title: Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD.
abstract: Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.
authors.affiliation: a Department of Ophthalmology ; Indiana University School of Medicine ; Indianapolis , IN USA.
authors.forename: Sayak K
authors.initials: SK
authors.lastname: Mitter
authors.forename: Chunjuan
authors.initials: C
authors.lastname: Song
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Qi
authors.forename: Haoyu
authors.initials: H
authors.lastname: Mao
authors.forename: Haripriya
authors.initials: H
authors.lastname: Rao
authors.forename: Debra
authors.initials: D
authors.lastname: Akin
authors.forename: Alfred
authors.initials: A
authors.lastname: Lewin
authors.forename: Maria
authors.initials: M
authors.lastname: Grant
authors.forename: William
authors.initials: W
authors.lastname: Dunn
authors.forename: Jindong
authors.initials: J
authors.lastname: Ding
authors.forename: Catherine
authors.initials: C
authors.lastname: Bowes Rickman
authors.forename: Michael
authors.initials: M
authors.lastname: Boulton
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D053327:Apolipoprotein E4 / Q000235:genetics; D001343:Autophagy; D002470:Cell Survival; D005786:Gene Expression Regulation; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D008062:Lipofuscin / Q000737:chemistry; D008268:Macular Degeneration / Q000473:pathology*; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology*
chemical_list: D053327:Apolipoprotein E4; D008062:Lipofuscin; D017382:Reactive Oxygen Species; C025946:3-methyladenine; D006861:Hydrogen Peroxide; D005978:Glutathione; D000225:Adenine
keywords: 3-MA, 3-methyladenine; ACTB, β-actin; AMD, age-related macular degeneration; APOE4, apolipoprotein E4; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; GFP, green fluorescent protein; GSH, glutathione, reduced; GSSG, glutathione, oxidized; H2O2, hydrogen peroxide; HFC, high fat, cholesterol-enriched diet; LC3, microtubule-associated protein 1 light chain 3; MMP, mitochondrial membrane potential; MTT, 3-(4 5-dimethylthiazol-3-yl)-2, 5-diphenyl tetrazolium bromide; ND, normal (rodent) diet; POS, photoreceptor outer segments; ROS, reactive oxygen species; RPE; RPE, retinal pigmented epithelium; SOD2/MnSOD, superoxide dismutase 2, mitochondrial; UPS, ubiquitin-proteasome system; age-related macular degeneration; aging; autophagy; oxidative stress