The long-term goal of this research is to identify and define the molecular pathways and cellular mechanisms that regulate retinal photoreceptor maintenance. Retinal photoreceptors are unique cells in that they are continually renewing a part of themselves - their outer segments, the light receptive organelle. Although this renewal process is central to the function of photoreceptors we still know very little about how these cell renew their outer segments. It is observed that the outer segments of photoreceptors progressively shorten before photoreceptors die during photoreceptor degeneration diseases and the question remains as to whether these cells might be dying because they lose their outer segments. This proposal focuses primarily on identifying the molecular mechanisms that control rod photoreceptor outer segment renewal. This proposal has one specific aim: perform a high content screen to identify compounds and molecular pathways affecting rod outer segment renewal, phagocytosis and digestion of shed rod outer segment material by the neighboring retinal pigmented epithelium, rod outer segment disc integrity, Rhodopsin trafficking and localization, and rod differentiation and morphogenesis. This project will use chemical libraries, containing many chemical compounds with known molecular targets. Although the primary objective of the proposal is to identify chemical compounds that stimulate outer segment growth, the design of the screen offers the unique and rich opportunity to identify compounds that affect additional aspects of rod photoreceptor cell biology and maintenance. This project will use young zebrafish larvae as a model organism. Zebrafish rod photoreceptors have the same morphology as mammalian species and similarly renew their outer segments. Zebrafish offers several advantages for this research. For example, this proposed screen requires large numbers of double transgenic larvae that also are homozygous mutants (albino) and large numbers of zebrafish offspring can be produced from a small number of matings. Rods are already maintaining their outer segments through renewal as soon as 6 days after fertilization, in comparison, it takes several weeks after fertilization for mouse rods to reach the same stage. In addition, unlike mammals, the retina continues to grow in zebrafish, which means that we also can examine new rods in the retinal margin to identify regulators of photoreceptor differentiation and initial photoreceptor morphogenesis, such as inner and outer segment formation at the same time that we screen for regulators of outer segment renewal, phagocytosis and digestion of shed rod outer segment material by the neighboring retinal pigmented epithelium, rod outer segment disc integrity, Rhodopsin trafficking and localization. If, as expected, chemical compounds are discovered that lengthen outer segments by either stimulating growth or suppressing shedding, these can then be tested in zebrafish and mouse models of human retinal degeneration disease for their ability to maintain outer segments, promote photoreceptor survival and prolong vision.
The outer segment is the specialized light receptive organelle in photoreceptor neurons and this structure appears to shrink and then is lost before photoreceptors die in photoreceptor degeneration diseases and vision is lost. The outer segment in photoreceptors is maintained through a unique and mysterious process of continual and daily renewal. This proposal primarily seeks to identify, using a chemical screen, molecular mechanisms that regulate the process of photoreceptor outer segment renewal and has the long term goal to test whether we can prolong vision by stimulating the growth of the outer segment.