Physiological and Optical Alterations Precede the Appearance of Cataracts in Cx46fs380 Mice

Abstract

Purpose: Cx46fs380 mice model a human autosomal-dominant cataract caused by a mutant lens connexin46, Cx46. Lenses from Cx46fs380 mice develop cataracts that are first observed at ∼2 months in homozygotes and at ≥4 months in heterozygotes. The present studies were conducted to determine whether Cx46fs380 mouse lenses exhibited abnormalities before there are detectable cataracts.

Methods: Lenses from wild-type and Cx46fs380 mice were studied at 1 to 3 months of age. Connexin levels were determined by immunoblotting. Gap junctional coupling was calculated from intracellular impedance studies of intact lenses. Optical quality and refractive properties were assessed by laser scanning and by photographing a 200-mesh electron microscopy grid through wild-type and Cx46fs380 mouse lenses.

Results: Connexin46 and connexin50 levels were severely reduced in mutant lenses. Gap junctional coupling was decreased in differentiating and mature fibers from Cx46fs380 lenses; in homozygotes, the mature fibers had no detectable coupling. Homozygous lenses were slightly smaller and had reduced focal lengths. Heterozygous and homozygous lenses significantly distorted the electron microscopy grid pattern as compared with wild-type lenses.

Conclusions: Before cataract appearance, Cx46fs380 lenses have decreased gap junctional conductance (at least in heterozygotes) and alterations in refractive properties (heterozygotes and homozygotes). The decreased focal distance of Cx46fs380 homozygous lenses is consistent with an increase in refractive index due to changes in cellular composition. These data suggest that Cx46fs380 lenses undergo a sequence of changes before the appearance of cataracts: low levels of connexins, decreased gap junction coupling, alterations in lens cell homeostasis, and changes in refractive index.

Figure 1

Cx46fs380 expression affects lens impedance. (A–C) Magnitude and phase angle of the impedance of intact lenses from wild-type (A), Cx46fs380 heterozygous, (B) and Cx46fs380 homozygous (C) mice recorded in differentiating fiber cells. The smooth curves are the best fits of Equations 1 and 2 to the data.

Figure 2

Gap junctional conductance is decreased in Cx46fs380 lenses. (A–C) Series resistance (R_s) data graphed as a function of normalized radial distance from the lens center (r/a) recorded from wild-type (A), heterozygous mutant, (B) and homozygous mutant (C) lenses. The effective resistivities, R_DF and R_MF, were determined by fitting Equation 3 to the data. (D) Comparison of the data from the three types of lenses. The inset shows an expanded view of the data in the differentiating fiber cells (r/a ≥ 0.85).

Figure 3

Levels of Cx46 and Cx50 are significantly decreased in Cx46fs380 lenses. (A, B) Immunoblots show the levels of immunoreactive Cx46 and Cx50 in total lens homogenates from 2-month-old wild-type (+/+) and Cx46fs380 heterozygous (+/fs380) and homozygous (fs380/fs380) mice. (C, D) Graphs show the densitometric values of the bands obtained in three independent experiments expressed as percentages of the values obtained in wild-type animals. Significant differences between wild-type and heterozygous Cx46fs380 or wild-type and homozygous Cx46fs380 lenses are indicated by asterisks (P < 0.05).

Figure 4

Examples of laser scan profiles from wild-type (+/+), and Cx46fs380 heterozygous (+/fs380) and homozygous (fs380/fs380) mouse lenses. The pathway of the laser beam (lines) and the data points (+) represent the focal point of each beam. The average BVD (white asterisk) was calculated from averaging the values of each of these data points, whereas the scatter of the points around the average is the variability in the BVD.

Figure 5

Expression of Cx46fs380 affects the refractive properties of the lens before the appearance of cataracts. Images were obtained by photographing lenses from 1- and 3.2-month-old wild-type (+/+) and Cx46fs380 heterozygous (+/fs380) and homozygous (fs380/fs380) mouse lenses against an electron microscopy grid. The pattern appears distorted at 3.2 months in heterozygotes and at both 1 and 3.2 months in homozygotes. Diagrams of the grid pattern and the barrel and pincushion deformations are shown at the bottom.

Figure 6

The warping index of the lens is altered in Cx46fs380 lenses before cataract appearance. The graph shows the warping index of wild-type (+/+), and mutant heterozygous (+/fs380) and homozygous (fs380/fs380) lenses from 1-month-old mice. The asterisks indicate significant differences between Cx46fs380 heterozygous or homozygous lenses versus wild-type lenses (P < 0.05).

Figure 7

Homozygous Cx46fs380 mice have slightly smaller lenses. (A, B) The graphs show the equatorial (A) and anteroposterior (B) diameters of wild-type (+/+), heterozygous (+/fs380), and homozygous (fs380/fs380) lenses from 1.6-month-old mice. (C, D) The graphs show the equatorial (C) and anteroposterior (D) diameters of wild-type (+/+) and homozygous (fs380/fs380) lenses from 6.4-month-old mice.