June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
mTOR signaling in zebrafish retinal pigment epithelium regeneration
Author Affiliations & Notes
  • fangfang lu
    Department of Ophthalmology, Louis J. Fox Center for Vision Restoration, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
    Department of Ophthalmology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
  • Jeffrey M Gross
    Department of Ophthalmology, Louis J. Fox Center for Vision Restoration, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Lyndsay Leach
    Department of Ophthalmology, Louis J. Fox Center for Vision Restoration, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   fangfang lu, None; Jeffrey Gross, None; Lyndsay Leach, #9458428 (P)
  • Footnotes
    Support  NIH R01 EY29410; P30 EY08098; Xiangya School of Medicine Funding; Research to Prevent Blindness Grand;
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 2589. doi:
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      fangfang lu, Jeffrey M Gross, Lyndsay Leach; mTOR signaling in zebrafish retinal pigment epithelium regeneration. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2589.

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

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Abstract

Purpose : Our previous work characterized zebrafish RPE (retina pigment epithelium) regeneration after genetic ablation and has begun to identify molecular and cellular regulators of the regenerative response. mTOR (mechanistic target of rapamycin) signaling has been shown to be activated after tissue injury and to regulate tissue regeneration in multiple contexts; however, whether mTOR signaling is involved in RPE regeneration is unknown. We hypothesized that mTOR signaling was involved in RPE regeneration and sought to identify mechanisms by which mTOR regulates RPE regeneration.

Methods : At 5 days post-fertilization (dpf), rpe65a:nfsB-eGFP larvae were exposed to 10mM metronidazole (MTZ) for 24 hours to ablate the RPE. Larvae were treated with mTOR antagonists (2μM rapamycin/0.9μM INK 128) or DMSO from 24 hours prior to RPE ablation until 4 days post-injury (dpi). BrdU incorporation and RPE pigment recovery were quantified to assess the effect of mTOR antagonists on RPE regeneration. Immunostaining of phosphorylated 40S ribosomal protein S6 (p-S6), a readout for mTORC1 activity, was performed at 3, 6 and 12 hours post-injury (hpi), and 1- 4dpi timepoints in RPE-ablated and age-matched unablated controls, as well as at 2dpi in larvae treated with mTOR antagonists or DMSO. p-S6 distribution in the RPE was quantified to evaluate mTOR activity during regeneration and to validate drug efficacy. To identify mechanisms by which mTOR modulates RPE regeneration, RNA-seq was performed on eGFP+ cells from rapamycin- or DMSO-treated larvae at 2 and 4dpi (n=3). Pathway enrichment analyses (STRING) were performed on groups of significantly differentially expressed genes (DEGs).

Results : Rapamycin and INK 128 treatment significantly decreased BrdU+ cells in the RPE (p≤0.0001) and impaired central pigment recovery at 4dpi, compared to controls (p≤0.0001). Quantification of p-S6 enrichment in the RPE showed significant increases from 6hpi-3dpi with peak expression at 12hpi (p≤0.01). p-S6 activity was abrogated in rapamycin/INK128 treated larvae at 2dpi. RNA-Seq results identified a variety of genes that were downregulated in the RPE at 4dpi.

Conclusions : mTOR signaling is required for RPE regeneration after genetic ablation. Putative mTOR targets have been identified and are being experimentally interrogated to determine the mechanisms by which mTOR regulates RPE regeneration.

This is a 2021 ARVO Annual Meeting abstract.

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