Age and disease-related structural changes in the retinal pigment epithelium

As the retinal pigment epithelium (RPE) ages, a number of structural changes occur, including loss of melanin granules, increase in the density of residual bodies, accumulation of lipofuscin, accumulation of basal deposits on or within Bruch’s membrane, formation of drusen (between the basal lamina of the RPE and the inner collagenous layer of Bruch’s membrane), thickening of Bruch’s membrane, microvilli atrophy and disorganization of the basal infoldings. Although these changes are well known, the basic mechanisms involved in them are frequently poorly understood. These age-related changes progress slowly and vary in severity in different individuals. These changes are also found in age-related macular degeneration (AMD), a late onset disease that severely impacts the RPE, but they are much more pronounced than during normal aging. However, the changes in AMD lead to severe loss of vision. Given the many supporting functions which the RPE serves for the retina, it is important to decipher the age-related changes in this epithelium in order to understand age-related changes in vision

DJ-1-Dependent Regulation of Oxidative Stress in the Retinal Pigment Epithelium (RPE)

Abstract Background: DJ-1 is found in many tissues, including the brain, where it has been extensively studied due to its association with Parkinson's disease. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress

Age-Related Changes in the Retinal Pigment Epithelium (RPE)

<div><h3>Background</h3><p>Age-related changes in the retina are often accompanied by visual impairment but their mechanistic details remain poorly understood.</p> <h3>Methodology</h3><p>Proteomic studies were pursued toward a better molecular understanding of retinal pigment epithelium (RPE) aging mechanisms. RPE cells were isolated from young adults (3–4 month-old) and old (24–25 month-old) F344BN rats, and separated into subcellular fractions containing apical microvilli (MV) and RPE cell bodies (CB) lacking their apical microvilli. Proteins were extracted in detergent, separated by SDS-PAGE, digested in situ with trypsin and analyzed by LC MS/MS. Select proteins detected in young and old rat RPE were further studied using immunofluorescence and Western blot analysis.</p> <h3>Principal Findings</h3><p>A total of 356 proteins were identified in RPE MV from young and 378 in RPE MV from old rats, 48% of which were common to each age group. A total of 897 proteins were identified in RPE CB from young rats and 675 in old CB, 56% of which were common to each age group. Several of the identified proteins, including proteins involved in response to oxidative stress, displayed both quantitative and qualitative changes in overall abundance during RPE aging. Numerous proteins were identified for the first time in the RPE. One such protein, collectrin, was localized to the apical membrane of apical brush border of proximal tubules where it likely regulates several amino acid transporters. Elsewhere, collectrin is involved in pancreatic β cell proliferation and insulin secretion. In the RPE, collectrin expression was significantly modulated during RPE aging. Another age-regulated, newly described protein was DJ-1, a protein extensively studied in brain where oxidative stress-related functions have been described.</p> <h3>Conclusions/Significance</h3><p>The data presented here reveals specific changes in the RPE during aging, providing the first protein database of RPE aging, which will facilitate future studies of age-related retinal diseases.</p> </div

Proteomics implicates peptidyl arginine deiminase 2 and optic nerve citrullination in glaucoma pathogenesis

PURPOSE. Proteomic analyses of normal and glaucomatous human optic nerve were pursued for insights into the molecular pathology of primary open-angle glaucoma (POAG). Peptidyl arginine deiminase 2 (PAD2), an enzyme that converts protein arginine to citrulline, was found only in POAG optic nerve and was probed further for a mechanistic role in glaucoma. METHODS. Protein identification used liquid chromatographytandem mass spectrometry. Northern, Western, and immunohistochemical analyses measured PAD2 expression and/or protein citrullination and arginyl methylation in human and mouse optic nerve and in astrocyte cultures before and after pressure treatment. Proteins were identified after anticitrulline immunoprecipitation. In vitro translation of PAD2 was monitored in polyA RNA depleted optic nerve extracts. PAD2 shRNA transfections were evaluated in pressure-treated astrocytes. RESULTS. Western and immunohistochemical analyses confirmed elevated PAD2 and citrullination in POAG optic nerve and decreased arginyl methylation. PAD2 was also detected in optic nerve from older, glaucomatous DBA/2J mice, but not in younger DBA/2J or control C57BL6J mice. Myelin basic protein was identified as a major citrullinated protein in POAG optic nerve. Pressure-treated astrocytes exhibited elevated PAD2 and citrullination without apparent change in PAD2 mRNA. Addition of exogenous polyA RNA to depleted optic nerve extracts yielded increased PAD2 expression in POAG but not in control extracts. Transfection with shRNA restored PAD2 and citrullination to control levels in pressure-treated astrocytes. CONCLUSIONS. Current results support translational modulation of PAD2 expression and a possible role for the enzyme in POAG optic nerve damage through citrullination and structural disruption of myelination. (Invest Ophthalmol Vis Sci

Protein Deimination in Aging and Age-Related Diseases with Ocular Manifestations

Deimination refers to the conversion of protein-bound arginines into citrulline. It has been established as a posttranslational modification due to the lack of any known tRNA carrier for citrulline, as well as the presence of deiminases that are capable of catalyzing this modification in vitro. There is no known enzyme that can revert protein-bound citrulline into arginine, rendering it a relatively long-term modification. Elevated deimination has been found in neuronal tissues in a number of neurodegenerative diseases including multiple sclerosis and glaucoma. Observations in the retina, a tissue where the retinal ganglion cell layer lacks a substantial presence of astroglial cells, demonstrated that elevated and reduced deimination occurs simultaneously in astroglial cells and neurons, respectively. Such opposite effects are expected to complicate therapeutic strategies, necessitating cell-specific delivery systems for perturbation of deiminases that catalyze deimination in neuronal tissues. In this review, we will briefly discuss the occurrence of deimination with normal aging, the importance of deimination in diseases, and the effect of deimination on mRNA transport in neuronal tissue. Elevated deimination induces proteolysis via modification of protein structures, while reduced deimination affects protein synthesis and the outgrowth of dendrites in neurons

Deimination in Ocular Tissues: Present and Future

Deimination refers to the posttranslational conversion of protein-bound arginines into protein-bound citrullines, catalyzed by peptidyl arginine deiminase (PAD) enzymes (Vossenaar et al. 2003). Lack of a carrier tRNA for citrulline has established deimination as a posttranslational modification. Deimination and citrullination are interchangeably used for conversion of arginine into citrulline. To distinguish free arginine citrullination from protein-bound citrullination, we prefer referring to the latter as deimination (Bhattacharya 2009). Throughout this chapter we use deimination to refer to the conversion of protein-bound arginine modification into citrulline

Oxidation of DJ-1 Cysteines in Retinal Pigment Epithelium Function

The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function under oxidative conditions. The present study was conducted to analyze the impact of DJ-1 expression changes and oxidation of its C residues on RPE function. Monolayers of the ARPE-19 cell line and primary human fetal RPE (hfRPE) cultures were infected with replication-deficient adenoviruses to investigate the effects of increased levels of DJ-1 in these monolayers. Adenoviruses carried the full-length human DJ-1 cDNA (hDJ) and mutant constructs of DJ-1, which had all or each of its three C residues individually mutated to serine (S). Alternatively, endogenous DJ-1 levels were decreased by transfection and transduction with shPARK7 lentivirus. These monolayers were then assayed under baseline and low oxidative stress conditions. The results were analyzed by immunofluorescence, Western blot, RT-PCR, mitochondrial membrane potential, and viability assays. We determined that decreased levels of endogenous DJ-1 levels resulted in increased levels of ROS. Furthermore, we observed morphological changes in the mitochondria structure of all the RPE monolayers transduced with all the DJ-1 constructs. The mitochondrial membrane potential of ARPE-19 monolayers overexpressing all DJ-1 constructs displayed a significant decrease, while hfRPE monolayers only displayed a significant decrease in their &Delta;&Psi;m when overexpressing the C2S mutation. Viability significantly decreased in ARPE-19 cells transduced with the C53S construct. Our data suggest that the oxidation of C53 is crucial for regulating endogenous levels of ROS and viability in RPE cells