Cataracts, or eye lens opacities, form due to congenital abnormalities as well modifications to lens constituents upon aging. Cataracts afflict ~ 85% of the elderly and blind 17,000,000 people. The cyclic process of cell division is required to elaborate the embryonic lens and continues in germinative epithelial lens cells. In contrast, elongating epithelial cells cease division and begin the unidirectional process of differentiation into lens fiber cells. Removal of nuclei from maturing lens fiber cells is crucial for establishing a transparent, functional lens. Failure of denucleation is causally associated with some congenital cataracts, a major genetic cause of blindness. Incomplete cell denucleation is also observed in many animal models that are used to elucidate mechanisms of senile cataractogenesis. The molecular mechanisms that govern lens cell denucleation have remained an enigma for over a century. Elucidation of these mechanisms is critical for development of strategies and new drug targets for prevention of cataracts. The proposed research will break new conceptual ground by demonstrating how processes that are used to regulate the cyclic process of cell proliferation have been appropriated for removing nuclei in lens fiber cells. We will also add to fundamental understanding about ubiquitin, Cdk and p27 biologies as well as bring new technologies to eye research. We will first corroborate our preliminary data indicating that the lens contains and appropriates the molecular machinery for denucleation from usual cell division or mitosis. Specifically, we hypothesize that cyclin dependent kinase-1 (Cdk1) that usually controls mitotic entry, also controls denucleation (Aim 1), and that Cdk1 is itself regulated by a recently discovered autoregulatory loop. We will both characterize the components of the Cdk1 autoregulatory loop that function in fiber cell denucleation, and utilize genetic and pharmacological approaches to inactivate and unambiguously identify the relevant Cdk1 regulators that initiate the denucleation process. Our published work suggests that Cdk1 activity in the lens is inhibited by the cell cycle inhibitor, p27Kip1 , in a location dependent manner.
Aim 2 tests the hypothesis that accumulation of p27Kip1 is a common governor of Cdk1 activity in mechanistically diverse models of cataract that involve failed lens fiber cell denucleation.
In Aim 3, we will utilize novel 3D imaging capacities to learn how Cdk1 functions at the nucleus to allow entry of the degradative machinery that executes the final steps of lens denucleation. None of these concepts or technologies have been previously studied or exploited within a lens context. To do this work we are also creating new animals that will be of great interest to cancer biologists and the drug developers charged with improving cataract surgery outcomes or delaying cataract. The information will also reveal new ways to control ?secondary? cataracts and cell proliferation, the latter also being of keen interest to cancer researchers. Additionally, our novel integration of pathways of control of cellular protein quality, cell replication and development/aging, will also find use in the neurodegeneration field.
Opacification of the normally clear lens, or cataract, is probably the most common human age-related disease, afflicting about 85% of the elderly and blinding 17,000,000 people; genetic cataracts also blind about 0.4% of infants. In order to create a clear lens, the inner lens cells must disassemble their nuclei and failure to do so results in cataracts. We must learn how to regulate the denucleation process because denucleation is critical, will also allow controlling cell proliferation, and this knowledge would also inform about ways to avoid ?secondary cataracts? that appear after cataract surgery, as well as have major ramifications for cancer and other diseases in which control of cell proliferation is desirable. Although an enigma for over 100 years, based upon preliminary data, literature, novel technologies and new animal models we will create and exploit, we think we can now solve this puzzle with the support of this grant.
