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Brigitte Muller, Franziska Wagner, Leonie Luise Laucke, Wilhelm Rheinhard, Birgit Lorenz, Knut Stieger; Unexpected Localization of the DNA Repair Protein Ku80 within the Murine Retina.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4933.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study was to characterize the spatial distribution of the DNA-double strand break (DSB) repair protein Ku80 in the murine retina.
Retinae of wild type C57Bl6, rd10, LBR2 mice, and of an XLRP mouse model (B6J.SV129-Rpgrtm1stie) were investigated with respect to the presence of Ku80. Neuroretina explants were prepared and evaluated after 1 to 8 days in culture by immunohistochemistry or qPCR. Un-cultured retinae were used as controls. Total RNA was isolated from whole explants or individual retinal layers after laser micro dissection (LMD) and processed for qPCR. For histological investigations, explants where harvested and prepared for cryo-sectioning. Ku80 antibodies were used in combination with markers for photoreceptor proteins and analyzed by confocal microscopy.
Strong Ku80 immunoreactivity was detected in the outer plexiform layer (OPL) of the retina in all mouse lines investigated. No nuclear staining was observed in the outer nuclear layer (ONL). Weak Ku80 immunoreactivity could be found in nuclei of the inner nuclear layer of retinal explants in all mouse lines at most culture time points. During retinal explant culture, Ku80-positive droplets got released into the ONL to some extent. Analysis of relative Ku80 expression by qPCR revealed a constant upregulation during retinal explant culture by up to three-fold compared to un-cultured retinae. In wildtype mice, this upregulation was higher compared to the LBR2 mouse model and 9-month-old RPGR mice. Only retinal explants of 3-month-old RPGR mice showed a downregulation from day 2 until day 6. With LMD, we were able to compare the Ku80 expression in the ONL and the inner retina with whole retina samples and found an increasing expression during retinal explant culture, especially in the ONL.
The Ku80 protein is a key player in the DNA-DSB-repair pathway called non-homologous end-joining (NHEJ). Direct Ku80 protein detection and upregulated relative Ku80 expression in whole retina preparations as well as in individual retinal layers (ONL) demonstrate the presence of this important protein in retinal neurons. The delocalization of Ku80 in the photoreceptor synaptic terminals in the OPL was rather unexpected and needs further investigation. The knowledge about the DSB repair mechanisms in all retinal neurons is the prerequisite for future genome editing strategies for IRD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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