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Autophagy alleviates hypoxia-induced blood-brain barrier injury via regulation of CLDN5 (claudin 5).

ABSTRACT: Blood-brain barrier (BBB) disruption is a key event in triggering secondary damage to the central nervous system (CNS) under stroke, and is frequently associated with abnormal macroautophagy/autophagy in brain microvascular endothelial cells (BMECs). However, the underlying mechanism of autophagy in maintaining BBB integrity remains unclear. Here we report that in BMECs of patients suffering stroke, CLDN5 (claudin 5) abnormally aggregates in the cytosol accompanied by autophagy activation. In vivo zebrafish and in vitro cell studies reveal that BBB breakdown is partially caused by CAV1 (caveolin 1)-mediated redistribution of membranous CLDN5 into the cytosol under hypoxia. Meanwhile, autophagy is activated and contributes mainly to the degradation of CAV1 and aggregated CLDN5 in the cytosol of BMECs, therefore alleviating BBB breakdown. Blockage of autophagy by genetic methods or chemicals aggravates cytosolic aggregation of CLDN5, resulting in severer BBB impairment. These data demonstrate that autophagy functions in the protection of BBB integrity by regulating CLDN5 redistribution and provide a potential therapeutic strategy for BBB disorder-related cerebrovascular disease.Abbreviations: BBB: blood-brain barrier; BECN1: beclin 1; BMEC: brain microvascular endothelial cell; CAV1: caveolin 1; CCA: common carotid artery; CLDN5: claudin 5; CNS: central nervous system; CQ: chloroquine; HIF1A: hypoxia inducible factor 1 subunit alpha; MCAO: middle cerebral artery occlusion-reperfusion; OCLN: occludin; ROS: reactive oxygen species; STED: stimulated emission depletion; TEER: trans-endothelial electrical resistance; TEM: transmission electron microscopy; TJ: tight junction; TJP1: tight junction protein 1; UPS: ubiquitin-proteasome system.

SUBMITTER: Yang Z

PROVIDER: S-EPMC8526012 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe blood-brain barrier (BBB) ensures central nervous system (CNS) homeostasis by strictly controlling the passage of molecules and solutes from the bloodstream into the CNS. Complex and continuous tight junctions (TJs) between brain endothelial cells block uncontrolled paracellular diffusion of molecules across the BBB, with claudin-5 being its dominant TJs protein. However, claudin-5 deficient mice still display ultrastructurally normal TJs, suggesting the contribution of other claudins or tight-junction associated proteins in establishing BBB junctional complexes. Expression of claudin-12 at the BBB has been reported, however the exact function and subcellular localization of this atypical claudin remains unknown.MethodsWe created claudin-12-lacZ-knock-in C57BL/6J mice to explore expression of claudin-12 and its role in establishing BBB TJs function during health and neuroinflammation. We furthermore performed a broad standardized phenotypic check-up of the mouse mutant.ResultsMaking use of the lacZ reporter allele, we found claudin-12 to be broadly expressed in numerous organs. In the CNS, expression of claudin-12 was detected in many cell types with very low expression in brain endothelium. Claudin-12lacZ/lacZ C57BL/6J mice lacking claudin-12 expression displayed an intact BBB and did not show any signs of BBB dysfunction or aggravated neuroinflammation in an animal model for multiple sclerosis. Determining the precise localization of claudin-12 at the BBB was prohibited by the fact that available anti-claudin-12 antibodies showed comparable detection and staining patterns in tissues from wild-type and claudin-12lacZ/lacZ C57BL/6J mice.ConclusionsOur present study thus shows that claudin-12 is not essential in establishing or maintaining BBB TJs integrity. Claudin-12 is rather expressed in cells that typically lack TJs suggesting that claudin-12 plays a role other than forming classical TJs. At the same time, in depth phenotypic screening of clinically relevant organ functions of claudin-12lacZ/lacZ C57BL/6J mice suggested the involvement of claudin-12 in some neurological but, more prominently, in cardiovascular functions.

Project description:BackgroundMammalian Claudin-5 is the main endothelial tight junction component maintaining blood-brain barrier (BBB) permeability, while Claudin-1 and -3 seal the paracellular space of choroid plexus (CP) epithelial cells contributing to the blood-cerebrospinal fluid barrier (BCSFB). In zebrafish, two paralogs of claudin-5a and -5b are expressed while their roles in the formation of BBB and BCSFB are unclear.MethodsThe expression patterns of Claudin-5a and -5b in zebrafish brains were systematically analyzed by immunofluorescence (IF) assay. The developmental functions of Claudin-5a and -5b were characterized by generating of claudin-5a and -5b mutants respectively. Meanwhile, the cerebral inflammation and cell apoptosis in claudin-5a-/- were assessed by live imaging of transgenic zebrafish, RT-qPCR, IF, and TUNEL assay. The integrity of BBB and BCSFB was evaluated by in vivo angiographic and dye permeation assay. Finally, RT-qPCR, whole-mount RNA in situ hybridization (WISH), and transmission electron microscopy (TEM) analyses were performed to investigate the development of cerebral vessels and choroid plexus.ResultsWe showed that Claudin-5a and -5b are both expressed in zebrafish cerebrovascular endothelial cells (ECs). In addition, Claudin-5a was strongly expressed in CP epithelial cells. Loss of Claudin-5b showed no effect on zebrafish vasculogenesis or BBB function. In contrast, the knockout of claudin-5a caused a lethal phenotype of severe whole-brain oedema, ventricular dilatation, and cerebral hernia in zebrafish larvae, although the cerebral vasculogenesis and the development of CP were not altered. In claudin-5a-/- , although ultrastructural analysis of CP and cerebral capillary showed intact integrity of epithelial and endothelial tight junctions, permeability assay indicated a disruption of both BBB and BCSFB functions. On the molecular level, it was found that ZO-1 was upregulated in the CP epithelium of claudin-5a-/-, while the notch and shh pathway responsible for CP development was not affected due to loss of Claudin-5a.ConclusionsOur findings verified a non-functional role of zebrafish Claudin-5b in the BBB and identified Claudin-5a as the ortholog of mammalian Claudin-5, contributing to the development and the functional maintenance of both BBB and BCSFB.

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Autophagy alleviates hypoxia-induced blood-brain barrier injury via regulation of CLDN5 (claudin 5).

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