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Anastasiia Strizhakova, Adithya Sreenivas, Victoria Koontz, Peng Shang, Haitao Liu, Sayan Ghosh, Olivia Chowdhury, Stacey L Hose, J. Samuel Zigler, Nadezda A Stepicheva, Debasish Sinha; βA3/A1-crystallin may link lysosomal clearance and the circadian rhythm in the RPE. Invest. Ophthalmol. Vis. Sci. 2022;63(7):778 – F0337.
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© ARVO (1962-2015); The Authors (2016-present)
It has been well-documented that lysosomal clearance in the retinal pigment epithelial (RPE) cells is tightly regulated by the circadian clock. However, the details of this regulation are still unclear. We hypothesize that βA3/A1-crystallin, a protein known to be important for maintaining lysosomal homeostasis in the RPE, may also provide an important link between lysosomal clearance and the circadian rhythm in the RPE.
Wild-type (C56Bl/6J) mice (6-8 weeks old) were maintained at LD (light-dark) 12:12 cycle and sacrificed at ZT0, ZT2, ZT8, ZT14, ZT20 (Zeitgeber time; hours after lights on). A second set of mice was kept in complete darkness for 3 days prior to euthanasia. Western blotting and qPCR were performed on WT RPE to detect oscillations in the expression of Cryba1 (gene encoding βA3/A1-crystallin) and known circadian genes. For the in vitro studies, human fetal RPE (fRPE) cells were cultured and entrained with serum shock. Samples were collected every 4 hours after entrainment. qPCR was performed to analyze the expression of Cryba1 and the known circadian genes. Bioinformatic analysis of the Cryba1 gene sequence was performed with MoLoTool and miRDB.
Cryba1 expression oscillates at both mRNA and protein levels during the day in the RPE from WT mice, with the peak of protein expression coinciding with the peak of RPE lysosomal activity. Entrainment of fRPE cells with serum shock resulted in successful entrainment of Cryba1 expression, consistent with the circadian regulation of Cryba1 transcription in the RPE. Bioinformatic analysis revealed potential regulation of Cryba1expression by the circadian machinery (through NR1D1 transcription factor and miR-206).
We have shown that Cryba1 expression is regulated by circadian rhythm. This work contributes to a more complete understanding of the circadian rhythm entrainment in the RPE cells and introduces a novel, RPE-specific member into the circadian network.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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