Abstract
Mouse Cx50D47A and human Cx50D47N are non-functional connexin mutants that cause dominantly-inherited cataracts. In tissue culture expression experiments, they both exhibit impaired cellular trafficking and gap junction plaque formation. Lenses of mice expressing Cx50D47A have cataracts, reduced size, drastically decreased levels of connexin50, and less severely reduced levels of connexin46. The PERK-dependent pathway of the ER response to misfolded proteins is activated, and they have impaired differentiation with retained cellular organelles. Since treatments that enhance protein folding improve trafficking and plaque formation by Cx50D47N and other mutant connexins in vitro, and they are successful therapeutics for some other diseases caused by misfolded proteins, we tested the efficacy of the chemical chaperone, 4-phenylbutyrate (4-PBA) in cultured cells and mice expressing Cx50D47A. 4-PBA treatment increased the formation of Cx50D47A-containing plaques at appositional membranes of transiently transfected HeLa cells. Heterozygous Cx50D47A mice were treated with 4-PBA by addition to the drinking water and parenteral injection of pregnant mice (starting 10 days after pairing of males and females) and their pups. Lenses from 1-month-old mice were examined by darkfield illumination and immunofluorescence microscopy. Protein levels were determined by immunoblotting. Cataract size and density were not detectably different between the control and the 4-PBA-treated groups. Lens size was not increased following treatment. Levels of connexin46 and connexin50 were significantly increased in lenses of 4-PBA-treated mice compared with saline-treated animals. Immunofluorescence showed an increased abundance of connexin46 immunoreactivity and puncta. The ratio of phosphorylated to total EIF2α was not altered, and levels of organellar proteins were not significantly reduced, suggesting that the ER response to misfolded proteins and differentiation were not changed. Thus, treatment with 4-PBA improved critical pathological issues in these mice (low connexin and gap junction abundance), but the magnitude of this recovery (especially for Cx50) was inadequate to impact the reduced size or the opacification of Cx50D47A lenses.