June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Rat CRISPR/Cas9 knockout of Interphotoreceptor retinoid-binding protein (IRBP, RBP3) suggests role in maintaining lamellar disc outer segment stability
Author Affiliations & Notes
  • Federico Gonzalez-Fernandez
    Research, G.V. (Sonny) Montgomery VAMC; Ophthamology and Pathology UMMC, Jackson, Mississippi, United States
  • John Kenneth Lippincott
    Research, G.V. (Sonny) Montgomery VAMC; Ophthamology and Pathology UMMC, Jackson, Mississippi, United States
  • Asyvia M. Powell
    Research, G.V. (Sonny) Montgomery VAMC; Ophthamology and Pathology UMMC, Jackson, Mississippi, United States
  • Taylor Coleman
    Research, G.V. (Sonny) Montgomery VAMC; Ophthamology and Pathology UMMC, Jackson, Mississippi, United States
  • Footnotes
    Commercial Relationships   Federico Gonzalez-Fernandez, None; John Lippincott, None; Asyvia Powell, None; Taylor Coleman, None
  • Footnotes
    Support  Start up awards from Research, Mississippi! Inc. and the Veterans Affairs Office of Research Development
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 1528. doi:
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      Federico Gonzalez-Fernandez, John Kenneth Lippincott, Asyvia M. Powell, Taylor Coleman; Rat CRISPR/Cas9 knockout of Interphotoreceptor retinoid-binding protein (IRBP, RBP3) suggests role in maintaining lamellar disc outer segment stability. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1528.

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

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Abstract

Purpose : Interphotoreceptor retinoid-binding protein (IRBP, RBP3), is the most abundant protein component of the interphotoreceptor matrix, and is also a component of the vitreous humor. IRBP has been implicated in a variety of important disease states including a form of recessive retinitis pigmentosa, a neuroprotective role in diabetic retinopathy, and its absence is associated with myopia. Biochemically, IRBP has free radical scavenging activity, and protects retinoids from photodecomposition. It has an important role in delivering and removing 11-cis retinal and all-trans retinol from the outer segments respectively. Our goal is to better understand its role in the retinoid cycle, and explore new functions for IRBP.

Methods : Rats were selected over mice as their larger size can facilitate tissue collection, surgical manipulation and pharmacokinetic studies. We generated a CRISPER/Cas9 knockout of the complete RBP3 gene in an albino Sprague Dawley background. The knockout was back crossed to obtain a homozygous IRBP(-/-) animals. IRBP(+/+) litter mates were used for dontrols. Characterization of the new model consisted of histopathology, ERG, immiunohistochemistry, and electron microscopy.

Results : Contol IRBP(+/+) liter mates showed prominent IRBP immunospecific staining restricted to the zone betweeen the apical RPE surface, and external limiting membrane. In contrast, IRBP(-/-) animals showed no staining for IRBP. Histopathology showed a time dependent thinning of the outer nuclear layer with sparing of the inner nuclear layer, and shortening of the outer segments compared to age matched controls starting at the age of weaning and progeresswing steadily over the next several months. The degeneration was assoicated with an equivalent reduction of the electroretinogram A and B waves. Ultrastructural studies showed abnormal outer segment structure. Instead of the normal uniform contour, the rod outer segments were irregular, disorganized and sometimes broken. Increased space betweeen the outer segment themsleves suggested the presence of edema.

Conclusions : Although its function is far from clear, IRBP is necessay for the health of the outer retina and for the structural integrity of the outer segments. A role in maintaining the stability of the lamellar discs/ outer segment structure is suggested by the present data.

This is a 2020 ARVO Annual Meeting abstract.

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