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Elora Vanoni, Emeline F Nandrot; Deregulation of POS phagocytosis and retinal adhesion rhythms in Prpf31-mutant mice: implication of the RPE circadian clock. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5979.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the ubiquitous Pre-mRNA Processing Factors 3, 8, and 31 (PRPF3, 8 and 31) constitute the second most prominent cause of autosomic dominant retinitis pigmentosa. Prpf-mutant primary retinal pigment epithelial (RPE) cells as well as Human ARPE-19 cells down-regulated for PRPF31 display a 40% decrease of photoreceptor outer segments (POS) phagocytosis, suggesting similar pathological processes occur in both species. In vivo, the diurnal rhythmicity of both POS phagocytosis and retinal adhesion is greatly attenuated at peak activity time while appearing normal at other times. Moreover, POS phagocytosis starts earlier in all 3 Prpf-mutant mice. We thus investigated whether the intracellular clock was directly affected in Prpf31-mutant mice.
The circadian expression of genes and proteins from the cellular clock were studied at different times of the light:dark cycle on 3-month-old animals. Analysis of the 3 components of the transcriptional feedback loop were performed using qRT-PCR and immunoblots on RPE/choroid and neural retina tissues. Investigated candidates were as follows: Clock, Bmal1 and Npas2 (activator complex), Per1-3 and Cry1-2 (repressor complex), and Rev-Erb-alpha, Ror-alpha and -gamma (secondary transcriptional loop).
As expected, all studied genes showed a circadian expression profile in wildtype animals. In Prpf31-mutant mice, all genes showed a perturbated profile at least at one time of the light:dark cycle. Notably, the Npas2 and Bmal1 activator genes, as well as Bmal1 protein, were overexpressed in mutant samples at light onset (8.00AM). In contrast, the Per1 and Cry1 repressors were overexpressed at the time of the phagocytic peak (10.00AM), both at the mRNA and protein levels.
All data together, the circadian clock appears to be deregulated in Prpf31-mutant RPE cells. Interestingly, current results are consistent with the phenotype of disrupted POS phagocytic rhythm featuring an earlier peak which overall intensity is attenuated. Further studies are underway investigating the potential disturbance of the required nocturnal re-localization of repressor complex members to the cell nucleus. Our results give us some insights on central molecular targets of Prpf31 dysfunction, and suggest that other rhythmic functions could be implicated in phenotype development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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