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Masoumeh Ashouri, Ankush Madaan, Michael Nahmou, Sui Wang, Jeffrey L Goldberg, Evan G. Cameron; Dynamic Transcriptional Profiling of Reactive Muller Glia Following Retinal Injury. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4398. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Muller glia (MG) span the entire retina and contact nearly all retinal cells and thus are poised to respond to various types of retinal insult. How do MG respond to injury and/or disease associated with inner versus outer retinal neurons? Here we characterize MG transcriptional changes associated with glaucomatous injury and light induced photoreceptor degeneration.
Thiol uracil-tagging is a molecular approach that enables labeling of nascent mRNA in living cells through the incorporation of a modified uracil base. Incorporation is facilitated by the uracil salvage enzyme uracil phosphoribosyltransferase (UPRT) that converts ribose-5-phosphate and 4-thiouracil (4sU) to 4-thioluridine-monophosphate (4-TU). The UPRT-TU tagging protocol was optimized in vitro. Briefly, COS cells were transfected with a UPRT-GFP fusion construct and treated with 20 uM 4-thiol-uracil for 16 hours. Following 4-TU incorporation, total RNA was isolated and 4-TU-tagged RNA was biotinylated and purified using a streptavidin column. The ratio of 4-TU incorporated nucleotides to non-thio-containing nucleotides was determined using the following equation: R= (A327/A260) x (9010cm-1/16,600cm-1M-1). UPRT was introduced into MG via subretinal injection of a novel AAV virus (AAV-MG) that specifically targets these cells. 4-TU incorporation was temporally controlled by the time and duration of 4sU administration. Following incorporation and RNA extraction, TU-tagged RNA was biotinylated on the reactive thiol group, purified and sequenced.
UPRT tagging optimization in COS cells indicated a 10-to-1 incorporation ratio compared to control cells. This approach was applied in vivo to assess transcriptional changes in MG using AAV-MG. Subretinal injection of AAV-MG selectively transduced MG with high transduction efficiency and specificity (>85%). MG transcriptional changes between glaucomatous injury and photoreceptor degeneration were compared to identify gene and pathway differences between inner and outer retinal cell injuries.
Identifying transcriptional changes in MG after various types of insult provides insights into the molecular mechanisms that underlie MG response to injury and may ultimately lead to new approaches to prevent vision loss associated with retinal injury or disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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