Abstract
Purpose :
Retinal Pigment Epithelium (RPE) functions, including energy absorption, photo-pigment recycling and oxidative stress regulation, depend on a healthy mitochondrial network. Notably, RPE mitochondria decline during aging and even more so Age-related Macular Degeneration (AMD). Healthy mitochondria pervade the entirety of the cell as an organelle network undergoing constant dynamic renewal through fusion, fission and mitophagy. Armadillo repeat-containing genes located on the X chromosome (ARMCX) form an eutharian specific cluster of 6 genes which are implicated in mitochondrial motility in axons and, more generally, in mitophagy (as substrates of PARK2). ARMCX6 is up-regulated in differentiated RPE whilst ARMCX3 is down-regulated in AMD, collectively suggesting an important role in the homeostastis of RPE. This study specifically analyzed the role of ARMCX proteins in RPE cells.
Methods :
ARMCX gene expression was analyzed by RNAseq, Q-PCR, immunoblotting and indirect immunofluorescence on cultured RPE cells and human paraffin embedded retinae. Cultured ARPE-19 cells were transfected with plasmids encoding green fluorescent protein (GFP)-tagged wild type and mutant ARMCXs, as well as siRNA targeting ARMCX. Functional consequences for mitochondrial dynamics and mitophagy were analyzed by indirect immunofluorescence of as well as time-lapsed microscopy.
Results :
ARMCX1, 2, 3 and 6 but not 4 or 5 were strongly up-regulated in differentiated human fetal RPE cells. This differentiation regulated expression was also seen in cultured ARPE-19 cells and further analyses were restricted to ARMCX1, 2, 3 and 6. Ectopic expression of GFP-tagged ARMCXs showed strong co-localization with mitochondria in cultured ARPE-19 cells. ARMCX overexpression resulted in aggregation and reduced mobility of mitochondria indicating disrupted dynamics. In contrast knock-down of ARMCX1 resulted in increased mitochondrial movements and led to fragmentation of the mitochondrial network, suggesting enhanced fission and/or mitophagy. Mitochondrial targeting of ARMCXs was confirmed by mutagenesis of putative targeting signals. For ARMCX1, expression of the mitochondrial targeting mutant led to mitochondria dispersion throughout the cells. Endogenous ARMCX1 co-localized with mitochondria in ARPE-19 and was strongly expressed in the RPE cell layer of the human retina.
Conclusions :
ARMCXs are important regulators of mitochondria in RPE.
This is a 2020 ARVO Annual Meeting abstract.