Photoreceptor Mitochondrial Protein Nitration in Uveitis

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DESCRIPTION (provided by applicant): Blindness in uveitis results from degeneration of photoreceptor cells. This degeneration is attributed to macrophage infiltration and the subsequent generation of various inflammatory mediators, including the nitric oxide-derived highly reactive oxidant peroxynitrite, which is known to cause photoreceptor damage via nitration of tyrosine residues. Our preliminary data, however, shows the nitration commences in the early phase of EAU, prior to infiltration by macrophages. Moreover, we found that the nitration selectively involves the mitochondrial proteins. These novel findings implicate photoreceptor cell mitochondrial protein nitration as a central molecular event in the initiation of photoreceptor degeneration. This nitration is certainly not mediated by macrophages. We propose a hypothesis: "Photoreceptor degeneration in autoimmune uveitis begins with selective mitochondrial protein nitration as a consequence of reactive nitric oxide species generation in the photoreceptor mitochondria." We will test this hypothesis with the following specific aims in animals with early EAU, prior to macrophage infiltration of the retina. 1. Evaluate nitric-oxide reactive species generation in the photoreceptor mitochondria (immunohistochemistry, immunoblotting, and mass spectrometry). 2. Evaluate mitochondrial protein nitration in the photoreceptors (immunohistochemical localization of nitrated proteins, immunoblotting of photoreceptor proteins, and capillary liquid chromatography-tandem mass spectrometry). 3. Evaluate functions of the photoreceptor mitochondria (oxygen consumption and activities of electron transport chain complexes, and mitochondrial transmembrane potential). To assure successful completion of the specific aims, we have assembled a multidisciplinary team of experts. Professor Terry Lee is an accomplished chemist with expertise in identifying nitrated proteins, and their site of nitration by mass spectrometry and other novel methods. Dr. Guey-Shuang Wu has expertise in PCR, Western blot, and separation of biological molecules. The principal investigator is experienced in the field of EAU, immunohistochemistry, free radical biology, and reactive nitric oxide species. Understanding the role of mitochondria in the pathogenesis of retinal degenerations could lead to the development of specific therapy to minimize mitochondrial generation of peroxynitrite.