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Jingyu Yao, Lin Jia, Kecia L Feathers, Cheng-mao Lin, Daniel Klionsky, Thomas A Ferguson, David N Zacks; The role of autophagy in catabolism of visual transduction proteins. Invest. Ophthalmol. Vis. Sci. 2016;57(12):185.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy plays important roles in maintaining cellular homeostasis, preventing the intracellular accumulation of toxic proteins. Selective knockout of the gene encoding an essential autophagy protein, ATG5, from the rod cells (Atg5ΔRod mouse) results in an accumulation of phototransduction proteins and a resultant degeneration of the photoreceptor. The purpose of this study is to test the hypothesis that autophagic degradation of visual cycle proteins prevents retinal degeneration and that these proteins can be found within the autophagosome of photoreceptors in vivo.
We knocked out both Gnat1 (transducin-α) and Atg5 in the rod photoreceptors of mice by crossing the gnat1-/- mouse with the Atg5ΔRod mouse. We performed ex vivo and in vivo analyses to assess retinal structure and function. To further identify the contents of the autophagosomes, we developed a technique to isolate autophagosomes by immunoprecipitation of green fluorescent protein (GFP)-tagged LC3 autophagosomes from GFP-LC3 transfected cell lines and from retinas of the GFP-LC3 transgenic mice.
The retinas of the gnat1-/- Atg5ΔRod mice have a significantly decreased rate of rod cell degeneration as compared to the Atg5ΔRod mouse retina, and considerable preservation of normal photoreceptor structure and contents. Immunoprecipitant prepared from both GFP-LC3-transfected cell lines and the GFP-LC3 mouse retinas resulted in accumulation of autophagosome-associated proteins, such as GFP-LC3 and SQSTM1, suggesting that we had enriched for the autophagosome fraction of the cell. Transmissions electron microscopy confirmed that our immunoprecipitation product contained mainly double-membraned autophagosomes. We confirmed the presence of the visual transduction proteins GNAT1 and arrestin within this autophagosome-enriched fraction.
Altogether, this study demonstrated that autophagy plays critical roles on regulating phototransduction protein degradation in photoreceptors. Alleviation of the amount of the phototransduction protein GNAT1 increased photoreceptor survival and decreased the rate of rod degeneration in Atg5ΔRod mice. In addition, for the first time, our results provided strong confirmation that our immunoisolation technique was sufficient for enrichment of autophagosomes from GFP-LC3 mouse retina, providing a novel application to the study of autophagosomal contents across different organs and specific cell types in vivo.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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