Parkin elimination of mitochondria is important for maintenance of lens epithelial cell ROS levels and survival upon oxidative stress exposure

Abstract

Age-related cataract is associated with oxidative stress and death of lens epithelial cells (LECs) whose survival is dependent on functional mitochondrial populations. Oxidative stress-induced depolarization/damage of LEC mitochondria results in increased reactive oxygen species (ROS) levels and cell death suggesting the need for a LEC mechanism to remove mitochondria depolarized/damaged upon oxidative stress exposure to prevent ROS release and LEC death. To date, a mechanism(s) for removal of depolarized/damaged LEC mitochondria has yet to be identified and the importance of eliminating oxidative stress-damaged mitochondria to prevent LEC ROS release and death has not been established. Here, we demonstrate that Parkin levels increase in LECs exposed to H₂O₂-oxidative stress. We establish that Parkin translocates to LEC mitochondria depolarized upon oxidative stress exposure and that Parkin recruits p62/SQSTM1 to depolarized LEC mitochondria. We demonstrate that translocation of Parkin results in the elimination of depolarized/damaged mitochondria and that Parkin clearance of LEC mitochondria is dependent on its ubiquitin ligase activity. Importantly, we demonstrate that Parkin elimination of damaged LEC mitochondria results in reduced ROS levels and increased survival upon oxidative stress exposure. These results establish that Parkin functions to eliminate LEC mitochondria depolarized/damaged upon oxidative stress exposure and that elimination of damaged mitochondria by Parkin is important for LEC homeostasis and survival. The data also suggest that mitochondrial quality control by Parkin could play a role in lens transparency.

Keywords: Cataract; Lens cell survival; Mitochondria; Oxidative stress; Reactive oxygen species.

Fig 1.0. Parkin levels increase in lens epithelial cells exposed to H₂O₂-oxidative stress

A. Immunoblot analysis of Parkin protein levels in 15 μg of total protein extract isolated from SRA 01/04 lens epithelial cells. SRA 01/04s were treated with indicated amounts of H₂O₂ in serum free media for 2 h and allowed to recover in complete media for 24 h. B. Immunoblot for GAPDH is shown as a control for equal protein loading. C. Densitometric analyses of the immunoblots plotted as Parkin levels as a ratio to GAPDH loading control.

Fig 2.0. Parkin translocates to lens epithelial cell mitochondria exposed to H₂O₂-oxidative stress

SRA 01/04 lens epithelial cells (A-C) or primary chick lens epithelial cells (D-F) were treated with either H₂O₂-induced oxidative stress (B,E) or the mitochondrial uncoupler CCCP (C,F) and compared to untreated cells (A,D). Cells were treated with H₂O₂ for 5 h and CCCP for 1 h. Following treatment, cells were immunostained for Parkin (green), the outer mitochondrial membrane protein TOMM20 (red) and counterstained with DAPI (blue). Images were obtained using a confocal fluorescent microscope. All images were obtained using the 40 × objective. In representative images arrows show co-localization of Parkin (green) and TOMM20 (red) resulting in yellow puncta in the overlay image (zoomed images). Areas with large numbers of yellow puncta are outlined.

Fig 2.0. Parkin translocates to lens epithelial cell mitochondria exposed to H₂O₂-oxidative stress

SRA 01/04 lens epithelial cells (A-C) or primary chick lens epithelial cells (D-F) were treated with either H₂O₂-induced oxidative stress (B,E) or the mitochondrial uncoupler CCCP (C,F) and compared to untreated cells (A,D). Cells were treated with H₂O₂ for 5 h and CCCP for 1 h. Following treatment, cells were immunostained for Parkin (green), the outer mitochondrial membrane protein TOMM20 (red) and counterstained with DAPI (blue). Images were obtained using a confocal fluorescent microscope. All images were obtained using the 40 × objective. In representative images arrows show co-localization of Parkin (green) and TOMM20 (red) resulting in yellow puncta in the overlay image (zoomed images). Areas with large numbers of yellow puncta are outlined.

Fig 3.0. Parkin levels increase in mitochondria of lens epithelial cells exposed to H₂O₂-oxidative stress

Immunoblot analysis of Parkin levels in mitochondria isolated from H₂O₂ treated SRA 01/04 lens epithelial cells. SRA 01/04 cells were equilibrated in serum free media for 2 h and treated with indicated amounts of H₂O₂ for 4 h. Immunoblot for mitochondrial protein SDHA is shown as a control for equal protein loading.

Fig 4.0. Wt-Parkin recruits p62/SQSTM1 to damaged mitochondria in lens epithelial cells exposed to H₂O₂-oxidative stress

SRA 01/04 lens epithelial cells (A-C) were transiently transfected with YFP-wt-Parkin and treated with either 200 μM H₂O₂ (B) or 10 μM CCCP (C) for 6 h and compared to untreated cells (A). Following treatment cells were immunostained for p62/SQSTM1 (pseudo green) and TOMM20 (pseudo red) and counterstained with DAPI (blue). Images were obtained using a confocal fluorescent microscope. All images were obtained using the 40 × objective. Cells overexpressing YFP-Parkin are outlined. In representative images, the overlay images show co-localization of p62/SQSTM1 (pseudo green) and TOMM20 (pseudo red) in YFP-wt-Parkin overexpressing cells (outline), resulting in yellow puncta. Examples of cells with yellow puncta are shown in large zoom for each treatment.

Fig 5.0. Mutant C431N-Parkin fails to recruit p62/SQSTM1 to damaged mitochondria in lens epithelial cells exposed to H₂O₂-oxidative stress

SRA 01/04 lens epithelial cells (A-C) were transiently transfected with mutant YFP-C431N-Parkin and treated with either 200 μM H₂O₂ (B) or 10 μM CCCP (C) for 6 h and compared to untreated cells (A). Following treatment cells were immunostained for p62/SQSTM1 (pseudo green) and TOMM20 (pseudo red) and counterstained with DAPI (blue). Images were obtained using a confocal fluorescent microscope. All images were obtained using the 40 × objective. Cells overexpressing mutant YFP-C431N-Parkin are outlined. In representative images, the overlay images demonstrate that p62/SQSTM1 (pseudo green) fails to co-localize with TOMM20 (pseudo red) in mutant YFP-C431N-Parkin overexpressing cells (outline). Examples of mutant YFP-C431N-Parkin cells are shown in large zoom for each treatment.

Fig 6.0. Parkin E3-ubiquitin ligase activity is required for clearance of damaged mitochondria in SRA 01/04 lens epithelial cells exposed to H₂O₂-oxidative stress

SRA 01/04 lens epithelial cells (A-F) were transiently transfected with either YFP-wt-Parkin (green) or YFP-C431N-Parkin (green) and treated with either 200 μM H₂O₂ (A&B) or 10 μM CCCP (C&D) for 48 h and compared to untreated cells (E,F). Primary chick lens epithelial cells (G-J) were treated with 10 μM CCCP only (G,H) for 48 h and compared to untreated cells (I,J). Following a 48 h exposure to stress, cells were fixed and immunostained for outer mitochondrial marker protein TOMM20 (red) and counterstained with nuclear stain DAPI. Images were obtained using fluorescent confocal microscope. All images were obtained using the 40 × objective. Outlined cells in YFP-wt-Parkin show absence of TOMM20 stain indicating clearance of mitochondria. Outlined cells in YFP-C431N-Parkin overexpressing SRA 01/04 lens epithelial cells treated with either 200 μM H₂O₂ or 10 μM CCCP for 48 h show large yellow puncta in the overlay image indicating co-localization of YFP-C431N-Parkin (green) with mitochondria marker TOMM20 (red).

Fig 7.0. Parkin E3-ubiquitin ligase activity is required for clearance of damaged mitochondria in primary chick lens epithelial cells exposed to the mitochondrial uncoupler CCCP

Primary chick lens epithelial (A-D) were transiently transfected with either YFP-wt-Parkin (green) or YFP-C431N-Parkin (green) and treated 10 μM CCCP (A&B) for 48 h and compared to untreated cells (C,D). Following a 48 h exposure to stress, cells were fixed and immunostained for outer mitochondrial marker protein TOMM20 (red) and counterstained with nuclear stain DAPI. Images were obtained using fluorescent confocal microscope. All images were obtained using the 40 × objective. Outlined cells in YFP-wt-Parkin show absence of TOMM20 stain indicating clearance of mitochondria. Outlined cells in YFP-C431N-Parkin overexpressing primary chick lens epithelial cells treated with10 μM CCCP for 48 h show large yellow puncta in the overlay image indicating co-localization of YFP-C431N-Parkin (green) with mitochondria marker TOMM20 (red).

Fig 8.0. Clearance of damaged mitochondria by Parkin results in decreased ROS levels in lens epithelial cells exposed to H₂O₂-oxidative stress

A. Ethidium bromide stained agarose gels showing Parkin transcript levels and control GAPDH transcript levels in 200 ng RNA isolated from SRA 01/04 lens epithelial cells stably overexpressing GFP2 (control), YFP-wt-Parkin or YFP-C431N Parkin. B. Immunoblot analysis of Parkin protein levels in 15 μg of total protein extract isolated from SRA 01/04 lens epithelial cells stably overexpressing GFP2 (control), YFP-wt-Parkin or YFP-C431N Parkin. Immunoblot for GAPDH is shown as a control for equal protein loading. C. SRA 01/04 lens epithelial cells stably overexpressing either YFP-wt-Parkin or YFP-C431N Parkin were treated with 200 μM or 300 μM H₂O₂ for 24 h and stained for ROS production using the superoxide specific dye DHE. Images were obtained using live cell fluorescent confocal microscope.

Fig 9.0. Clearance of damaged mitochondria by Parkin results in increased survival of lens epithelial cells exposed to H₂O₂-oxidative stress

Representative graphs depicting viability upon exposure to H₂O₂-induced oxidative stress or CCCP-induced mitochondrial depolarization in SRA 01/04 lens epithelial cells stably overexpressing either YFP-wt-Parkin or YFP-C431N-Parkin using MTS cell-viability assays. H₂O₂ (A) treatments were conducted for 24 h in serum-free media, (B) CCCP treatments were conducted in serum containing media for 24 h. Error bars represent standard deviations of eight separate cell-viability assays. Viability levels are shown as mean abs at 492 nm ± standard deviation. Differences among treatments were determined using Students T-test assuming equal variance where p < 0.01 was considered statistically significant. * p<.01 compared to YFP-wt-Parkin overexpressing SRA 01/04s.