June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Interaction between disheveled 1 and claudin-5 is required for norrin-induction of blood-retinal barrier
Author Affiliations & Notes
  • Laura Gonzalez
    Ophtalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan, United States
  • Monica Diaz Coranguez
    Ophtalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan, United States
  • Xuwen Liu
    Ophtalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan, United States
  • David A Antonetti
    Ophtalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan, United States
  • Footnotes
    Commercial Relationships   Laura Gonzalez None; Monica Diaz Coranguez None; Xuwen Liu None; David Antonetti EyeBiotech, Code C (Consultant/Contractor)
  • Footnotes
    Support  NH Grant EY012021, NH Grant EY007003, NH Grant DK20572, RPB Grant N029083, NH Grant S10OD28612
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3255. doi:
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    • Get Citation

      Laura Gonzalez, Monica Diaz Coranguez, Xuwen Liu, David A Antonetti; Interaction between disheveled 1 and claudin-5 is required for norrin-induction of blood-retinal barrier. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3255.

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

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Abstract

Purpose : Purpose: Previous studies demonstrated that norrin restores blood-retinal barrier (BRB) properties after vascular endothelial growth factor-induced permeability providing a potentially important new therapeutic approach to treat VEGF-driven macular edema. The norrin-induced barrier restoration requires disheveled 1 (Dvl1) as knock-down of Dvl1 prevents the norrin response. Here, we explore the contribution of Dvl1 to bovine retinal endothelial cell (BREC) barrier properties induced by norrin. We hypothesize that norrin signals throughDvl1 promoting claudin-5 interaction and stabilizing tight junctions (TJs).

Methods : Methods: The localization of Dvl1 at the TJ was analyzed by confocal microscopy. The interaction between Dvl1 with claudin-5 was determined by co-immunoprecipitation in BREC and capture assays in HEK293 cells transfected with Dvl1 mutants. In BREC cells transfected with the C-terminal region of Dvl1 the expression of TJ proteins was evaluated by western blot and barrier properties were analyzed by measurement of the solute flux of 70kDa RITC-dextran.

Results : Results: Dvl1 immunofluorescence showed co-localization with ZO-1 and claudin-5 at the TJ complex. The co-immunoprecipitation assays demonstrated that Dvl co-precipitates with both ZO-1 and claudin-5 and the interaction with claudin-5 appeared specific for Dvl1. Further, the interaction of Dvl1 with claudin-5 is stronger after the co-stimulation with VEGF/norrin. Dvl1 has previously been shown to form intramolecular binding between the C-terminal PDZ-binding domain (PDZ-bd) with an internal PDZ domain. Co-transfection of HEK293 cells with Dvl1 mutants and claudin-5 revealed that Dvl1 interacts with claudin-5 through the Dvl PDZ domain and claudin-5 PDZ-bd since this interaction increases with deletion of the Dvl1 PDZ-bd and is blocked by expressing the C-terminal fragment of Dvl1. In BREC cells, transfection of the C-terminal fragment of Dvl1 downregulates the expression of claudin-5 but has no effect on expression of other proteins of the TJs including: ZO-1, occludin, claudin-1, or VE-cadherin. Blocking Dvl1/claudin-5 interaction increased basal permeability and prevents norrin induction of barrier properties after VEGF.

Conclusions : Conclusions: Altogether, these results demonstrate that norrin promotes the interaction between Dvl1 and claudin-5, stabilizing claudin-5 and inducing barrier properties.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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