Abstract
Purpose :
While mitochondrial biogenesis and autophagy dysfunction have been implicated in the pathogenesis of age-related macular degeneration, the molecular regulation of RPE metabolism remains unclear. We previously identified the transcription co-factor, PGC-1α, as an important inducer of RPE oxidative metabolism and anti-oxidant capacity. The aim of this study was to further characterize PGC-1α roles in RPE cells metabolism, integrity and function.
Methods :
PGC-1α expression was silenced in ARPE-19 cells by lentivirus-mediated delivery of pGIPZ-encoded shRNAs specific to the human PGC-1α sequence (shPGC-1α). A pGIPZ non-silencing shRNA (shControl) was used as negative control. Transduced cells were positively selected by FACS sorting and confirmed at the mRNA and protein level. ShPGC-1α and shControl cell lines were maturated under serum starvation for up to 21 days. PGC-1α gain of function was performed by adenoviral transduction. Gene expression of PGC-1 isoforms, mitochondrial dynamic and autophagy was analyzed by qPCR. Mitochondrial morphology and redox status were measured by Mitotracker and MitoSox respectively. Cellular organization and autophagy markers were evaluated by IHC. Barrier function was assessed by transepithelial electrical resistance (TEER).
Results :
Following 7 days of maturation, severe disorganization of the mitochondrial network associated with increase in toroidal (donut-shaped) mitochondria and increased mitochondrial superoxide production (p=0.006) was observed in shPGC-1α cells. Evaluation of the RPE barrier function revealed that loss of PGC-1α leads to significant decrease in TEER by day 14 (p=0.03). LAMP-1 positive organelles appeared abnormally dilated at 7 days suggesting that PGC-1α also regulates RPE autophagy. This concept was supported by gene expression analysis showing that PGC-1α is upregulated by autophagy-inducing treatments such as amino acid starvation (p=0.05 at 24 hrs), exposure to 1 mM H2O2 (p=0.04 at 6 and 12 hrs) and to 60 nM tunicamycin (p=0.004 at 24 hrs). Gain of function experiments confirmed that PGC-1α induces the expression of autophagosomes biogenesis associated genes such as WIPI1, MAP1LC3B (LC3) and ATG4D.
Conclusions :
Our findings indicate that lack of PGC-1α alters mitochondrial redox state and dynamics in RPE and leads to mitochondrial dysfunction, defective autophagy and loss of epithelial integrity.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.