Abstract
Purpose:
Accumulation of DNA damage and subsequent G1/S cell cycle phase transition of post mitotic neural cell is associated with pathologies of some neurodegenerative disorders. Among responsible polymorphisms for primary open angle glaucoma (POAG), several genes have their functions involved in G1/S transition. Here we have investigated the relevance of DNA damage and G1/S transition associated with RGC death under hypoxia.
Methods:
RGCs extracted from five to eight days-old rats were cultivated under hypoxia of 5% oxygen. The number of nuclear foci of γH2AX and 53BP1 per cell, markers of DNA double-strand breaks and the repair, were counted. AnnexinV staining was employed for cell death analysis. The length of the neurite per cell was also measured. To investigate the cell cycle phase, fluorescence intensity of DAPI for chromatin was analyzed. One hour before hypoxia load, a prostaglandin F2α analogue, latanoprost was added to the culture medium and its effects were evaluated. In addition to in vitro experiment, to examine 53BP1 nuclear foci in vivo, rat optic nerve (ON) crush models were generated.
Results:
The number of 53BP1 nuclear foci decreased under hypoxia from 0.60 ± 0.17 under normoxia to 0.37 ± 0.14 pcs / cell under hypoxia after 24 hours (p < 0.01). Neurite length after three hours and twelve hours of hypoxia shortened (p < 0.05). G1/S transition and cell death was facilitated under hypoxia. G1 cells were 81.2 ± 1.55% under normoxia and reduced to 63.8 ± 2.67% by hypoxia. Latanoprost reversed these effects significantly; 53BP1 (p < 0.05), apoptotic cell (p < 0.05), axon length (p < 0.001). In ON crush model, 53BP1 foci positive cells were significantly decreased (p < 0.005) as well as those in vitro experiment.
Conclusions:
DNA damage response including function of 53BP1and G1/S transition might play a significant role in RGC fate, in vitro and in vivo. Hypoxia exacerbates RGC death related to DNA damage response, which was partially canceled by latanoprost