Abstract
Purpose:
Parkin is an E3 ubiquitin ligase that targets damaged mitochondria for degradation. Mitochondria are particularly prone to irreversible damage upon exposure to UV-light and other cataract-associated environmental insults resulting in release of reactive oxygen species, oxidative stress, induction of cytochrome c-mediated apoptosis and cell death. Since Parkin directs removal of damaged mitochondria we hypothesized that Parkin could have a key role in the survival of lens and other cells exposed to oxidative stress insults.
Methods:
Parkin expression levels were examined by RT-PCR and immunoblotting. Translocation of Parkin to the mitochondria was examined by immunoblotting of mitochondrial fractions of treated cells and immunocytochemistry and confocal imaging of Parkin and mitochondrial marker TOMM20. ROS levels were examined using MitoSox reagent to measure mitochondrial superoxide levels and induction of apoptosis was examined by TUNEL assay and immunostaining with the M30 cytodeath antibody. MTS viability assays were used to examine cell survival upon exposure to oxidative stress.
Results:
Consistent with an important role for Parkin in cell survival, the Parkin gene was rapidly induced in lens epithelial cells upon exposure to peroxide-induced oxidative stress. Parkin was concomitantly detected to translocate to damaged and depolarized lens cell mitochondria followed by increased mitophagy. Overexpression of wild-type Parkin resulted in complete elimination of mitochondria in peroxide-treated lens epithelial cells by 48h. By contrast identically treated lens epithelial cells overexpressing a mutant form of Parkin that lacks E3 ubiquitin ligase activity (C341N-Parkin) accumulated damaged mitochondria along with increased ROS release and induction of apoptosis. C341N-Parkin overexpressing cells exhibited decreased survival relative to wild-type Parkin overexpressing cells upon exposure to oxidative stress.
Conclusions:
These data identify cell survival as a new function for Parkin and they establish Parkin as a key requirement for lens cell resistance against environmental damage that is likely important for resistance to cataract.<br />