Abstract
Purpose: :
GSH is the most abundant anti-oxidative compound found in the lens. It fulfils numerous cellular functions, including protection against oxidative stress, control of cellular redox potential and detoxification of xenobiotics. In age-related nuclear cataract (ARNC), GSH levels are impaired and accompanied with increased oxidation, protein disulfide formation and protein cross-link. Last year at ARVO, we reported preliminary data regarding our lens conditional Gclc knockout mouse, a key enzyme in GSH de novo synthesis. Here we present follow-up studies
Methods: :
The LEGSKO mouse was created by crossbreeding of Gclcflox/flox with MRL-10 Cre mouse. The mouse was continuously bred into C57BL/6 background. The lens opacification and cataract formation were monitored by Slit-lamp. Lens protein disulfide formation was determined by 2-D SDS-PAGE gel and the proteins were identified by tandem mass sequencing. Gamma crystallin and its disulfide crosslink were confirmed by western-blot. Gene expression was determined by real-time PCR.
Results: :
Homozygous knockout mouse had a complete block of Gclc mRNA and protein production. The GSH level dropped 50-70% compared to wild type lenses. 30 homozygous LEGSKO mice and 30 age- matched wild type control mice were monitored for lens opacification and cataract formation using Slit-lamp. 4 of 30 mice lenses were found with light nuclear opacification at 4 mos, and those mice quickly progressed to nuclear cataract at 7mos, while no wild type mice lenses were found any degree of opacification at age of 9mos. At age of 8mos, 16 mice demonstrated nuclear cataract and 12 mice diagnosed with various degree of lens opacification. There are significant numbers of lens inter-protein disulfide in LEGSKO mice compared to age-matched wild type mice at 4mos, and such crosslink increased with advancing age. Moreover, the changes in the 4mos LEGSKO were similar with those in 24 months old wild type lens. Major proteins found in disulfide bonds were alpha A, alpha B, beta B1, B2 and gamma crystallin. Interestingly young wild type and LEGSKO mice lens crystallin formed mostly intramolecular disulfide bonds. With aging, these intra-disulfide bonds disappeared, and were converted into protein-protein crosslinks. These results indicate that intermolecular crystallin disulfides play a critical role in ARNC via oxidation.
Conclusions: :
Homozygous LEGSKO mouse, like old human lenses, have 50% lenticular GSH levels and develop ARNC that is characterized by protein-protein disulfides. This animal model is expected to be very fruitful for the development of pharmacological agents against ARNC.
Keywords: cataract • aging • oxidation/oxidative or free radical damage