Abstract
The tight junction protein claudin-3 has been identified as a transcriptional target of the Wnt/β-catenin signaling pathway regulating blood-brain barrier (BBB) maturation. In neurological disorders loss of claudin-3 immunostaining is observed at the compromised BBB and blood-cerebrospinal fluid barrier (BCSFB). Although these observations support a central role of claudin-3 in regulating brain barriers’ tight junction integrity, expression of claudin-3 at the brain barriers has remained a matter of debate. This prompted us to establish claudin-3−/− C57BL/6J mice to study the role of claudin-3 in brain barrier integrity in health and neuroinflammation. Bulk and single cell RNA sequencing and direct comparative qRT-PCR analysis of brain microvascular samples from WT and claudin-3−/− mice show beyond doubt that brain endothelial cells do not express claudin-3 mRNA. Detection of claudin-3 protein at the BBB in vivo and in vitro is rather due to junctional reactivity of anti-claudin-3 antibodies to an unknown antigen still detected in claudin-3−/− brain endothelium. We confirm expression and junctional localization of claudin-3 at the BCSFB of the choroid plexus. Our study clarifies that claudin-3 is not expressed at the BBB and shows that absence of claudin-3 does not impair brain barrier function during health and neuroinflammation in C57BL/6J mice.
Highlights
The tight junction protein claudin-3 has been identified as a transcriptional target of the Wnt/β-catenin signaling pathway regulating blood-brain barrier (BBB) maturation
Barrier function at the BBB is established at the level of highly specialized microvascular endothelial cells, whereas the blood-cerebrospinal fluid barrier (BCSFB) is established by the choroid plexus epithelium[1]
Correct gene targeting in embryonic stem cells (ES) cells and germline transmission were confirmed by Southern blotting (Fig. 1b) and absence of claudin-3 protein in claudin-3−/− C57BL/6J mice was confirmed by Western blotting and immunofluorescence staining (Fig. 1c and Supplementary Fig. S5a)
Summary
The tight junction protein claudin-3 has been identified as a transcriptional target of the Wnt/β-catenin signaling pathway regulating blood-brain barrier (BBB) maturation. In neurological disorders loss of claudin-3 immunostaining is observed at the compromised BBB and blood-cerebrospinal fluid barrier (BCSFB). These observations support a central role of claudin-3 in regulating brain barriers’ tight junction integrity, expression of claudin-3 at the brain barriers has remained a matter of debate. This prompted us to establish claudin-3−/− C57BL/6J mice to study the role of claudin-3 in brain barrier integrity in health and neuroinflammation. With claudin-1 forming a paracellular barrier and claudin-2 forming a paracellular water channel, BCSFB TJs may be adapted to the role of the choroid plexus in producing cerebrospinal fluid (CSF)[19,20,21]. Little is known about specific alterations in the junctional architecture of the BCSFB under neuroinflammatory conditions[16,29]
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