The role of ferroportin in retinal iron homeostasis

Age-related macular degeneration (AMD) is the leading cause of blindness in Western Nations. The etiology of AMD is multifactorial and has been associated with auto-immunity, the complement cascade, and oxidative stress. Iron is a potent generator of free radicals that contribute to oxidative stress. We have previously demonstrated increased iron levels in AMD-maculas and macular degeneration in a patient with aceruloplasminemia, an iron overload disease. Mice lacking the ferroxidases Ceruloplasmin (Cp) and Hephaestin (Heph) accumulate iron within the retina and develop retinal degeneration with features of AMD. Ferroportin (Fpn), an iron transport protein, functions with Cp/Heph to transport iron out of cells. Furthermore, the presence of Fpn on the cell membrane is regulated by hepcidin, a protein produced by both the liver and the retina. To study the role of iron accumulation in retinal degeneration and the function of Fpn within the retina, we undertook several studies. The expression of Fpn and response to hepcidin was studied in vitro using a human retinal cell line, ARPE-19. We used Polycythaemia and Flatiron mice to study changes in retinal iron homeostasis in mice that have genomic mutations in ferroportin. Finally, we developed a transgenic mouse line, BEST1-cre, that has retinal pigment epithelium specific ocular cre expression. These mice were use to generate RPE specific knockout of Fpn, and then analyzed for retinal degeneration, and changes in iron homeostasis. The following results suggest Fpn plays an important role in retinal iron homeostasis. Fpn protein and message increased in serum withdrawn ARPE-19 as they became more differentiated. Hepcidin treatment of ARPE-19 decreased cell surface Fpn levels, and, 60min after application decreased iron export and increased intracellular iron. Polycythaemia (Pcm) mice had an age-dependant thinning of the outer nuclear layer of the retina indicating that Fpn is important for retinal health. Pcm retinas had increased transferrin receptor immunofluorescence, suggesting retinal iron deficiency. Similarly, comparison of retinal gene expression in 6 month old Flatiron retinas vs. age-matched control showed an increase in transferrin receptor message, indicating that Flatiron retinas were iron deficient. BEST1-cre mice generated herein had RPE-specific cre expression causing the deletion of floxed sequences, in several mouse lines, including the floxed Fpn line. However, this line was not informative regarding RPE Fpn function in adult mice. Overall, experiments performed herein indicate that RPE ferroportin is regulated by intracellular iron levels and by hepcidin. It is important for retinal iron homeostasis and long-term photoreceptor survival

The role of ferroportin in retinal iron homeostasis

Age-related macular degeneration (AMD) is the leading cause of blindness in Western Nations. The etiology of AMD is multifactorial and has been associated with auto-immunity, the complement cascade, and oxidative stress. Iron is a potent generator of free radicals that contribute to oxidative stress. We have previously demonstrated increased iron levels in AMD-maculas and macular degeneration in a patient with aceruloplasminemia, an iron overload disease. Mice lacking the ferroxidases Ceruloplasmin (Cp) and Hephaestin (Heph) accumulate iron within the retina and develop retinal degeneration with features of AMD. Ferroportin (Fpn), an iron transport protein, functions with Cp/Heph to transport iron out of cells. Furthermore, the presence of Fpn on the cell membrane is regulated by hepcidin, a protein produced by both the liver and the retina. To study the role of iron accumulation in retinal degeneration and the function of Fpn within the retina, we undertook several studies. The expression of Fpn and response to hepcidin was studied in vitro using a human retinal cell line, ARPE-19. We used Polycythaemia and Flatiron mice to study changes in retinal iron homeostasis in mice that have genomic mutations in ferroportin. Finally, we developed a transgenic mouse line, BEST1-cre, that has retinal pigment epithelium specific ocular cre expression. These mice were use to generate RPE specific knockout of Fpn, and then analyzed for retinal degeneration, and changes in iron homeostasis. The following results suggest Fpn plays an important role in retinal iron homeostasis. Fpn protein and message increased in serum withdrawn ARPE-19 as they became more differentiated. Hepcidin treatment of ARPE-19 decreased cell surface Fpn levels, and, 60min after application decreased iron export and increased intracellular iron. Polycythaemia (Pcm) mice had an age-dependant thinning of the outer nuclear layer of the retina indicating that Fpn is important for retinal health. Pcm retinas had increased transferrin receptor immunofluorescence, suggesting retinal iron deficiency. Similarly, comparison of retinal gene expression in 6 month old Flatiron retinas vs. age-matched control showed an increase in transferrin receptor message, indicating that Flatiron retinas were iron deficient. BEST1-cre mice generated herein had RPE-specific cre expression causing the deletion of floxed sequences, in several mouse lines, including the floxed Fpn line. However, this line was not informative regarding RPE Fpn function in adult mice. Overall, experiments performed herein indicate that RPE ferroportin is regulated by intracellular iron levels and by hepcidin. It is important for retinal iron homeostasis and long-term photoreceptor survival

Serum And Forskolin Cooperate To Promote G1 Progression In Schwann Cells By Differentially Regulating Cyclin D1, Cyclin E1, And P27\u3csup\u3eKip\u3c/sup\u3e Expression

Proliferation of Schwann cells in vitro, unlike most mammalian cells, is not induced by serum alone but additionally requires cAMP elevation and mitogenic stimulation. How these agents cooperate to promote progression through the G1 phase of the cell cycle is unclear. We studied the integrative effects of these compounds on receptor-mediated signaling pathways and regulators of G1 progression. We show that serum alone induces strong cyclical expression of cyclin D1 and E1, 6 and 12 h after addition, respectively. Serum also promotes strong but transient erbB2, ERK, and Akt phosphorylation, but Schwann cells remain arrested in G1 due to high levels of the inhibitor, p27Kip. Forskolin with serum promotes G1 progression in 22% of Schwann cells between 18 and 24 h by inducing a steady decline in p27Kip levels that reaches a nadir at 12 h coinciding with peak cyclin E1 expression. Forskolin also delays neuregulin-induced loss of erbB2 receptors allowing strong acute activation of PI3K, sustained erbB2 phosphorylation and G1 progression in 31% of Schwann cells. We find that the ability of forskolin to decrease p27Kip is associated with its ability to decrease Krox-20 expression that is induced by serum and further increased by neuregulin. Our results explain why serum is required but insufficient to stimulate proliferation and identify two routes by which forskolin promotes proliferation in the presence of serum and neuregulin. These findings provide insights into how G1 progression and, cell cycle arrest leading to myelination are regulated in Schwann cells. © 2007 Wiley-Liss, Inc

PGC-1α Induces Human RPE Oxidative Metabolism and Antioxidant Capacity

Purpose Oxidative stress and metabolic dysregulation of the RPE have been implicated in AMD; however, the molecular regulation of RPE metabolism remains unclear. The transcriptional coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) is a powerful mediator of mitochondrial function. This study examines the ability of PGC-1α to regulate RPE metabolic program and oxidative stress response. Methods: Primary human fetal RPE (hfRPE) and ARPE-19 were matured in vitro using standard culture conditions. Mitochondrial mass of RPE was measured using MitoTracker staining and citrate synthase activity. Expression of PGC-1 isoforms, RPE-specific genes, oxidative metabolism proteins, and antioxidant enzymes was analyzed by quantitative PCR and Western blot. Mitochondrial respiration and fatty-acid oxidation were monitored using the Seahorse extracellular flux analyzer. Expression of PGC-1α was increased using adenoviral delivery. ARPE-19 were exposed to hydrogen peroxide to induce oxidative stress. Reactive oxygen species were measured by CM-H2DCFDA fluorescence. Cell death was analyzed by LDH release. Results: Maturation of ARPE-19 and hfRPE was associated with significant increase in mitochondrial mass, expression of oxidative phosphorylation (OXPHOS) genes, and PGC-1α gene expression. Overexpression of PGC-1α increased expression of OXPHOS and fatty-acid β-oxidation genes, ultimately leading to the potent induction of mitochondrial respiration and fatty-acid oxidation. PGC-1α gain of function also strongly induced numerous antioxidant genes and, importantly, protected RPE from oxidant-mediated cell death without altering RPE functions. Conclusions: This study provides important insights into the metabolic changes associated with RPE functional maturation and identifies PGC-1α as a potent driver of RPE mitochondrial function and antioxidant capacity

Age-Dependent Retinal Iron Accumulation and Degeneration in Hepcidin Knockout Mice

Hepcidin is an iron regulatory hormone expressed in the retina. In the present study, evidence from mice and tissue culture suggest that hepcidin is upregulated in response to increased retinal iron levels and normally serves to prevent retinal iron excess

The oral iron chelator deferiprone protects iron overload-induced retinal degeneration. Invest Ophthalmol Vis Sci 52:959–968

PURPOSE. Iron-induced oxidative stress may exacerbate agerelated 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). METHODS. Cultured 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. RESULTS. DFP at 60 M 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. CONCLUSIONS. Oral 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. (Invest Ophthalmol Vis Sci. 2011;52:959 -968) DOI:10.1167/iovs.10-6207 I ron is crucial for optimal cellular metabolism, but is also a potent generator of oxidative stress if present in excess, especially in the form of labile ferrous iron. Inability of the body to actively excrete excess iron leads to age-dependent iron accumulation in certain tissues, including the macula. 1 Excess tissue iron generates reactive oxygen species (ROS) via the Fenton reaction, leading to oxidative damage. Free radicals and oxidative stress have been implicated in a growing number of conditions, from normal aging to cancer, diabetes, and neurodegenerative diseases, making iron overload or metabolic mishandling of iron an important target for therapeutic intervention. 2-6 Since iron catalyzes the production of the hydroxyl radical, the most damaging of the free radicals, it is likely to exacerbate oxidative damage in a tissue that is already prone to oxidative insult. Retinal pigment epithelial (RPE) cells and photoreceptors are especially vulnerable to oxidative damage due to high oxygen tension, ROS production by large numbers of mitochondria, and abundant, easily oxidized polyunsaturated fatty acids in photoreceptor membranes. 7 Indeed, several neurodegenerative disorders with iron dysregulation feature retinal degeneration. 8 These include the rare hereditary disorders aceruloplasminemia, Friedreich's ataxia, and pantothenate kinase-associated neurodegeneration. Further, traumatic siderosis causes rapid retinal degeneration. 9 Similarly, retinal degeneration in several mouse models is associated with retinal iron dysregulation. -12 Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly worldwide. Although the pathogenesis of AMD is incompletely understood, growing evidence suggests that, in addition to inflammation, complement activation, and other hereditary and environmental influences, 9 Supporting this hypothesis, patients lacking the ferroxidase ceruloplasmin (Cp) as a result of the autosomal recessive condition aceruloplasminemia, have retinal iron accumulation and early-onset macular degeneration. From th

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

The authors characterize a new transgenic mouse line, BEST1-cre, that provides RPE-specific ocular cre expression. These mice begin expressing cre at postnatal day 10 and maintain expression into adulthood without causing retinal dysfunction. Cre expression is present in up to 90% of RPE nuclei. Therefore, these mice provide a useful tool for studying the postnatal function of genes within the RPE