Activation of the unfolded protein response by a cataract-associated αA-crystallin mutation

Abstract

αA-crystallin is a lens chaperone that plays an essential role in the transparency and refractive properties of the lens. Mutations in αA-crystallin have been associated with the development of hereditary cataracts. The R49C mutation of αA-crystallin (αA-R49C) was identified in a four-generation Caucasian family with hereditary cataracts. The αA-R49C protein forms larger-than-normal oligomers in the lens and has decreased solubility. This aberrant αA-R49C oligomerization suggests that protein folding is altered. However, whether activation of the unfolded protein response (UPR) occurs during crystallin mutation-induced cataract formation and whether the UPR causes cell death under these conditions is unclear. We investigated UPR activation in an in vivo mouse model of αA-R49C using immunoblot analysis of lens extracts. We found that expression of the endoplasmic reticulum (ER) chaperone, BiP, was 5-fold higher in homozygous αA-R49C lenses than in wild type lenses. Analysis of proteins typically expressed during the UPR revealed that ATF-4 and CHOP levels were also higher in homozygous lenses than in wild type lenses, while the opposite was true of ATF-6 and XBP-1. Taken together, these findings show that mutation of αA-crystallin induces activation of the UPR during cataract formation. They also suggest that the UPR is an important mediator of cell death observed in homozygous αA-R49C lenses.

Fig. 1

Histology of wild type, αA-R49C heterozygous mutant, and αA-R49C homozygous mutant eye lenses. (A) Wild type lens, (B) αA-R49C heterozygous lens, and (C) αA-R49C homozygous lens. Note that the wild type and heterozygous mutant lenses were similar in size, but the αA-R49C homozygous lenses were smaller and had a highly disrupted morphology.

Fig. 2

Immunoblot analysis of UPR-associated proteins in wild type, heterozygous αA-R49C (R49C Het), and homozygous αA-R49C (R49C Homo) lenses. (A) BiP expression. Note that BiP upregulation was detected in both the water-soluble and water-insoluble fractions of homozygous mutant lenses. The protein level was quantified by densitometry (see graph to right). (B) ATF-6 expression in the insoluble fraction. Loss of ATF-6 expression (50-kDa band) was accompanied by an increase in a 20-kDa species in homozygous αA-R49C lenses. (C) XBP-1 expression in the insoluble fraction. Note the loss of XBP-1 expression in αA-R49C homozygous mutant lenses. (D) CHOP expression in the soluble fraction. CHOP expression was 5-fold greater in homozygous αA-R49C lenses than in wild type lenses. (E) ATF-4 expression in the soluble fraction. Discrete bands with molecular weights between 55 kDa and 170 kDa were observed only in αA-R49C homozygous lenses. For each gel, equal amounts of wild type, αA-R49C heterozygous, and αA-R49C homozygous lens proteins were loaded in each lane. Error bars represent standard deviations from the mean of two to five experiments. Statistical analysis was performed by t-test. Asterisks indicate statistical significance with p ≤values 0.05.

Fig. 3

Immunofluorescence labeling for BiP in wild type and αA-R49C homozygous lenses. (A) Equatorial region of a wild type lens at 40× magnification. BiP labeling (red) was localized primarily to the region around the nuclei (stained with DRAQ5, blue) in lens epithelial cells. Some labeling was detected around the nuclei just before nuclear breakdown (arrow). (B–D) BiP labeling in the equatorial region (B), anterior region (C), and posterior region (D) of an αA-R49C homozygous lens. All images are 40× magnification. BiP was localized in lens epithelial and fiber cells (arrows). Bar = 20 μm for all images.