Glaucoma is no longer viewed simply as elevated intraocular pressure that damages the optic nerve. In addition to high intraocular pressure, evidence is rapidly accumulating that suggests damage to the optic nerve may be initiated or sustained by any number of factors including excitotoxicity, neurotrophin insufficiency, peroxynitrite damage or others yet undefined. These different harmful influences then likely act through common final pathways that eventually disturb ion transport and activate the cellular proteases that accompany neuronal programmed cell death. In this application, the investigators provide evidence that one form of glaucoma may be an autoimmune neuropathy. This mechanism, which they believe is operative in a subset of patients most commonly with normal intraocular pressure, thus represents an important and previously unfund3d area of glaucoma investigation. More importantly, they believe the study of the cell death machinery that mediates this form of glaucomatous optic neuropathy has broad relevance to all forms of glaucoma. They have shown that many normal pressure glaucoma patients have aberrant serum autoantibodies to retinal proteins. In particular, they found that these patients have elevated serum antibodies to heat shock proteins (hsp's). Heat shock proteins are a family of cellular proteins of varying molecular weights that are considered neuroprotective since their expression is induced in neurons to ameliorate damage in response to a variety of stress conditions such as ischemia and excitotoxicity. They have further shown that the application of exogenous small hsp antibodies to human retina and retinal cells in culture, can lead to apoptotic cell death. They believe this is an important clue towards discovering the biochemical mechanism that underlies the optic neuropathy in many patients with glaucoma. They therefore propose a series of experiments to study hsp-antibody induced retinal cell death. They will perform biochemical and immunocytochemical studies to investigate the protective effects of retinal hsp's, and the harmful effects induced by antibodies against these hsp's. They will study the effects of hsp's and their antibodies on the viability of retinal cells utilizing several model systems including isolated human retina, isolated embryonal chick retinal (ex vivo), an established retinal cell line (E1A.NR3) and dissociated rat retinal ganglion cells (in vitro). They will also perform experiments to gain insight into the specific role played by """"""""caspases"""""""" in the retinal apoptotic cell death induced by antibodies against hsp's. In addition, they will continue their search for other potential retinal autoantigens in their patients by performing partial amino acid sequencing of proteins that are immuunoprecipitated with retinal hsp's using monoclonal antibodies. They will also perform molecular cloning of putative autoantigen genes from a human retina cDNA expression library probed with nucleotide sequences based on candidate proteins; or alternatively, with purified antibodies from sera of patients with normal pressure glaucoma. Finally, they will utilize a yeast """"""""two-hydrid"""""""" system to identify genes encoding potentially novel proteins that bind and interact with retinal heat shock proteins.
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