Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability

Abstract

The refractivity and transparency of the ocular lens is dependent on the stability and solubility of the crystallins in the fiber cells. A number of mutations of lens crystallins have been associated with dominant cataracts in humans and mice. Of particular interest were γB- and γD-crystallin mutants linked to dominant cataracts in mouse models. Although thermodynamically destabilized and aggregation-prone, these mutants were found to have weak affinity to the resident chaperone α-crystallin in vitro To better understand the mechanism of the cataract phenotype, we transgenically expressed different γD-crystallin mutants in the zebrafish lens and observed a range of lens defects that arise primarily from the aggregation of the mutant proteins. Unlike mouse models, a strong correlation was observed between the severity and penetrance of the phenotype and the level of destabilization of the mutant. We interpret this result to reflect the presence of a proteostasis network that can "sense" protein stability. In the more destabilized mutants, the capacity of this network is overwhelmed, leading to the observed increase in phenotypic penetrance. Overexpression of αA-crystallin had no significant effects on the penetrance of lens defects, suggesting that its chaperone capacity is not limiting. Although consistent with the prevailing hypothesis that a chaperone network is required for lens transparency, our results suggest that αA-crystallin may not be efficient to inhibit aggregation of lens γ-crystallin. Furthermore, our work implicates additional inputs/factors in this underlying proteostasis network and demonstrates the utility of zebrafish as a platform to delineate mechanisms of cataract.

Keywords: aggregation; cataract; chaperone; crystallin; lens; proteostasis; small heat shock protein (sHsp); zebrafish.

FIGURE 1.

Transgenic expression of human CRYGD mutants induce lens defects in 4-dpf zebrafish embryos. A, embryos of lens-specific Hsa.CRYGD transgenes displayed Cerulean marker in the heart. Panel a, an overlay image of DIC and Cerulean fluorescence. Panel b, a white arrow marks the Cerulean marker in the heart. B, comparison of percentage of embryos showing lens defects between WT and three Hsa.CRYGD transgenes: I4F, V76D, and I4/F/V76D.

FIGURE 2.

The lens defects and reflectance in zebrafish embryos expressing human CRYGD mutants. A, compared with WT siblings (non-transgenic carriers), embryos carrying Tg(cryaa:Hsa.CRYGD_I4F) and Tg(cryaa:Hsa.CRYGD_I4F/V76D) at 4 dpf exhibited various degrees of lens defects (panels d–f, arrows), as well as changes in reflectance (panels a–c). B, reflectance analysis of WT lens and Hsa.CRYGD_I4F and Hsa.CRYGD_I4F/V76D lens at 4 dpf suggested that the lens of embryos expressing γD-crystallin variants scattered more lights than WT lens (t test; p < 0.001). C, histological sections of the lens in Tg(cryaa:Hsa.CRYGD_I4F/V76D) embryos stained by toluidine blue showed distinct dark inclusion bodies (panels b and d) compared with WT siblings (panels a and c). Panels a and b, transverse section; panels c and d, sagittal section.

FIGURE 3.

Destabilized γd-crystallin proteins forms aggregation in the lens. A, panel a, schematic of the experiment utilizing the Gal4/UAS targeted gene expression system. Double transgenic line, Tg[cryaa:Gal4]; Tg[UAS:GFP], males were outcrossed to AB females. Fertilized zygotes were injected with tol2 mRNA and UAS responder Tol2 constructs expressing fluorescently tagged human γD-crystallin (Hsa.CRYGD-mCherry), including wild type and three variants: I4F, V76D, and I4F/V76D double mutant. Panel b, embryos possessing lens fibers positive for mCherry expression were selected and imaged at 4 dpf. Panel b′, examples of green lens (from UAS:GFP) and mosaic expression of mCherry-tagged γD-crystallin. B, mosaic expression of mCherry-tagged human γD-crystallins, wild type (panels a–c) and I4F/V76D double mutant (panels d–f). Apparent mCherry punctuates/aggregates were observed in the lens expressing Hsa.CRYGD_I4F/V76D-mCherry (white arrow in panels e and f), but not in those expressing wild-type γD-crystallin construct (mostly shown a diffused and elongated pattern, which is consistent with the shape of lens fiber cells). DIC images (panels a and d), fluorescent images (red channel; panels b and e), and merged images (panels c and f) are shown. C, the frequency of embryos showing mCherry punctuates in the lens (wild type and three variants: I4F, V76D, and I4F/V76D double mutant).

FIGURE 4.

Human CRYGD mutants show different solubility in adult lens. In the 15-month-old adult lens analyzed by iTRAQ quantitative proteomics, there was a significantly increased amount of human γD-crystallin peptides detected in the WIF in the fish lens expressing the γD-crystallin I4F/V76D compared with I4F, based on the ratio between ΔWIF and ΔWSF. The ΔWIF and ΔWSF from each sample (γD-crystallin I4F/V76D and γD-crystallin I4F) were calculated as fold changes of the expression level relative to the lens expressing γD-crystallin WT.

FIGURE 5.

Percentages of the lens defects induced by human CRYGD_I4F expression at 4-dpf embryos were not suppressed by exogenous expression of αA-crystallin but were exacerbated by reducing dosage of endogenous αA-crystallin. A, adult Tg(cryaa:Hsa.CRYGD_I4F/V76D) and Tg(cryaa:Rno.Cryaa) transgenic zebrafish lines were crossed. The lens defects of resulting embryos (I4F/V76D) were examined and classified into three severity classes as previously mentioned. The proportion of I4F/V76D embryos showing lens defects showed no significant changes with or without the presence of Rno.Cryaa transgene. B, a significantly higher proportion of Tg(cryaa:Hsa.CRYGD_I4F) embryos showed major lens defects when losing one copy of cryaa (I4F; cryaa^+/−; ∼39%), compared with those in wild-type background (I4F; ∼14%), as well as to the non-transgenic cryaa^+/− or cryaba^+/− embryos.

FIGURE 6.

Denucleation failure or apoptotic cell death was not induced by transgenic expression of human CRYGD mutants. Unlike in cloche mutant lens, an incomplete denucleation process was clearly observed; either by nuclear-localized H2B-GFP visualization (C) or by DAPI staining (F), the lens of Tg(cryaa:Hsa.CRYGD_I4F/V76D) embryos (B and E) showed no signs of denucleation failure and appeared similar to WT siblings (A and D). G and H, the TUNEL staining revealed no significant increase of apoptosis in Tg(cryaa:Hsa.CRYGD_I4F/V7D) embryos compared with WT siblings.