Abstract
Purpose:
Mitochondrial function is essential for the homeostasis and transparency of the eye lens but those mechanisms that maintain functional mitochondria under oxidative stress conditions have yet to be elucidated. Parkin translocates to and thereby targets damaged mitochondria for degradation through recruitment to LC3B-labelled autophagosomes where they are degraded. Since oxidative stress is associated with damage to lens mitochondrial proteins, lens mitochondrial depolarization, loss of lens mitochondrial function and cataract formation, we hypothesized that Parkin is required to maintain functional lens cell mitochondria under oxidative stress conditions and thereby be important for lens oxidative stress defense.
Methods:
cDNA microarrays, high throughput RNA sequencing, RT-PCR and western analysis were used to detect Parkin pathway components in human and chick lens epithelial and fiber cells. Dual-label fluorescent confocal microscopy was used to establish translocation of Parkin to the mitochondria and to co-localize mitochondrial and autophagosome markers. EYFP-labelled Parkin was used to identify Parkin-overexpressing cells and the mitochondrial outer membrane protein TOM20 used to track mitochondrial degradation.
Results:
Parkin, Pink1, p62 and Ambra1 were detected at the transcript and protein levels throughout the human and embryonic chicken lens. Treatment of human SRA04/01 and cultured primary chick epithelial cells with the membrane depolarizing agent CCCP or H202-oxidative stress resulted in depolarization of lens mitochondria followed by the translocation of Parkin to the mitochondrial membrane. Overexpression of Parkin in lens epithelial cells exposed to CCCP or H202-oxidative stress resulted in complete elimination of lens cell mitochondria.
Conclusions:
The lens expresses the components of the Parkin mitophagy pathway. Parkin translocates to depolarized and damaged lens mitochondria to eliminate dysfunctional lens cell mitochondria through mitophagy. Since functional mitochondria are critical for lens homeostasis and transparency, and oxidative stress is associated with loss of mitochondrial function and cataract, the results suggest that loss of Parkin-regulated mitophagy could contribute to cataract formation.
Keywords: 634 oxidation/oxidative or free radical damage •
445 cataract •
600 mitochondria