Autophagy machinery is functional in human pluripotent stem cell-derived retinal pigment epithelial cells

Abstract

Purpose : The impairment in autophagic and proteasomal cleansing has been documented in retinal pigment epithelial (RPE) cells and age-related macular degeneration (AMD) pathology, but not studied with human embryonic stem cell (hESC)-RPE cells.

Methods : Here the role of autophagy and proteolytic machinery was assessed in the regulation of melanocytic pigmentation with a tandem fluorescently tagged LC3 (GFP-mCherry-LC3) transfected or non-transfected mature hESC-RPE cells which were treated either with MG-132, AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), bafilomycin A1 or their combinations. The effects were analysed by western blotting (WB), confocal and electron microscopy (EM) and calcein fluorescence.

Results : Inhibition of proteasomal function with MG-132, or autophagy with bafilomycin A1 increased the accumulation of premelanosomes seen in EM. The upregulation of autophagy markers SQSTM/p62 and MAP1LC3A/LC3-II, and the perinuclear localization of them was observed in WB and confocal microscopy, respectively. The AMP-dependent protein kinase activator AICAR treatment decreased the proteasome inhibitor -induced accumulation of melanosomes observed in EM, and also eradicated the SQSTM/p62 and LC3-II detected in WB. The autophagy flux was shown to be induced during the MG-132 proteasome inhibition, and was further enhanced by simultaneous AICAR treatment, observed in confocal microscopy with the GFP-mCherry-LC3. Bafilomycin A1 increased amount of autophagosomes, premelanosomes and LC3-II observed in EM, calcein fluorescence or WB.

Conclusions : Our result suggests that proteasomal inhibition increases the expression of the autophagy flux markers SQSTM1/p62 and LC3-II and lead to elevated accumulation of autophagosomes and melanosomal granules in the hESC-RPE. Furthermore, the AMPK activator AICAR promoted cleansing of up-regulated melanin granules, SQSTM/p62 and LC3-II, indicating autophagic cleansing. These results reveal that autophagy machinery is functional in hESC-RPE cells and regulates cellular pigmentation with proteasomes.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.