Advisor(s)
Om Srivastava
Committee Member(s)
Jillian Ziemanski
Kirsten Lampi
Lawrence Sincich
Marina Gorbatyuk
School
School of Optometry
Document Type
Dissertation
Department (new version)
Vision Science
Date of Award
1-7-2025
Abstract
The Deletion of Glycine 91 is a mutational hotspot in the CRYβA1 gene (mutant named βA3ΔG91), which causes autosomal dominant congenital nuclear cataracts in several ethnically diverse populations worldwide. Previous in-vitro studies of recombinant βA3ΔG91 protein revealed that this mutation results in defective protein folding, reduced solubility, and oligomerization with other crystallins. While βA3/A1-crystallin is primarily known as a structural protein in the lens, it also plays a role in autophagy regulation in the retinal pigment epithelium (RPE) by controlling endolysosomal acidification through the V-ATPase proton pump. This study employs a novel βA3ΔG91 knock-in mouse model to investigate the pathogenic mechanism of βA3ΔG91 induced congenital cataracts. Phenotypic characterization was conducted to determine cataract onset and its progression. Lens fiber cell arrangement was examined via suture line analysis, and various stages of lens epithelial-to-fiber cell differentiation were assessed using the BrdU (proliferation) assay, scratch- and transwell (migration) assays, immunostaining, and transmission electron microscopy (TEM). Transcriptomic changes were evaluated using mRNA sequencing and Ingenuity pathway analysis to identify affected genes and pathways. To explore the relationship between βA3/A1-crystallin and autophagy in the lens, the up- or down-regulation of autophagic markers was examined through immunoassays, qPCR, and TEM. Proteomic analysis was performed to determine the crystallin solubility profiles by HPLC (high performance liquid chromatography) and mass spectrometry. Our results show that similar to humans, βA3ΔG91 mice showed progressive nuclear cataracts which were accompanied by disrupted suture line patterns, reduced lens epithelial cell proliferation and migration, and altered differentiation to fiber cells in βA3ΔG91 lenses relative to WT lenses. Additionally, βA3ΔG91 lenses showed disorganization of the cytoskeletal F-actin protein, delayed nuclei clearance in the organelle-free zone (OFZ), and cellular apoptosis. These lenses also showed elevated levels of autophagy markers at the transcriptional and translational levels, which include ubiquitin, p62, LC3, and LAMP2. TEM analysis revealed large undegraded autophagosomes containing organelles in the lens, which might contribute to the lens opacity development. Increased insolubilization of αA-, αB-, and βB1-crystallins, which are known to interact with βA3/A1-crystallin was observed. Taken together, the results suggest that the disruption of autophagy and altered lens fiber cell organization in βA3ΔG91 lenses lead to cellular apoptosis, which could be causative factors for cataract development. These findings provide insight into understanding the mechanisms of βA3ΔG91-induced congenital cataract development and provide clues for therapeutic intervention.
ProQuest ID
Recommended Citation
Boateng, Akosua Konadu, "Molecular Mechanism Of Congenital Cataract Caused By G91 Deletion In Cryβa1" (2025). All ETDs from UAB. 7280.
https://digitalcommons.library.uab.edu/etd-collection/7280
