Detection of DNA Double Strand Breaks by γH2AX Does Not Result in 53bp1 Recruitment in Mouse Retinal Tissues

Abstract

Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB) repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30) in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months) retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL), suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted.

RAG-2 deficiency results in fewer phosphorylated histone H2AX foci, but increased retinal ganglion cell death and altered axonal growth

“…These results support the involvement of RAG-2 in retinal development. Although our experimental approach allowed only indirect assessment, our findings are compatible with the interpretation that RAG-2 is at least partially responsible for the generation of naturally occurring DSBs in the developing retina 18–20,36 .

Figure 2DSB number is reduced in the retinas of E13.5 rag2 −/− mice. ( a,b ) Presence of DSBs in WT and rag2 −/− mice as determined by γH2AX immunostaining (green).

…”

“…The origins of DSBs in the developing retina remain unclear 18–20,36 . Characterization of potential sources of DSBs could provide important clues as to their physiological relevance.…”

Mouse Models of Inherited Retinal Degeneration with Photoreceptor Cell Loss

“…Nevertheless, mechanisms are present in rod cells to repair DNA damage, as evidenced by the robust levels of DNA repair factors [241] and by our results indicating that a network of DNA damage response genes is required for maintaining PR cell viability. Together, these results support the idea that a non-canonical DNA damage response pathway exists in rod PR cells [512]. Further study of the DNA damage response genes linked to PR cell loss in mice may be useful for elucidating this pathway.…”

Differences in the Response to DNA Double-Strand Breaks between Rod Photoreceptors of Rodents, Pigs, and Humans

“…Cones also displayed 53BP1 foci but much fewer and less intense compared with the INL (Figure S2B). The lower foci number/intensity in the cones compared to the INL might be related to the tissue preparation protocol since 53BP1 signals in cones can be more readily detected in cryosections in comparison to paraffin sections [30]. Next, we analyzed DSB repair by the enumeration of γH2AX foci and observed a similar induction level at 15 min after 1 Gy in rods, cones, and cells of the INL (Figure 3C,D).…”