An oxygen-adaptive interaction between SNHG12 and occludin maintains blood-brain barrier integrity.

Tight junctions (TJs) of brain microvascular endothelial cells (BMECs) play a pivotal role in maintaining the blood-brain barrier (BBB) integrity; however, precise regulation of TJs stability in response to physiological and pathological stimuli remains elusive. Here, using RNA immunoprecipitation with next-generation sequencing (RIP-seq) and functional characterization, we identify SNHG12, a long non-coding RNA (lncRNA), as being critical for maintaining the BBB integrity by directly interacting with TJ protein occludin. The interaction between SNHG12 and occludin is oxygen adaptive and could block Itch (an E3 ubiquitin ligase)-mediated ubiquitination and degradation of occludin in human BMECs. Genetic ablation of endothelial Snhg12 in mice results in occludin reduction and BBB leakage and significantly aggravates hypoxia-induced BBB disruption. The detrimental effects of hypoxia on BBB could be alleviated by exogenous SNHG12 overexpression in brain endothelium. Together, we identify a direct TJ modulator lncRNA SNHG12 that is critical for the BBB integrity maintenance and oxygen adaption.

Caspr1 Facilitates sAPPα Production by Regulating α-Secretase ADAM9 in Brain Endothelial Cells.

The expression of contactin-associated protein 1 (Caspr1) in brain microvascular endothelial cells (BMECs), one of the major cellular components of the neurovascular unit (NVU), has been revealed recently. However, the physiological role of Caspr1 in BMECs remains unclear. We previously reported the nonamyloidogenic processing of amyloid protein precursor (APP) pathway in the human BMECs (HBMECs). In this study, we found Caspr1 depletion reduced the levels of soluble amyloid protein precursor α (sAPPα) in the supernatant of HBMECs, which could be rescued by expression of full-length Caspr1. Our further results showed that ADAM9, the α-secretase essential for processing of APP to generate sAPPα, was decreased in Caspr1-depleted HBMECs. The reduced sAPPα secretion in Caspr1-depleted HBMECs was recovered by expression of exogenous ADAM9. Then, we identified that Caspr1 specifically regulates the expression of ADAM9, but not ADAM10 and ADAM17, at transcriptional level by nuclear factor-κB (NF-κB) signaling pathway. Caspr1 knockout attenuated the activation of NF-κB and prevented the nuclear translocation of p65 in brain endothelial cells, which was reversed by expression of full-length Caspr1. The reduced sAPPα production and ADAM9 expression upon Caspr1 depletion were effectively recovered by NF-κB agonist. The results of luciferase assays indicated that the NF-κB binding sites are located at -859 bp to -571 bp of ADAM9 promoter. Taken together, our results demonstrated that Caspr1 facilitates sAPPα production by transcriptional regulation of α-secretase ADAM9 in brain endothelial cells.

A CASPR1-ATP1B3 protein interaction modulates plasma membrane localization of Na+/K+-ATPase in brain microvascular endothelial cells.

Contactin-associated protein 1 (CASPR1 or CNTNAP1) was recently reported to be expressed in brain microvascular endothelial cells (BMECs), the major component of the blood-brain barrier. To investigate CASPR1's physiological role in BMECs, here we used CASPR1 as a bait in a yeast two-hybrid screen to identify CASPR1-interacting proteins and identified the ß3 subunit of Na+/K+-ATPase (ATP1B3) as a CASPR1-binding protein. Using recombinant and purified CASPR1, RNAi, GST-pulldown, immunofluorescence, immunoprecipitation, and Na+/K+-ATPase activity assays, we found that ATP1B3's core proteins, but not its glycosylated forms, interact with CASPR1, which was primarily located in the endoplasmic reticulum of BMECs. CASPR1 knockdown reduced ATP1B3 glycosylation and prevented its plasma membrane localization, phenotypes that were reversed by expression of full-length CASPR1. We also found that the CASPR1 knockdown reduces the plasma membrane distribution of the α1 subunit of Na+/K+-ATPase, which is the major component assembled with ATP1B3 in the complete Na+/K+-ATPase complex. The binding of CASPR1 with ATP1B3, but not the α1 subunit, indicated that CASPR1 binds with ATP1B3 to facilitate the assembly of Na+/K+-ATPase. Furthermore, the activity of Na+/K+-ATPase was reduced in CASPR1-silenced BMECs. Interestingly, shRNA-mediated CASPR1 silencing reduced glutamate efflux through the BMECs. These results demonstrate that CASPR1 binds with ATP1B3 and thereby contributes to the regulation of Na+/K+-ATPase maturation and trafficking to the plasma membrane in BMECs. We conclude that CASPR1-mediated regulation of Na+/K+-ATPase activity is important for glutamate transport across the blood-brain barrier.

Caspr1 is a host receptor for meningitis-causing Escherichia coli.

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but the pathogenesis of E. coli meningitis remains elusive. E. coli penetration of the blood-brain barrier (BBB) is the critical step for development of meningitis. Here, we identify Caspr1, a single-pass transmembrane protein, as a host receptor for E. coli virulence factor IbeA to facilitate BBB penetration. Genetic ablation of endothelial Caspr1 and blocking IbeA-Caspr1 interaction effectively prevent E. coli penetration into the brain during meningitis in rodents. IbeA interacts with extracellular domain of Caspr1 to activate focal adhesion kinase signaling causing E. coli internalization into the brain endothelial cells of BBB. E. coli can invade hippocampal neurons causing apoptosis dependent on IbeA-Caspr1 interaction. Our results indicate that E. coli exploits Caspr1 as a host receptor for penetration of BBB resulting in meningitis, and that Caspr1 might be a useful target for prevention or therapy of E. coli meningitis.