in lens epithelial cells from both the embryonic and adult mice at different ages.

Fig 2. Lgr4 deletion leads to the early onset of age-related cataract.

At the age of 6 to 8 weeks (wild-type: n = 24; Lgr4 ^−/−: n = 16), Lgr4 ^−/− mice showed no increased incidence of cataract formation. In the second stage (9 to 16 weeks, wild-type: n = 36; Lgr4 ^−/−: n = 22), the incidence of cataract formation was increased in Lgr4 ^−/− mice. The incidence of cataracts in Lgr4 ^−/− mice was significantly increased by maturity (17 to 24 weeks, wild-type: n = 42; Lgr4 ^−/−: n = 29) and older than 24 weeks (wild-type: n = 62; Lgr4 ^−/−: n = 45). *P < 0.001, **P < 0.0001.

Fig 3. Morphologic changes of murine lenses cultured in the presence of H₂O₂.

Lenses removed from 30-week-old mice were subjected to peroxidase damage (H₂O₂) and incubated for 24 hours. Lens images were captured on culture plates at 0, 12, and 24 hours after H₂O₂ treatment using dark field microscopy. Representative images show that the degree of lens opacity in the knockout mice was significantly more than in wild-type mice. All images are representative of at least 3 independent experiments.

Fig 4. Oxidation-induced denaturation of proteins in Lgr4 knockout lenses.

Lens supernatants of 10 weeks (10 W) and >18 weeks (>18 W) diluted to 1 mg/mL protein using the TC-199 medium were subjected to oxidative damage with 0.1 mM H₂O₂ and 2.31 units of glucose oxidase (GO) for indicated times. Sample absorbance was measured at 360 nM. Supernatants from Lgr4 knockout lenses showed a more rapid increase in turbidity (and therefore, protein denaturation), suggesting that the knockout lens had less overall resistance to oxidation than wild-type. Data are expressed as mean ± SD (n = 3). These results are representative of 3 independent experiments.

Fig 5. The gene expression levels of antioxidant enzymes are reduced in Lgr4 ^−/− mice older than 24 weeks.

The lens epithelium from Lgr4 ^−/− mice and their wild-type littermates was isolated as described in Materials and Methods. Total RNA was extracted and the gene expression was measured by real-time PCR. The data were normalized to the level in wild-type mice. Results are expressed as mean ± SEM. n = 3, *P < 0.01.