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

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.