Autophagy-mediated catabolism of visual transduction proteins prevents retinal degeneration

Abstract

Autophagy is a lysosomal degradation pathway critical to preventing the accumulation of cytotoxic proteins. Deletion of the essential autophagy gene Atg5 from the rod photoreceptors of the retina (atg5 Drod mouse) results in the accumulation of the phototransduction protein transducin and the degeneration of these neurons. The purpose of this study is to test the hypothesis that autophagic degradation of visual transduction proteins prevents retinal degeneration. Targeted deletion of both Gnat1 (a gene encoding the a subunit of the heterotrimeric G-protein transducin) and Atg5 in the rod photoreceptors resulted in a significantly decreased rate of rod cell degeneration as compared to the atg5Drod mouse retina, and considerable preservation of photoreceptors. Supporting this we used a novel technique to immunoprecipitate green fluorescent protein (GFP)-tagged autophagosomes from the retinas of the GFP-LC3 mice and demonstrated that the visual transduction proteins transducin and ARR/ arrestin are associated with autophagosome-specific proteins. Altogether, this study shows that degradation of phototransduction proteins by autophagy is necessary to prevent retinal degeneration. In addition, we demonstrate a simple and easily reproducible immunoisolation technique for enrichment of autophagosomes from the GFP-LC3 mouse retina, providing a novel application to the study of autophagosome contents across different organs and specific cell types in vivo.

Light‐responsive microRNA miR‐211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance

“…A subset of autophagy‐related proteins, including Beclin1 and ATG5, are engaged cyclically in the RPE to act in LC3‐associated phagocytosis, a non‐canonical form of autophagy (Kim et al , ). Once lipidated, LC3II is recruited to the POS‐ingested phagosomes, which then fuse with the lysosomes forming phagolysosomes for the degradation and recycling of the ingested POS cargo (Yao et al , ). However, this process does not require the autophagy initiation complex containing Ulk1/Atg13/Fip200 proteins.…”

Inhibition of MicroRNA-96 Ameliorates Cognitive Impairment and Inactivation Autophagy Following Chronic Cerebral Hypoperfusion in the Rat

“…Autophagy is a lysosomal degradation pathway for the removal of damaged cellular components and plays a critical role in cellular homeostasis, especially in neurons [19,20]. However, autophagy is a double-edged sword since defective or excessive autophagy may induce cell death [21,22].…”

Autophagosome immunoisolation from GFP-LC3B mouse tissue

“…3.4.7. Once the grid is dry, observe the sample with a transmission electron microscope (in the work presented here, a JEOL 1400-plus TEM [9] was used).…”

“…Previously described methods for isolation of autophagosomes [3][4][5][6][7], require complex centrifugation protocols and demand relatively large amounts of starting material; for example, a single rat liver, which makes it very challenging for researchers who study organs in smaller animals such as mouse. Based on a study of immunoisolation of autophagosomes from GFP-LC3-transfected cultured cells [8], we recently described a simple, fast and reproducible method to enrich for autophagosomes from retinas of GFP-LC3 mice by immunoprecipitation using superparamagnetic microbeads conjugated to an anti-GFP antibody [9]. The purpose of this protocol paper is to provide step-by-step instructions for this procedure.…”

“…We have used this enriched autophagosome fraction to perform a number of analyses on their contents. We have previously published the protein content of autophagosomes from retinas of the GFP-LC3 mouse (as analyzed by mass spectroscopy), as well as showing the difference in the levels of the visual transduction proteins ARR3 (arrestin 3, retinal) and transducin in the retinal autophagosomes of dark versus light-adapted mice [9]. We have also demonstrated the differential level of proteasome subunits being consumed in the retinas of the P23H mouse model of hereditary retinal degeneration as compared to wild-type [16].…”