title: Repressed SIRT1/PGC-1α pathway and mitochondrial disintegration in iPSC-derived RPE disease model of age-related macular degeneration.
abstract: Study of age related macular degeneration (AMD) has been hampered by lack of human models that represent the complexity of the disease. Here we have developed a human in vitro disease model of AMD to investigate the underlying AMD disease mechanisms.
  
  
  
  Generation of iPSCs from retinal pigment epithelium (RPE) of AMD donors, age-matched normal donors, skin fibroblasts of a dry AMD patient, and differentiation of iPSCs into RPE (AMD RPE-iPSC-RPE, normal RPE-iPSC-RPE and AMD Skin-iPSC-RPE, respectively). Immunostaining, cell viability assay and reactive oxygen species (ROS) production under oxidative stress conditions, electron microscopy (EM) imaging, ATP production and glycogen concentration assays, quantitative real time PCR, western blot, karyotyping.
  
  
  
  The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE present functional impairment and exhibit distinct disease phenotypes compared to RPE-iPSC-RPE generated from normal donors (Normal RPE-iPSC-RPE). The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE show increased susceptibility to oxidative stress and produced higher levels of reactive oxygen species (ROS) under stress in accordance with recent reports. The susceptibility to oxidative stress-induced cell death in AMD RPE-iPSC-RPE and Skin-iPSC-RPE was consistent with inability of the AMD RPE-iPSC-RPE and Skin-iPSC-RPE to increase SOD2 expression under oxidative stress. Phenotypic analysis revealed disintegrated mitochondria, accumulation of autophagosomes and lipid droplets in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE. Mitochondrial activity was significantly lower in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE compared to normal cells and glycogen concentration was significantly increased in the diseased cells. Furthermore, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis and function was repressed, and lower expression levels of NAD-dependent deacetylase sirtuin1 (SIRT1) were found in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE as compared to normal RPE-iPSC-RPE.
  
  
  
  Our studies suggest SIRT1/PGC-1α as underlying pathways contributing to AMD pathophysiology, and open new avenues for development of targeted drugs for treatment of this devastating neurodegenerative disease of the visual system.
authors.affiliation: Department of Ophthalmology, Georgetown University Medical Center, 3900 Reservoir Road NW, Medical-Dental Building, Room NE203, Washington, DC, 20057, USA. ncg8@georgetown.edu.|Department of Neurology, Georgetown University Medical Center, Washington, DC, USA. ncg8@georgetown.edu.|Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC, USA. ncg8@georgetown.edu.
authors.forename: Nady
authors.identifier: 0000-0001-7573-0962
authors.initials: N
authors.lastname: Golestaneh
authors.affiliation: Department of Ophthalmology, Georgetown University Medical Center, 3900 Reservoir Road NW, Medical-Dental Building, Room NE203, Washington, DC, 20057, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Chu
authors.affiliation: Department of Ophthalmology, Georgetown University Medical Center, 3900 Reservoir Road NW, Medical-Dental Building, Room NE203, Washington, DC, 20057, USA.
authors.forename: Shuk Kei
authors.initials: SK
authors.lastname: Cheng
authors.affiliation: Department of Ophthalmology, Georgetown University Medical Center, 3900 Reservoir Road NW, Medical-Dental Building, Room NE203, Washington, DC, 20057, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Cao
authors.affiliation: Retinal Cell and Molecular Biology (LRCMB), National Eye Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Poliakov
authors.affiliation: Retina Group of Washington, Chevy Chase, MD, 20815, USA.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Berinstein
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002454:Cell Differentiation; D005260:Female; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D008268:Macular Degeneration / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D018384:Oxidative Stress; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha / Q000378:metabolism*; D010587:Phagocytosis; D010641:Phenotype; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000473:pathology* / Q000648:ultrastructure; D015398:Signal Transduction; D056564:Sirtuin 1 / Q000378:metabolism*; D012867:Skin / Q000473:pathology; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: C486497:PPARGC1A protein, human; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; C447939:SIRT1 protein, human; D056564:Sirtuin 1
keywords: AMD; Cell viability; Mitochondria; Oxidative stress; PGC-1α; ROS; RPE; SIRT1