June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Prom1 is a Novel Regulator of Autophagy in the Human Retinal Pigment Epithelium
Author Affiliations & Notes
  • Sujoy Bhattacharya
    Ophthalmology, University of Tennessee Health Science Center, Memphis, TN
  • Jinggang Yin
    Ophthalmology, University of Tennessee Health Science Center, Memphis, TN
  • Christina Winborn
    Ophthalmology, University of Tennessee Health Science Center, Memphis, TN
  • Edward Chaum
    Ophthalmology, University of Tennessee Health Science Center, Memphis, TN
  • Footnotes
    Commercial Relationships Sujoy Bhattacharya, None; Jinggang Yin, None; Christina Winborn, None; Edward Chaum, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2395. doi:
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      Sujoy Bhattacharya, Jinggang Yin, Christina Winborn, Edward Chaum, ; Prom1 is a Novel Regulator of Autophagy in the Human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2395.

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

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Abstract

Purpose: The transmembrane glycoprotein Prom1 (CD133) localizes to membrane protrusions but it is best known as a biomarker for cancer stem cells. Mutations in the PROM1 gene have been shown to disrupt photoreceptor disk morphogenesis and cause an autosomal dominant form of Stargardt disease (STGD4), but is also seen in families with retinal macular dystrophy and cone-rod dystrophy. A specific PROM1 variant, c.1170C>A, is associated with central areolar choroidal dystrophy. Despite the known involvement of Prom1 in photoreceptors, its role in the retinal pigment epithelium (RPE) where the phenotype is manifested is unknown.

Methods: To explore the role of Prom1 in RPE function, we constructed wild type (WT) and mutant (Mut)-Prom1-GFP-lentiviral constructs using site-directed mutagenesis to produce the c.1170C>A mutant and overexpress Prom1 in ARPE-19, primary RPE, and HEK cell cultures. In addition, we transfected RPE cells with a selectable Prom1-siRNA lentiviral construct and used a CRISPR/cas9 PROM1 lentiviral construct to knockout PROM1 in vitro. Western analysis, qPCR, and IHC by confocal microscopy was performed. Apoptosis and autophagy were measured using Western assays.

Results: ARPE-19 and primary RPE cultures contained similar amounts of Prom1. Polarization of the cells decreased the level of Prom1 expression. Endogenous Prom1 localizes in the cytoplasmic, perinuclear, and nuclear regions in RPE cells, but surprisingly, failed to localize to tight and adherens junctions or the apical microvilli in the polarized cells. In RPE cells treated with siRNA, knockdown of Prom1 decreased levels of autophagy-associated proteins LC3-1 and LC3-II but had no impact on apoptosis, as evidenced by the lack of caspase-3 activation. Lentiviral transduction of RPE cells with WT-Prom1 increased expression of LC3-I, whereas Mut-Prom1 reduced LC3-1 and LC3-II levels. Transfection of HEK cells with WT- and Mut-Prom1 constructs robustly increased Prom1 expression without affecting autophagy, suggesting that it is the loss of WT Prom1 function that is associated with aberrant regulation of autophagy in RPE cells.

Conclusions: Our results demonstrate for the first time that Prom1 plays a role beyond organizing photoreceptor cell membrane architecture in the retina. PROM1 mutations may play a role in regulating health and homeostasis in the RPE through the modulation of autophagy.

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