Roles for methylation of H4K20 and 53BP1 in hypoxia-induced retinal ganglion cell death

Abstract

 

Purpose

 

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.

 

Methods

 

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.

 

Results

 

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).

 

Conclusions

 

Methylation of H4K20 and 53BP1 play roles in RGC survival and the mechanism is involved in neuroprotection by latanoprost.

 

 

Left: Nuclear focus of 53BP1 localizes to DNA damage site in primary cultured RGC Right: Suppression of 53BP1 foci formarion was reversed by latanoprost

 

 

Left: Suppression of H4K20me1 under hypoxia was recovered by latanoprost Right: Hypoxia induced apoptosis was canceled by latanoprost

 

Keywords: 531 ganglion cells • 426 apoptosis/cell death • 615 neuroprotection