Only through a better understanding of the pathogenic mechanisms of glaucoma will improved and innovative treatments evolve. Preliminary studies using different series of in vitro and in vivo experiments, as well as histopathological studies using human donor eyes, have provided evidence that alterations in protein expression, protein protein interactions, post translational protein modifications, and proteolytic cleavage change the protein complement of retinal ganglion cells (RGCs) during glaucomatous neurodegeneration with important implications in pathogenic mechanisms. The proposed experiments in this application are based on the hypothesis that an innovative analytical approach using the proteomics technology can identify time dependent alterations in the RGC protein complement on a large scale, which characterize precise mechanisms of the RGC response to injury from initial insult to execution of cell death in glaucoma.
The specific aims that will address this hypothesis include the identification of alterations in the RGC protein expression and post translational protein modifications during the course of glaucomatous neurodegeneration in an experimental rat model of chronic pressure induced glaucoma. Time dependent alterations of the RGC proteome, such as up regulation or down regulation of protein expression, will be quantitatively evaluated by comparing the proteomic datasets between ocular hypertensive and control eyes using RGC protein samples obtained at different time points by pooling from rat eyes matched for intraocular pressure (IOP) exposure and axon loss. Relationship between the differential protein expression and the level of IOP exposure and axon loss will be statistically determined. To improve the sensitivity of the detection, identification, and relative quantification of protein expression, complementary proteomic approaches will be utilized, which will include 2D PAGE based and gel free techniques using mass spectrometry. Oxidatively modified RGC proteins will be identified through 2D oxylot analysis, and phosphorylated RGC proteins and their interacting proteins in enriched phosphoprotein complexes will be identified through complementary approaches of the targeted proteomics. Parallel experiments will also determine cellular localization of the identified proteins using immunohistochemistry. In addition to histological sections obtained from ocular hypertensive and control rat eyes, glaucomatous and normal retinas obtained from human donor eyes will also be utilized to better validate the relevance of new findings to human disease. These studies should provide comprehensive information about the cellular mechanisms associated with RGC death at the protein level, thereby offering biomarkers and novel treatment targets for neuroprotective interventions in glaucoma, a leading cause of blindness. The information obtained from these studies is also expected to be useful in multiple disciplines to characterize pathogenic processes of various other neurodegenerative diseases leading to RGC death. In glaucoma, a specific type of nerve cells, the retinal ganglion cells, progressively die leading to gradual loss of visual function. Since the current treatment of this blinding disease is not sufficient to prevent disease progression, additional treatment strategies need to be developed to protect these cells in glaucomatous eyes. As a requirement to accomplish this goal, this project aims to identify the precise mechanisms associated with the nerve cell death in glaucoma using a powerful technology in animal experiments, as well as performing experiments utilizing human donor eyes. These studies are expected to provide comprehensive information about the nerve cell death process, thereby allowing new treatment possibilities for 3 million Americans suffering from glaucoma.
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