Characterization of a Novel Gja8 (Cx50) Mutation in a New Cataract Rat Model

Abstract

Purpose: To describe a novel spontaneous cataract inbred strain isolated from large-scale breeding SD rats, identify the responsible gene mutation, and understand how this mutation affects lens function.

Methods: Exome sequencing of 12 cataract-associated genes was performed in the affected and healthy relatives. Sequences of rat wild-type or mutant gap junction protein alpha 8 gene (Gja8) were transfected into cells. The expression level of protein was assayed by Western blot analysis. Subcellular localization of connexin 50 (Cx50) was analyzed in confocal fluorescent images. Wound-healing, 5-ethynyl-2'-deoxyuridine incorporation, and attachment assay were performed to characterize the cell migration, proliferation and adhesion.

Results: The abnormality was found to be inheritable in an autosomal semi-dominant pattern through different mating patterns. We found a G to T transversion at codon 655 in Gja8, leading to a substitution of valine by phenylalanine (p.V219F). Gja8V219F/+ heterozygotes expressed nuclear cataract while Gja8V219F/V219F homozygotes manifested microphthalmia in addition to cataract. Histology revealed fiber disorders and loss of organelle-free zone in the mutant lens. Cx50V219F altered its location in HeLa cells and inhibited the proliferation, migration and adhesion abilities of HLEB3 cells. The mutation also reduced the expression of focal adhesion kinase and its phosphorylation.

Conclusions: The c.655G>T mutation (p.V219F) is a novel mutation in Gja8, inducing semi-dominant nuclear cataracts in a new spontaneous cataract rat model. The p.V219F mutation altered Cx50 distribution, inhibited lens epithelial cell proliferation, migration, and adhesion, and disrupted fiber cell differentiation. As a consequence, the nuclear cataract and small lens formed.

Figure 1.

The distinct phenotypes and genotypes of the TSC rats. (A) The photos of rat eyes and lenses obtained from 6-week-6-day-old littermates. The Gja8^+/+ rats were with transparent lenses. The Gja8^V219F/+ heterozygotes exhibited typical nuclear opacity in the lens center. Opaque center with ashes-like opacity around was observed in small lenses from Gja8^V219F/V219F rats. The cataractous lenses were smaller than the wild type ones, with a major difference between homozygous lenses and wild-type ones. Scale bar: 1 mm. (B) Partial nucleotide sequence of Gja8 from affected and unaffected individuals. The sequence in rats with cataract and smaller sized eyeballs showed a heterozygous G→T transversion (indicated by the arrow), resulting in a substitution of valine by phenylalanine at amino acid residue 219. The sequence in animals with pulverulent nuclear cataract and microphthalmia showed a homozygous G→T transversion (indicated by the arrow), resulting in a substitution of valine by phenylalanine at amino acid residue 219. Unaffected members and the healthy controls lacked this nucleotide change. (C) The partial alignments of Gja8 sequence with the corresponding segments in diverse species. The 219th valine is highly conserved in connexin 50 proteins, indicated by the arrow.

Figure 2.

Eyes of F1 intercross rats aged from six weeks one days to six weeks three days. (A) Whole eyeballs provided by Gja8^V219F/V219F homozygotes, Gja8^V219F/+ heterozygotes, and Gja8^+/+ unaffected members are shown. The cataractous eyeballs were smaller than the wild-type eyes. The difference was obvious between the Gja8^V219F/V219F eyes and Gja8^+/+ eyes. Scale bar: 1 mm. (B) The average weight of left and right eyes is shown for each genotype. All values are represented as the mean ± SD. There are no significant differences between males and females with the same genotype (P = 0.935, 0.302 and 0.560 for Gja8^V219F/V219F, Gja8^V219F/+, and Gja8^+/+, respectively) whereas the differences between any two of the three groups are significant (P < 0.001 among all pairs). Statistical differences between genders of the same genotype were calculated by two-tailed t tests. Analysis of variance testing was used to compare the statistical significance in three phenotypes while Dunnett t test was used to compare two of the three groups. ***P < 0.001; ns, not significant. P < 0.05 was considered statistically significant.

Figure 3.

Progression of lens opacity in Gja8^V219F/+ heterozygotes. The eyes of rats were photographed and the intact lenses were pictured at different time points. The opacity occurred initially around three weeks after birth. The central opacity worsened with increasing age and finally led to a totally opaque center before six weeks. White arrow indicates the clear ring-shaped zone in cataractous lenses. Scale bar: 1 mm.

Figure 4.

Histological sections of lenses obtained from 6-week-old littermates. Images A-L were stained by hematoxylin and eosin. Images M and N were stained by toluidine blue. Histological images of lenses were from Gja8^+/+ rats (A-D, M), Gja8^V219F/+ rats (E-H) and Gja8^V219F/V219F rats (I-L, N), respectively. The positions of the images are indicated in image O. (A, E, I) cover the anterior subcapsular region of the lens. (B, F, J) show the equatorial region while (C, G, K) are close to the nucleus of lens. (D, H, L, M, N) are in the lens nucleus. Asterisk indicates abnormal rounded lens fiber cells. Arrowhead marks the vacuoles distributed among fiber cells. Scale bar: 50 µm.

Figure 5.

Impaired expression of Cx50^V219F protein in cellular membranes. (A) Cx50^WT-Flag and Cx50^V219F-Flag protein were stably expressed in transfected HeLa cells. HeLa cells transfected with vector showed no Cx50-Flag expression. (B) The relative Cx50-Flag amount compared to GAPDH. Protein expression of Cx50^V219F-Flag was not significantly different from that of Cx50^WT-Flag (P = 0.700). Statistical differences were calculated by Mann-Whitney test. Each bar represents the quantification (mean ± SD) of Western blots from three independent experiments. ns, not significant. P < 0.05 was considered statistically significant. (C) Immunofluorescent imaging of Cx50^WT-Flag and Cx50^V219F-Flag in HeLa cells. Cells were immunostained with anti-Flag monoclonal antibody (red). DAPI shows nuclear DNA staining (blue). The merged panels show the superposition of the Flag and DAPI fluorescence signals. The arrows show junction plaques formed at areas of cell-cell contact and the arrowheads indicate areas of cell to cell apposition. Scale bar: 20 µm.

Figure 6.

The impaired functions of HLEB3 cells with Cx50^V219F expression. (A) Representative pictures of EdU-labeled cells. Scale bar: 250 µm. (B) The ratio of EdU-labeled cell counts to DAPI-labeled cell counts for cells transfected with Cx50^WT, Cx50^V219F or vector. The ratio of Cx50^V219F group is less than that of Cx50^WT group (P = 0.003). (C) The number of remaining cells on Matrigel-coated plates was evaluated by CCK8 assay. The adhesion pattern of Cx50^V219F group is lower than Cx50^WT (P < 0.001). (D) Cells were scraped to create wounds of similar sizes. Representative images were pictured at timepoints 0, 12 and 24 h after cell scratches. Scale bar, 250 µm. E. The wound closure ratios were calculated at each timepoint by ImageJ. The ratio of Cx50^V219F group is lower than Cx50^WT group (P < 0.001 for 12 and 24 hours, respectively). Statistical differences were calculated by two-tailed t tests. Each bar represents the quantification (mean ± SD) of three independent experiments. **P < 0.01; ***P < 0.001. P < 0.05 was considered statistically significant.

Figure 7.

Protein level of FAK and pFAK-Y³⁹⁷ in HLEB3 cells. (A) FAK protein level in HLEB3 cell clones stably transfected with Cx50^V219F-Flag, Cx50^WT-Flag or vector. Cx50^WT-Flag and Cx50^V219F-Flag protein were stably expressed in transfected HLEB3 cells. Expression of Cx50^V219F-Flag reduced protein expression of total FAK. (B) Protein level of pFAK-Y³⁹⁷ in HLEB3 cell clones stably transfected with Cx50^V219F-Flag, Cx50^WT-Flag or vector. Cx50^WT-Flag and Cx50^V219F-Flag protein were stably expressed in transfected HLEB3 cells. Expression of Cx50^V219F-Flag reduced protein expression of pFAK-Y³⁹⁷. (C) The relative FAK amount compared to GAPDH. Cx50^V219F reduced protein expression of total FAK, compared with Cx50^WT group (P = 0.044). (D) The V219F mutation reduced expression level of pFAK-Y³⁹⁷ (compared with Cx50^WT group, P < 0.001). Statistical differences were calculated by two-tailed t tests. Each bar represents the quantification (mean ± SD) of Western blots from three independent experiments. *P < 0.05; ***P < 0.001. P < 0.05 was considered statistically significant.