Claudin-3-deficient C57BL/6J mice display intact brain barriers

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.

The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis

“…As first step for the comprehensive barrier target analysis the relative expression of these targets was assessed in hCMEC/D3 under standard growth conditions. In the BBB research field the claudins-1, -3, -5, -11 and -12 are mostly investigated as surrogate markers for TJs, whereas recent studies with knock-out mice raised doubts about the presence and role of claudin-3 and -12 at the BBB [7,8]. In the last 2 to 3 years some reports with expression data of primary human BCECs and hiPSC-BCECs have been published.…”

“…At the moment, however, there is a lively discussion about the role of individual claudins at the BBB. For example, it was recently shown that the claudins-3 and -12 in the BBB of the mouse do not have the previously believed importance for BBB function [7,8]. In addition, there are postulated species differences that make it difficult to get a clear picture of which claudins are present in the human BBB and which function they fulfill [3].…”

Role of Claudins in Renal Branching Morphogenesis

“…We removed multiple claudins using the C‐CPE reagent because removal of single C‐CPE‐sensitive claudins has not generated severe renal developmental phenotypes, suggesting there may be functional redundancy amongst members and/or different requirements depending on the model organism. For example, removal of Claudin‐3, −4, −6, or −8 has not led to any defect in mouse kidney development based on the fact that the offspring survive and do not succumb to renal failure postnatally (Anderson et al., 2008; Castro Dias et al., 2019; Fujita, Hamazaki, Noda, Oshima, & Minato, 2012; Gong et al., 2015). In the absence of comprehensive counts of nephron number, however, we cannot rule out the possibility that the mice may have mild to moderate defects in nephron number from a deficiency in renal branching morphogenesis.…”

Mechanisms Ensuring Endothelial Junction Integrity Beyond VE-Cadherin

“…Central components of tight junctions are claudins (Cldn), tetraspanning membrane proteins, of which Cldn5 is found in vessels in all organs ( Morita et al, 1999 ). Expression of Cldn3, Cldn12, and Cldn1 in brain capillary endothelial cells is rather controversial ( Ohtsuki et al, 2008 ; Tietz and Engelhardt, 2015 ; Vanlandewijck et al, 2018 ), and while Cldn3 is inducible during mouse brain angiogenesis ( Liebner et al, 2008 ) it is clearly absent in adult brain endothelium ( Castro Dias et al, 2019 ). Occludin is another bona fide tight junction component of endothelial and epithelial cells, however, its gene inactivation does not cause obvious defects in the vasculature ( Saitou et al, 2000 ).…”