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Camille Dejos, Ted Han, Sharee Kuny, Rebecca Matthew, Vladimir Rancic, Helene Lemieux, Yves Sauve; Phagocytic and mitochondrial respiration defects in RPE cells presented with outer segments from photoreceptors expressing human mutated ELOVL4. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5792.
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© ARVO (1962-2015); The Authors (2016-present)
To decipher the mechanisms through which expression of human mutant ELOVL4 by photoreceptors (causing Stargardt-like juvenile maculopathy, STGD3) leads to retinal pigmented epithelium (RPE) cytotoxicity.
Photoreceptor outer segments (POS) were isolated from WT and ELOVL4 transgenic (TG) mice at 2 months of age, prior to photoreceptor death. Phagocytosis activity of RPE cells (human ARPE-19 cell line) was then evaluated in vitro, after incubation with isolated POS. First, we relied on a pulse-chase phagocytosis assay to assess the binding, internalization and degradation of FITC-labeled POS (fluorescence scanning). Second, the motility of fluorescent labeled phagosomes (TexasRed-labeled POS) along microtubules was recorded by live-cell imaging. In parallel, transient expression of tandem-fluorescent LC3 (tagged with mCherry and GFP, tfLC3) was used to monitor fusion of autophagosomes (yellow fluorescence) with lysosomes (red fluorescence). Finally, we relied on high-resolution respirometry (OROBOROS, O2k) to assess mitochondrial function in retinas and eyecups freshly dissected from WT and TG mice.
The amount of FITC-POS bound and internalized by ARPE-19 cells was similar between POS derived from WT and TG mice. However, degradation by ARPE-19 cells of POS was slower when these were isolated from TG mice. This delayed POS processing was correlated with both impairment in phagosomes motility and their delay in fusing with lysosomes. In addition, high-resolution respirometry showed a significant decrease in complex I respiration in both retina and RPE as well as a strong defect in complex IV respiration (40%) in the RPE when comparing ELOVL4 transgenic with aged-matched WT mice.
Our results suggest that by 2 months of age, photoreceptor outer segments isolated from the ELOVL4 transgenic mouse exert detrimental effects on healthy RPE cells. Consequently, the resultant RPE pathology may negatively impact outer segment renewal and lead to photoreceptor death. In addition, defects in mitochondrial electron transport systems may also contribute to increased production of reactive oxygen species, further contributing to cell death.
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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