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Sriganesh Ramachandra Rao, Nestor Mas Gomez, Bruce A. Pfeffer, Aryn Rowsam, Claire H Mitchell, Steven J Fliesler; Compromised phagosome maturation underlies defective RPE clearance in an in vitro model of Smith-Lemli-Opitz Syndrome. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6035. doi: https://doi.org/.
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The retinal pigmented epithelium (RPE) in the AY9944 rat model of Smith-Lemli-Opitz syndrome (SLOS), a genetic disorder of cholesterol biosynthesis, exhibits accumulation of phagosomes and other inclusions, compared to controls [Fliesler et al., 2004, Arch. Ophthalmol.]. We examined SLOS patient iPSC-derived (SLOS RPE), vs. normal human embryonic stem cell-derived (nhRPE), cells in vitro to determine the underlying mechanism of this defect.
SLOS RPE (harboring both T93M and IVS8 G-C DHCR7 mutations) and nhRPE were treated with bovine rod outer segments (ROS) for 48 h; rhodopsin levels were quantified by Western blotting/densitometry (WB/D; probed with 1D4 MAb) to assess phagosome clearance. Lysosomal protease (mature Cathepsin-D), and markers of autophagic flux (p62 and LC3-I/II) were assessed by WB/D, normalized to GAPDH levels, with corresponding antibodies. SLOS RPE and nhRPE lysosomal pH was measured using LysoSensor Yellow/Blue DND-160, and lysosomal Cathepsin-D activity was estimated using a BODIPY-Pepstatin assay kit (Molecular Probes). Statistical significance of mean/S.E. values was determined by paired Student’s t-test (criterion, p ≤ 0.05).
Autophagic marker levels in SLOS RPE (expressed as % change, vs. nhRPE) were as follows (p<0.05): Beclin-1, +62%; p62, +18%; LC3-II, -50%. Mature Cathepsin-D levels were unaltered. Lysosomal pH in SLOS hRPE was statistically more acidic (4.50 ± 0.02, p<0.05) compared to nhRPE (4.56 ± 0.01), but Cathepsin-D activity was not significantly altered. However, heterophagic clearance of exogenous ROS was defective in SLOS RPE, as evidenced by persistence of 1D4+ material (rod opsin C-terminus), compared to nhRPE cells.
Decreased LC3-II levels, elevated p62 levels, and persistence of 1D4+ material indicate compromised phagosome maturation in SLOS RPE compared to nhRPE. Elevated Beclin1 levels obviate defective initiation of autophagy/heterophagy; also, compromised lysosomal physiology was not involved. However, increased 7-dehydrocholesterol (7DHC) levels (a SLOS hallmark) and/or oxysterols derived therefrom may underlie the SLOS-associated RPE defect.
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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