Polarized Secretion of Mitochondrial DNA by Retinal Pigment Epithelial cells

Mitochondrial DNA (mtDNA) damage is strongly implicated in age-related macular degeneration (AMD), the most frequent cause of age-mediated visual impairment in developed countries. Here, we investigated and compared the fate of acutely induced oxidation damage in primary retinal pigment epithelial (RPE) cells. The individual RPE clones responded heterogeneously to hydrogen peroxide, both with respect to cell sensitivity and mtDNA damage formation. Peroxide-induced mtDNA damage in human RPE (hRPE) was fully repaired within 4 hours. In parallel, hRPE cells secreted mtDNA from both apical and basolateral surfaces (AP-mtDNA and BL-mtDNA, respectively) independent of peroxide exposure and that could not be explained by detached cells. Most mtDNA was released apically, and the quality of AP-mtDNA was comparable to that of intracellular mtDNA (c-mtDNA). In contrast, BL-mtDNA constituted only 3% of AP-mtDNA, and exhibited a 10-fold higher damage burden. The difference in mtDNA quality suggests a non-random selection of mtDNA molecules to be targeted for export in apical versus basolateral direction. To investigate this, we analyzed epigenetic marks (m.545 methylation) and SNPs (heteroplasmies) in extracellular and cellular mtDNA. We detected differences Extracellular mtDNA in general appeared to be more modified than c-mtDNA and that AP-mtDNA differs from BL-mtDNA, which is indicative of a targeted secretion. Porcine RPEs (pRPEs) from a minipig model of Huntington's Disease associated with impaired epithelial polarity (TgHD) displayed approximately 2-fold higher leakage of AP-mtDNA than control pRPEs, but lower than hRPEs. Together, our data imply that extracellular mtDNA originating from RPE shapes the outer retina and the implications for polar secretion are discussed.