Loss of DJ-1 elicits retinal abnormalities, visual dysfunction, and increased oxidative stress in mice.

DJ-1/PARK7 mutations or deletions cause autosomal recessive early onset Parkinson's disease (PD). Thus, DJ-1 protein has been extensively studied in brain and neurons. PD patients display visual symptoms; however, the visual symptoms specifically attributed to PD patients carrying DJ-1/PARK7 mutations are not known. In this study, we analyzed the structure and physiology of retinas of 3- and 6-month-old DJ-1 knockout (KO) mice to determine how loss of function of DJ-1 specifically contributes to the phenotypes observed in PD patients. As compared to controls, the DJ-1 KO mice displayed an increase in the amplitude of the scotopic ERG b-wave and cone ERG, while the amplitude of a subset of the dc-ERG components was decreased. The main structural changes in the DJ-1 KO retinas were found in the outer plexiform layer (OPL), photoreceptors and retinal pigment epithelium (RPE), which were observed at 3 months and progressively increased at 6 months. RPE thinning and structural changes within the OPL were observed in the retinas in DJ-1 KO mice. DJ-1 KO retinas also exhibited disorganized outer segments, central decrease in red/green cone opsin staining, decreased labeling of ezrin, broader distribution of ribeye labeling, decreased tyrosine hydroxylase in dopaminergic neurons, and increased 7,8-dihydro-8-oxoguanine-labeled DNA oxidation. Accelerated outer retinal atrophy was observed in DJ-1 KO mice after selective oxidative damage induced by a single tail vein injection of NaIO3, exposing increased susceptibility to oxidative stress. Our data indicate that DJ-1-deficient retinas exhibit signs of morphological abnormalities and physiological dysfunction in association with increased oxidative stress. Degeneration of RPE cells in association with oxidative stress is a key hallmark of age-related macular degeneration (AMD). Therefore, in addition to detailing the visual defects that occur as a result of the absence of DJ-1, our data is also relevant to AMD pathogenesis.

Retinal deimination and PAD2 levels in retinas from donors with age-related macular degeneration (AMD).

Deimination is a form of protein posttranslational modification carried out by the peptidyl arginine deiminases (PADs) enzymes. PAD2 is the principal deiminase expressed in the retina. Elevated levels of PAD2 and protein deimination are present in a number of human neurological diseases, with or without ocular manifestation. To define the association of deimination with the pathogenesis of age-related macular degeneration (AMD), we studied protein deimination and PAD2 levels in retinas of AMD donor eyes compared to age-matched non-AMD retinas. Eyes from non-AMD and AMD donors were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer. Retina and retinal pigment epithelium (RPE) from donor eyes were processed for immunohistochemical detection and western blotting using antibodies to PAD2 and citrulline residues. The ganglion cell, inner plexiform, inner nuclear and outer nuclear layers were labeled by both PAD2 and citrulline antibodies. Changes in the localization of deiminated residues and PAD2 were evident as the retinal layers were remodeled coincident with photoreceptor degeneration in AMD retinas. Immunodetection of either PAD2 or citrulline residues could not be evaluated in the RPE layer due to the high autofluorescence levels in this layer. Interestingly, higher deimination immunoreactivity was detected in AMD retinal lysates. However, no significant changes in PAD2 were detected in the AMD and non-AMD retinas and RPE lysates. Our observations show increased levels of protein deimination but not PAD2 in AMD retinas and RPE, suggesting a reduced rate of turnover of deiminated proteins in these AMD retinas.

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

[This corrects the article DOI: 10.1371/journal.pone.0067983.].

DJ-1-dependent regulation of oxidative stress in the retinal pigment epithelium (RPE).

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. METHODOLOGY: 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. PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

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 (SgI) and II (SgII) 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 SgI was analyzed in retina and RPE total lysates and SgI was detected by western blot in human retina and RPE. The intensity of immunoreactivity was significantly reduced in the AMD eyes. SgI 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 SgI in the AMD retinas substantially lower than observed in normal retina. SgI localization to photoreceptors and the RPE suggests a possible function related to the ability of these cells to sequester zinc.

Oxidative damage-induced inflammation initiates age-related macular degeneration.

Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues and in plasma samples from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.

Clathrin and adaptin accumulation in drusen, Bruch's membrane and choroid in AMD and non-AMD donor eyes.

Clathrin was identified in a recent proteomic analysis of Bruch's membrane from age-related macular degeneration (AMD) donor eyes. The present study was conducted to determine the localization of clathrin in AMD tissues and to compare this distribution and relative content with that in non-AMD control tissues. The distribution of adaptin, which is functionally linked to clathrin, was also evaluated. Human eyes were from donors between 66 and 94 years of age; 13 eyes were from donors with AMD and 13 from non-AMD donors. Bruch's membrane and choroid from the macula of each donor eye were prepared for immunohistochemistry and Western blotting. Differences in immunoreactivity were quantitated. Drusen, Bruch's membrane and choroid from AMD tissues showed greater immunoreactivity for clathrin and adaptin than did non-AMD tissues. Western blots also showed more intense clathrin and adaptin immunoreactivity in AMD tissues than were present in non-AMD samples. This study suggests that accumulation of clathrin and adaptin in drusen, Bruch's membrane and choroid may reflect a higher rate of clathrin mediated endocytosis in AMD tissues. Alternatively, the accumulation of these proteins in these extracellular compartments may reflect a higher susceptibility to oxidative damage.

Immunohistochemical detection and Western blot analysis of nitrated protein in stored human corneal epithelium.

While the production of nitric oxide by human corneas in storage has recently been demonstrated, protein nitration as a result of this production has not been demonstrated. In this study, nitrated protein accumulation in the epithelium of stored human corneas was assessed. One half of five donor corneas maintained in storage media for 3 days were prepared for immunohistochemical studies. The other halves remained in storage media for 7 additional days and were also processed for immunohistochemistry. Mouse monoclonal antibody to nitrotyrosine adducts was used to define the localisation of these epitopes. The density of antibody staining was observed and quantified on a digital camera system and statistically analysed. Immunostaining in the epithelium was greater in tissues recovered after 10 days in storage compared to the intensity of staining after 3 days of storage (p<0.0001). No staining was evident in the epithelium in sections exposed to non-immune mouse IgG. Western blot analysis was performed on epithelial cells scraped from corneal surfaces of one-half of four donor corneas in storage for 3 days and from the other half at 10 days of storage. Nitrated BSA was used as a positive control. After extraction and homogenisation, identical protein concentrations of each sample were loaded per lane on 10% gels and subjected to SDS-PAGE. Proteins were blotted and probed with the anti-nitrotyrosine antibody. Western blot immunoreactivity was detected in epithelial samples at the 3 and 10 day recovery times with the latter samples showing greater staining intensity. Nitrated protein, thought to indicate toxic peroxynitrite formation, accumulates in the human corneal epithelium with time of storage. Our study shows that there is an association between increased nitrated protein and storage time.

Spacrcan binding to hyaluronan and other glycosaminoglycans. Molecular and biochemical studies.

Photoreceptors project from the outer retinal surface into a specialized glycocalyx, the interphotoreceptor matrix (IPM), which contains hyaluronan (HA) and two novel proteoglycans, Spacr and Spacrcan. This matrix must be stable enough to function in the attachment of the retina to the outer eye wall yet porous enough to allow movement of metabolites between these tissues. How this matrix is organized is not known. HA is a potential candidate in IPM organization since biochemical studies show that these proteoglycans bind HA. RHAMM (receptor for HA-mediated motility)-type HA binding motifs (HABMs) are present in their deduced amino acid sequence and may be the sites of this HA interaction. To test this hypothesis, we subcloned three fragments of mouse Spacrcan that contain the putative HABMs. We found that each recombinant fragment binds HA. Binding decreased when residues in the HABMs were mutated. This provides direct evidence that the RHAMM-type HABMs in Spacrcan are involved in hyaluronan binding. Since chondroitin sulfate and heparan sulfate proteoglycans are important for retinal development and function, we also evaluated the binding of these recombinant proteins to heparin and chondroitin sulfates, the glycosaminoglycan side chain of these proteoglycans. We found that each recombinant protein bound to both heparin and chondroitin sulfates. Binding to chondroitin sulfates involved these HABMs, because mutagenesis reduced binding. Binding to heparin was probably not mediated through these HABMs since heparin binding persisted following their mutagenesis. These studies provide the first evidence defining the sites of protein-carbohydrate interaction of molecules present in the IPM.

SPACRCAN in the interphotoreceptor matrix of the mouse retina: molecular, developmental and promoter analysis.

SPACRCAN is a novel proteoglycan present in the interphotoreceptor matrix (IPM) of the rat and human retina that resists aqueous extraction through its binding to hyaluronan. The purpose of this study was: to clone mouse Spacrcan; to characterize the promoter elements; to define the deduced amino acid sequence; to establish the time of Spacrcan expression during retinal development; and to determine the time of appearance and distribution of SPACRCAN protein. Spacrcan cDNA clone was obtained through PCR amplification of a mouse retina cDNA library, and RT-PCR amplification and 5'RACE of mouse retina RNA. The deduced polypeptide sequence of mouse SPACRCAN contains a signal peptide at the N-terminal, seven N-link glycosylation sites, numerous potential O-linked glycosylation sites in a central mucin-like domain, two glycosaminoglycan attachment sites, five potential hyaluronan-binding motifs, two epidermal growth factor-like domains, and a hydrophobic stretch of 23 amino acids near the C-terminal. Comparison of the genomic structure of mouse and human SPACRCAN showed significant structure conservation. Analysis of the promoter region revealed several important putative regulatory elements including a Ret-1/PCE-1 element, an 11 base motif for Crx binding, six copies of PIRE, a Ret-4 element, three copies of AP-1, a CRE element, and five copies of GATA3. Northern blot analysis and immunohistochemistry were used to determine the tissue specificity of Spacrcan mRNA and to localize SPACRCAN in developing retina. Spacrcan mRNA is expressed in both retina and pineal gland and was detectable as early as embryonic day 15. The protein is first detectable in the IPM at postnatal day 8 where it increases in concert with the extension of photoreceptor inner and outer segments from the outer retinal surface. The presence of several unique regulatory elements in the promoter region and characteristic molecular features shared with the orthologue in human and rat suggest an important functional role of SPACRCAN in the IPM. The time of appearance of the SPACRCAN protein during retinal development suggests that this matrix protein may establish the extracellular microenvironment into which photoreceptor outer segments are elaborated.

Inhibition of nitric oxide synthesis in corneas in storage media.

The nitrate/nitrite content in storage media was determined after nitric oxide synthase inhibition by adding 400 microl of 100 mm N(G)-monomethyl-l-arginine (LMMA) to four chambers of Optisol GS corneal storage media, each containing one viable human cornea. The companion corneas in storage media without LMMA served as controls. Four hundred microlitre aliquots obtained at baseline (day 0) and at one-day intervals for 20 more days for both groups were analyzed for nitrate and nitrite (breakdown products of nitric oxide) concentration levels using a spectrophotometric method based on the Greiss reaction. Average nitrate/nitrite concentrations, statistically analyzed using a polynomial random coefficients model, showed a statistically significant marked reduction in the levels of nitrate and nitrite accumulation in the study chambers as compared to control chambers for days 1-20(P < 0.001) There was also a reduction in the accumulation rate of nitrate and nitrite concentrations, as compared to controls (P < 0.05) until around day 8 when the differences in rates were no longer statistically significant. The progressive increase in nitrate and nitrite accumulation in corneal storage media can be blunted by the addition of a nitric oxide synthase inhibitor. Given the toxic free radical properties of nitric oxide, corneas in storage awaiting transplantation may benefit from having a nitric oxide synthase inhibitor added to storage media.