Hashimoto, Yosuke and Porkoláb, Gergő and Hudson, Natalie and O'Callaghan, Jeffrey and Hanley, Nicole and Delaney, Conor and Deli, Mária Anna and Westenskow, Peter and Campbell, Matthew (2025) EHD4 and ASAP2 are critical negative regulators of the claudin‐5‐based endothelial barrier. FEBS JOURNAL. ISSN 1742-464X
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Abstract
Disruption to barriers of the central nervous system (CNS) has been shown in both prime and drive pathologies observed across numerous neurological and ophthalmological conditions. These barriers are composed of well evolved endothelial tight junctions, and the key junctional component, claudin‐5 (CLDN‐5), is responsible for maintaining homeostasis of brain and retinal tissues. Indeed, decreased CLDN‐5 expression has now been observed across many neurological and retinal diseases. Additionally, methods aimed at stabilising and upregulating CLDN‐5 expression may have profound efficacy in treating a vast array of these conditions. However, very few targeted and specific methods can enhance CLDN‐5 expression levels, and none of these have detailed its localisation and stability on the cell surface. In an effort to discover unknown and specific regulators of CLDN‐5 expression, we performed a genome‐wide cell‐sorting‐based phenotypic screen using CRISPR/Cas9. Sorting cells based on the phenotype of ‘barrier tightness’ revealed two candidate genes, EH domain‐containing protein 4 ( EHD4 ) and Arf‐GAP with SH3 domain, ANK repeat, and PH domain‐containing protein 2 ( ASAP2 ), which, when suppressed, led to significant upregulation of CLDN‐5 protein on the cell surface. EHD4 appeared to regulate the transcriptional activity of CLDN5 , whereas ASAP2 controlled junctional localisation of CLDN‐5. Identification of these candidate genes suggests that pharmacological inhibitors of EHD4 or ASAP2 may represent profound approaches to regulating CLDN‐5 in neural endothelial cells.
| Item Type: | Article |
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| Additional Information: | Funding Agency and Grant Number: Science Foundation Ireland (SFI) [Eye-D-21/SPP/3732]; Irish Research Council (IRC); SFI [21/RC/10294_P2]; European Regional Development fund by Future Neuro industry partners; European Research Council (ERC) grant, 'Retina-Rhythm' [864522]; Eye Research Foundation for the Aged (ERFA); Japan Society for the Promotion of Science (JSPS) [JPJSBP120259931]; HIRAKU-Global Program by MEXT's 'Strategic Professional Development Program for Young Researchers' Funding text: This work was supported by grants from Science Foundation Ireland (SFI), (Eye-D-21/SPP/3732), the Irish Research Council (IRC) and by a research grant from SFI under grant number 21/RC/10294_P2, and co-funded under the European Regional Development fund by FutureNeuro industry partners. The Campbell lab is also supported by a European Research Council (ERC) grant, 'Retina-Rhythm' (864522). YH is supported by a research grant from the Eye Research Foundation for the Aged (ERFA), the Japan Society for the Promotion of Science (JSPS) (JPJSBP120259931) and the HIRAKU-Global Program, which is funded by MEXT's 'Strategic Professional Development Program for Young Researchers'. |
| Subjects: | Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia R Medicine / orvostudomány > R1 Medicine (General) / orvostudomány általában |
| SWORD Depositor: | MTMT SWORD |
| Depositing User: | MTMT SWORD |
| Date Deposited: | 11 Feb 2026 13:33 |
| Last Modified: | 11 Feb 2026 14:13 |
| URI: | https://real.mtak.hu/id/eprint/233698 |
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