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Sriganesh Ramachandra Rao, Lara A. Skelton, Bruce A Pfeffer, Steven J Fliesler; Considerations in monitoring autophagy in the neural retina: Utility of CAG-RFP-GFP-LC3 transgenic mice.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1672.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the in vivo effects of CAG promoter-driven overexpression of RFP-GFP-LC3 in the neural retina, and the utility of the CAG-RFP-GFP-LC3 mouse line for studying autophagy in the retina.
Adult CAG-RFP-GFP-LC3 mice were maintained under standard 12 h:12 h cyclic light/dark conditions, and were selected by PCR tail snip analysis. A GFP reporter mouse line served as a positive control for GFP expression. Eyes were enucleated, fixed, and cryosections were obtained and subjected to confocal fluorescence microscopy. Particle analysis was carried out utilizing ImageJ® software. Split channel images were threshold-modified to retain critical puncta while minimizing background fluorescence, and thresholded images were particle-filtered. Retinas were harvested 6 h post-light onset, in parallel, and LC3B-I (cytosolic) and LC3B-II (phagosome-bound) isoforms and ATG5/12 levels were determined by Western blot analysis. Immunohistochemical analysis (utilizing anti-RFP) was performed to monitor the distribution of LC3B isoforms. Student’s t-test (P<0.05 threshold) was used for all statistical analyses.
Low-magnification images revealed GFP-positive streaks, consistent with cytosolic GFP, in the photoreceptor layer (inner segments (IS), outer nuclear layer (ONL), and outer plexiform layer). Punctate RFP-positive (phagolysosomes) and RFP-GFP-positive (immature phagosomes) signals were observed in photoreceptor IS and ONL. Anti-RFP immunostaining revealed the total phagosome population, as well as the cytosolic GFP signal. ImageJ® particle analysis identified and differentiated mature from immature phagosomes, vs. non-specific hyperfluorescence. Western blot analysis of retinas from CAG-RFP-GFP-LC3 vs. age-matched C57BL6/J control mice (n=3 each) demonstrated a significant (3-fold, P<0.05) increase in LC3B-I/LC3B-II ratio, but with no alteration in endogenous ATG5/12 levels.
Autophagy in the neural retina can be directly detected using the CAG-RFP-GFP-LC3 mouse line, without the need to alter autophagy by starvation or chloroquine treatment. Hence, the RFP-GFP-LC3 reporter line serves as a technical improvement over GFP-LC3 mice to monitor autophagy in the neural retina.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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