The Cataract-linked Mutant Connexin50D47A Causes Endoplasmic Reticulum Stress in Mouse Lenses.

Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation. Cx50D47A does not traffic properly, and homozygous mutant lenses show increased levels of the stress-responsive αB-crystallins. Therefore, we assessed whether expression of Cx50D47A led to endoplasmic reticulum (ER) stress in the lens in vivo Although pharmacologic induction of ER stress can be transduced by three different pathways, we found no evidence for activation of the IRE1α or ATF6 pathways in Cx50D47A-expressing lenses. In contrast, heterozygous and homozygous Cx50D47A lenses showed an increase in phosphorylated PERK immunoreactivity and in the ratio of phosphorylated to total EIF2α (2.4- and 3.3-fold, respectively) compared with wild type. Levels of ATF4 were similar in wild type and heterozygous lenses but elevated in homozygotes (391%). In both heterozygotes and homozygotes, levels of calreticulin protein were increased (184 and 262%, respectively), as was Chop mRNA (1.9- and 12.4-fold, respectively). CHOP protein was increased in homozygotes (384%). TUNEL staining was increased in Cx50D47A lenses, especially in homozygous mice. Levels of two factors that may be pro-survival, Irs2 and Trib3, were greatly increased in homozygous lenses. These results suggest that expression of Cx50D47A induces ER stress, triggering activation of the PERK-ATF4 pathway, which potentially contributes to the lens pathology and leads to increased expression of anti-apoptotic factors, allowing cell survival.

Connexin23 deletion does not affect lens transparency.

While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and growth, the contributions of a more recently identified lens fiber connexin, Cx23, are poorly understood. Therefore, we studied the consequences of absence of Cx23 in mouse lenses. Cx23-null mice were generated by homologous Cre recombination. Cx23 mRNA was abundantly expressed in wild type lenses, but not in Cx23-null lenses. The transparency and refractive properties of Cx23-null lenses were similar to wild type lenses when examined by darkfield microscopy. Neither the focusing ability nor the light scattering was altered in the Cx23-null lenses. While both Cx46 and Cx50 localized to appositional fiber cell membranes (as in wild type lenses), their levels were consistently (but not significantly) decreased in homozygous Cx23-null lenses. These results suggest that although Cx23 expression can influence the abundance of the co-expressed lens fiber connexins, heterozygous or homozygous expression of a Cx23-null allele does not alter lens transparency.

Roles and regulation of lens epithelial cell connexins.

The avascular lens of the eye is covered anteriorly by an epithelium containing nucleated, metabolically active cells. This epithelium contains the first lens cells to encounter noxious external stimuli and cells that can develop compensatory or protective responses. Lens epithelial cells express the gap junction proteins, connexin43 (Cx43) and connexin50 (Cx50). Cx43 and Cx50 form gap junction channels and hemichannels with different properties. Although they may form heteromeric hemichannels, Cx43 and Cx50 probably do not form heterotypic channels in the lens. Cx50 channels make their greatest contribution to intercellular communication during the early postnatal period; subsequently, Cx43 becomes the predominant connexin supporting intercellular communication. Although epithelial Cx43 appears dispensable for lens development, Cx50 is critical for epithelial cell proliferation and differentiation. Cx43 and Cx50 hemichannels and gap junction channels are regulated by multiple different agents. Lens epithelial cell connexins contribute to both normal lens physiology and pathology.

Connexin46fs380 causes progressive cataracts.

PURPOSE: Although many connexin46 (Cx46) mutants have been linked to inherited human cataracts, there are no adequate animal models for their study. The current experiments were designed to characterize the consequences of expression of one such mutant, Cx46fs380, in the mouse lens. METHODS: Mice expressing Cx46fs380 were generated by a knockin strategy. Levels and distribution of specific proteins were analyzed by immunoblotting and immunofluorescence. RESULTS: Dark-field microscopy revealed that lenses of young heterozygous and homozygous Cx46fs380 mice did not have opacities, but they developed anterior nuclear cataracts that became more severe with age. Immunofluorescence and immunoblotting showed that Cx46 was severely reduced in both heterozygous and homozygous Cx46fs380 lenses at 1 month of age, whereas immunoreactive connexin50 (Cx50) was moderately decreased. The reduction in Cx50 became more severe in older lenses. The solubilities of crystallins from young wild-type and fs380 mice were similar, but older fs380 lenses exhibited abnormalities of abundance, solubility, and modification of some crystallins. CONCLUSIONS: Major decreases in connexin levels precede the development of cataracts. These mice represent a useful model for elucidation of the progression of lens abnormalities during cataractogenesis especially as caused by a mutant connexin.

Connexin50D47A decreases levels of fiber cell connexins and impairs lens fiber cell differentiation.

PURPOSE: Substitutions of aspartate-47 (D47) of Connexin50 (Cx50) have been linked to autosomal dominant congenital cataracts in several human pedigrees. To elucidate the lens abnormalities caused by a substitution at this position, we studied No2 mice, which carry the Cx50D47A mutation and parallel the human pathology. METHODS: Lenses from mice of different ages (neonatal to 4 months) were examined by dark-field and immunofluorescence microscopy. Protein levels were determined by immunoblotting using primary antibodies directed against connexins, other membrane proteins, crystallins, and proteins residing in different organelles. RESULTS: Lenses of both heterozygous and homozygous Cx50D47A mice had cataracts and were smaller than those of wild-type littermates. Levels of Cx50 were severely reduced in mutant animals as compared with those in wild-type mice (<20% in heterozygotes and ≤3% in homozygotes). Levels of Cx46 and aquaporin0 were also decreased, but to a lesser extent. The immunostaining pattern of lens connexins was altered in mutant animals. The lenses of Cx50D47A mice showed persistence of nuclear remnants in deep regions of the lens and elevated levels of H3 histone and the mitochondrial protein, Tom20. γ-Crystallin levels were decreased in lenses of all mutant mice, and ß-crystallins were reduced in homozygotes. CONCLUSIONS: These data suggest that mice expressing Cx50D47A develop cataracts due to a severe decrease in the abundance of functional connexin channels. They also implicate Cx50 in fiber cell differentiation, since mutant lenses showed impaired degradation of organelles and decreased levels of some crystallins.