Natalie Wolkow

The oral iron chelator deferiprone protects against iron overload-induced retinal degeneration.

PURPOSEnIron-induced oxidative stress may exacerbate age-related macular degeneration (AMD). Ceruloplasmin/Hephaestin double-knockout (DKO) mice with age-dependent retinal iron accumulation and some features of AMD were used to test retinal protection by the oral iron chelator deferiprone (DFP).nnnMETHODSnCultured retinal pigment epithelial (ARPE-19) cells and mice were treated with DFP. Transferrin receptor mRNA (Tfrc), an indicator of iron levels, was quantified by qPCR. In mice, retinal oxidative stress was assessed by mass spectrometry, and degeneration by histology and electroretinography.nnnRESULTSnDFP at 60 μM decreased labile iron in ARPE-19 cells, increasing Tfrc and protecting 70% of cells against a lethal dose of H(2)O(2). DFP 1 mg/mL in drinking water increased retinal Tfrc mRNA 2.7-fold after 11 days and also increased transferrin receptor protein. In DKOs, DFP over 8 months decreased retinal iron levels to 72% of untreated mice, diminished retinal oxidative stress to 70% of the untreated level, and markedly ameliorated retinal degeneration. DFP was not retina toxic in wild-type (WT) or DKO mice, as assessed by histology and electroretinography.nnnCONCLUSIONSnOral DFP was not toxic to the mouse retina. It diminished retinal iron levels and oxidative stress and protected DKO mice against iron overload-induced retinal degeneration. Further testing of DFP for retinal disease involving oxidative stress is warranted.

Iron Chelation Protects the Retinal Pigment Epithelial Cell Line ARPE-19 against Cell Death Triggered by Diverse Stimuli

PURPOSEnCell death can be induced by exogenous reactive oxygen species (ROS). Endogenous ROS can also play a role in cell death triggered by agents that are not themselves ROS. One of the most potent ROS-generating systems is the iron-catalyzed Fenton reaction. Herein, the authors tested whether iron plays an important role in cell death induced by diverse stimuli in retinal pigment epithelial (RPE) cells.nnnMETHODSnThe ability of the iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) to chelate intracellular labile iron was tested in the human cell line ARPE-19. The ability of SIH to protect against RPE cell death induced by hydrogen peroxide, staurosporine, anti-Fas, and exposure to A2E plus blue light was determined. ROS production by staurosporine was assessed in the presence and absence of SIH. The protective activity of SIH was compared with that of other iron chelators and an antioxidant.nnnRESULTSnAcute exposure to SIH was nontoxic and at least partially protective against cell death induced by all tested agents. On a molar basis, SIH was more protective against hydrogen peroxide than other iron chelators and an antioxidant. SIH decreased levels of staurosporine-induced ROS.nnnCONCLUSIONSnIron chelation with SIH can decrease levels of ROS and protect RPE cells against cell death induced by diverse stimuli. These results suggest a central role for iron in cell death pathways, potentially involving the generation of oxidative stress. SIH or related iron chelators may prove useful for protection against diseases involving RPE death, such as AMD.

Aceruloplasminemia: Retinal Histopathologic Manifestations and Iron-Mediated Melanosome Degradation

OBJECTIVEnTo examine the retinal histopathologic manifestation of aceruloplasminemia, an autosomal recessive disease caused by mutation of the ferroxidase ceruloplasmin, resulting in tissue iron overload.nnnMETHODSnThe morphologic features of the human aceruloplasminemic retina were studied with light and electron microscopy. Retinal iron accumulation was assessed with Perls Prussian blue staining, immunohistochemistry, and secondary ion mass spectrometry.nnnRESULTSnLight and electron microscopic analysis revealed several ocular pathologic findings that resembled age-related macular degeneration, including retinal pigment epithelium (RPE) depigmentation, atrophy and hypertrophy, nodular and diffuse drusen, and lipofuscin and melanolipofuscin granules. Complement deposition was detected in drusen. The RPE cells and neural retina had increased levels of iron. Two major types of RPE cells were observed: melanosome rich and melanosome poor. Melanosome-rich cells had increased levels of iron and melanolipofuscin. The melanolipofuscin granules were observed in large aggregates, where some of the melanosomes were degrading. Melanosome-poor cells lacked melanosomes, melanolipofuscin, and lipofuscin but contained electron-dense aggregates high in iron, phosphorus, and sulfur.nnnCONCLUSIONSnThe findings in the aceruloplasminemic retina resemble some of those found in age-related macular degeneration. Also, they suggest that melanosomes in the RPE can be degraded via iron-mediated reactive oxygen species production.nnnCLINICAL RELEVANCEnMechanisms underlying the pathologic mechanisms found in aceruloplasminemia also may be important in age-related macular degeneration.

The multicopper ferroxidase hephaestin enhances intestinal iron absorption in mice

Hephaestin is a vertebrate multicopper ferroxidase important for the transfer of dietary iron from intestinal cells to the blood. Hephaestin is mutated in the sex-linked anemia mouse, resulting in iron deficiency. However, sex-linked anemia mice still retain some hephaestin ferroxidase activity. They survive, breed, and their anemia improves with age. To gain a better understanding of the role of hephaestin in iron homeostasis, we used the Cre-lox system to generate knockout mouse models with whole body or intestine-specific (Villin promoter) ablation of hephaestin. Both types of mice were viable, indicating that hephaestin is not essential and that other mechanisms, multicopper ferroxidase-dependent or not, must compensate for hephaestin deficiency. The knockout strains, however, both developed a microcytic, hypochromic anemia, suggesting severe iron deficiency and confirming that hephaestin plays an important role in body iron acquisition. Consistent with this, the knockout mice accumulated iron in duodenal enterocytes and had reduced intestinal iron absorption. In addition, the similarities of the phenotypes of the whole body and intestine-specific hephaestin knockout mice clarify the important role of hephaestin specifically in intestinal enterocytes in maintaining whole body iron homeostasis. These mouse models will serve as valuable tools to study the role of hephaestin and associated proteins in iron transport in the small intestine and other tissues.

Iron prochelator BSIH protects retinal pigment epithelial cells against cell death induced by hydrogen peroxide

Dysregulation of localized iron homeostasis is implicated in several degenerative diseases, including Parkinsons, Alzheimers, and age-related macular degeneration, wherein iron-mediated oxidative stress is hypothesized to contribute to cell death. Inhibiting toxic iron without altering normal metal-dependent processes presents significant challenges for standard small molecule chelating agents. We previously introduced BSIH (isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene]-hydrazide) prochelators that are converted by hydrogen peroxide into SIH (salicylaldehyde isonicotinoyl hydrazone) chelating agents that inhibit iron-catalyzed hydroxyl radical generation. Here, we show that BSIH protects a cultured cell model for retinal pigment epithelium against cell death induced by hydrogen peroxide. BSIH is more stable than SIH in cell culture medium and is more protective during long-term experiments. Repetitive exposure of cells to BSIH is nontoxic, whereas SIH and desferrioxamine induce cell death after repeated exposure. Combined, our results indicate that cell protection by BSIH involves iron sequestration that occurs only when the cells are stressed by hydrogen peroxide. These findings suggest that prochelators discriminate toxic iron from healthy iron and are promising candidates for neuro- and retinal protection.

Generation of Cre Transgenic Mice with Postnatal RPE-Specific Ocular Expression

PURPOSEnTo generate and characterize a constitutively active, RPE-specific, cre-expressing transgenic mouse line. This line can be used to create RPE-specific knockouts by crossing with mice harboring loxP-flanked (floxed) genes.nnnMETHODSnA transgene construct was assembled with the BEST1 promoter driving cre expression. Transgenic mice were generated on a C57BL/6 background. Cre expression was assessed by immunofluorescence and Western blot analysis. Cre enzymatic activity was tested by crossing to three lines with floxed DNA regions and detecting deletion of the intervening sequences or through histochemical detection of lacZ activity. Potential cre-mediated toxicity was assessed by retinal histology up to 24 months of age and by electroretinography.nnnRESULTSnThe BEST1-cre line with expression in the highest percentage of RPE cells displayed a patchy mosaic expression pattern, with 50% to 90% of RPE cells expressing cre. In mice outcrossed to a mixed B6/129 background, expression was consistently found in 90% of RPE cells. Within the eye, only the RPE cells were immunoreactive with an anti-cre antibody. Maximum cre expression quantified by Western blot analysis occurred at P28. Crosses with three lines containing floxed sequences revealed RPE-specific cre activity in the eye and extraocular expression limited to the testes. Histology and electroretinography showed no cre-mediated RPE toxicity.nnnCONCLUSIONSnThis BEST1-cre transgenic line enables generation of RPE-specific knockout mice. The mosaic expression pattern provides an internal control; the non-cre-expressing RPE cells continue to express the floxed genes. These mice should facilitate study of the multifunctional RPE and the generation of mouse models of human retinal disease.

Bmp6 Regulates Retinal Iron Homeostasis and Has Altered Expression in Age-Related Macular Degeneration

Iron-induced oxidative stress causes hereditary macular degeneration in patients with aceruloplasminemia. Similarly, retinal iron accumulation in age-related macular degeneration (AMD) may exacerbate the disease. The cause of retinal iron accumulation in AMD is poorly understood. Given that bone morphogenetic protein 6 (Bmp6) is a major regulator of systemic iron, we examined the role of Bmp6 in retinal iron regulation and in AMD pathogenesis. Bmp6 was detected in the retinal pigment epithelium (RPE), a major site of pathology in AMD. In cultured RPE cells, Bmp6 was down-regulated by oxidative stress and up-regulated by iron. Intraocular Bmp6 protein injection in mice up-regulated retinal hepcidin, an iron regulatory hormone, and altered retinal labile iron levels. Bmp6(-/-) mice had age-dependent retinal iron accumulation and degeneration. Postmortem RPE from patients with early AMD exhibited decreased Bmp6 levels. Because oxidative stress is associated with AMD pathogenesis and down-regulates Bmp6 in cultured RPE cells, the diminished Bmp6 levels observed in RPE cells in early AMD may contribute to iron build-up in AMD. This may in turn propagate a vicious cycle of oxidative stress and iron accumulation, exacerbating AMD and other diseases with hereditary or acquired iron excess.

Prochelators Triggered by Hydrogen Peroxide Provide Hexadentate Iron Coordination to Impede Oxidative Stress

Prochelators are agents that have little affinity for metal ions until they undergo a chemical conversion. Three new aryl boronate prochelators are presented that are responsive to hydrogen peroxide to provide hexadentate ligands for chelating metal ions. TRENBSIM (tris[(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)-2-aminoethyl]amine), TRENBSAM (tris[(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-2-aminoethyl]amine), and TB (tris[(2-boronic acid-benzyl)2-aminoethyl]amine) convert to TRENSIM (tris[(salicylideneamino)ethyl]amine), TRENSAM (tris[(2-hydroxybenzoyl)-2-aminoethyl]amine), and TS (tris[2-hydroxybenzyl)2-aminoethyl]amine), respectively. The prochelators were characterized by (11)B NMR, and the structures of TRENBSAM, TRENBSIM, and the Fe(III) complex of TS were determined by X-ray crystallography. Of the three prochelator/chelator pairs, TB/TS was identified as the most promising for biological applications, as they prevent iron and copper-induced hydroxyl radical generation in an in vitro assay. TB has negligible interactions with metal ions, whereas TS has apparent binding constants (log K) at pH 7.4 of 15.87 for Cu(II), 9.67 Zn(II) and 14.42 for Fe(III). Up to 1 mMTB was nontoxic to retinal pigment epithelial cells, whereas 10 μM TS induced cell death. TS protected cells against H(2)O(2)-induced death, but only within a 1-10 μM range. TB, on the other hand, had a much broader window of protection, suggesting that it may be a useful agent for preventing metal-promoted oxidative damage.

Ferroxidase Hephaestin's Cell-Autonomous Role in the Retinal Pigment Epithelium

Hephaestin (Heph) is a ferroxidase protein that converts ferrous to ferric iron to facilitate cellular iron export by ferroportin. Many tissues express either Heph or its homologue, ceruloplasmin (Cp), but the retina expresses both. In mice, a combined systemic mutation of Heph and systemic knockout of Cp (Cp(-/-), Heph(sla/sla)) causes retinal iron accumulation and retinal degeneration, with features of human age-related macular degeneration; however, the role of Heph and Cp in the individual retinal cells is unclear. Herein, we used conditional knockout mice to study Hephs role in retinal pigment epithelial (RPE) and photoreceptor cells. Loss of both Heph and Cp from RPE cells alone results in RPE cell iron accumulation and degeneration. We found, however, that RPE iron accumulation in these conditional knockout mice is not as great as in systemic knockout mice. Photoreceptor-specific Heph knockout indicates that the additional iron in the RPE cells does not result from loss of ferroxidases in the photoreceptors, and Cp and Heph play minor roles in photoreceptors. Instead, loss of ferroxidases in other retinal cells causes retinal iron accumulation and transfer of iron to the RPE cells. Cp and Heph are necessary for iron export from the retina but are not essential for iron import into the retina. Thus, our studies, revise how we think about iron import and export from the retina.

Transcription Factor SOX9 Plays a Key Role in the Regulation of Visual Cycle Gene Expression in the Retinal Pigment Epithelium

Background: The visual cycle is an enzymatic cascade that regenerates the visual chromophore. Results: Visual cycle gene expression is regulated by SOX9 in combination with OTX2 or LHX2 and can be modulated by common microRNAs. Conclusion: A core transcriptional network involving SOX9 regulates visual cycle genes. Significance: Understanding visual cycle gene regulation may have implications for treating retinal degenerative diseases. The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible regulation of visual cycle genes by common miRNAs.