Investigating the Molecular Mechanisms Dictating Lens Stiffness and Growth: Functional Studies of Gap Junction and α-Crystallin Mutant Lenses

Our vision depends on several key optical properties of the eye lens including: transparency, high refractive index, and accommodation, which relies on the elastic process of lens deformation to focus light clearly onto the retina. The lens is composed of a monolayer of epithelial cells that cover the anterior hemisphere of bulk elongated fibers and is wrapped by a basement membrane called the lens capsule. Genetic studies reveal that mutation in members of key lens proteins such as gap junctions, cytoskeletal proteins, and crystallins impair lens growth, homeostasis, transparency, and / or other optical properties. However, the underlining mechanisms of how mutated genes lead to lens pathology remain not well understood. Moreover, it is unknown how mutated gap junctions, consisting of several different connexin subunits, affect lens elasticity, or how mutated αA-crystallin impairs lens growth or disrupts the cytoskeleton./nMy thesis project, consisting of three parts, aims to elucidate the roles of gap junctions, the lens cytoskeleton, and crystallins in the development and maintenance of lens elasticity and lens growth. In part one, connexin mutant mouse lenses are subject to the measurement of lens stiffness by a novel muscle lever system. The results show that knockout of gap junction gene Gja3, also known as α3-connexin or Cx46, caused a marked increase in lens stiffness across almost all genotypes and ages. In addition, it was shown that mouse strain backgrounds, associated with variant periaxin, a cytoskeletal organization protein, had significant effects on the bulk lens stiffness, with the C57BL/6J strain background resulting in softer lenses, while the 129 strain background produced stiffer lenses. Part two investigates lenses with mutated αA-Y118D crystallin protein, which present a unique lens growth defect. Mutant lenses stop growing after 8 weeks of age and mutant lens fiber cells exhibit abnormal growth and are unable to elongate. Next, studies of αA-crystallin mutations in cultured lens epithelial cells reveal the interaction between αA-crystallin with the lens cytoskeleton. Results show that αA-crystallin is associated with lamellar actin in wild-type cells, while αA-Y118D mutant protein tends to form aggregates with actin. Finally, part three reveals a novel regulation of lens connexin expression in cultured lens epithelium with a new cell culture medium. Treatment with SB431542 (SB), a transforming growth factor β (TGF-β) inhibitor, is important to maintain the expression of gap junction gene Gja8, also known as α8-connexin or Cx50, while fibroblast growth factor (FGF) treatment suppresses its expression./nMy dissertation reveals the novel role of gap junctions and cytoskeletal proteins in the regulation of lens elasticity and further elucidates the unique role of αA-crystallin in the regulation of lens cytoskeletal structures in vivo and in vitro. Finally, the expression of gap junction proteins in lens epithelial cells is negatively regulated by both the TGF-β and FGF signaling pathways. This work further improves our understanding of the regulation and maintenance of lens optical properties during lens development.

Advisor: Gong, Xiaohua