Do connexin mutants cause cataracts by calcium precipitation?

Abstract

Purpose : Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. In mice, expression of Cx46fs380 leads to decreased gap junctional coupling, and formation of calcium precipitates with opacities of similar distributions. To test whether Cx50 mutants also cause cataracts by calcium precipitation, we studied the lens circulation, intracellular concentration of free calcium ions ([Ca²⁺]_i), cataract morphology and calcium precipitate distribution in lenses from Cx50D47A mice.

Methods : Lenses from wild type and Cx50D47A mice were studied at 3-10 weeks of age. Connexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Refractive properties were assessed by photographing a 200-mesh electron microscopy grid through the lenses. [Ca²⁺]_i was measured using Fura-2 and fluorescence imaging. The presence of calcium precipitates was assessed by whole mount Alizarin red staining and compared to the distribution of opacities in darkfield images.

Results : Levels of Cx50 and Cx46 in Cx50D47A lenses were 5% and 55% in heterozygotes and 0.27% and 16% in homozygotes as compared to wild type. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% in heterozygotes and 29% in homozygotes, and in mature fibers 24% in heterozygotes and 4% in homozygotes compared to wild type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. Heterozygous and homozygous lenses distorted the electron microscopy grid pattern as compared to wild type lenses. [Ca²⁺]_i was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses, and corresponded closely to their cataracts as visualized by darkfield microscopy.

Conclusions : Expression of Cx50D47A altered the internal circulation of the lens by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calcium ions accumulated, precipitated and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates that correspond to the cataracts.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.