Oxidative stress induces Z-DNA-binding protein 1–dependent activation of microglia via mtDNA released from retinal pigment epithelial cells

Oxidative stress, inflammation, and aberrant activation of microglia in the retina are commonly observed in ocular pathologies. In glaucoma or age-related macular degeneration, the chronic activation of microglia affects retinal ganglion cells and photoreceptors, respectively, contributing to gradual vision loss. However, the molecular mechanisms that cause activation of microglia in the retina are not fully understood. Here we show that exposure of retinal pigment epithelial (RPE) cells to chronic low-level oxidative stress induces mitochondrial DNA (mtDNA)-specific damage, and the subsequent translocation of damaged mtDNA to the cytoplasm results in the binding and activation of intracellular DNA receptor Z-DNA-binding protein 1 (ZBP1). Activation of the mtDNA/ZBP1 pathway triggers the expression of proinflammatory markers in RPE cells. In addition, we show that the enhanced release of extracellular vesicles (EVs) containing fragments of mtDNA derived from the apical site of RPE cells induces a proinflammatory phenotype of microglia via activation of ZBP1 signaling. Collectively, our report establishes oxidatively damaged mtDNA as an important signaling molecule with ZBP1 as its intracellular receptor in the development of an inflammatory response in the retina. We propose that this novel mtDNA-mediated autocrine and paracrine mechanism for triggering and maintaining inflammation in the retina may play an important role in ocular pathologies. Therefore, the molecular mechanisms identified in this report are potentially suitable therapeutic targets to ameliorate development of ocular pathologies. Oxidative stress, inflammation, and aberrant activation of microglia in the retina are commonly observed in ocular pathologies. In glaucoma or age-related macular degeneration, the chronic activation of microglia affects retinal ganglion cells and photoreceptors, respectively, contributing to gradual vision loss. However, the molecular mechanisms that cause activation of microglia in the retina are not fully understood. Here we show that exposure of retinal pigment epithelial (RPE) cells to chronic low-level oxidative stress induces mitochondrial DNA (mtDNA)-specific damage, and the subsequent translocation of damaged mtDNA to the cytoplasm results in the binding and activation of intracellular DNA receptor Z-DNA-binding protein 1 (ZBP1). Activation of the mtDNA/ZBP1 pathway triggers the expression of proinflammatory markers in RPE cells. In addition, we show that the enhanced release of extracellular vesicles (EVs) containing fragments of mtDNA derived from the apical site of RPE cells induces a proinflammatory phenotype of microglia via activation of ZBP1 signaling. Collectively, our report establishes oxidatively damaged mtDNA as an important signaling molecule with ZBP1 as its intracellular receptor in the development of an inflammatory response in the retina. We propose that this novel mtDNA-mediated autocrine and paracrine mechanism for triggering and maintaining inflammation in the retina may play an important role in ocular pathologies. Therefore, the molecular mechanisms identified in this report are potentially suitable therapeutic targets to ameliorate development of ocular pathologies. Normal retina function is facilitated by an orchestrated interplay between several cell types. The retinal pigment epithelium (RPE) is a highly specialized monolayer of cells with pigmented microvilli, lining the Bruch's membrane located between the neural retina and the choroid in the eye. RPE cells perform several critical functions to maintain retinal homeostasis, including phagocytosis of photoreceptor outer segments, recycling vitamin A, and maintaining the blood–retinal barrier (1Lakkaraju A. Umapathy A. Tan L.X. Daniele L. Philp N.J. Boesze-Battaglia K. Williams D.S. The cell biology of the retinal pigment epithelium.Prog. Retin. Eye Res. 2020; 78: 100846Crossref Scopus (83) Google Scholar). Polarized, mature, and terminally differentiated RPE cells are mitotically quiescent under normal physiological conditions. The RPE is also characterized by an apical-basolateral polarity, with the basolateral surface sitting on the Bruch's membrane and the apical microvilli interfacing with the photoreceptors outer segments. The polarized architecture of the RPE together with tight junctions between cells enables the RPE to form a blood–retinal barrier that is critical for transport of nutrients, ions, and water across the RPE and for directional secretion of growth factors and extracellular matrix components to help maintain structure and function of the photoreceptors and choroids (1Lakkaraju A. Umapathy A. Tan L.X. Daniele L. Philp N.J. Boesze-Battaglia K. Williams D.S. The cell biology of the retinal pigment epithelium.Prog. Retin. Eye Res. 2020; 78: 100846Crossref Scopus (83) Google Scholar, 2Caceres P.S. Rodriguez-Boulan E. Retinal pigment epithelium polarity in health and blinding diseases.Curr. Opin. Cell Biol. 2020; Scopus Google Scholar). the critical role of RPE in of retinal homeostasis, RPE is observed in several The of ocular is age-related macular and the that to RPE cells by enhanced oxidative stress is a in development Oxidative stress and the in age-related macular Biol. Scopus Google Scholar, A. K. oxidative stress in the retinal pigment epithelium to retinal Scopus Google Scholar, signaling is in the RPE an oxidative Eye Res. Scopus Google Scholar, in the of age-related macular Scopus Google Scholar). in the RPE are also in as of and is with RPE E. and Scopus Google Scholar). to mitochondrial DNA together with mitochondrial is in are as a of K. E. A. of mitochondrial DNA in of age-related macular Scopus Google Scholar, A. in mitochondrial DNA retinal function and the retina to Scopus Google Scholar, in mitochondrial DNA and of DNA in the neural Scopus Google Scholar, A. K. and DNA and in age-related macular Scopus Google Scholar, A. of retinal pigment epithelium in age-related macular S