Abstract
We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization.MethodsLenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.ResultsCompared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice.ConclusionsThe results show that an increased lens membrane association of αAN101D-−relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.
Highlights
The cornea is the primary refractive tissue performing 70–80% of refraction of the eye, the major function of the lens is in accommodation and to partly help in the refraction
Age-related protein Insolubilization in lenses of CRYαAN101D and Wild-type alpha-A crystallins (CRYαAWT) mice To determine at what age there is change in the protein profiles in lenses of CRYαAN101D and CRYαAWT mice occurred, a comparative analysis of water soluble (WS)-proteins and water insoluble (WI)-proteins from the lenses of the two types of mice of different ages was carried out (Fig. 1)
The results suggested a greater aggregation with higher Mr of the HMW-proteins in CRYαAN101D lenses compared to the identical aged CRYαAWT lenses
Summary
The cornea is the primary refractive tissue performing 70–80% of refraction of the eye, the major function of the lens is in accommodation and to partly help in the refraction. Additional cataract-causative factors are identified, which include mutations in crystallins [10], oxidative insults of crystallins, the loss of redox balance of glutathione [11], extensive truncations of α-, β-, and γ-crystallins [12,13,14,15,16,17,18,19,20], a variety of post-translational modifications with deamidation as being the most abundant [21,22,23,24,25], and the loss of membrane integrity [7, 26, 27] These factors individually or in combination cause lens opacity through altered lens cellular structures and contents, ionic imbalance, increased water and oxygen levels, loss of natural interactions among crystallins, and crystallins’ unfolding, degradation and cross-linking
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