March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Human Embryonic Stem Cell (hESC)-derived Retinal Pigment Epithelium (RPE) as a Model to Screen Agents that Induce Autophagy
Author Affiliations & Notes
  • Tave van Zyl
    Ophthalmology/Surgery,
    Yale University School of Medicine, New Haven, Connecticut
  • Shawn M. Ferguson
    Cell Biology,
    Yale University School of Medicine, New Haven, Connecticut
  • Caihong Qiu
    Cell Biology,
    Yale University School of Medicine, New Haven, Connecticut
  • Lilangi S. Ediwickrema
    Ophthalmology/Surgery,
    Yale University School of Medicine, New Haven, Connecticut
  • Lina Li
    Cell Biology,
    Yale University School of Medicine, New Haven, Connecticut
  • Ron A. Adelman
    Ophthalmology,
    Yale University School of Medicine, New Haven, Connecticut
  • Lawrence J. Rizzolo
    Ophthalmology/Surgery,
    Yale University School of Medicine, New Haven, Connecticut
  • Footnotes
    Commercial Relationships  Tave van Zyl, None; Shawn M. Ferguson, None; Caihong Qiu, None; Lilangi S. Ediwickrema, None; Lina Li, None; Ron A. Adelman, None; Lawrence J. Rizzolo, None
  • Footnotes
    Support  Yale Endowed Research Fund,Connecticut Innovations, 10SBC02, Leir Foundation, Newman’s Own Foundation
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1125. doi:https://doi.org/
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      Tave van Zyl, Shawn M. Ferguson, Caihong Qiu, Lilangi S. Ediwickrema, Lina Li, Ron A. Adelman, Lawrence J. Rizzolo; Human Embryonic Stem Cell (hESC)-derived Retinal Pigment Epithelium (RPE) as a Model to Screen Agents that Induce Autophagy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1125. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Pharmacological enhancement of autophagy in RPE has been proposed as a strategy to reduce the burden of immunogenic debris associated with drusen formation and Age-Related Macular Degeneration (AMD). In order to investigate compounds altering autophagic flux in human RPE, we sought to establish a method for monitoring autophagy in hESC-derived RPE cell lines, as well as assess nuclear translocation of Microphthalmia Transcription Factor (MITF) as a novel readout of autophagy.

Methods: : RNA-sequencing identified hESC-derived RPE that most closely resembled native RPE on a whole transcriptome level. The effects of starvation and treatment with the mTOR inhibitors rapamycin and Torin were compared in hfRPE and hESC-derived RPE, both in the presence and absence of the lysosomal inhibitor, chloroquine. Degradation of long-lived proteins (standard autophagy substrates) was quantified by pulse-chase labeling with 14[C]-valine, and overall autophagosome formation was assessed by morphometric quantitation of immunofluorescent LC3-positive punctate structures as well as immunoblotting of LC3-I to LC3-II conversion. Additionally, changes in autophagy-related gene transcription were measured via qPCR. Specific changes in nuclear abundance of MITF were monitored by both immunofluorescence and subcellular fractionation.

Results: : hESC-derived RPE was found to express 72% of autophagy-associated mRNAs in similar levels as hfRPE. Induction of autophagy in response to either nutrient starvation or pharmacologic inhibition of mTOR was observed in both hESC-RPE and hfRPE as demonstrated by time- and dose-dependent increases in degradation of long-lived proteins, LC3-positive punctate staining and LC3 conversion and turnover. Detection was enhanced by use of chloroquine to block autophagosome turnover. In parallel with increases in standard autophagy markers, we also observed a dramatic translocation of MITF to the nucleus.

Conclusions: : We have established an effective system for monitoring autophagy in hfRPE and hESC-derived RPE using a combination of classic (LC3-based) and novel (MITF-based) approaches. This system will facilitate investigation of small-molecule autophagy enhancers as ocular therapeutics to protect against and/or repair age-related damage in the retina.

Keywords: retinal pigment epithelium • age-related macular degeneration • protective mechanisms 
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