This is a revision of a grant to understand the cellular basis for the formation of human age-related nuclear cataracts, the most common form of human cataract and a leading cause of worldwide blindness. The project has expanded to include detailed studies of fiber cell differentiation and formation of the organelle-free zone that supports initial transparency of the lens nucleus. Significant progress has been made in characterizing the extensive cellular modifications that fiber cells in primate lenses undergo during terminal differentiation to form cells that can generate the radial gradient of refractive index, support the high refractive index of the nucleus and lead to cellular compaction in the adult nucleus. We were able to demonstrate that autophagy and mitophagy occur in the lens and participate in the removal of membranous organelles except the nucleus. Using a chick embryo animal model, we have identified a macromolecular complex that selectively degrades the nuclear envelope in the final stages of formation of the organelle-free zone. We have preliminary evidence that this new organelle operates in human and monkey lenses. Our evidence suggests that this complex is a new organelle specific to fiber cells and a goal is to determine its molecular composition and complex organization, using microscopy, gene silencing of specific components and imaging mass spectrometry. These studies were made possible because a modified fixation protocol allowed excellent preservation of entire lenses. An important observation in the study of autophagy was that some autophagic vesicles appeared to contain dense regions of cytoplasm covered by multiple membranes, which resembled the multilamellar bodies we described previously as potential sources of forward light scattering in age-related nuclear cataracts. Scattering from these large particles is complementary to the light scattering commonly seen from cataracts caused by post- translational modifications and aggregation of lens-specific cytoplasmic crystallins. Our evidence suggests that autophagy is a general process in vertebrate lenses that begins in the embryo and fetus where vesicular debris that is not completely cleared could be precursors to multilamellar bodies. In collaboration with a major eye institute in India, we plan to explore the role of autophagy as possible sources of multilamellar bodies and the structure of a variety of nuclear cataracts analyzed employing transmission electron microscopy, fluorescent scanning confocal microscopy, antibody labeling approaches and theoretical treatments to predict the expected light scattering from particles and cellular defects. These ultrastructural studies and theoretical analyses are expected to lead to better understanding of the mechanisms of cell damage that produce excessive light scattering and allow us to suggest and evaluate non-surgical methods to prevent or delay cataract formation.
Cataracts are a major source of visual impairment throughout the world. Many patients can be effectively treated with surgery, although services may not be available or affordable. Moreover, the aging population is developing new cataracts at a significant rate. This research supports the search for the underlying cellular damage that leads to age-related cataracts and alternative treatment strategies for preventing or delaying the progression of cataracts.
