July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Identifying intracellular signaling disrupted by loss of MFRP in human iPSC derived RPE
Author Affiliations & Notes
  • Steven Henle
    Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Ross F Collery
    Department of Ophthalmology of Visual Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
    Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Amy Ludwig-Kubinski
    Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Joseph Fogerty
    Cleveland Clinic, Cleveland, Ohio, United States
  • Joseph C Besharse
    Department of Ophthalmology of Visual Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
    Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Brian A Link
    Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Steven Henle, None; Ross Collery, None; Amy Ludwig-Kubinski, None; Joseph Fogerty, None; Joseph Besharse, None; Brian Link, None
  • Footnotes
    Support  R01EY014167 and K99EY027467
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4006. doi:
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      Steven Henle, Ross F Collery, Amy Ludwig-Kubinski, Joseph Fogerty, Joseph C Besharse, Brian A Link; Identifying intracellular signaling disrupted by loss of MFRP in human iPSC derived RPE. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4006.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Mutations in MFRP in humans can lead to a spectrum of disorders including microphthalmia and retinitis pigementosa. In the eye MFRP is greatly enriched in the RPE, but the molecular mechanisms by which it regulates RPE function and development are unclear. To better understand the role of MFRP in cell signaling in RPE we created induced pluripotent stem cells (iPSC) with a deletion mutation leading to loss of the MFRP protein.

Methods : Using Crispr technology we mutated the MFRP gene in an IMR90 iPSC line to prevent production of functional MFRP protein. We then differentiated MFRP deficient and wildtype iPSCs into RPE. To compare the wildtype and MFRP deficient lines we used a combination of RNA-seq/bioinformatics analysis, immunofluorescence, and super resolution microscopy.

Results : Little is known about the signaling downstream of MFRP. MFRP mutant RPE develop a disorganized actin cytoskeleton, as has been previously reported. Additionally, MFRP mutant RPE take longer to display the mature pattern of Ezrin staining. We are currently investigating if other markers of RPE maturity show similar defects. After performing RNA-seq analysis we performed a pathway analysis of this data that identified MFRP as a key regulatory of the visual cycle and retinal biosynthesis. Additional factors were also identified as potential mediators of MFRP signaling. To address phylogenetic conservation in MFRP signaling, we compared transcriptomic data from MFRP knockout in 3 species (humans, mice, and zebrafish). Loss of MRFP in these species each show eye phenotypes that are similar, but have distinct characteristics, and we are working to identify the relationships between these phenotypes and their transcriptomic changes.

We also looked at the role of C1QTNF5 as a potential ligand for MFRP. C1QTNF5 can bind to the extracellular portion of MFRP, but a potential ligand-receptor based signaling between these two proteins is unknown. Application of exogenous C1QTNF5 to cultures of RPE differentiated from human iPSCs showed similar changes in expression to that of loss of MFRP alone.

Conclusions : We have begun to build a model of what cellular events MFRP controls, and how they might be regulated. Overall, these findings provide insight into the signaling controlled by MFRP within RPE cells and how MFRP might be controlled, and thus provides a better understanding of the disease states caused by mutation of this gene.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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