Enhanced autophagy drives endothelial tight junction loss, BBB disruption, and behavioral deficits during inflammation

  • Autophagy. 2026 Apr 25:1-18. doi: 10.1080/15548627.2026.2662440.
Milin Peng  1  2  3  4  5 ,  Wei Chen  1  2  3 ,  Qingtao Gao  6 ,  Xuejing Zhu  1  2  3 ,  Laidong Yu  1  4  7  8 ,  Lin Zhang  1  2  3  4 ,  Xinying Zou  9 ,  Zhonghua Hu  1  2  3  4  7  8 ,  Lina Zhang  1  2  3
Affiliations
  • 1. Department of Critical Care Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China.
  • 2. National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China.
  • 3. Hunan Provincial Clinical Research Center for Critical Care Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China.
  • 4. Institute of Molecular Precision Medicine and Hunan Key Laboratory of Molecular Precision Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China.
  • 5. Department of Intensive Care Medicine, Hunan University of Medicine General Hospital, Huaihua, Hunan, China.
  • 6. School of Life Science and Technology, Central South University of Forestry and Technology, Changsha, Hunan, China.
  • 7. MOE Key Laboratory of Rare Pediatric Diseases & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China.
  • 8. Hunan Key Laboratory of Animal Models for Human Diseases, School of Life Sciences, Central South University, Changsha, Hunan, China.
  • 9. Department of Pharmacy, School of Medicine, Hunan Normal University, Changsha, Hunan, China.
PMID: 42003242 DOI: 10.1080/15548627.2026.2662440
Abstract

The blood-brain barrier (BBB) protects the brain but becomes compromised during systemic inflammatory conditions such as sepsis. The mechanisms driving BBB disruption remain incompletely understood. Here, we identified a significant enrichment of the macroautophagy/autophagy-lysosome-related pathway in the upregulated proteome using quantitative proteomics on brain microvessels from mice after cecal ligation and puncture (CLP) that induces polymicrobial sepsis. CLP progressively induced autophagic flux in brain endothelial cells, peaking at 24 h post-procedure before subsiding. Similarly, an mRFP-GFP-LC3 reporter assay and immunoblotting showed that lipopolysaccharide (LPS) treatment increased autophagic flux in bEnd.3 cells in a time- and dose-dependent manner. Mice intraperitoneally (IP) injected with the Autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA) were resistant to BBB disruption caused by CLP or IP injection of LPS, whereas those injected with the Autophagy Inducer rapamycin (Rapa) were more susceptible. CQ and 3-MA reduced, while Rapa increased, CLP-induced lethality in mice. These effects were confirmed in vitro using a dextran infiltration assay on bEnd.3 cell transwell cultures. CQ alleviated both the acute disruption of the tight junction proteins TJP1/ZO-1 and CLDN5 in brain microvessels and the long-term memory and anxiety deficits in LPS-challenged mice. siRNA-mediated knockdown of the SNARE protein STX17, which inhibits autophagosome-lysosome fusion, attenuated LPS-induced tight junction protein degradation in bEnd.3 cells. Importantly, inhibition of TLR4 or its downstream kinase TBK1 reduced LPS-induced Autophagy and preserved tight junction proteins, implicating TLR4-TBK1 signaling in endothelial Autophagy activation. These results suggest that excessive Autophagy in endothelial cells drives BBB damage and cognitive dysfunction in sepsis.Abbreviations: 3-MA: 3-methyladenine; ATG5: Autophagy related 5; BBB: blood-brain barrier; bEnd.3 cells: brain-derived endothelial cells.3; CLP: cecal ligation and puncture; CQ: chloroquine; EPM, elevated plus maze; GO: Gene Ontology; IP: intraperitoneal; LPS: lipopolysaccharide; MAP1LC3/LC3-II: microtubule associated protein 1 light chain 3-II; MWM: Morris Water Maze; NOR: novel object recognition test; OFT: open field test; Rapa: rapamycin; SAE: sepsis-associated encephalopathy; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TBK1: TANK binding kinase 1; TICAM1/TRIF: TIR domain containing adaptor molecule 1; TLR4: toll like receptor 4; TMT: tandem mass tag.

Keywords
Autophagy; blood-brain barrier; brain endothelial cell; sepsis; tight junction.
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