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

Cost Effectiveness of Preoperative Screening for Healthy Patients Undergoing Robotic Hysterectomy

The objective of this study was to determine whether routine preoperative type and screen blood testing is cost effective and medically warranted for benign diagnosis in healthy patients undergoing robotic hysterectomy. The study was designed as a cross sectional retrospective descriptive study. Four hundred and twenty-two medical records of American Society of Anesthesiologists (ASA) Classifications I and II patients undergoing robotically-assisted laparoscopic hysterectomy between 1 June 2011 and 31 May 2014 at a 211 bed regional medical center were analysed. The results from this study paralleled the findings of other published research. Preoperative type and screen testing was performed on 249 (59%) of the patients in the study. Ten patients (2.4% of the group) converted to open laparotomy. Mean estimated blood loss was 59.59ml. No perioperative transfusions were required. The results indicate that preoperative type and screen testing is not warranted for patients meeting the inclusion criteria

Semenogelins in the human retina: Differences in distribution and content between AMD and normal donor tissues

The two cellular targets of interest in age-related macular degeneration (AMD) are the photoreceptors and the RPE. However, the mechanisms involved in AMD pathology are not yet fully understood. In the present report, we extend our previous studies on semenogelin proteins (Sgs) in normal human retina and compare these with the distribution in retinas from AMD donor eyes. Semenogelins I (SaI) and II (SaII) are the major structural protein components of semen coagulum, but have been recently found in non-genital tissues as well. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with a specific antibody. The presence of Sal was analyzed in retina and RPE total lysates and SaI was detected by western blot in human retina and RPE. The intensity of immunoreactivity was significantly reduced in the AMD eyes. Sal is expressed in the normal human retina and in the retina of AMD donor eyes, where localization was detected in the photoreceptors and in a few ganglion cells. We find the distribution of Sal in the AMD retinas substantially lower than observed in normal retina. Sal localization to photoreceptors and the RPE suggests a possible function related to the ability of these cells to sequester zinc

Age-related reduction in retinal deimination levels in the F344BN rat

Increased deimination and peptidyl arginine deiminase type 2 (PAD2) expression has been observed in age-related neurodegenerative diseases without discrimination between their aging and disease component. Here, we describe reduced levels of deimination commensurate with reduced protein, mRNA and activity of peptidylarginine deiminase type 2 in the retina, optic nerve and plasma of aged rats when compared to young rats. The decrease was significant in the ganglion cell layer, inner plexiform layer and inner nuclear layer. Because our observations suggest reduced deimination is a consequence of aging, we conclude that increased deimination must be a consequence of disease. Our findings are important to understand late-onset and progressive diseases such as glaucoma, pseudoexfoliation syndrome, age-related macular degeneration and Oguchi’s disease

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

<div><p>Background</p><p>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.</p><p>Methodology</p><p>Retinal pigment epithelial (RPE) cultures were treated with H₂O₂ for various times followed by biochemical and immunohistological analysis. Cells were transfected with adenoviruses carrying the full-length human DJ-1 cDNA and a mutant construct, which has the cysteine residues at amino acid 46, 53 and 106 mutated to serine (C to S) prior to stress experiments. DJ-1 localization, levels of expression and reactive oxygen species (ROS) generation were also analyzed in cells expressing exogenous DJ-1 under baseline and oxidative stress conditions. The presence of DJ-1 and oxidized DJ-1 was evaluated in human RPE total lysates. The distribution of DJ-1 was assessed in AMD and non-AMD cryosectionss and in isolated human Bruch’s membrane (BM)/choroid from AMD eyes.</p><p>Principal Findings</p><p>DJ-1 in RPE cells under baseline conditions, displays a diffuse cytoplasmic and nuclear staining. After oxidative challenge, more DJ-1 was associated with mitochondria. Increasing concentrations of H₂O₂ resulted in a dose-dependent increase in DJ-1. Overexpression of DJ-1 but not the C to S mutant prior to exposure to oxidative stress led to significant decrease in the generation of ROS. DJ-1 and oxDJ-1 intensity of immunoreactivity was significantly higher in the RPE lysates from AMD eyes. More DJ-1 was localized to RPE cells from AMD donors with geographic atrophy and DJ-1 was also present in isolated human BM/choroid from AMD eyes.</p><p>Conclusions/Significance</p><p>DJ-1 regulates RPE responses to oxidative stress. Most importantly, increased DJ-1 expression prior to oxidative stress leads to decreased generation of ROS, which will be relevant for future studies of AMD since oxidative stress is a known factor affecting this disease.</p></div

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

<p>Correction: DJ-1-Dependent Regulation of Oxidative Stress in the Retinal Pigment Epithelium (RPE)</p

Presence of oxDJ-1 in RPE cells subjected to oxidative stress.

<p>ARPE-19 monolayers were treated with increasing concentrations (0 to 800 µM) of H₂O₂ for 1 hr (A) and 18 hs (B), harvested, and analyzed by immunoblot assay with oxDJ-1 antibody (upper panel). Protein loadings were confirmed in replicate blots probed with GAPDH (lower panel). Each lane contained 20 µg of protein. A dose response is observed when cells are exposed to increasing concentrations of H₂O₂ for 1 h (A, lanes 1 to 6) and 18 hrs (B, lanes 7 to 12). Confocal immunofluorescence staining of baseline ARPE-19 cultures (C) fixed before extraction with Triton X-100 and labeling with oxDJ-1 antibodies revealed absence of oxDJ-1. However, oxDJ-1 is observed in the cytoplasm (arrows) and perinuclear area (arrowheads) of RPE cells exposure to 400 µM H₂O₂ for 1 h (D) and 18 hrs (E). Cell nuclei were labeled with TO-PRO-3. Scale bar = 20 µm.</p

DJ-1 is present in RPE cell cultures.

<p>Lysates of the human RPE cell lines ARPE-19 (A, lane 1) and D407 (A, lane 2), the mouse RPE cell line B6-RPE07 (A, lane 3), mouse primary RPE (A, lane 4) and mouse brain lysates (A, lane 5) were harvested and analyzed by immunoblot assay with DJ-1 antibody. The DJ-1 signal varied in intensity in each RPE cell culture. Each lane contained 20 µg of protein. Protein loads were confirmed in replicate blots probed with GAPDH (B). Alternatively, monolayers of ARPE-19 (C), D407 (D), B6-RPE07 (E) and mouse primary RPE (F) were fixed with paraformaldehyde before extraction with Triton X-100 and labeling with DJ-1 antibodies. Cell nuclei were labeled with TO-PRO-3. DJ-1 labeling was diffusely distributed in the cytoplasm (arrows) and in the nuclei (*) of some of the cells (C-F, arrows). Scale bar = 20 µm.</p