An Autosomal Dominant Mutation in AlphaB-Crystallin Causes Cataracts and Myopathy in Mice

Abstract

Purpose: : An autosomal dominant missense mutation in αB-crystallin (αB-R120G) causes cataracts and desmin-related myopathy. We analyzed knock-in mice carrying the R120G mutation to examine development of lens opacities and myopathy in these mice.

Methods: : Knock-in mice were generated by homologous recombination in 129SvJ male embryonic stem (ES) cells (SCC-10), modifying the αB-crystallin gene (cryab) such that exon 3 contained the R120G mutation in one allele, while the second copy of the gene was wild type. Cataract formation was scored by slit lamp biomicroscopy. High performance liquid chromatography (HPLC)-gel permeation chromatography was performed to determine light scattering and molecular mass changes in crystallins. Lens proteins were analyzed by 2-DIGE. A co-immunoprecipitation assay was used to investigate the effect of the αB-R120G mutation on the association of vimentin with αB-crystallin. Quantitative grip strength measurements, αB-crystallin solubility and muscle histology were analyzed to assess muscle changes.

Results: : αB-R120G knock-in mice developed cataracts, myopathy, weakness and loss of αB-crystallin solubility. Cataract severity increased with age and mutant gene dosage. The total mass of α-crystallin oligomer increased from 1.13 x 10⁶ Da in wild type to 2.01 x 10⁶ Da in heterozygous and 2.85 x 10⁶ Da in homozygous 4-month old lenses. The hydrodynamic radius of α-crystallin increased from 10.1 nm in wild type to 13.5 nm in heterozygous and 14 nm in homozygous mutant lenses. Light scattering of the α-crystallin fraction as well as the interaction of αB-crystallin with vimentin increased in mutant lenses, whereas the αB-crystallin solubility decreased. 2D-DIGE suggested that the pI of αB-R120G was shifted to more acidic range. The mass of the β-crystallin fraction also increased in mutant lenses. Forelimb grip strength was significantly lower in 6-month-old homozygous mutant mice compared with wild type controls. H&E staining of the tibialis anterior muscle of αB-R120G mutant mice exhibited myopathy with dark basophilic fibers, internal nuclei, scattered necrosis, and fibrosis by 10 months of age, although the degree of myopathy was greater in homozygous mutant mouse muscle. In skeletal muscle, αB-R120G co-aggregated with desmin, became detergent insoluble, and was ubiquitinated in mutant mice.

Conclusions: : The αB-crystallin R120G knock-in mouse model recapitulates the key symptoms of cataract disease pathology and human hereditary myopathy.

Keywords: chaperones • cataract • protein structure/function