In vivo lens deficiency of the R49C alphaA-crystallin mutant

Abstract

The R49C mutation of alphaA-crystallin (alphaA-R49C) causes hereditary cataracts in humans; patients in a four-generation Caucasian family were found be heterozygous for this autosomal dominant mutation. We previously generated knock-in mouse models of this mutation and found that by 2 months of age, heterozygous mutant mice exhibited minor lens defects including reduced protein solubility, altered signaling in epithelial and fiber cells, and aberrant interactions between alphaA-crystallin and other lens proteins. In contrast, homozygous mutant alphaA-R49C knock-in mice displayed earlier and more extensive lens defects including small eyes and small lenses at birth, death of epithelial and fiber cells, and the formation of posterior, nuclear, and cortical cataracts in the first month of life. We have extended this study to now show that in alphaA-R49C homozygous mutant mice, epithelial cells failed to form normal equatorial bow regions and fiber cells continued to die as the mice aged, resulting in a complete loss of lenses and overall eye structure in mice older than 4 months. These results demonstrate that expression of the hereditary R49C mutant of alphaA-crystallin in vivo is sufficient to adversely affect lens growth, lens cell morphology, and eye function. The death of fiber cells caused by this mutation may ultimately lead to loss of retinal integrity and blindness.

Figure 1

Histology of eyes from wild type and αA-R49C mutant mice. Above: Eyes from 6-day-old wild type (A), heterozygous (B), and homozygous mutant (C) mice. Below: Eyes from 4-month-old wild type (D), heterozygous (E), and homozygous mutant (F) mice. Mid-sagittal sections were stained with hematoxylin and eosin. At least three sections were analyzed for each age and genotype. Scale bar in (F) = 1 mm for each image (A–F).

Figure 2

Ki67 labeling of interior lens fiber cells in αA-R49C homozygous mutant mice. (A) Lens fiber cells showed significant Ki67 labeling (red arrows) in homozygous mutant mice, but not in wild type or heterozygous mice. (B) The histogram shows the number of Ki67-labeled cells present in each 15° sector normalized against the total number of Ki67-labeled cells in the lens. The data are representative of three mid-sagittal sections per lens, for at least two lenses of each genotype. Overlayed lines in (A) indicate the boundaries of the 15° sectors used for quantifying the angular distribution of labeled cells given in (B). Note that the homozygous mutant lens was labeled in the interior fiber cells (A, B), whereas wild type and heterozygous lenses were both labeled in only in the equatorial region (B).

Figure 3

Failure to form the normal epithelial bow region in 160-day-old αA-R49C homozygous mutant mice. Homozygous mutant lenses lacked the normal equatorial bow region seen in the wild type lenses. Posterior migration of epithelial cell nuclei in R49C homozygous mutant lenses: (A) low magnification image of the homozygous mutant eye, (B) epithelial cell nuclei at the equator, (C) epithelial cell nuclei migrating along the posterior capsule, and (D) epithelial cells at the posterior pole. (B, inset) The inset shows the lens bow region of an age-matched wild-type lens. Arrows indicate migration of nuclei from the equator towards the posterior of the lens.