Abstract
Abstract: :
Purpose: In mice, light damage susceptibility (LDS) is controlled by several genetic factors. One has been identified in RPE65, a pigment epithelial specific protein involved in the (re)generation of rhodopsin during the visual cycle. A sequence variant (Danciger et al. (2000) Mamm Genome 11, 422) in Rpe65 influences RPE65 protein levels and the rate of rhodopsin regeneration after bleaching. Fast regeneration leads to increased photon absorptions per unit time and to high LDS whereas slow regeneration kinetics render mouse photoreceptors more resistant against light damage (Wenzel et al. (2001), J. Neurosci. 21,53). Here we analyzed different rat strains to determine whether a similar system may influence LDS also in rats. Methods: Four rat strains were screaned for RPE65 protein levels by Western blotting. A high and a low expressing strain was analyzed further. Rpe65 mRNA levels were assesssed by RT-PCR and rhodopsin regeneration kinetics were determined spectrophotometrically. Sequences of the Rpe65 cDNA were determined. LDS was analzed by exposure to white light (3000 lux) for various times. Apoptotic cell death of photoreceptors was tested by TUNEL staining and by light microscopy of retinal sections. Results: RPE65 protein levels were higher in Lewis and Brown Norway rats compared to Wistar and Long Evans. The albino strains Wistar and Lewis were investigated further. Lewis had higher Rpe65 mRNA levels than Wistar. Sequence analysis of the Rpe65 cDNA revealed no sequence variations in the two strains. Content and regeneration of rhodopsin after bleaching were comparable in both strains. However, light damage occurred in Wistar already after 20' of exposure whereas Lewis rats had to be exposed for 30' minutes to achieve comparable damage. Conclusion: Lower RPE65 protein levels in Wistar may have been caused by decreased gene expression and not by a sequence variation as observed in mice. In rats, RPE65 may not be the limiting factor that determines rhodopsin regeneration kinetics. Since LDS in rats did not directly correlate with RPE65 protein levels and rhodopsin regeneration, other yet unidentified (genetic) factors may account for the susceptibility differences observed in rats.
Keywords: 341 cell death/apoptosis • 561 retinal degenerations: cell biology • 517 photoreceptors