June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Photoreceptor outer segment phagocytosis couples lysosome biogenesis to autophagy in the retinal pigment epithelium
Author Affiliations & Notes
  • Jin Xu
    Ophthalmology and Visual Science, UW-Madison, Madison, WI
  • Kimberly Toops
    Ophthalmology and Visual Science, UW-Madison, Madison, WI
  • Aparna Lakkaraju
    Ophthalmology and Visual Science, UW-Madison, Madison, WI
  • Footnotes
    Commercial Relationships Jin Xu, None; Kimberly Toops, None; Aparna Lakkaraju, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1773. doi:
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      Jin Xu, Kimberly Toops, Aparna Lakkaraju; Photoreceptor outer segment phagocytosis couples lysosome biogenesis to autophagy in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1773.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To investigate (a) the interplay between outer segment (OS) phagocytosis, lysosome biogenesis and autophagy in the retinal pigment epithelium (RPE) and (b) the role of the transcription factor EB (TFEB) in these processes. In macrophages, two proteins critical for autophagy, LC3 and beclin-1, are transiently recruited to phagosomes during phagocytosis of pathogens. This LC3-associated phagocytosis may be required for phagosome acidification. TFEB is a master regulator of lysosome biogenesis: cytosolic TFEB senses lysosomal state and translocates to the nucleus to induce expression of genes involved in lysosome function and autophagy. Since effective clearance of phagocytosed OS depends on lysosomes, we investigated TFEB translocation, lysosome enzyme activities and autophagy after OS phagocytosis in healthy RPE and in RPE with the major lipofuscin fluorophore A2E.

Methods: Polarized cultures of primary pig RPE (± A2E) were fed pig rod OS and harvested at various times. Nuclear and cytosolic fractions were immunoblotted for TFEB to measure TFEB translocation. Autophagic flux was assessed by LC3-I to LC3-II ratio, beclin-1 and lactate dehydrogenase (LDH) levels. OS clearance was measured by opsin immunoblotting. Colocalization of opsin and LC3 was used to follow recruitment of LC3 to phagosomes. Live imaging of lysosome-autophagosome interactions was performed in cells expressing mCherry-LC3 and GFP-Lamp2 by spinning disk confocal microscopy. Lysosomal enzyme activities were measured using commercial substrates.

Results: OS phagocytosis increases conversion of LC3-I to LC3-II and induces beclin-1 expression. In cells with A2E, LC3-II levels are lower although rhodopsin clearance is unaffected. A2E also interferes with basal autophagy in the RPE and leads to LDH accumulation. OS phagocytosis induces TFEB translocation and this is decreased in cells with A2E. TFEB overexpression increases the activity of lysosomal enzymes in the RPE. Analysis of live imaging data is ongoing.

Conclusions: Our results show that the RPE appears to co-opt the autophagic machinery to degrade phagocytosed OS. Lysosome biogenesis orchestrated by TFEB is likely critical for OS clearance. A2E interferes with lysosome biogenesis and autophagy supporting the hypothesis that declining degradative functions with age contributes to the pathogenesis of diseases like age-related macular degeneration.

Keywords: 701 retinal pigment epithelium • 582 ipofuscin • 412 age-related macular degeneration  
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