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Zhiqin Huang, Samuel McLenachan, Dan Zhang, Fred Kuanfu Chen; Disease modelling and functional characterisation using iPSC derived RPE in RCBTB1-associated retinopathy. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2450 – F0394.
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© ARVO (1962-2015); The Authors (2016-present)
RCBTB1 has been hypothesized to be involved in biological processes associated with the ubiquitin proteasome system (UPS) and oxidative stress pathways in retinal cells. We sought to expand our understanding of the physiological and pathological role RCBTB1 in retinal cells using an iPSC derived RPE cellular model of RCBTB1-associated retinopathy.
Expression of RCBTB1 was quantified via qRT-PCR and western blotting in patient derived iPSC-RPE cells (c.[170delG];[707delA]) compared to healthy controls. Co-immunoprecipitation (Co-IP) was conducted in health control iPSC-RPE cells to characterize interactions between RCBTB1 and other proteins including CUL3, UBE2E3 and NFE2L2. Human ubiquitin array analysis in these cells using an R&D Systems Proteome Profiler Human Ubiquitin Array Kit (ARY027, R&D Systems) was performed to measure the ubiquitination level of 49 different proteins. Furthermore, mitochondrial integrity and structure in iPSC-RPE were investigated using transmission electron microscopy.
Reduced RCBTB1 expression level was confirmed in patient derived iPSC-RPE cells compared to healthy controls. Co-IP experiment demonstrated that RCBTB1 interacts with CUL3 and UBE2E3 but not NFE2L2 in iPSC-RPE cells. Analysis of ubiquitin array identified two protein with significant change of ubiquitination level, including PDGFR β (platelet derived growth factor receptor β) and p53. Ultrastructural analysis not only showed a thinner RPE cell layer displaying shorter microvilli, but also showed disrupted cristae formation and architecture of mitochondria in patient derived RPE compared to control, indicating mitochondrial dysfunction and consequent excessive ROS production may play a role in cellular damage in RCBTB1 deficient RPE cells.
We identified RCBTB1 deficiency and its effect on UPS and mitochondria ultrastructure using an iPSC derived RPE cellular model. Furthermore, we identified two potential RCBTB1 interacting proteins, CUL3 and UBE2E3. Our findings shed light on the physiological role RCBTB1 plays in human RPE cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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