Molecular and Cellular Mechanisms of Glaucoma

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DESCRIPTION (provided by applicant): The gene, GLC1A, was found to be associated with juvenile onset glaucoma and some adult onset glaucoma. GLC1A encodes myocilin, which is expressed in several key ocular cell types. The structure(s) and function(s) of myocilin remain unknown. Analysis of endogenous human myocilin reveals that several sizes are produced. Because alternative RNA splicing has not been observed in myocilin, the size differences are presumed to be due to use of different start sites and post-translational modifications. I hypothesize that the various sizes ere produced by differential regulation in different tissues and cell types. I want to solve the structure(s) of myocilin and learn its function(s). To do this, I will analyze myocilin synthesis, processing, and secretion in healthy cells from different cell types. Using native secreted myocilin from cultured cells and recombinant myocilin, my specific aims are: (1) To select the richest source of native myocilin. I will analyze the synthesis of the different kinds of native myocilin from different ocular cell types using cultures of human oligodendrocytes, astrocytes, trabecular meshwork cells and vascular endothelial cells. Different forms of myocilin will be purified independently in order to analyze the properties of each. (2) To determine the biochemical and biophysical properties of myocilin synthesized by human ocular cells using purified soluble, native myocilin proteins. (3) To screen crystallization conditions using both native secreted and recombinant myocilin and to obtain good, diffracting crystals. Given the functional importance of ocular cells in determining neuronal survival or degeneration, analysis of the regulation, synthesis and structure of myocilin will provide valuable information to elucidate the functions of myocilin in normal and glaucomatous eyes.