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Haijiang Lin, Bo Tian, ahmad Al-moujahed, Joan W Miller, Demetrios G. Vavvas; Damaged nuclear DNA accumulates in retinal pigment epithelium cells of age-related macular degeneration patients. Invest. Ophthalmol. Vis. Sci. 2018;59(9):795.
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Chronic inflammation plays an important role in the pathogenesis of AMD. Recently, there is increasing evidence to support that endogenous damaged nuclear DNA (nDNA), as a DAMP (damage-associated molecular pattern), can activate nucleic-acid sensing pattern-recognition receptors (PRRs) and initiate inflammatory in different tissues and cells. In this study, we investigated whether the damaged nDNA accumulates in the RPE cells of AMD patients.
AMD and age-matched healthy control donor eyes (65-91 years) were obtained from NDRI and Georgia Eye Bank. The tissue blocks were subsequently sectioned to 5μm by a microtome. Damaged nDNA was detected in RPE cells by immunohistochemistry (IHC) staining for γH2AX, an established marker of damaged double stranded DNA (dsDNA). Further, RPE cells from donor globes were isolated under sterile conditions. An 8-mm sterile trephine punch was used to acquire macular RPE cells while remaining area were isolated as peripheral RPE cells. Immunofluorescence was adopted to detect dsDNA and γH2AX in cytosol using anti-dsDNA and anti- γH2AX antibodies. The average number of positive staining cells from different groups was counted from 10 random microscope fields.
Damaged nuclear DNA was detected in macular RPE cells from AMD patient donors by IHC staining for γH2AX. It showed that the number of RPE cells with accumulation of damaged nDNA from macula of AMD patients (33%) is significantly higher than that from both age-matched healthy controls’ macula (6%) and AMD peripheral retina (5%). Furthermore, the immunofluorescence staining using anti-dsDNA antibody clearly showed that dsDNA also locates in cytoplasm in isolated macular RPE cells from AMD patient donor eyes. The percentage of RPE cells harboring cytosolic dsDNA was significantly higher (60%) in macular region of AMD donor eyes compare to macular region from healthy donor eyes (19%). Importantly, γH2AX co-localized with dsDNA form the large cytosolic aggregates proximal to the nucleus but rarely co-localized with mitochondria, supporting this dSDNA origin mainly from the nucleus not mitochodria.
Our studies demonstrated that damaged nuclear DNA accumulated in macular RPE cells from AMD patients. This study contributes to explore the molecular mechanism in the development of AMD.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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