Abstract
Purpose :
The circadian clock plays an important role in the regulation of photoreceptor’s functions. Previous studies indicate that the removal of the clock gene Bmal1 from the neural retina alters visual sensitivity, spectral identity of the cone and cone photoreceptors functioning and viability in mice. Our laboratories have also shown that 661W cells (a cone photoreceptor-like cell line) contain a functional circadian clock and survival of the cells showed a circadian rhythm when cells were subjected to an oxidative stress challenge. In this study we investigated the mechanisms by which the circadian clock may modulate the response of the cell to oxidative stress.
Methods :
Bmal1 knock-out 661W (BKO) cells were generated from 661W (WT) cells using CRISPR/Cas9. Both WT and BKO cells were synchronized by medium exchange. Then cells were collected at six hours interval. The expression of Nuclear factor erythroid 2-related factor 2 (Nrf2), Glutathione Peroxidase (Gpx)1, Gpx4, aquaporin (Aqp)1 and Aqp5 were measured by q-PRC. GPX and Catalase activity were also measured at two different time points (i.e. at the point in which we observed high and low cells survival) using activity assay kits (Cayman).
Results :
Nrf2, Gpx1, Gpx4 and Aqp5 showed a rhythmic expression in 661W cells (One-Way ANOVA, p < 0.05 in all cases). In contrast, no rhythmicity was observed in BKO cells (One-Way ANOVA, p > 0.05 in all cases). The results from GPX assay revealed that GPX activity was significantly higher (Two-way ANOVA followed by Tukey test p< 0.05) at the time in which the cells showed and increased survival rate after an oxidative stress challenge. In BKO cells GPX activity was low and no different between the two time points. Catalase activity was not detected in both 661W and BKO cells.
Conclusions :
Our data indicate that the circadian clock in 661W cells controls the transcription of genes involved in cellular antioxidative defense and the membrane water channel. The circadian clock also controls the level of GPX activity. These results may elucidate circadian alteration of the sensitivity to oxidative response in 661W cell.
This is a 2020 ARVO Annual Meeting abstract.