J or H mtDNA haplogroups in retinal pigment epithelial cells: Effects on cell physiology, cargo in extracellular vesicles, and differential uptake of such vesicles by naïve recipient cells

• Published in: Biochimica et biophysica acta. General subjects Vol. 1865; no. 4; p. 129798
• Main Authors: Nicholson, Crystal, Ishii, Masaaki, Annamalai, Balasubramaniam, Chandler, Kyrie, Chwa, Marilyn, Kenney, M. Cristina, Shah, Navjot, Rohrer, Bärbel
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2021
• Subjects: Age-related macular degeneration; biomarkers; blood serum; endocytosis; energy metabolism; epithelium; Extracellular vesicles; fibronectins; integrins; Ligands; mitochondria; mtDNA cybrids; OXPHOS; proteoglycans; Retinal pigment epithelium; risk; Extracellular vesicles; OXPHOS; Ligands; Age-related macular degeneration; Retinal pigment epithelium; mtDNA cybrids
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129798

Extracellular vesicles (EVs) are predicted to represent the internal state of cells. In polarized RPE monolayers, EVs can mediate long-distance communication, requiring endocytosis via protein-protein interactions. EV uptake from oxidatively stressed donor cells triggers loss in transepithelial resistance (TER) in recipient monolayers mediated by HDAC6. Here, we examine EVs released from RPE cells with identical nuclear genes but different mitochondrial (mt)DNA haplogroups (H, J). J-cybrids produce less ATP, and the J-haplogroup is associated with a higher risk for age-related macular degeneration. Cells were grown as mature monolayers to either collect EVs from apical surfaces or to serve as naïve recipient cells. Transfer assays, transferring EVs to a recipient monolayer were performed, monitoring TER and EV-uptake. The presence of known EV surface proteins was quantified by protein chemistry. H- and J-cybrids were confirmed to exhibit different levels of TER and energy metabolism. EVs from J-cybrids reduced TER in recipient ARPE-19 cells, whereas EVs from H-cybrids were ineffective. TER reduction was mediated by HDAC6 activity, and EV uptake required interaction between integrin and its ligands and surface proteoglycans. Protein quantifications confirmed elevated levels of fibronectin and annexin A2 on J-cybrid EVs. We speculate that RPE EVs have a finite set of ligands (membrane proteoglycans and integrins and/or annexin A2) that are elevated in EVs from stressed cells; and that if EVs released by the RPE could be captured from serum, that they might provide a disease biomarker of RPE-dependent diseases. •RPE cells with identical nuclear DNA but different mtDNA haplogroups (H, J) and differences in AMD risk (H<J) are studied.•Barrier function and energy metabolism are reduced in J- when compared to H-cybrids.•EVs from J-, but not H-cybrids, are taken up by naïve RPE cell monolayers and induce barrier function loss.•EV uptake involves ligands (fibronectin, annexin A2) previously shown to characterize EVs of oxidatively stressed RPE.•Characterization of EV bioactive molecules from diseased RPE cells might provide for eye disease biomarker discovery.