Abstract
Purpose :
CRYβA1-ΔG91 (βA3ΔG91) is a mutational hotspot in CRYβA1, which causes autosomal dominant congenital nuclear cataract in humans. The aim of this study was to determine the phenotypic characteristics of lenses of transgenic βA3ΔG91 mice to gain understanding about the molecular mechanism of congenital cataract development.
Methods :
The βA3ΔG91 mouse model was generated using CRISPR-Cas9 technology at the UAB Transgenic & Genetically Engineered Models facility. Slit-lamp analyses were conducted at postnatal days (P) 0, 15, and 1-month to examine cataract development in both wildtype (WT) and mutant mice. Proliferation and migration of lens epithelial cells (LECs) were assessed using BrdU-, scratch-, and transwell migration assays. Immunohistochemistry (IHC) and transmission electron microscopy (TEM) were employed to analyze organelle-degradation while autophagy was assessed by markers, p62 and LC3 using IHC and Western blot at P0, P15, and 1-month. Additionally, RNA-sequencing-, Ingenuity Pathway- and mass spectrometry techniques were employed for comprehensive gene- and protein profiling. For mass spectrometry analyses, the proteins from both types of lenses were separated into water-soluble (WS)-, water insoluble-urea soluble (WS-US)- and water insoluble-urea-insoluble (WI-UI)-protein fractions.
Results :
Relative to WT lenses, the βA3ΔG91 lenses showed: (1) Microphakia. (2) Congenital cataract development which progressed from P0 to 1-month. (3) Significant reduction in migration and proliferation of LECs. (4) Downregulation of genes associated with LECs proliferation and migration. (5) Attenuation of lens organelle-degradation, particularly nuclei. (6) Altered expression of autophagy-related genes (Atg3, Atg4, LC3, Lamp2, Vamp8 and V0-ATPase) and genes of lysosomal enzymes (Ctsb, Ctsh, Ctsk). (7) Upregulation of P62 and LC3 at P0 in the entire lens, which progressively localized to the inner cortex by P15 and 1-month. (8) Insolubilization of βA3/A1-crystallin, polyubiquitin, major lens intrinsic protein (MIP), and histone H2A.
Conclusions :
Relative to WT lenses, the deletion of G91 in βA3ΔG91 lenses resulted in reduced proliferation of LECs and caused the insolubilization of βA3/A1-crystallin in the lens. Several autophagy-associated proteins were upregulated, suggesting attenuation of organelle-degradation and autophagy disruption. The disruption of autophagy might play a role in congenital nuclear cataract development.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.