Biosynthesis and physiological roles of extremely long chain lipids in the eye

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 DESCRIPTION (provided by applicant): The fundamental goal of this project is to investigate metabolic pathways that lead to generation of lipid-rich secretion (called meibum) by Meibomian glands (MG). MG are embedded into the tarsal plates of humans and animals. Meibum protects the ocular surface from hazardous environmental factors and desiccation through its participation in formation of the tear film (TF). Abnormal quality, and/or insufficient quantity of meibum destabilize the human TF, which is one of the underlying factors of dry eye disease (DE), particularly of one of its major forms - Meibomian gland dysfunction. Numerous reports indicated that DE affects up to 30% of the general population of the USA and other countries. As a practical outcome of our research, we expect to discover new, more effective treatments for DE than those that are available now. Currently, no detailed information on the biosynthetic pathways that lead to formation of meibum is available. Our plan is to fill in this important gap i knowledge using mice as animal models. Earlier, we have discovered that lipid compositions of meibum of humans and mice are very close. Also, our pilot experiments with human and mouse MG tissue specimens demonstrated that all tested lipid-synthesizing enzymes are expressed in both species. Thus, mice are convincing (and convenient) models for studies that are ultimately targeting human ocular pathologies related to MG. As Aim 1, we propose to investigate biosynthetic pathways that lead to formation of these specific lipids, and determine which enzymatic steps and lipid products are critical for making healthy meibum. We will determine expression patterns and cellular and subcellular localization of major enzymes responsible for producing meibum, in mouse MG and adnexa. Our Aim 2 is to elucidate changes in meibum, MG, and adnexa in response to suppression or activation of specific genes related to lipid metabolism. To test our ideas, we will make use of various currently available gene knockout and transgene mouse models with known lipid abnormalities, whose ocular features have not been characterized yet because those mutant mice were developed for other purposes. Also, we will develop new approaches for temporary silencing the genes of interest using anti-sense RNA. As Aim 3, we will determine whether dietary lipids can have a major impact on meibum by tracing the uptake and metabolism of 2H- and 13C-labeled dietary lipid tracers by MG. Our goals will be achieved using a combination of chemical, biochemical, physiological and genomic approaches, which include, but are not limited to, liquid and gas chromatography, mass spectrometry, microcalorimetry, hot stage polarized light microscopy, in vivo confocal microscopy, proteomics, in-situ hybridization, immuno(histo)chemical staining, gene expression analysis, in vivo confocal microscopy, and clinical evaluation of the ocular structures of experimental mice.