Posttranslational modification of differentially expressed mitochondrial proteins in the retina during early experimental autoimmune uveitis.

PURPOSE: Posttranslational modification of proteins plays an important role in cellular functions and is a key event in signal transduction pathways leading to oxidative stress and DNA damage. In this study, we used matrix-assisted laser desorption/ionization- time of flight (MALDI-TOF) to investigate the posttranslational modifications of the differentially expressed proteins in the retinal mitochondria during early experimental autoimmune uveitis (EAU). METHODS: EAU was induced in 18 B10RIII mice with 25 µg of inter-photoreceptor retinoid-binding protein (IRBP) emulsified with complete Freund's adjuvant (CFA); 18 mice treated with CFA without IRBP served as controls. Retinas were removed from the experimental and control groups on day 7 post immunization; mitochondrial fractions were extracted and subjected to 2 dimentional-difference in gel electrophoresis (2D-DIGE); and the protein spots indicating differential expression were subjected to MALDI-TOF for protein identification and indication of any posttranslational modifications. RESULTS: Of the 13 proteins found to be differentially expressed by 2D-DIGE (including upregulated aconitase, mitochondrial heat shock protein (mtHsp) 70, lamin-1, syntaxin-binding protein, αA crystallin, ßB2 crystallin, along with downregulated guanine nucleotide-binding protein and ATP synthase) nine were found to undergo posttranslational modification. Oxidation was a common modification found to occur on aconitase, mtHsp 70, ATP synthase, lamin-1, ßB2-crystallin, guanine nucleotide-binding protein, and manganese superoxide dismutase (MnSOD). In addition, aconitase hydratase, mtHsp 70, guanine nucleotide-binding protein, ATP synthase, syntaxin-binding protein, ßB2-crystallin, and lamin-1 were also modified by carbamidomethylation. αA-crystallin had a pyro-glu modification. CONCLUSIONS: Several proteins present in the retinal mitochondria are posttranslationally modified during early EAU, indicating the presence of oxidative stress and mitochondrial DNA damage. The most common modifications are oxidation and carbamidomethylation. A better understanding of the proteins susceptible to posttranslational modifications in the mitochondria at the early stage of the disease may serve to advance therapeutic interventions to attenuate disease progression.

The role of TLR4 in photoreceptor {alpha}a crystallin upregulation during early experimental autoimmune uveitis.

Purpose. Previous studies indicate that the upregulation of alphaA crystallin prevents photoreceptor mitochondrial oxidative stress-mediated apoptosis in experimental autoimmune uveitis (EAU). In this study, the role of TLR4 was investigated in the upregulation of alphaA crystallin in the retinas of animals with EAU. Methods. TLR4(-/-), iNOS(-/-), TNF-alpha(-/-), MyD88(-/-), wild-type (WT) control (C57BL/6), and nude mice (B6.Cg-Foxn1(nu)) were immunized with IRBP mixed with complete Freund's adjuvant; eyes were enucleated on day 7 after immunization. Real-time polymerase chain reaction was first used to detect upregulated inflammatory cytokines and alphaA crystallin in retinas with EAU; confirmed with Western blot analysis, and the site of upregulation was localized by immunohistochemistry. Oxidative stress was localized using 8-OHdG, and TUNEL staining was used to detect apoptosis. Results. In early EAU, increased expression of TNF-alpha, iNOS, and alphaA crystallin genes were detected in the retinas of WT mice, whereas such upregulation was absent in TLR4-deficient mice (P < 0.001). alphaA Crystallin was not elevated in MyD88(-/-), TNF-alpha(-/-), and iNOS(-/-) mice with EAU. Immunostaining revealed TNF-alpha, iNOS, and alphaA crystallin localization in the photoreceptor inner segments and outer plexiform layer in the WT controls with EAU; but such staining was absent in TLR4-deficient mice with EAU. 8-OHdG staining showed oxidative stress in the photoreceptors in WT mice with EAU and there was no apoptosis. Conclusions. TLR4 plays an important role in the upregulation of alphaA crystallin through the interaction of MyD88 and the subsequent generation of TNF-alpha and iNOS in the EAU retina. Such crystallin upregulation may prevent oxidative stress-mediated apoptosis of photoreceptors in uveitis.

Mitochondrial oxidative DNA damage in experimental autoimmune uveitis.

PURPOSE: In experimental autoimmune uveitis (EAU), recent work has demonstrated that retinal damage involves oxidative stress early in uveitis, before macrophage cellular infiltration. The purpose of this study was to determine whether oxidative mitochondrial DNA damage occurs early in EAU, before leukocyte infiltration. METHODS: Lewis rats were immunized with S-antigen mixed with complete Freund adjuvant (CFA) to induce EAU. Nonimmunized animals and animals injected with CFA served as controls. Animals were killed on days 3, 4, 7, and 12 after immunization. Damage to mitochondrial DNA and nuclear DNA was assessed using a novel long quantitative polymerase chain reaction technique. TUNEL staining to detect apoptosis and immunohistochemical detection of leukocyte infiltration in EAU retinas were also performed at these times. RESULTS: Mitochondrial DNA damage occurred early in EAU, from day 4 to day 12. In the early phase of EAU (days 4-7), there was no inflammatory cell infiltration. On day 12 inflammatory cells infiltrated the retina and uvea. Nuclear DNA damage occurred later in EAU at day 12. Neither mitochondrial nor nuclear DNA damage was detected in the controls. TUNEL-positive staining for apoptosis was detected only at day 12 in EAU retina. CONCLUSIONS: Oxidative mitochondrial DNA damage begins at day 4 in EAU, supporting the view that oxidative stress selectively occurs in the mitochondria in the early phase of EAU, before leukocyte infiltration. Such oxidative damage in the mitochondria may be the initial event leading to retinal degeneration in EAU.