Functional characterization of Cat(Tohm), a mouse AQP0 mutation that causes oxidative stress, cytotoxicity, dominant congenital lens cataract and microphthalmia.

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.