June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017

Microtubule-associated protein 1 light chain 3 (LC3) isoforms in RPE: expression and function
Author Affiliations & Notes
  • Anuradha Dhingra
    Department of Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Juan Reyes-Reveles
    Department of Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Desiree Alexander
    Department of Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Rachel C Sharp
    Department of Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Aditi Swarup
    Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
  • Hye Jin Kim
    Department of Pathology and Cell Biology, Columbia University, New York , New York, United States
  • Janet R Sparrow
    Department of Pathology and Cell Biology, Columbia University, New York , New York, United States
  • Kathleen Boesze-Battaglia
    Department of Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Anuradha Dhingra, None; Juan Reyes-Reveles, None; Desiree Alexander, None; Rachel C Sharp, None; Aditi Swarup, None; Hye Jin Kim, None; Janet Sparrow, None; Kathleen Boesze-Battaglia, None
  • Footnotes
    Support  NIH EY010420 (KBB), EY026525 (KBB and NJP), EY12951 (JRS)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3022. doi:
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    • Get Citation

      Anuradha Dhingra, Juan Reyes-Reveles, Desiree Alexander, Rachel C Sharp, Aditi Swarup, Hye Jin Kim, Janet R Sparrow, Kathleen Boesze-Battaglia;
      Microtubule-associated protein 1 light chain 3 (LC3) isoforms in RPE: expression and function. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3022.

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

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Abstract

Purpose : Microtubule-associated protein 1 light chain 3 (LC3) is an essential component of autophagy as well as LC3 associated phagocytosis (LAP), a hybrid degradation pathway utilizing components of canonical autophagy and phagocytosis. Autophagy dependent processes, collectively termed heterophagy, play a key role in RPE homeostasis. LC3 exists as three isoforms in human (LC3A, B, and C), with two of these (LC3A and B) in mouse. Here, we tested the hypothesis that LC3B plays a critical role in maintaining lipid homeostasis in the RPE through degradation of photoreceptor outer segment and recycling of visual pigments.

Methods : C57Bl6/J (WT) and LC3BKO mice (from 2 month to >2 years old) were used. LC3 isoform expression was analyzed by RTPCR and immunoblotting. Biopotigen Spectral Domain Optical Coherence Tomography at 840nm was used to image retina. Bisretinoid levels were measured by HPLC. Fixed frozen retinal sections were stained for neutral lipids with Bodipy 493/503, or immunostained for various antibodies followed by confocal imaging. Lipid turnover as reflected in, β-Hydroxybutyarte (β-HB) released from mouse RPE explants was measured using LiquiColor reagent kit (StanBio).

Results : Quantitative RT-PCR experiments showed that LC3B is the predominant isoform in human RPE, with both LC3A and LC3B expressed at equivalent levels in mouse RPE. In an LC3BKO RPE, there appears to be no compensatory upregulation of LC3A transcripts. The levels of bisretinoids A2E and atRALdi-PE were higher in the LC3BKO RPE compared to the WT. Consistent with this, there appeared to be an increase in neutral lipid staining in the LC3BKO RPE. In addition, lipid turn-over, as inferred from mitochondrial ketogenesis was altered: RPE explants from WT animals show a peak of β-HB at 2 hours after light onset, whereas in LC3BKO, this peak was delayed by 4 hours relative to the WT. LC3BKO mice show normal retinal layering and thickness, however there was an increase in Iba1 positive microglia in the outer retina in older mice.

Conclusions : LC3B is the predominant LC3 isoform in human RPE, both LC3A and B are expressed in mouse RPE at similar levels and there is no compensatory upregulation of LC3A in the absence of LC3B. Our study provides evidence for the role played by LC3B in lipid homeostasis, as lack of LC3B causes defective phagocytosis, leading to metabolic imbalance, lipid deposition, and pro-inflammatory microenvironment.

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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