June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
The alternative RPGR isoform RPGRCONST, not implicated in X-linked RP, plays a crucial role in mammalian retina.
Author Affiliations & Notes
  • bhubanananda sahu
    Department of Ophthalmology & Visual Sciences, University of Massachusetts Medical School, Worcester, Massachusetts, United States
  • Laura Moreno Leon
    Department of Ophthalmology & Visual Sciences, University of Massachusetts Medical School, Worcester, Massachusetts, United States
  • Linjin Li
    Department of Ophthalmology & Visual Sciences, University of Massachusetts Medical School, Worcester, Massachusetts, United States
  • Wei Zhang
    Department of Ophthalmology & Visual Sciences, University of Massachusetts Medical School, Worcester, Massachusetts, United States
  • Manisha Anand
    Department of Ophthalmology & Visual Sciences, University of Massachusetts Medical School, Worcester, Massachusetts, United States
  • Michael Brodsky
    Molecular, Cell and Cancer Biology, Umass medical school, Worcester, Massachusetts, United States
  • Raju V S Rajala
    Dean McGee Eye institute, University of Oklahoma health science center, Oklahoma, Oklahoma, United States
  • Hemant Khanna
    Department of Ophthalmology & Visual Sciences, University of Massachusetts Medical School, Worcester, Massachusetts, United States
  • Footnotes
    Commercial Relationships   bhubanananda sahu, None; Laura Moreno Leon, None; Linjin Li, None; Wei Zhang, None; Manisha Anand, None; Michael Brodsky, None; Raju Rajala, None; Hemant Khanna, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4420. doi:
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      bhubanananda sahu, Laura Moreno Leon, Linjin Li, Wei Zhang, Manisha Anand, Michael Brodsky, Raju V S Rajala, Hemant Khanna; The alternative RPGR isoform RPGRCONST, not implicated in X-linked RP, plays a crucial role in mammalian retina.. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4420.

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

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Abstract

Purpose : Mutations in Retinitis pigmentosa GTPase regulator (RPGR) account for majority of X-linked retinitis pigmentosa (XLRP). The RPGR gene encodes two major isoforms: RPGRCONST (19 exons) and RPGRORF15 (terminating in intron 15; ORF15). Exon ORF15 of RPGR is a mutational hotspot, making RPGRORF15 a major disease-associated isoform. No mutations have yet been reported that specifically affect the RPGRCONST isoform. Although the role of RPGRCONST is unclear, its carboxyl-terminal isoprenylation motif modulates ciliary localization of phosphoinositide phosphatase INPP5E. Our aim is to delineate the role of RPGRCONST in vision.

Methods : To generate a RpgrCONST-mutant mouse, we ablated the isoprenylation domain using the CRISPR-Cas9 strategy in C57BL6/J mice. Retinas were processed for immunofluorescence and immunoblotting using standard procedures. Retinal structure and light responses were evaluated by electron microscopy, histology and electroretinography (ERG). Phosphoinisotide substrates of INPP5E were measured using PH-domain coupled ELISA.

Results : The RpgrCONST-mutant mice carry a 23bp deletion in the carboxyl-terminus leading to a frameshift and ablation of the isoprenylation motif. The RPGRCONST-mutant protein expression was significantly reduced whereas RPGRORF15 levels were unaffected in the mutant mice. The RPGRCONST-mutant protein accumulated in the inner segment as compared to a predominant ciliary localization of the wild-type RPGRCONST protein. Moreover, the levels of INPP5E in the outer segment and the phosphoinositide PI4P, a product of INPP5E activity were significantly reduced while the levels of an INPP5E substrate, PI-3,4,5-P3 were significantly elevated in the mutant mice. Although the photoreceptor outer segment morphology appeared normal, the mutant retinas displayed abnormal outer segment-RPE interface. Such anomalies were more prominent at later stages and correlated with reduced scotopic ERGs in the RpgrCONST-mutant mice.

Conclusions : Our studies show that the RPGRCONST isoform is critical for photoreceptor maintenance. We propose that the RPGRCONST isoform is regulates the cellular levels of phosphoinositides and their interaction with effector phospholipid-binding proteins for signaling in the retina. The RPGRCONST likely critical for normal photoreceptor-RPE interaction and photoreceptor function.

This is a 2020 ARVO Annual Meeting abstract.

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