Mitochondrial Proteins in the Photoreceptor Inner Segments are Selectively Nitrated in the Early Phase of Experimental Autoimmune Uveitis

Abstract

Abstract: : Purpose: Previously we have reported that at the peak of experimental uveitis (EAU), tyrosine (Tyr) nitration occurred mainly in three mitochondria–related proteins namely, cytochrome c, mitochondria import stimulation factor and phosphoglycerate mutase. In this study, we attempted to localize these nitrated proteins in the course of EAU and compare them with the emergence of other pathogenic factors such as retinal microglia, inflammatory infiltrates, and retinal lipid peroxidation. Methods: EAU was induced in Lewis rats by bovine S–antigen. At days 5 (D5), 10 (D10), and 14 (D14) postimmunization, six rats each were killed. The controls (D0) were six non–immunized rats. In each period, two eyes were processed for H & E. Frozen sections were stained with anti–nitroTyr and anti–rat CD11b as primary antibodies for detecting nitrated Tyr and microglia, respectively. Lipid peroxidation was detected by coupling the peroxide–derived carbonyl products with fluorescent 3–hydroxy–2–naphthoic acid hydrazide for confocal microscopy and by reacting with fast blue B for light microscopy. Results: Infiltration of phagocytes was not seen in D5 or D10, and retinal morphology was preserved. Intense Tyr–nitration was exclusively localized in the photoreceptor inner segments at D5 and D10, then extended to the entire photoreceptor layer and vessels in the nerve fiber layer (NFL) on D14. No staining was noted in D0. At D0 and D5, microglia were in their normal locations in NFL, inner and outer plexiform layers. Extensive hydroperoxide–derived cellular carbonyls were seen in the photoreceptors at D14, but were absent in D0, D5 and D10. Conclusions: The photoreceptor inner segments are richly endowed with mitochondria due to unusually high metabolic requirement for visual processes. The intense staining of nitroTyr only observed in the inner segments in the absence of lipid peroxidation strongly suggests that the photoreceptor inner segments are the early source of peroxynitrite that leads to nitration of cellular proteins at the proximity. This phenomenon, detected at D5 in the absence of macrophage infiltration and microglia migration further indicates that the initial oxidative insult takes place in the photoreceptor mitochondria, resulting in subsequent amplification of EAU.

Keywords: nitric oxide • oxidation/oxidative or free radical damage • mitochondria