Abstract
Purpose: :
The ARMS2 locus in the 10q26.13 chromosomal region has been consistently associated with AMD. However, functional characterization of ARMS has been difficult since it is only expressed in primates and the localization of the ARMS2 protein has been controversial. The purpose of this study was to determine the extent to which ARMS2 localizes to mitochondria or other subcellular organelles and how this localization may be affected in oxidative stress and AMD.
Methods: :
Normal and AMD human donor eyes were fixed, sectioned and prepared for either immunohistochemistry or immunogold electron microscopy. Tissues were immunolabeled for ARMS2 using rabbit polyclonal ARMS2 antibody raised against GST fusion full-length ARMS2 protein or mitochondrial protein markers (MnSOD, porin). Primary human RPE cultures were grown to confluence and treated with 400µM hydrogen peroxide, 5µM rotenone or vehicle control for varying times up to 6 hours. Spatial and temporal changes in the localization of ARMS2 were achieved by live cell staining of mitochondria with Mitotracker red, followed by fixation and immunostaining for ARMS2. In addition, association between mitochondrial membrane potential and ARMS2 was achieved by live cell staining with JC-I followed by fixation and immunostaining for ARMS2. Some cell cultures were also prepared for immunogold electron microscopy. Omission of the primary antibody and mouse tissue, which does not express ARMS2, were used as negative controls.
Results: :
Immunoelectron microscopy and routine immunohistochemistry demonstrated that ARMS2 was both localized to the cytosol and associated with mitochondria in RPE cells. No ARMS2 staining was observed in mouse tissue. ARMS2 appeared to be preferentially localized to the mitochondrial outer membrane. In normal tissue, around 85% of mitochondria were associated with ARMS2, with the remaining mitochondria negative for ARMS2. However, this distribution showed cell-to-cell variations. The RPE in AMD tissue demonstrated no overall change in ARMS2 expression, but there was decreased association with mitochondria compared to non-AMD tissue. Exposure of cultured RPE cells to acute oxidative stress demonstrated lesser ARMS2 expression, loss of mitochondrial membrane potential and a reduced association of ARMS2 with mitochondria.
Conclusions: :
ARMS2 is present both in the cytoplasm and associated with the mitochondrial outer membrane. Oxidative stress and AMD result in reduced association of ARMS2 with mitochondria, and this correlated with loss of mitochondrial membrane potential. Further studies are necessary to dissect the role of ARMS2 in mitochondrial pathophysiology and in AMD.
Keywords: mitochondria • age-related macular degeneration • oxidation/oxidative or free radical damage