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Seifollah Azadi, Robert E Anderson, Raju V S Rajala; Isolation and Characterization of Autophagosomes from the Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1338.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy is a normal cellular process that prevents the formation of protein deposits in the central nervous system from large protein aggregates that are inefficiently degraded by proteasomes. Class III phosphoinositide 3-kinase (PI3K) generated PI-3-P is a known regulator of autophagy. However, when we deleted class I PI3K in cones, we found an accumulation of double membranous autophagosomes followed by age-related cone degeneration. Earlier studies have used PI3K inhibitors to study autophagy, but they inhibit all classes of PI3K. Another major limitation in studying autophagy in the retina is the lack of suitable methods to isolate the autophagosomes. There are no studies available on the role of class I PI3K-generated PI-3-P in autophagy, so we characterized class I PI3K in retinal autophagosomes.
We have successfully developed a micro-method to isolate highly enriched autophagosomes from small amounts of retina. This procedure involves several sedimentations and centrifugation steps. We used vinblastine to suppress the microtubule-dependent translocation to lysosomes, which causes the accumulation of autophagosomes. Nuclei were removed based on their size. We used a sucrose gradient to remove mitochondria; endoplasmic reticulum, endosomes, and other small organelles were separated from the autophagosomes by Percoll sieving; lysosomes by substrate-induced osmotic destruction; and cytosol by being non-sedimentable. The isolated fractions were examined for the accumulation of autophagosome markers, microtubule-associated protein 1 light chain 3 (LC3A), autophagy-linked FYVE protein (Alfy), and p85 alpha subunit of PI3K, the regulatory subunit of class I PI3K, by immunoblot analysis.
We found an accumulation of autophagosome makers LC3A and Alfy in retinal autophagosomes. We also found the localization of p85 alpha subunit of PI3K in autophagosomes.
Our study shows that class I PI3K is present in retinal autophagosomes, which suggests that class I PI3K generated PI-3-P may be involved in the normal functions of autophagy in the retina. This is the first study demonstrating the successful isolation of a retinal autophagosomes, and will enable us to identify specific protein(s) by proteomic analysis.
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