Unbalanced redox status network as an early pathological event in congenital cataracts

Abstract

The lens proteome undergoes dramatic composition changes during development and maturation. A defective developmental process leads to congenital cataracts that account for about 30% of cases of childhood blindness. Gene mutations are associated with approximately 50% of early-onset forms of lens opacity, with the remainder being of unknown etiology. To gain a better understanding of cataractogenesis, we utilized a transgenic mouse model expressing a mutant ubiquitin protein in the lens (K6W-Ub) that recapitulates most of the early pathological changes seen in human congenital cataracts. We performed mass spectrometry-based tandem-mass-tag quantitative proteomics in E15, P1, and P30 control or K6W-Ub lenses. Our analysis identified targets that are required for early normal differentiation steps and altered in cataractous lenses, particularly metabolic pathways involving glutathione and amino acids. Computational molecular phenotyping revealed that glutathione and taurine were spatially altered in the K6W-Ub cataractous lens. High-performance liquid chromatography revealed that both taurine and the ratio of reduced glutathione to oxidized glutathione, two indicators of redox status, were differentially compromised in lens biology. In sum, our research documents that dynamic proteome changes in a mouse model of congenital cataracts impact redox biology in lens. Our findings shed light on the molecular mechanisms associated with congenital cataracts and point out that unbalanced redox status due to reduced levels of taurine and glutathione, metabolites already linked to age-related cataract, could be a major underlying mechanism behind lens opacities that appear early in life.

Keywords: Cataracts; Glutathione; Proteomics; Redox status; Taurine; Ubiquitin.

Graphical abstract

Fig. 1

Cataract formation in K6W-Ub lenses. Photographs of dissected WT and K6W-Ub lenses at E15, P1, and P30 show nuclear cataracts (arrows) in K6W-Ub lenses.

Fig. 2

Developmental proteomic changes in the lens overlap with K6W-Ub proteomic alterations. Comparison of DEPs between WT and K6W-Ub at P1 (X-axis) versus developmental changes in WT lenses between E15 and P1 (Y-axis). Dashed axis lines mark boundaries of statistical significance. Colored dots represent different functional groups of proteins.

Fig. 3

Quantitative proteomics reveals changes in amino acid and glutathione metabolism in congenital cataracts. (A–C) Grid visualization of downregulated DEPs identified in K6W-Ub lenses at (A) E15, (B) P1, and (C) P30 and. (D–F) Grid visualization of upregulated DEPs identified in K6W-Ub lenses at (D) E15, (E) P1, and (F) P30. Shown in columns are the top 10 associated KEGG pathways, which are ordered by p-values. KEGG pathways involved in glutathione and amino acid metabolism (highlighted in red). Rows indicate DEPs, based on comparisons of K6W-Ub relative to WT. In A-C, blue shades indicate the degree of downregulation in K6W-Ub lenses, with darker shades corresponding to more downregulation, according to the key next to each grid. In D-F, red shades indicate the degree of upregulation in K6W-Ub lenses, with darker shades corresponding to more upregulation, according to the key next to each grid. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

CMP reveals altered spatiotemporal expression patterns for free amino acids in congenital cataracts. (A) Representative CMP pictures of WT and K6W-Ub lenses collected at E15 (top), P1 (middle), and P30 (bottom) and stained against alanine, cysteine, aspartate, lysine, glutamate, glutamine, glycine, arginine, glutathione, valine, taurine, and serine. (B) Metabolic signatures of WT and K6W-Ub lenses collected at P1 and colored according to metabolic classes derived from K-means clustering. (C) Partial least squares regression analysis discriminated metabolomes of WT (green) and K6W-Ub (red) lenses at all ages. (D) Variable importance in projection (VIP) scores for different amino acids responsible for the separation in the regression plot. All amino acids were higher in WT than K6W-Ub except for cysteine. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Changes in abundance and spatial distributions of taurine and glutathione in the WT and K6W-Ub lenses. (A–C) Representative CMP pictures for taurine staining in WT and K6W-Ub lenses collected at (A) E15, (B) P1, and (C) P30. (D–F) Representative CMP pictures for glutathione staining in WT and K6W-Ub lenses collected at (D) E15, (E) P1, and (F) P30. Quantification of average intensities is shown as arbitrary units (a.u.). All values are mean ± SEM. and differences with WT were significant for (*) p < 0.05; (**) p < 0.01; and (***) p < 0.001.

Fig. 6

HPLC revealed altered levels of amino acids in K6W-Ub lenses. (A–F) Lens content of (A) taurine, (B) GSH/GSSG, (C) cysteine, (D) leucine, (E) tryptophan and (F) isoleucine in wild types and K6W-Ub lenses collected at P1 (left) and P30 (right) are shown. All values are mean ± SEM. and differences with WT were significant for (*) p < 0.05; (**) p < 0.01; and (***) p < 0.001. Sample size is n = 8.

Fig. 7

Downregulated proteins in K6W-Ub lenses associated with glutathione and amino acids metabolism. (A–B) Representative images of immunofluorescence (A) in E15 and P1 WT and K6W-Ub lenses and (B) Western blotting of lysates of P1 WT and K6W-Ub lenses using antibodies against γ-glutamylcyclotransferase (GGCT). (C–D) Representative images of immunofluorescence (C) in E15 and P1 WT and K6W-Ub lenses and (D) Western blotting of lysates of P1 lenses using antibodies against glutathione s-transferase pi 1 (GSTP1). (E–F) Representative immunoblot against heme oxygenase 1 (HMOX1) (E), guanidinoacetate N-methyltransferase (GAMT) (E), and phosphoglycerate mutase 2 (PGAM2) (F) in P30 WT and K6W-Ub lenses. Actin-normalized densitometric quantitation is shown for each sample. For all Western blots, two biological replicates are shown.