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Mari Katsura, Reiko Yamagishi, Makoto Aihara, Glaucoma; Roles for methylation of H4K20 and 53BP1 in hypoxia-induced retinal ganglion cell death. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2397.
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© ARVO (1962-2015); The Authors (2016-present)
Understanding mechanisms of retinal ganglion cell (RGC) death is required for the prevention of blindness. Recently the roles of DNA damage responses and epigenetic modification of chromatin in apoptosis of neuron have been focused. Here we have investigated the alteration of DNA damage responses leading apoptosis induced by hypoxia using rat retinal ganglion cells.
RGCs were extracted from five to eight days old Wister rats with two-step panning procedure. After three days of incubation, the cultured RGCs were grown under 5% oxygen of hypoxia and analyzed. RGC-5 cells were also used for western blotting and cell cycle analysis after two days of serum starvation. Immunofluorescences were employed for analysis of nuclear foci formation by DNA damage response proteins, 53BP1 and γH2AX. Western blotting using anti-histone H4K20me1 and H4K20me2 analyzed the methylation of histone H4K20.
First, in primary cultured rat RGC, the number of nuclear foci of 53BP1 from 0.60 per cell under normoxia to 0.37 per cell under hypoxia (p < 0.01). Correspondingly, apoptotic cells increased from 49.8% under normoxia to 78.2% under hypoxia (p < 0.05). Second, KU55933; a specific inhibitor for ataxia telangiectasia mutated (ATM), the upstream of 53BP1, decreased the number of 53BP1 nuclear foci from 0.71 per cell to 0.42 per cell (p < 0.05). Apoptotic cells increased from 49.8% with DMSO to 64.0% with KU55933 (p < 0.05). Under hypoxia, the numbers of 53BP1 foci were also decreased and apoptotic cells increased and H4K20me1 was down-regulated both by hypoxia and KU55933. Latanoprost, one of the PGF2alpha agonist, increased the methylation of H4K20 and reversed the 53BP1 foci from 0.35 per cell to 0.51 per cell (p < 0.05). Hypoxia-induced RGC death was suppressed from 87.6% under hypoxia to 58.7% with addition of 100 nM of latanoprost (p < 0.05).
Methylation of H4K20 and 53BP1 play roles in RGC survival and the mechanism is involved in neuroprotection by latanoprost.
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