June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Dynamic Transcriptional and Translational Profiling of Reactive Muller Glia Following Retinal Injury
Author Affiliations & Notes
  • Masoumeh Ashouri
    Spencer Center For Vision Research, Byers Eye Institute, Stanford University School of Medicine, Stanford, California, United States
  • Mohajeet Balveer Bhuckory
    Spencer Center For Vision Research, Byers Eye Institute, Stanford University School of Medicine, Stanford, California, United States
  • Michael Nahmou
    Spencer Center For Vision Research, Byers Eye Institute, Stanford University School of Medicine, Stanford, California, United States
  • Sarah hay
    Spencer Center For Vision Research, Byers Eye Institute, Stanford University School of Medicine, Stanford, California, United States
  • Cara M Knasel
    Spencer Center For Vision Research, Byers Eye Institute, Stanford University School of Medicine, Stanford, California, United States
  • Evan G. Cameron
    Spencer Center For Vision Research, Byers Eye Institute, Stanford University School of Medicine, Stanford, California, United States
  • Sui Wang
    Spencer Center For Vision Research, Byers Eye Institute, Stanford University School of Medicine, Stanford, California, United States
  • Daniel V Palanker
    Spencer Center For Vision Research, Byers Eye Institute, Stanford University School of Medicine, Stanford, California, United States
  • Jeffrey L Goldberg
    Spencer Center For Vision Research, Byers Eye Institute, Stanford University School of Medicine, Stanford, California, United States
  • Footnotes
    Commercial Relationships   Masoumeh Ashouri, None; Mohajeet Bhuckory, None; Michael Nahmou, None; Sarah hay, None; Cara Knasel, None; Evan Cameron, None; Sui Wang, None; Daniel Palanker, None; Jeffrey Goldberg, None
  • Footnotes
    Support  NEI (P30-EY026877 to JLG), and Research to Prevent Blindness, Inc.
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1690. doi:
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      Masoumeh Ashouri, Mohajeet Balveer Bhuckory, Michael Nahmou, Sarah hay, Cara M Knasel, Evan G. Cameron, Sui Wang, Daniel V Palanker, Jeffrey L Goldberg; Dynamic Transcriptional and Translational Profiling of Reactive Muller Glia Following Retinal Injury. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1690.

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

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Abstract

Purpose : Muller glia (MG) respond to retinal injury through a process referred to as reactive gliosis, driven by changes in gene expression that promote both neuroprotective and neurotoxic functions. Differences in the MG reactivity induced by inner and outer retinal injury suggest MG locally respond to insults via differential gene expression and local translation. Here, we describe a novel molecular approach to label newly synthesized mRNA in vivo to characterize transcriptional changes in MG associated with glaucomatous injury and photoreceptor degeneration. Further, we show inner and outer MG processes can be isolated by laser ablation to assess local transcriptional and translational changes.

Methods : Thiol uracil (TU) tagging of nascent mRNA is facilitated by introduction of the uracil salvage enzyme uracil phosphoribosyltransferase (UPRT) that converts 4-thio-uracil (4sU) to 4-thio-uradine-5’-monophosphate (4TU). A transgenic mouse line, UPRT/GFAPcre25/TdTom, was used to express the UPRT enzyme in MG cells specifically. UPRT-expressing mice were injected with 4-sU every 3 hours over a 6-hour period and assessed TU-tagged RNA (TU-RNA) levels by HPLC. TU-RNA was treated with iodoacetamide to alkylate, enabling identification of labeled transcripts by RNA seq. To assess translational changes, a transgenic line, Ribo/GFAPcre25/TdTom, was used to obtain Ribo-Tag expression in MG and an excimer laser was used to ablate the inner or outer retina to isolate MG processes.

Results : Comparison of 4sU/RNA sequencing and MG single cell sequencing reveals that 85% of identified genes overlap in both RNA sequencing results. Inner and outer retinal injury were induced by optic nerve crush and photocoagulation, respectively. Both optic nerve injury and photocoagulation resulted in MG reactivity and peak GFAP expression was detected 4 days after insult. The inner and outer retina were successfully ablated using an excimer laser with a desirable precision. Tissue sections confirmed the ablation of the inner nuclear layer. Eight excimer treated retinas were required to obtain the minimum amount of RNA for sequencing

Conclusions : Identifying transcriptional and translational changes in MG after various types of retinal insult provides insights into the molecular mechanisms that underlie MG reactivity and may ultimately lead to new approaches to prevent vision loss associated with retinal injury or disease.

This is a 2021 ARVO Annual Meeting abstract.

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