Abstract
Purpose: :
The R120G mutation in alpha B-crystallin causes desmin-related myopathy and cataracts. The mechanism of cataract and myopathy disease is not understood. Autophagy is a fundamental cellular process in which protein aggregates, excess and/or damaged organelles are cleared from the cell. We examined whether mutant alphaB-crystallin protein aggregates induce autophagy in a knock-in mouse model for the alpha B-crystallin R120G mutation linked with cataract and myopathy. In this study we assessed basal and rapamycin-induced autophagy in this mouse model.
Methods: :
AlphaB-R120G knock-in mice were bred to generate wild type, heterozygous and homozygous mutant mice. Adult mice at ages 3 months and 12 months were analyzed. In each age group, 8 mice each of wild type, R120G heterozygous and R120G homozygous mutant were used. Mice were weighed prior to injection and intraperitoneally injected daily for a week with rapamycin (10 µg/g body weight) and colchicine (16 µg/g body weight). Vehicle only and colchicine plus vehicle were used as controls. Skeletal muscle and lenses were extracted and homogenized in RIPA buffer. Autophagy was assessed by western blotting with antibodies to LC3 and p62 proteins.
Results: :
AlphaB-R120G homozygous mutant mice that were injected with rapamycin plus colchicine displayed a 40% increase in lens LC3-II expression compared to similarly injected wild type mice, indicating an increase in rapamycin-induced autophagy. Expression of p62 in homozygous mouse lenses was increased by 36% when treated with rapamycin plus colchicine over that of wild type control levels. This increase in p62 also suggests an increase in autophagy in homozygous mutant lenses. Older αB-R120G mutant mouse lenses showed a decrease in basal autophagy as compared with wild type controls. Analyses of skeletal muscle indicated that autophagy was stimulated by rapamycin plus colchicine in wild type mice. The mutation induced autophagy to become highly upregulated even in the absence of rapamycin, indicating that basal autophagy is upregulated in skeletal muscle of αB-R120G mutant mice.
Conclusions: :
These studies suggest that autophagy is modulated by the αB-R120G mutation in lens and skeletal muscle. Autophagy may play a significant role in pathology caused by this mutation.
Keywords: chaperones • cataract • protein structure/function