June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Loss of Prom1 Impairs Autophagy via mTORC1-TFEB signaling and Promotes RPE Degeneration in a Mouse Model
Author Affiliations & Notes
  • Sujoy Bhattacharya
    Ophthalmology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Jinggang Yin
    Ophthalmology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Weihong Huo
    Ophthalmology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Tonia S Rex
    Ophthalmology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Edward Chaum
    Ophthalmology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Footnotes
    Commercial Relationships   Sujoy Bhattacharya None; Jinggang Yin None; Weihong Huo None; Tonia Rex None; Edward Chaum None
  • Footnotes
    Support  International Retina Research Foundation; Research to Prevent Blindness
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3888. doi:
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      Sujoy Bhattacharya, Jinggang Yin, Weihong Huo, Tonia S Rex, Edward Chaum; Loss of Prom1 Impairs Autophagy via mTORC1-TFEB signaling and Promotes RPE Degeneration in a Mouse Model. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3888.

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

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Abstract

Purpose : There are no current effective treatments for retinal pigment epithelial (RPE) cell loss in atrophic AMD (aAMD). Autophagy regulates human RPE homeostasis, and impairment of this process contributes to AMD pathophysiology. We showed earlier that CD133/Prom1 knockout (KO) in the human RPE impairs autophagy due to mTORC1/2 activation. Although Prom1 is a structural protein in mouse photoreceptors, its expression and function in mouse RPE are not well characterized.

Methods : To explore the role of Prom1 in mouse RPE (mRPE), we used CRISPR/Cas9-mediated gene editing to delete Prom1 in vitro. Autophagy and epithelial-mesenchymal transition (EMT) pathways in Prom1-KO cells were assessed by western blotting and qPCR. Using polarized mRPE cells, we investigated phagocytosis of bovine photoreceptor outer segments (POS) and a rhodopsin antibody to detect engulfed POS. To test whether RPE-specific Prom1-KO recapitulates features of retinal degeneration in aAMD, we used purified viral suspension (1μl) for suprachoroidal injection of single-vector AAV2/1-saCas9-control-sgRNA or Prom1-sgRNA (6.76x1012GC/ml) in male and female adult C57/BL6J mice eyes. Ten weeks later, we characterized visual function in RPE-specific Prom1-KO mice by electroretinography (ERG). abnormal retinal morphology by fundus photography, and post-mortem by histology.

Results : Prom1-KO decreases autophagy flux, increases p62 levels, activates mTORC1, and concomitantly decreases TFEB and Cathepsin-D activities in mRPE cells. In addition, Prom1-KO reduces the clearance of bovine POS by mRPE cells due to increased mTORC1 and reduced TFEB activities. Dysfunction of Prom1-dependent autophagy correlates with a decrease in ZO-1 and E-cadherin with a simultaneous increase in Vimentin and Snail levels, suggesting EMT induction in Prom1-KO cells. Our data show that AAV2/1-gRNA-mediated Prom1-KO in the mRPE in vivo causes abnormal RPE morphology, sub-retinal fluid accumulation, and RPE cell loss. These changes correlate with hyperfluorescent signals by fundus imaging, confirming RPE pathology and a significant reduction in A-wave amplitude during ERG, showing retinal degeneration.

Conclusions : Our results demonstrate that Prom1-mTORC1-TFEB signaling is a central driver of cell-autonomous mRPE homeostasis. We show that RPE-specific deletion of Prom1 leads to aAMD-like RPE defects and retinal degeneration in a mouse model.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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