Functional characterization of CatTohm, a mouse AQP0 mutation that causes oxidative stress, cytotoxicity, dominant congenital lens cataract and microphthalmia

Abstract

A natural AQP0 mutation, Cat^Tohm, resulted in smaller eyes, and lenses with bilateral dominant cataracts in mice. Our objective was to characterize this mutation and explore the possible reasons for Cat^Tohm causing dominant cataracts. We studied lens morphology, transparency, functional alterations and cytotoxicity. Lens morphology and nuclear fiber cell organization were severely affected. Water permeability (Pw) of oocytes expressing Cat^Tohm-AQP0 cRNA (12 ± 2 μm/s) reduced markedly (P < 0001) compared with WT-cRNA-expressing oocytes (25 ± 2 μm/s); co-expression of both cRNAs decreased the Pw significantly (20 ± 3 μm/s; P < 0.001). Pw of membrane vesicles of heterozygous (16 ± 4 μm/s), or homozygous (7 ± 3 μm/s) fiber cells was considerably lower (P < 0.001) than that of the WT (37 ± 6 μm/s). The hydrogen peroxide permeability of the Cat^Tohm lens was remarkably lesser (P < 0.0001) than that in the WT. The oxidative stress test revealed a significant (P < 0.001) increase in Reactive Oxygen Species in Cat^Tohm lenses. In oocytes and cultured cells, transfected WT-AQP0 trafficked and expressed at the plasma membranes; mutant Cat^Tohm-AQP0 protein remained in the cytoplasm, and partly co-localized with the WT-AQP0. Cells transfected with Cat^Tohm-AQP0 showed more necrosis than apoptosis. The cultured cells expressing mutant AQP0, or ex vivo cultured lenses of Cat^Tohm displayed a substantial (P < 0.001) rise in the discharge of lactate dehydrogenase in the culture medium, corroborating necrosis. A transgenic mouse lens expressing Cat^Tohm mutant AQP0 along with the WT-AQP0 had more severe microphthalmia than that of Cat^Tohm mouse. Overall, the Cat^Tohm mutation exerted a dominant negative effect affecting protein localization and functionality, and causing cellular stress, necrosis, lens cataracts and microphthalmia.

Keywords: AQP0; Cat(Tohm); Congenital lens cataract; Cytotoxicity; Dominant negative effect; H(2)O(2) transport; Lens; Membrane water permeability; Microphthalmia; Oxidative stress; Tg4.