Abstract
Purpose: :
Pharmacological enhancement of autophagy in RPE has been proposed as a strategy to reduce the burden of immunogenic debris associated with drusen formation and Age-Related Macular Degeneration (AMD). In order to investigate compounds altering autophagic flux in human RPE, we sought to establish a method for monitoring autophagy in hESC-derived RPE cell lines, as well as assess nuclear translocation of Microphthalmia Transcription Factor (MITF) as a novel readout of autophagy.
Methods: :
RNA-sequencing identified hESC-derived RPE that most closely resembled native RPE on a whole transcriptome level. The effects of starvation and treatment with the mTOR inhibitors rapamycin and Torin were compared in hfRPE and hESC-derived RPE, both in the presence and absence of the lysosomal inhibitor, chloroquine. Degradation of long-lived proteins (standard autophagy substrates) was quantified by pulse-chase labeling with 14[C]-valine, and overall autophagosome formation was assessed by morphometric quantitation of immunofluorescent LC3-positive punctate structures as well as immunoblotting of LC3-I to LC3-II conversion. Additionally, changes in autophagy-related gene transcription were measured via qPCR. Specific changes in nuclear abundance of MITF were monitored by both immunofluorescence and subcellular fractionation.
Results: :
hESC-derived RPE was found to express 72% of autophagy-associated mRNAs in similar levels as hfRPE. Induction of autophagy in response to either nutrient starvation or pharmacologic inhibition of mTOR was observed in both hESC-RPE and hfRPE as demonstrated by time- and dose-dependent increases in degradation of long-lived proteins, LC3-positive punctate staining and LC3 conversion and turnover. Detection was enhanced by use of chloroquine to block autophagosome turnover. In parallel with increases in standard autophagy markers, we also observed a dramatic translocation of MITF to the nucleus.
Conclusions: :
We have established an effective system for monitoring autophagy in hfRPE and hESC-derived RPE using a combination of classic (LC3-based) and novel (MITF-based) approaches. This system will facilitate investigation of small-molecule autophagy enhancers as ocular therapeutics to protect against and/or repair age-related damage in the retina.
Keywords: retinal pigment epithelium • age-related macular degeneration • protective mechanisms