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1.
Mol Ther ; 29(7): 2227-2238, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-33677092

RESUMO

mRNA vaccines induce potent immune responses in preclinical models and clinical studies. Adjuvants are used to stimulate specific components of the immune system to increase immunogenicity of vaccines. We utilized a constitutively active mutation (V155M) of the stimulator of interferon (IFN) genes (STING), which had been described in a patient with STING-associated vasculopathy with onset in infancy (SAVI), to act as a genetic adjuvant for use with our lipid nanoparticle (LNP)-encapsulated mRNA vaccines. mRNA-encoded constitutively active STINGV155M was most effective at maximizing CD8+ T cell responses at an antigen/adjuvant mass ratio of 5:1. STINGV155M appears to enhance development of antigen-specific T cells by activating type I IFN responses via the nuclear factor κB (NF-κB) and IFN-stimulated response element (ISRE) pathways. mRNA-encoded STINGV155M increased the efficacy of mRNA vaccines encoding the E6 and E7 oncoproteins of human papillomavirus (HPV), leading to reduced HPV+ TC-1 tumor growth and prolonged survival in vaccinated mice. This proof-of-concept study demonstrated the utility of an mRNA-encoded genetic adjuvant.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Neoplasias Pulmonares/terapia , Proteínas de Membrana/imunologia , Proteínas E7 de Papillomavirus/imunologia , RNA Mensageiro/imunologia , Vacinas de mRNA/imunologia , Adjuvantes Imunológicos , Animais , Apoptose , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Proliferação de Células , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Lipossomos/química , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , RNA Mensageiro/genética , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas , Vacinas de mRNA/administração & dosagem , Vacinas de mRNA/genética
2.
J Biol Chem ; 289(18): 12908-21, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24634209

RESUMO

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.


Assuntos
Proteínas do Olho/genética , Regulação da Expressão Gênica , Epitélio Pigmentado da Retina/metabolismo , Fatores de Transcrição SOX9/fisiologia , Animais , Sítios de Ligação/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Proteínas do Olho/metabolismo , Redes Reguladoras de Genes , Células HEK293 , Humanos , Imuno-Histoquímica , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , Modelos Genéticos , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Epitélio Pigmentado da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo
3.
J Neurochem ; 135(5): 958-74, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26303407

RESUMO

Brain iron accumulates in several neurodegenerative diseases and can cause oxidative damage, but mechanisms of brain iron homeostasis are incompletely understood. Patients with mutations in the cellular iron-exporting ferroxidase ceruloplasmin (Cp) have brain iron accumulation causing neurodegeneration. Here, we assessed the brains of mice with combined mutation of Cp and its homolog hephaestin. Compared to single mutants, brain iron accumulation was accelerated in double mutants in the cerebellum, substantia nigra, and hippocampus. Iron accumulated within glia, while neurons were iron deficient. There was loss of both neurons and glia. Mice developed ataxia and tremor, and most died by 9 months. Treatment with the oral iron chelator deferiprone diminished brain iron levels, protected against neuron loss, and extended lifespan. Ferroxidases play important, partially overlapping roles in brain iron homeostasis by facilitating iron export from glia, making iron available to neurons. Above: Iron (Fe) normally moves from capillaries to glia to neurons. It is exported from the glia by ferroportin (Fpn) with ferroxidases ceruloplasmin (Cp) and/or Hephaestin (Heph). Below: In mice with mutation of Cp and Heph, iron accumulates in glia, while neurons have low iron levels. Both neurons and glia degenerate and mice become ataxic unless given an iron chelator.


Assuntos
Ceruloplasmina/genética , Quelantes de Ferro/uso terapêutico , Ferro/metabolismo , Proteínas de Membrana/genética , Mutação/genética , Doenças Neurodegenerativas , Piridonas/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Ceruloplasmina/metabolismo , Deferiprona , Modelos Animais de Doenças , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Força Muscular/efeitos dos fármacos , Força Muscular/genética , Proteína Básica da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Tirosina 3-Mono-Oxigenase/metabolismo
4.
Exp Eye Res ; 128: 92-101, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25277027

RESUMO

The purpose of our studies was to examine the relationship between iron and melanogenesis in retinal pigment epithelial cells, as prior observations had suggested that iron may promote melanogenesis. This relationship has potential clinical importance, as both iron overload and hyperpigmentation are associated with age-related macular degeneration (AMD). Human fetal retinal pigment epithelial cells and ARPE-19 cells were treated with iron in the form of ferric ammonium citrate, after which quantitative RT-PCR and electron microscopy were performed. Melanogenesis genes tyrosinase, tyrosinase-related protein 1, Hermansky-Pudlak Syndrome 3, premelanosome protein and dopachrome tautomerase were upregulated, as was the melanogenesis-controlling transcription factor, microphthalmia-associated transcription factor (MITF). Iron-treated cells had increased pigmentation and melanosome number. Multiple transcription factors upstream of MITF were upregulated by iron.


Assuntos
Compostos Férricos/farmacologia , Melaninas/biossíntese , Melanossomas/metabolismo , Compostos de Amônio Quaternário/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Regulação para Cima/fisiologia , Western Blotting , Proteínas de Transporte/genética , Células Cultivadas , Idade Gestacional , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Oxirredutases Intramoleculares/genética , Glicoproteínas de Membrana/genética , Monofenol Mono-Oxigenase/genética , Oxirredutases/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/embriologia , Epitélio Pigmentado da Retina/metabolismo , Doadores de Tecidos , Antígeno gp100 de Melanoma/genética
5.
Mol Metab ; 86: 101965, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38871178

RESUMO

OBJECTIVE: Interleukin (IL)-22 is a potential therapeutic protein for the treatment of metabolic diseases such as obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease due to its involvement in multiple cellular pathways and observed hepatoprotective effects. The short serum half-life of IL-22 has previously limited its use in clinical applications; however, the development of mRNA-lipid nanoparticle (LNP) technology offers a novel therapeutic approach that uses a host-generated IL-22 fusion protein. In the present study, the effects of administration of an mRNA-LNP encoding IL-22 on metabolic disease parameters was investigated in various mouse models. METHODS: C57BL/6NCrl mice were used to confirm mouse serum albumin (MSA)-IL-22 protein expression prior to assessments in C57BL/6NTac and CETP/ApoB transgenic mouse models of metabolic disease. Mice were fed either regular chow or a modified amylin liver nonalcoholic steatohepatitis-inducing diet prior to receiving either LNP-encapsulated MSA-IL-22 or MSA mRNA via intravenous or intramuscular injection. Metabolic markers were monitored for the duration of the experiments, and postmortem histology assessment and analysis of metabolic gene expression pathways were performed. RESULTS: MSA-IL-22 was detectable for ≥8 days following administration. Improvements in body weight, lipid metabolism, glucose metabolism, and lipogenic and fibrotic marker gene expression in the liver were observed in the MSA-IL-22-treated mice, and these effects were shown to be durable. CONCLUSIONS: These results support the application of mRNA-encoded IL-22 as a promising treatment strategy for metabolic syndrome and associated comorbidities in human populations.


Assuntos
Interleucina 22 , Interleucinas , Doenças Metabólicas , Camundongos Endogâmicos C57BL , RNA Mensageiro , Animais , Camundongos , Interleucinas/metabolismo , Interleucinas/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Masculino , Doenças Metabólicas/metabolismo , Doenças Metabólicas/genética , Nanopartículas , Meia-Vida , Camundongos Transgênicos , Fígado/metabolismo , Modelos Animais de Doenças , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Lipídeos/sangue , Lipossomos
6.
Am J Pathol ; 180(4): 1614-24, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22342521

RESUMO

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 Heph's 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.


Assuntos
Degeneração Macular/metabolismo , Proteínas de Membrana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Células Cultivadas , Ceruloplasmina/metabolismo , Modelos Animais de Doenças , Ferro/metabolismo , Degeneração Macular/genética , Degeneração Macular/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Mutação , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Neurônios Retinianos/metabolismo , Epitélio Pigmentado da Retina/patologia
7.
Nat Genet ; 31(4): 354-62, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12118253

RESUMO

Neurofibromatosis type 2 is an autosomal dominant disorder characterized by tumors, predominantly schwannomas, in the nervous system. It is caused by mutations in the gene NF2, encoding the growth regulator schwannomin (also known as merlin). Mutations occur throughout the 17-exon gene, with most resulting in protein truncation and undetectable amounts of schwannomin protein. Pathogenic mutations that result in production of defective schwannomin include in-frame deletions of exon 2 and three independent missense mutations within this same exon. Mice with conditional deletion of exon 2 in Schwann cells develop schwannomas, which confirms the crucial nature of exon 2 for growth control. Here we report that the molecular adaptor paxillin binds directly to schwannomin at residues 50-70, which are encoded by exon 2. This interaction mediates the membrane localization of schwannomin to the plasma membrane, where it associates with beta 1 integrin and erbB2. It defines a pathogenic mechanism for the development of NF2 in humans with mutations in exon 2 of NF2.


Assuntos
Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Éxons , Integrina beta1/metabolismo , Camundongos , Mutação , Neurofibromatose 2/genética , Neurofibromatose 2/fisiopatologia , Paxilina , Isoformas de Proteínas , Ratos , Receptor ErbB-2/metabolismo , Células de Schwann/citologia , Células de Schwann/metabolismo
8.
Am J Pathol ; 179(1): 335-48, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21703414

RESUMO

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.


Assuntos
Proteína Morfogenética Óssea 6/fisiologia , Ferro/metabolismo , Degeneração Macular/etiologia , Degeneração Macular/patologia , Estresse Oxidativo , Epitélio Pigmentado da Retina/patologia , Animais , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Sobrecarga de Ferro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Degeneração Retiniana , Epitélio Pigmentado da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Biomaterials ; 272: 120786, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33839625

RESUMO

Restoring numbers and function of regulatory T cells (Tregs) is a novel therapeutic strategy for neurodegenerative disorders. Whether Treg function is boosted by adoptive cell transfer, pharmaceuticals, or immune modulators, the final result is a robust anti-inflammatory and neuronal sparing response. Herein, a newly developed lipid nanoparticle (LNP) containing mRNA encoding granulocyte-macrophage colony-stimulating factor (Gm-csf mRNA) was developed to peripherally induce Tregs and used for treatment in preclinical Parkinson's disease (PD) models. Administration of Gm-csf mRNA to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and rats overexpressing alpha-synuclein produced dose-dependent increases in plasma GM-CSF levels and peripheral CD4+CD25+FoxP3+ Treg populations. This upregulation paralleled nigrostriatal neuroprotection, upregulated immunosuppression-associated mRNAs that led to the detection of a treatment-induced CD4+ T cell population, and decreased reactive microgliosis. The current findings strengthen prior works utilizing immune modulation by harnessing Gm-csf mRNA to augment adaptive immune function by employing a new delivery platform to treat PD and potentially other neurodegenerative disorders.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Doença de Parkinson , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Camundongos , Camundongos Endogâmicos C57BL , Neuroproteção , Doença de Parkinson/genética , Doença de Parkinson/terapia , RNA Mensageiro/genética , Ratos
10.
Life Sci Alliance ; 2(3)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101737

RESUMO

The retinal pigment epithelium (RPE) supports visual processing and photoreceptor homeostasis via energetically demanding cellular functions. Here, we describe the consequences of repressing peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α), a master regulator of mitochondrial function and biogenesis, on RPE epithelial integrity. The sustained silencing of PGC-1α in differentiating human RPE cells affected mitochondria/autophagy function, redox state, and impaired energy sensor activity ultimately inducing epithelial to mesenchymal transition (EMT). Adult conditional knockout of PGC-1 coactivators in mice resulted in rapid RPE dysfunction and transdifferentiation associated with severe photoreceptor degeneration. RPE anomalies were characteristic of autophagic defect and mesenchymal transition comparable with the ones observed in age-related macular degeneration. These findings demonstrate that PGC-1α is required to maintain the functional and phenotypic status of RPE by supporting the cells' oxidative metabolism and autophagy-mediated repression of EMT.

11.
Prog Retin Eye Res ; 26(6): 649-73, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17921041

RESUMO

Iron is essential for many metabolic processes but can also cause damage. As a potent generator of hydroxyl radical, the most reactive of the free radicals, iron can cause considerable oxidative stress. Since iron is absorbed through diet but not excreted except through menstruation, total body iron levels buildup with age. Macular iron levels increase with age, in both men and women. This iron has the potential to contribute to retinal degeneration. Here we present an overview of the evidence suggesting that iron may contribute to retinal degenerations. Intraocular iron foreign bodies cause retinal degeneration. Retinal iron buildup resulting from hereditary iron homeostasis disorders aceruloplasminemia, Friedreich's ataxia, and panthothenate kinase-associated neurodegeneration cause retinal degeneration. Mice with targeted mutation of the iron exporter ceruloplasmin have age-dependent retinal iron overload and a resulting retinal degeneration with features of age-related macular degeneration (AMD). Post mortem retinas from patients with AMD have more iron and the iron carrier transferrin than age-matched controls. Over the past 10 years much has been learned about the intricate network of proteins involved in iron handling. Many of these, including transferrin, transferrin receptor, divalent metal transporter-1, ferritin, ferroportin, ceruloplasmin, hephaestin, iron-regulatory protein, and histocompatibility leukocyte antigen class I-like protein involved in iron homeostasis (HFE) have been found in the retina. Some of these proteins have been found in the cornea and lens as well. Levels of the iron carrier transferrin are high in the aqueous and vitreous humors. The functions of these proteins in other tissues, combined with studies on cultured ocular tissues, genetically engineered mice, and eye exams on patients with hereditary iron diseases provide clues regarding their ocular functions. Iron may play a role in a broad range of ocular diseases, including glaucoma, cataract, AMD, and conditions causing intraocular hemorrhage. While iron deficiency must be prevented, the therapeutic potential of limiting iron-induced ocular oxidative damage is high. Systemic, local, or topical iron chelation with an expanding repertoire of drugs has clinical potential.


Assuntos
Homeostase , Ferro/metabolismo , Ferro/intoxicação , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Animais , Transporte Biológico , Células Epiteliais/metabolismo , Humanos , Cristalino/metabolismo , Estresse Oxidativo , Retina/metabolismo
12.
Invest Ophthalmol Vis Sci ; 47(5): 2135-40, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16639025

RESUMO

PURPOSE: Iron can cause oxidative stress, and elevated iron levels have been associated with several neurodegenerative diseases including age-related macular degeneration (AMD). Transferrin, an iron transport protein, is expressed at high levels in the retina. The purpose of this study was to assess transferrin involvement in AMD by determining the expression profile of transferrin in retinas with AMD compared with retinas without evidence of disease. METHODS: Postmortem retinas were obtained from AMD and non-AMD eyes. Expression of transferrin was assessed in a microarray dataset from 33 retinas of unaffected donors and 12 retinas of patients with AMD (six with neovascular AMD and six with non-neovascular AMD). Quantitative real-time RT-PCR (QPCR) was used to confirm the microarray results. Transferrin protein expression was assessed by semiquantitative Western blot analysis and immunohistochemistry. RESULTS: In comparison to unaffected retinas, mean transferrin mRNA levels, as measured by microarray analysis were elevated 3.5- and 2.1-fold in non-neovascular and neovascular AMD retinas, respectively. Semiquantitative Western blot analysis demonstrated a 2.1-fold increase in transferrin protein in AMD eyes. Immunohistochemistry showed more intense and widespread transferrin label in AMD maculas, particularly in large drusen, Müller cells, and photoreceptors. CONCLUSIONS: These data demonstrate that transferrin expression is increased in the retinas of patients with AMD relative to those of healthy control patients of comparable age. Along with previous studies that have demonstrated elevated iron levels in AMD retinas, early onset drusen formation in a patient with retinal iron overload resulting from aceruloplasminemia, and retinal degeneration with some features of macular degeneration in the iron-overloaded retinas of ceruloplasmin/hephestin knockout mice, the present study suggests that altered iron homeostasis is associated with AMD.


Assuntos
Degeneração Macular/metabolismo , Retina/metabolismo , Transferrina/genética , Transferrina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
13.
Invest Ophthalmol Vis Sci ; 57(3): 1038-51, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26962700

RESUMO

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 ß-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.


Assuntos
Regulação da Expressão Gênica , Degeneração Macular/genética , Estresse Oxidativo , RNA/genética , Epitélio Pigmentado da Retina/metabolismo , Fatores de Transcrição/genética , Western Blotting , Morte Celular , Linhagem Celular , Proteínas de Choque Térmico , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/patologia , Fatores de Transcrição/biossíntese
14.
Invest Ophthalmol Vis Sci ; 55(3): 1941-53, 2014 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-24481259

RESUMO

PURPOSE: To systematically characterize the effects of NaIO3 on retinal morphology and function. METHODS: NaIO3 at 10, 20, or 30 mg/kg was administered by retro-orbital injection into adult C57BL/6J mice. Phenotypic and functional changes of the retina were assessed at 1, 3, 5, and 8 days postinjection by fundus imaging, optical coherence tomography (OCT), ERG, and histology. Direct NaIO3 cytotoxicity on ARPE-19 and 661W cells was quantified using lactate dehydrogenase (LDH) apoptosis assay. Effect of NaIO3 on RPE and photoreceptor gene expression was assessed in vitro and in vivo by quantitative PCR. RESULTS: While little to no change was observed in the 10 mg/kg NaIO3-injected group, significant retinal anomalies, such as RPE atrophy and retinal thinning, were observed in both 20 and 30 mg/kg NaIO3-injected groups. Gene expression analysis showed rapid downregulation of RPE-specific genes, increase in heme oxygenase 1 expression, and induction of the ratio of Bax to Bcl-2. Electroretinographic response loss and photoreceptor gene repression preceded gross morphological changes. High NaIO3 toxicity on 661W cells was observed in vitro along with reactive oxygen species (ROS) induction. NaIO3 treatment also disrupted oxidative stress, phototransduction, and apoptosis gene expression in 661W cells. Exposure of ARPE-19 cells to NaIO3 increased expression of neurotrophins and protected photoreceptors from direct NaIO3 cytotoxicity. CONCLUSIONS: Systematic characterization of changes associated with NaIO3 injection revealed a large variability in the severity of toxicity induced. Treatment with >20 mg/kg NaIO3 induced visual dysfunction associated with rapid suppression of phototransduction genes and induced oxidative stress in photoreceptors. These results suggest that NaIO3 can directly alter photoreceptor function and survival.


Assuntos
Iodatos/toxicidade , Estresse Oxidativo , Epitélio Pigmentado Ocular/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Degeneração Retiniana/metabolismo , Animais , Apoptose , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/patologia , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/patologia , Tomografia de Coerência Óptica/métodos
16.
Transl Vis Sci Technol ; 1(2): 7, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-24049707

RESUMO

PURPOSE: To investigate the effect of the iron chelator deferiprone (DFP) on sodium iodate (NaIO3)-induced retinal degeneration and on the hereditary retinal degeneration caused by the rd6 mutation. METHODS: Retinas from NaIO3-treated C57BL/6J mice, with or without DFP cotreatment, were analyzed by histology, immunofluorescence, and quantitative PCR to investigate the effect of DFP on retinal degeneration. To facilitate photoreceptor quantification, we developed a new function of MATLAB to perform this task in a semiautomated fashion. Additionally, rd6 mice treated with or without DFP were analyzed by histology to assess possible protection. RESULTS: In NaIO3-treated mice, DFP protected against retinal degeneration and significantly decreased expression of the oxidative stress-related gene heme oxygenase-1 and the complement gene C3. DFP treatment partially protected against NaIO3-induced reduction in the levels of mRNAs encoded by visual cycle genes rhodopsin (Rho) and retinal pigment epithelium-specific 65 kDa protein (Rpe65), consistent with the morphological data indicating preservation of photoreceptors and RPE, respectively. DFP treatment also protected photoreceptors in rd6 mice. CONCLUSIONS: The oral iron chelator DFP provides significant protection against retinal degeneration induced through different modalities. This suggests that iron chelation could be useful as a treatment for retinal degeneration even when the main etiology does not appear to be iron dysregulation. TRANSLATIONAL RELEVANCE: These data provide proof of principle that the oral iron chelator DFP can protect the retina against diverse insults. Further testing of DFP in additional animal retinal degeneration models at a range of doses is warranted.

17.
Transl Vis Sci Technol ; 1(3): 2, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-24049709

RESUMO

PURPOSE: To investigate the effect of the iron chelator deferiprone (DFP) on sodium iodate (NaIO3)-induced retinal degeneration and on the hereditary retinal degeneration caused by the rd6 mutation. METHODS: Retinas from NaIO3-treated C57BL/6J mice, with or without DFP cotreatment, were analyzed by histology, immunofluorescence, and quantitative PCR to investigate the effect of DFP on retinal degeneration. To facilitate photoreceptor quantification, we developed a new function of MATLAB to perform this task in a semiautomated fashion. Additionally, rd6 mice treated with or without DFP were analyzed by histology to assess possible protection. RESULTS: In NaIO3-treated mice, DFP protected against retinal degeneration and significantly decreased expression of the oxidative stress-related gene heme oxygenase-1 and the complement gene C3. DFP treatment partially protected against NaIO3-induced reduction in the levels of mRNAs encoded by visual cycle genes rhodopsin (Rho) and retinal pigment epithelium-specific 65 kDa protein (Rpe65), consistent with the morphological data indicating preservation of photoreceptors and RPE, respectively. DFP treatment also protected photoreceptors in rd6 mice. CONCLUSIONS: The oral iron chelator DFP provides significant protection against retinal degeneration induced through different modalities. This suggests that iron chelation could be useful as a treatment for retinal degeneration even when the main etiology does not appear to be iron dysregulation. TRANSLATIONAL RELEVANCE: These data provide proof of principle that the oral iron chelator DFP can protect the retina against diverse insults. Further testing of DFP in additional animal retinal degeneration models at a range of doses is warranted.

18.
Invest Ophthalmol Vis Sci ; 52(1): 109-18, 2011 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-20811044

RESUMO

PURPOSE: Iron dysregulation can cause retinal disease, yet retinal iron regulatory mechanisms are incompletely understood. The peptide hormone hepcidin (Hepc) limits iron uptake from the intestine by triggering degradation of the iron transporter ferroportin (Fpn). Given that Hepc is expressed in the retina and Fpn is expressed in cells constituting the blood-retinal barrier, the authors tested whether the retina may produce Hepc to limit retinal iron import. METHODS: Retinas of Hepc(-/-) mice were analyzed by histology, autofluorescence spectral analysis, atomic absorption spectrophotometry, Perls' iron stain, and immunofluorescence to assess iron-handling proteins. Retinal Hepc mRNA was evaluated through qPCR after intravitreal iron injection. Mechanisms of retinal Hepc upregulation were tested by Western blot analysis. A retinal capillary endothelial cell culture system was used to assess the effect of exogenous Hepc on Fpn. RESULTS: Hepc(-/-) mice experienced age-dependent increases in retinal iron followed by retinal degeneration with autofluorescent RPE, photoreceptor death, and subretinal neovascularization. Hepc(-/-) mice had increased Fpn immunoreactivity in vascular endothelial cells. Conversely, in cultured retinal capillary endothelial cells, exogenous Hepc decreased both Fpn levels and iron transport. The retina can sense increased iron levels, upregulating Hepc after phosphorylation of extracellular signal regulated kinases. CONCLUSIONS: These findings indicate that Hepc is essential for retinal iron regulation. In the absence of Hepc, retinal degeneration occurs. Increases in Hepc mRNA levels after intravitreal iron injection combined with Hepc-mediated decreases in iron export from cultured retinal capillary endothelial cells suggest that the retina may use Hepc for its tissue-specific iron regulation.


Assuntos
Envelhecimento/fisiologia , Peptídeos Catiônicos Antimicrobianos/fisiologia , Apoferritinas/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Receptores da Transferrina/metabolismo , Retina/metabolismo , Degeneração Retiniana/metabolismo , Animais , Apoferritinas/genética , Apoptose , Western Blotting , Proteínas de Transporte de Cátions/genética , Bovinos , Células Cultivadas , Endotélio Vascular/metabolismo , Fluorescência , Técnica Indireta de Fluorescência para Anticorpo , Hepcidinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Receptores da Transferrina/genética , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Espectrofotometria Atômica
19.
Invest Ophthalmol Vis Sci ; 52(2): 959-68, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21051716

RESUMO

PURPOSE: Iron-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). 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 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. 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.


Assuntos
Quelantes de Ferro/administração & dosagem , Sobrecarga de Ferro/prevenção & controle , Piridonas/administração & dosagem , Degeneração Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/efeitos dos fármacos , Administração Oral , Animais , Antígenos CD/metabolismo , Morte Celular , Linhagem Celular , Ceruloplasmina/genética , Deferiprona , Eletrorretinografia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Peróxido de Hidrogênio/toxicidade , Quelantes de Ferro/farmacologia , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Estresse Oxidativo/efeitos dos fármacos , Piridonas/farmacologia , RNA Mensageiro/genética , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Invest Ophthalmol Vis Sci ; 52(3): 1378-83, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21212186

RESUMO

PURPOSE: To 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: This 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.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Integrases/genética , Epitélio Pigmentado da Retina/enzimologia , Animais , Bestrofinas , Western Blotting , Eletrorretinografia , Proteínas do Olho/genética , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Canais Iônicos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética
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