Characterization of Double-Strand Break Repair Protein Ku80 Location Within the Murine Retina.

Abstract

PurposeTo characterize the spatial distribution of the DNA-double strand break-repair protein Ku80 in the murine retina. Even though robust data exist on the complexity of DNA repair mechanisms in dividing cells in vitro, almost nothing is known about it in post-mitotic neurons or photoreceptors (PRs). This knowledge is an important prerequisite for in vivo therapeutic approaches by genome editing in retina and PRs. Recently, it was shown that mouse rod PRs are incapable of repairing double-strand breaks induced by radiation.Material and MethodsRetinae from wild-type, rd10, and RPGR-KI mouse lines were obtained and stained with antibodies against Ku80, and cellular markers CtBP2, beta-Dystropglycan, Lamin B, and peanut agglutinin. Organotypic explant cultures were generated and maintained for up to 10 days. Laser microdissection was performed to obtain photoreceptor nuclei, and Ku80 expression was compared to whole retina by real-time PCR (RT-PCR).ResultsStrong Ku80 immunoreactivity was observed in rod but not cone photoreceptor terminals localized in the outer plexiform layer of the retina in all three mouse lines. During retinal explant culture, we observed that Ku80-positive globules translocate into the heterochromatin region of nuclei in the outer nuclear layer (ONL). By quantitative PCR, we showed upregulation of relative Ku80 expression in the ONL during wild-type retinal explant culture.DiscussionThe unexpected localization of Ku80 to murine rod terminals indicates another tissue-specific modification to the canonical DNA repair mechanisms and warrants further investigation.