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Vera L Bonilha, Mary E Rayborn, Karen G Shadrach, Joe G Hollyfield, Arvydas Maminishkis, Sheldon S Miller; DJ-1 Levels Alter Autophagy in the Retinal Pigment Epithelium (RPE).. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2408.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy maintains cell homeostasis through turnover of proteins, lipids, and<br /> organelles via the lysosomal pathway. This process is induced by both endogenous and exogenous stresses. DJ-1 is a multifunctional protein that regulates oxidative stress response in RPE cells through distinct cellular pathways. Therefore, we wanted to analyze the effects of DJ-1 expression of autophagic proteins of RPE cells.
The eyes of adult (6 month old) DJ-1 knockout (KO) and control mice were fixed in 2% paraformaldehyde + 2.5% glutaraldehyde in 0.1M cacodylate buffer and processed for transmission electron microscopy (TEM). For tissue immunohistology, eyes were fixed in 4% paraformaldehyde and processed for cryosectioning using autophagy-specific antibodies. Proteins were quantified from whole RPE lysates isolated from control and DJ-1 KO mice eyes. Monolayers of primary human fetal RPE (hfRPE) cultures were infected with a replication-deficient adenovirus carrying the full-length human DJ-1 cDNA (hDJ), a mutant construct of DJ-1, which has its cysteine (C) residues mutated to serine (S) (CtoS), and a control adenovirus vector prior to experiments. A parallel group of cells was also treated with 50uM chloroquine (CQ) or 100 nM Rapamycin (RAPA) for 24 hrs. Results were analyzed by immunofluorescence and Westerns.
TEM analysis indicated an increase in the number of phagolysosomes in the RPE cytoplasm of DJ-1 KO mice. RPE cells of the DJ-1 KO mice displayed decreased labeling for LC3 and p62 when compared to control mice. These findings were confirmed by quantification of the signal intensity of both proteins in lysates from isolated RPE. Under baseline condition, p62 localized to small vesicular cytoplasmic compartments in hfRPE cells. Treatment of hfRPE cells with CQ lead to aggregated perinuclear localization but RAPA treatment lead to a decrease in p62 labeling. No significant changes in p62 distribution were observed in hfRPE cultures overexpressing both hDJ and CtoS constructs at baseline conditions. Overexpression of RPE cells with both hDJ and CtoS constructs prior to CQ treatment lead to the decreased presence of p62 perinuclear aggregates in the cytoplasm.
In the RPE autophagy increases with the loss of DJ-1 and decreases when DJ-1 is overexpressed.
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