Purchase this article with an account.
Vera L Bonilha, Karen G Shadrach, Mary E Rayborn; Levels of DJ-1 Perturb Mitochondrial Structure in the Retinal Pigment Epithelium (RPE).. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4562.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Increased localization of DJ-1 in RPE mitochondria is associated with reduced ROS generation under oxidative stress conditions. Therefore, we wanted to evaluate the effects of DJ-1 expression in the mitochondrial structure of RPE cells.
ARPE-19 cultures were treated with H2O2 (0.100 mM) for various times. In addition, cells were infected with a replication-deficient adenovirus carrying the full-length human DJ-1 cDNA (hDJ) and a mutant construct, which has its cysteine (C) residues mutated to serine (S) (CtoS) prior to stress experiments. A parallel group of cells was also transfected with a set of human short hairpin (sh)RNA DJ-1 plasmids using Lipofectamin 2000. In addition, knockout (KO) mice and control mice RPE were analyzed. Results were analyzed by electron microcopy, immunofluorescence and Westerns using mitochondria specific antibodies.
Oxidative stress produced a fragmented mitochondrial phenotype in RPE cells. A similar phenotype was observed in RPE cultures overexpressing both hDJ and CtoS mutant both at baseline and under oxidative stress conditions. Transfection of RPE cells with DJ-1 shRNAs decreased DJ-1 levels and led to changes in mitochondria structure when labeled with Tom20 antibody. In RPE cells transfected with control shRNA mitochondria were significantly shorter than in RPE cells transfected with DJ-1 shRNAs. Mitochondria in the RPE from control mice displayed distinct outer and inner membranes, with intact cristae and matrix. In contrast, mitochondria in the RPE from DJ-1 KO mice displayed loss of cristae associated with increases in matrix density and in some instances, interruption of the mitochondrial internal and external membranes. Labeling with COX IV antibody and quantification of the signal revealed that there is less labeling to this COX subunit in DJ-1 KO then in control. Next, an analysis of the expression of 7,8- dihydro-8-oxoguanine (8-oxoG), the most abundant oxidized base generated in vivo by various types of ROS, and COX IV in mice RPE cells was performed. In control mice, 8-oxoG labeling was observed in the choriocapillaris but it was present in the RPE cell bodies in DJ-1 KO mice, which colocalized with COX IV labeling.
Mitochondrial morphology is altered when DJ-1 is absent and overexpressed. The mitochondria of DJ-1 KO RPE cells in vivo display: altered ultrastructure, decreased mitochondrial mass, and increased DNA oxidation.
This PDF is available to Subscribers Only