June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Diurnal regulation of peroxisome function in RPE cells, a role for autophagy-dependent turnover
Author Affiliations & Notes
  • Lauren Lee Daniele
    Biochemistry, SDM, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Jennifer Caughey
    School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Anuradha Dhingra
    Biochemistry, SDM, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Desiree Alexander
    Program in Cell and Molecular Biology, Biomedical Graduate Studies, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Nancy J Philp
    Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
  • Kathleen Boesze-Battaglia
    Biochemistry, SDM, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Lauren Daniele, None; Jennifer Caughey, None; Anuradha Dhingra, None; Desiree Alexander, None; Nancy Philp, None; Kathleen Boesze-Battaglia, None
  • Footnotes
    Support  NIH Grant EY010420
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3021. doi:
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      Lauren Lee Daniele, Jennifer Caughey, Anuradha Dhingra, Desiree Alexander, Nancy J Philp, Kathleen Boesze-Battaglia; Diurnal regulation of peroxisome function in RPE cells, a role for autophagy-dependent turnover. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3021.

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

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Abstract

Purpose : On a daily basis, photoreceptors shed ~10% of their lipid-rich outer segments (OS), for uptake and degradation by the retinal pigment epithelium (RPE). Ingested OS contain an abundance of very long chain fatty acid (VLCFA) which is oxidized in peroxisomes. β –oxidation of VLCFA produces H2O2 that is decomposed by antioxidant enzymes (e.g. catalase). Despite their important role in lipid metabolism, regulation of the biogenesis, activity, and turnover of peroxisomes is not well characterized. The purpose of this study is twofold; we tested the hypothesis that RPE peroxisomes are degraded by selective autophagy by examining peroxisome number and function in RPE of mice lacking ATG5 or LC3B. We also investigated whether peroxisome function and turnover depends on time of day.

Methods : Catalase activity was measured in RPE lysates of Atg5ΔRPE, LC3B-/- and WT (C57Bl6/J) mice at various times relative to light onset using a fluorescence-based assay and normalized to total catalase as assessed by dot blot. The expression of peroxisome specific membrane proteins Pex14 and PMP70 was assessed by immunohistochemistry (IHC) and Western blot analysis (WB) in RPE from WT, LC3B-/- and Atg5ΔRPE mice. Pex14 mediates the import of peroxisomal enzymes, and PMP70 is an ATP binding cassette transporter for long chain fatty acyl CoA intermediates. Dysregulated autophagy was determined by comparing expression of p62, LC3I, and LC3II in Atg5ΔRPE by WB.

Results : In WT RPE, the expression of Pex14 at 3 PM was reduced to ~65% of that at light onset as estimated by WB. In contrast, catalase activity nearly doubled at 3PM relative to light onset. There was no change in Pex14 expression or catalase activity 1 hr before, or after light onset. Pex14 and PMP70-positive puncta appeared more numerous in RPE of LC3B-/- and ATG5ΔRPE mouse eyes. Pex14 expression increased by ~20% in ATG5ΔRPE compared to WT in WB analysis. Additionally, p62 immunoreactivity was increased ~6-fold, and the LC3II/LC3I ratio decreased by >50%, in WB of ATG5ΔRPE vs. WT. Like WT, catalase activity was elevated in LC3B-/- at 3PM vs. light onset. In contrast to WT, Pex14 levels in LC3B-/- did not decrease at 3PM.

Conclusions : Our data suggest that RPE peroxisomes rely on selective autophagy for degradation that depends on time of day, and that diurnal catalase activity may be regulated independently of peroxisome turnover.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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