title: Inhibition or Stimulation of Autophagy Affects Early Formation of Lipofuscin-Like Autofluorescence in the Retinal Pigment Epithelium Cell.
abstract: The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is dependent on the effectiveness of photoreceptor outer segment material degradation. This study explored the role of autophagy in the fate of RPE lipofuscin degradation. After seven days of feeding with either native or modified rod outer segments, ARPE-19 cells were treated with enhancers or inhibitors of autophagy and the autofluorescence was detected by fluorescence-activated cell sorting. Supplementation with different types of rod outer segments increased lipofuscin-like autofluorescence (LLAF) after the inhibition of autophagy, while the induction of autophagy (e.g., application of rapamycin) decreased LLAF. The effects of autophagy induction were further confirmed by Western blotting, which showed the conversion of LC3-I to LC3-II, and by immunofluorescence microscopy, which detected the lysosomal activity of the autophagy inducers. We also monitored LLAF after the application of several autophagy inhibitors by RNA-interference and confocal microscopy. The results showed that, in general, the inhibition of the autophagy-related proteins resulted in an increase in LLAF when cells were fed with rod outer segments, which further confirms the effect of autophagy in the fate of RPE lipofuscin degradation. These results emphasize the complex role of autophagy in modulating RPE autofluorescence and confirm the possibility of the pharmacological clearance of RPE lipofuscin by small molecules.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, No.54 South Xianlie Road, Guangzhou 510060, China. leilei@gzzoc.com.|Department of Ophthalmology, University of Massachusetts Medical School, 381 Plantation Street, Worcester, MA 01605, USA. leilei@gzzoc.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Lei
authors.affiliation: Department of Ophthalmology, University of Massachusetts Medical School, 381 Plantation Street, Worcester, MA 01605, USA. rtzekov@health.usf.edu.|Department of Ophthalmology, University of South Florida, 13127 USF Magnolia Drive, Tampa, FL 33612, USA. rtzekov@health.usf.edu.|The Roskamp Institute, 2040 Whitfield Avenue, Sarasota, FL 34243, USA. rtzekov@health.usf.edu.
authors.forename: Radouil
authors.initials: R
authors.lastname: Tzekov
authors.affiliation: Gene Therapy Center, University of Massachusetts Medical School, 381 Plantation Street, Worcester, MA 01605, USA. Huapeng.Li@umassmed.edu.
authors.forename: Huapeng
authors.initials: H
authors.lastname: Li
authors.affiliation: The Department of Ophthalmology, University of Florida Health Science Center, 1600 SW Archer Road, Gainesville, FL 32610, USA. jmcdowel@ufl.edu.
authors.forename: J Hugh
authors.initials: JH
authors.lastname: McDowell
authors.affiliation: Gene Therapy Center, University of Massachusetts Medical School, 381 Plantation Street, Worcester, MA 01605, USA. Guangping.Gao@umassmed.edu.
authors.forename: Guangping
authors.initials: G
authors.lastname: Gao
authors.affiliation: The Department of Ophthalmology, University of Florida Health Science Center, 1600 SW Archer Road, Gainesville, FL 32610, USA. wcsmith@ufl.edu.
authors.forename: W Clay
authors.initials: WC
authors.lastname: Smith
authors.affiliation: Aier School of Ophthalmology, Central South University, Floor 4, New Century Building, 198# Furong Middle Road, Changsha 410015, China. tangshibo@vip.163.com.
authors.forename: Shibo
authors.initials: S
authors.lastname: Tang
authors.affiliation: Department of Ophthalmology, University of Massachusetts Medical School, 381 Plantation Street, Worcester, MA 01605, USA. skaushal108@gmail.com.|VRMI, 6205 NW 81st Drive, Gainesville, FL 32653, USA. skaushal108@gmail.com.
authors.forename: Shalesh
authors.initials: S
authors.lastname: Kaushal
mesh_terms: D001343:Autophagy; D002460:Cell Line; D005453:Fluorescence; D006801:Humans; D008062:Lipofuscin / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology
chemical_list: D008062:Lipofuscin; D020123:Sirolimus
keywords: age-related macular degeneration; autofluorescence; autophagy; degradation; lipofuscin; mTOR; retinal pigment epithelium