Abstract
Purpose :
CRYßA3/A1 is a lens-specific structural protein. However, in the RPE, recent evidence suggests that CRYßA3/A1 is also a lysosomal-resident protein where it controls autophagy and phagocytosis by regulating endolysosomal acidification via modulating the V-ATPase proton pump. G91-deletion (βA3ΔG91) is a common mutation in CRYßA3/A1 that causes autosomal dominant congenital cataract in humans. In this study, the purpose was to explore the molecular mechanism of βA3ΔG91-induced congenital cataract development.
Methods :
We generated a βA3ΔG91 mouse model using CRISPR-Cas9 methodology at the UAB Transgenic & Genetically Engineered Models Core (TGEMs) facility. Comparative phenotypic- and biochemical characterizations of lenses from 1-month-old βA3ΔG91- and wild-type (WT) mice were performed. The methodologies used were non-invasive slit lamp examination, cellular migration assay, hematoxylin and eosin (H&E) staining, immunohistochemical (IHC) and western blot analyses and TUNEL assay. During the analyses, anti-βA3-, anti-p62-, anti-LC3- and anti-GAPDH antibodies were utilized.
Results :
Slit-lamp examinations of lenses of 15-days- and 1-month old lenses showed that the βA3ΔG91 mice developed nuclear cataract and microphakia relative to age-matched WT mice. Scratch assay showed that βA3ΔG91 LECs had a relatively slower cellular migration rate than the WT cells. Comparative H&E- and Hoechst staining exhibited abnormally high number of nuclei and their debris in the inner and outer cortex of lenses of βA3ΔG91 than WT mice, suggesting an impaired nuclear degradation process. The IHC analyses showed ring-shaped overlapping expressions of LC3 A/B and CRYßA3 that were limited to the inner cortex of the βA3ΔG91 lenses. Additionally, βA3ΔG91 lenses also exhibited relatively higher p62 expression, lower CRYßA3 expression and greater apoptotic cells in the lens cortex in the TUNEL assay. These findings were further confirmed by western blot analyses.
Conclusions :
The study shows an association of impaired autophagy in βA3ΔG91-lenses relative to WT lenses. It is probable that the presence of the greater autophagic cargo might be due to G91-deletion in βA3-crystallin and a lysosomal defect.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.