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.

Brain microvascular endothelial cell exosome-mediated S100A16 up-regulation confers small-cell lung cancer cell survival in brain.

Small cell lung cancer (SCLC) is the most aggressive histologic subtype of lung cancer, with a strong predilection for early brain metastases. Despite efforts and advances in new therapeutics for SCLC, the prognosis of patients with SCLC with brain metastases is consistently poor. Therefore, a better understanding of the mechanisms of SCLC brain metastasis is important in improving current treatments. In this study, elevated S100A16 levels were associated with SCLC brain metastases, which was a possible secondary event arising from the brain metastatic microenvironment. Using an in vitro cell coculture system, we found that the coculturing of SCLC cells with human brain microvascular endothelial cells (HBMECs) led to an increased expression of S100A16 in SCLC cells. Conversely, treatment of HBMECs with GW4869, an inhibitor of exosome release, significantly blocked this effect in the cocultured SCLC cells. Alternatively, the results from Western blot analyses and immunofluorescence indicated that the HBMEC exosomes purified by ultracentrifugation also induced the elevation and translocation from the cytoplasm to the nucleus of S100A16 in the recipient SCLC cells. The inhibition experiments demonstrated that elevated S100A16 contributed a benefit of HBMEC exosomes for the survival of the recipient SCLC cells under stress. Moreover, the elevation of S100A16 in SCLC cells prevented the loss of mitochondrial membrane potential (Δψm) and enhanced resistance to apoptosis under stressful conditions, which were determined by Annexin V/propidium iodide and JC-1 assay. Further results showed that the S100A16-mediated protective effect was caused by the presence of an important element in Δψm, prohibitin (PHB)-1, a protein in the mitochondrial inner membrane. Conversely, the delivery of PHB-1 siRNAs into S100A16 overexpressing SCLC cells weakened these protective effects. Our findings suggest that elevated S100A16 plays an active role in facilitating the survival of SCLC cells through modulating the mitochondrial function, identifying S100A16 as an important potential target in SCLC brain metastasis.-Xu, Z.-H., Miao, Z.-W., Jiang, Q.-Z., Gan, D.-X., Wei, X.-G., Xue, X.-Z., Li, J.-Q., Zheng, F., Qin, X.-X., Fang, W.-G., Chen, Y.-H., Li. B. Brain microvascular endothelial cell exosome-mediated S100A16 up-regulation confers small cell lung cancer cell survival in brain.

CXCL1 promotes the proliferation of neural stem cells by stimulating the generation of reactive oxygen species in APP/PS1 mice.

PURPOSE: Elevated levels of CXCL1 were observed in the cerebrospinal fluid of patients with early Alzheimer's disease, which may affect neural stem cells in the subventricular zone. We used APP/PS1 mice and neural stem cells to elucidate the role of CXCL1 in Alzheimer's disease. METHODS & RESULTS: We detected CXCL1 in cerebrospinal fluid (CSF), activated macrophages, and microglia suggesting that macrophages may contribute to elevated CXCL1 in the CSF of middle-aged APP/PS1 mice. Proliferation and differentiation of neural stem cells were further analyzed and the results suggested that CXCL1 promotes the proliferation of neural stem cells and inhibits their differentiation into astrocytes. In order to determine how CXCL1 exerts these effects, we analyzed intracellular reactive oxygen species, cell signaling, and performed in vivo recovery experiments. Our results suggest that CXCL1 promotes neural stem cell proliferation through a mechanism involving the production of reactive oxygen species and the PI3K/Akt pathway. CONCLUSION: In APP/PS1 mice, macrophage-derived CXCL1 can promote the proliferation of neural stem cells in the subventricular zone via the NOX2-ROS-PI3K/Akt pathway.

Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production.

The formation of brain vasculature is an essential step during central nervous system development. The molecular mechanism underlying brain angiogenesis remains incompletely understood. The role of Atg7, an autophagy-related protein, in brain angiogenesis was investigated in this study. We found that the microvessel density in mice brains with endothelial-specific knockout of Atg7 (Atg7 EKO) was significantly decreased compared to wild-type control. Consistently, in vitro angiogenesis assays showed that Atg7 knockdown impaired angiogenesis in brain microvascular endothelial cells. Further results indicated that knockdown of Atg7 reduced interleukin-6 (IL-6) expression in brain microvascular endothelial cells, which is mediated by NF-κB-dependent transcriptional control. Interestingly, exogenous IL-6 restored the impaired angiogenesis and reduced cell motility caused by Atg7 knockdown. These results demonstrated that Atg7 has proangiogenic activity in brain angiogenesis which is mediated by IL-6 production in a NF-κB-dependent manner.

Visfatin Mediates SCLC Cells Migration across Brain Endothelial Cells through Upregulation of CCL2.

Small-cell lung cancer (SCLC) is characterized as an aggressive tumor with brain metastasis. Although preventing SCLC metastasis to the brain is immensely important for survival, the molecular mechanisms of SCLC cells penetrating the blood-brain barrier (BBB) are largely unknown. Recently, visfatin has been considered as a novel pro-inflammatory adipocytokine involved in various cancers. Herein, we present evidence that elevated levels of visfatin in the serum of SCLC patients were associated with brain metastasis, and visfain was increased in NCI-H446 cells, a SCLC cell line, during interacting with human brain microvascular endothelial cells (HBMEC). Using in vitro BBB model, we found that visfatin could promote NCI-H446 cells migration across HBMEC monolayer, while the effect was inhibited by knockdown of visfatin. Furthermore, our findings indicated that CC chemokine ligand 2 (CCL2) was involved in visfatin-mediated NCI-H446 cells transendothelial migtation. Results also showed that the upregulation of CCL2 in the co-culture system was reversed by blockade of visfatin. In particular, visfatin-induced CCL2 was attenuated by specific inhibitor of PI3K/Akt signaling in NCI-H446 cells. Taken together, we demonstrated that visfatin was a prospective target for SCLC metastasis to brain, and understanding the molecular mediators would lead to effective strategies for inhibition of SCLC brain metastasis.

The involvement of CXCL11 in bone marrow-derived mesenchymal stem cell migration through human brain microvascular endothelial cells.

Bone marrow-derived mesenchymal stem cells (MSCs) transplant into the brain, where they play a potential therapeutic role in neurological diseases. However, the blood-brain barrier (BBB) is a native obstacle for MSCs entry into the brain. Little is known about the mechanism behind MSCs migration across the BBB. In the present study, we modeled the interactions between human MSCs (hMSCs) and human brain microvascular endothelial cells (HBMECs) to mimic the BBB microenvironment. Real-time PCR analysis indicated that the chemokine CXCL11 is produced by hMSCs and the chemokine receptor CXCR3 is expressed on HBMECs. Further results indicate that CXCL11 secreted by hMSCs may interact with CXCR3 on HBMECs to induce the disassembly of tight junctions through the activation of ERK1/2 signaling in the endothelium, which promotes MSCs transendothelial migration. These findings are relevant for understanding the biological responses of MSCs in BBB environments and helpful for the application of MSCs in neurological diseases.

Endothelial lincRNA-p21 alleviates cerebral ischemia/reperfusion injury by maintaining blood-brain barrier integrity.

Blood-brain barrier (BBB) disruption is increasingly recognized as an early contributor to the pathophysiology of cerebral ischemia/reperfusion (I/R) injury, and is also a key event in triggering secondary damage to the central nervous system. Recently, long non-coding RNA (lncRNA) have been found to be associated with ischemic stroke. However, the roles of lncRNA in BBB homeostasis remain largely unknown. Here, we report that long intergenic non-coding RNA-p21 (lincRNA-p21) was the most significantly down-regulated lncRNA in human brain microvascular endothelial cells (HBMECs) after oxygen and glucose deprivation/reoxygenation (OGD/R) treatment among candidate lncRNA, which were both sensitive to hypoxia and involved in atherosclerosis. Exogenous brain-endothelium-specific overexpression of lincRNA-p21 could alleviate BBB disruption, diminish infarction volume and attenuate motor function deficits in middle cerebral artery occlusion/reperfusion (MCAO/R) mice. Further results showed that lincRNA-p21 was critical to maintain BBB integrity by inhibiting the degradation of junction proteins under MCAO/R and OGD/R conditions. Specifically, lincRNA-p21 could inhibit autophagy-dependent degradation of occludin by activating PI3K/AKT/mTOR signaling pathway. Besides, lincRNA-p21 could inhibit VE-cadherin degradation by binding with miR-101-3p. Together, we identify that lincRNA-p21 is critical for BBB integrity maintenance, and endothelial lincRNA-p21 overexpression could alleviate cerebral I/R injury in mice, pointing to a potential strategy to treat cerebral I/R injury.

Microglial TNF-α-dependent elevation of MHC class I expression on brain endothelium induced by amyloid-beta promotes T cell transendothelial migration.

The blood-brain barrier (BBB) normally bars peripheral T lymphocytes from entering the cerebrum. Interestingly, activated T cells exist as infiltrates in the brains of Alzheimer's disease (AD) patients, but little is known about the mechanisms involved. In this study, we observed significantly higher MHC class I expression in rat brain endothelial cells compared with controls following the induction of experimental AD models. An in vitro BBB model, which was constructed with human brain microvascular endothelial cells, was established to study the mechanisms underlying the transendothelial migration of T cells. Using in vitro studies, we demonstrated that secretion of TNF-α from Aß1-42-treated BV2 microglia contributes to the elevated expression of MHC class I on the brain microvessel endothelium. Transmigration assays and adhesion assays confirmed that the upregulation of MHC class I molecules was associated with T cell transendothelial migration. MHC class I knock-down in HBMECs significantly attenuated the migratory and adhesive capability of the T cells. Interestingly, a TNF-α neutralizing antibody effectively blocked the transendothelial migration of T cells triggered by treatment with the supernatant from Aß1-42-treated BV2 microglia. We propose that microglia-derived TNF-α upregulates MHC class I molecule expression on brain endothelial cells, which represents a mechanism of T cell migration into the brain. This study may provide a new insight into the potential pathomechanism of Alzheimer's disease.

Endothelial depletion of Atg7 triggers astrocyte-microvascular disassociation at blood-brain barrier.

Microvascular basement membrane (BM) plays a pivotal role in the interactions of astrocyte with endothelium to maintain the blood-brain barrier (BBB) homeostasis; however, the significance and precise regulation of the endothelial cell-derived BM component in the BBB remain incompletely understood. Here, we report that conditional knockout of Atg7 in endothelial cells (Atg7-ECKO) leads to astrocyte-microvascular disassociation in the brain. Our results reveal astrocytic endfeet detachment from microvessels and BBB leakage in Atg7-ECKO mice. Furthermore, we find that the absence of endothelial Atg7 downregulates the expression of fibronectin, a major BM component of the BBB, causing significantly reduced coverage of astrocytes along cerebral microvessels. We reveal Atg7 triggers the expression of endothelial fibronectin via regulating PKA activity to affect the phosphorylation of cAMP-responsive element-binding protein. These results suggest that Atg7-regulated endothelial fibronectin production is required for astrocytes adhesion to microvascular wall for maintaining the BBB homeostasis. Thus, endothelial Atg7 plays an essential role in astrocyte-endothelium interactions to maintain the BBB integrity.

cPLA2α-mediated actin rearrangements downstream of the Akt signaling is required for Cronobacter sakazakii invasion into brain endothelial cells.

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that causes sepsis and meningitis in neonate. The molecular mechanism involved in the pathogenesis of C. sakazakii meningitis remains unclear. In this study, we found that C. sakazakii invasion was significantly decreased in human brain microvascular endothelial cells (HBMEC) treated with cytosolic phospholipases A(2)α (cPLA(2)α) inhibitor. Increased phosphorylation of cPLA(2)α was observed in HBMEC infected with C. sakazakii, which was prevented by treatment with cPLA(2)α inhibitor. cPLA(2)α knockdown in HBMEC significantly attenuated C. sakazakii invasion into HBMEC. Immunofluorescence demonstrated that the rearrangements of actin filaments in HBMEC induced by C. sakazakii were effectively blocked by either treatment with cPLA(2)α inhibitor or transfection with cPLA(2)α siRNA. Interestingly, we found that C. sakazakii infection promoted the aggregation of phosphorylated cPLA(2)α, which was associated with depolymerized actin filaments in HBMEC. Furthermore, our data revealed that cPLA(2)α acts downstream of Akt signaling pathway in HBMEC stimulated with C. sakazakii. Taken together, our results illustrated that cPLA(2)α-mediated actin filament rearrangements downstream of Akt activation is required for C. sakazakii invasion into brain endothelial cells.

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.

Perfluorooctane sulfonate triggers tight junction "opening" in brain endothelial cells via phosphatidylinositol 3-kinase.

Perfluorooctane sulfonate (PFOS), an environmental pollutant, is widely distributed in humans and wildlife. Accumulation of PFOS in the brain and its neurotoxicity has been reported. Whether PFOS has any effect on the blood-brain barrier (BBB) remains unknown. In this study, human brain microvascular endothelial cells (HBMEC), which are the major components of BBB, were treated with PFOS and indicators of endothelial permeability were measured. Disassembly of endothelial tight junction (TJ) and increase of permeability were observed in response to PFOS. The PFOS-induced TJ disassembly in HBMEC was attenuated by pretreatment with PI3K inhibitors, whereas Rho kinase inhibitor had no such effect. Further results demonstrated that PFOS promoted the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in HBMEC. We found that overexpression of PI3K dominant-negative mutant in HBMEC abolished the PFOS-induced TJ disassembly. These data demonstrated that PFOS can trigger the "opening" of tight junction in brain endothelial cells through PI3K signaling pathway.

Inactivation of EphA2 promotes tight junction formation and impairs angiogenesis in brain endothelial cells.

Eph receptor tyrosine kinases and ephrin ligands participate in the regulation of a wide variety of biological processes, such as axon guidance, synaptic plasticity, angiogenesis, and tumorigenesis. The role of Eph receptors and ephrin ligands in brain endothelial cells remains unknown. Here, we examined the expression profile of EphA receptors and ephrin-A ligands in human brain microvascular endothelial cell line (HBMEC). Our results showed that multiple EphA receptors and ephrin-A ligands are expressed in HBMEC. We found that the phosphorylation of EphA2, but not other EphA receptors, was significantly increased in HBMEC treated with recombinant ephrin-A1/Fc. Meanwhile, elevated EphA2 phosphorylation was accompanied by disassembly of tight junctions in HBMEC. Furthermore, EphA2 RNAi in HBMEC could promote tight junction formation and prevent the ephrin-A1-induced tight junction disruption. Also, when a kinase-inactive mutant of EphA2 (EphA2-K646M) was expressed in HBMEC, the tight junction was enhanced and the ephrin-A1-induced tight junction disruption was blocked. In addition, EphA2 RNAi and expression of EphA2-K646M in HBMEC inhibited in vitro cell migration and angiogenesis of HBMEC. These data indicated an important role of EphA2 in regulating both tight junction formation and angiogenesis in brain endothelial cells.

Amyloid beta interaction with receptor for advanced glycation end products up-regulates brain endothelial CCR5 expression and promotes T cells crossing the blood-brain barrier.

How circulating T cells infiltrate into the brain in Alzheimer disease (AD) remains unclear. We previously reported that amyloid beta (Abeta)-dependent CCR5 expression in brain endothelial cells is involved in T cell transendothelial migration. In this study, we explored the signaling pathway of CCR5 up-regulation by Abeta. We showed that inhibitors of JNK, ERK, and PI3K significantly decreased Abeta-induced CCR5 expression in human brain microvascular endothelial cells (HBMECs). Chromatin immunoprecipitation assay revealed that Abeta-activated JNK, ERK, and PI3K promoted brain endothelial CCR5 expression via transcription factor Egr-1. Furthermore, neutralization Ab of receptor for advanced glycation end products (RAGE; an Abeta receptor) effectively blocked Abeta-induced JNK, ERK, and PI3K activation, contributing to CCR5 expression in HBMECs. Abeta fails to induce CCR5 expression when truncated RAGE was overexpressed in HBMECs. Transendothelial migration assay showed that the migration of MIP-1alpha (a CCR5 ligand)-expressing AD patients' T cells through in vitro blood-brain barrier model was effectively blocked by anti-RAGE Ab, overexpression of truncated RAGE, and dominant-negative PI3K, JNK/ERK, or Egr-1 RNA interference in HBMECs, respectively. Importantly, blockage of intracerebral RAGE abolished the up-regulation of CCR5 on brain endothelial cells and the increased T cell infiltration in the brain induced by Abeta injection in rat hippocampus. Our results suggest that intracerebral Abeta interaction with RAGE at BBB up-regulates endothelial CCR5 expression and causes circulating T cell infiltration in the brain in AD. This study may provide a new insight into the understanding of inflammation in the progress of AD.

Vascular endothelial growth factor receptor 1 contributes to Escherichia coli K1 invasion of human brain microvascular endothelial cells through the phosphatidylinositol 3-kinase/Akt signaling pathway.

Escherichia coli is the most common Gram-negative organism causing neonatal meningitis. Previous studies demonstrated that E. coli K1 invasion of brain microvascular endothelial cells (BMEC) is required for penetration into the central nervous system, but the microbe-host interactions that are involved in this process remain incompletely understood. Here we report the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) expressed on human brain microvascular endothelial cells (HBMEC) in E. coli K1 invasion of HBMEC. Our results showed that treatment of confluent HBMEC with pan-VEGFR inhibitors significantly inhibited E. coli K1 invasion of HBMEC. Immunofluorescence results indicated the colocalization of VEGFR1 with E. coli K1 during bacterial invasion of HBMEC. The E. coli-induced actin cytoskeleton rearrangements in HBMEC were blocked by VEGFR inhibitors but not by VEGFR2-specific inhibitors. The small interfering RNA (siRNA) knockdown of VEGFR1 in HBMEC significantly attenuated E. coli invasion and the concomitant actin filament rearrangement. Furthermore, we found an increased association of VEGFR1 with the p85 subunit of phosphatidylinositol 3-kinase (PI3K) in HBMEC infected with E. coli K1 and that E. coli K1-triggered Akt activation in HBMEC was blocked by VEGFR1 siRNA and VEGFR inhibitors. Taken together, our results demonstrate that VEGFR1 contributes to E. coli K1 invasion of HBMEC via recruitment of the PI3K/Akt signaling pathway.

PI3K-dependent host cell actin rearrangements are required for Cronobacter sakazakii invasion of human brain microvascular endothelial cells.

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that can cause neonatal sepsis and meningitis. The mechanism involved in the pathogenesis of C. sakazakii meningitis remains largely unknown. Previous studies indicated that bacterial invasion of brain microvascular endothelial cells is required for penetration into the central nervous system. In this study, we found that C. sakazakii invasion of human brain microvascular endothelial cells (HBMEC) was significantly inhibited by cytochalasin D, a disrupting agent of actin microfilaments. Disassembly of actin stress fibers and cortical actin fibers was observed in HBMEC infected with C. sakazakii. C. sakazakii infection leads to increased Akt phosphorylation in HBMEC, which was blocked by treatment with PI3K inhibitors. Meanwhile, PI3K and Akt inhibitors significantly inhibited C. sakazakii invasion of HBMEC. Our further results illustrated that the C. sakazakii-induced Akt activation and C. sakazakii invasion were attenuated in HBMEC transfected with dominant-negative PI3K (Δp110). More importantly, the actin filaments rearrangements in HBMEC induced by C. sakazakii were effectively blocked by PI3K inhibitors treatment and transfection with Δp110. Taken together, our findings demonstrated that PI3K-mediated actin rearrangements are required for C. sakazakii invasion of HBMEC.

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.

Tentative identification of glycerol dehydrogenase as Escherichia coli K1 virulence factor cglD and its involvement in the pathogenesis of experimental neonatal meningitis.

Escherichia coli (E. coli) is the most common gram-negative organism causing meningitis during the neonatal period. The mechanism involved in the pathogenesis of E. coli meningitis remains unclear. We previously identified a pathogenicity island GimA (genetic island of meningitic E. coli containing ibeA) from the genomic DNA library of E. coli K1, which may contribute to the E. coli invasion of the blood-brain barrier (BBB). CglD is one of the genes in GimA, and its function remains unknown. In order to characterize the role of cglD in the E. coli meningitis, an isogenic in-frame cglD deletion mutant of E. coli K1 was generated. The results showed that the median lethal dose of the cglD deletion mutant strain was significant higher than that of parent E. coli K1 strain, and the cglD deletion in E. coli K1 prolonged survival of the neonatal rats in experimental meningitis. However, deletion of cglD has no effect on the penetration of E. coli K1 through BBB in vitro and in vivo. Furthermore, our results showed that deletion of cglD in E. coli K1 attenuated cerebrospinal fluid changes, meningeal thickening, and neutrophil infiltration in the cerebral cortex in the neonatal rats with experimental meningitis. Additional results showed that the role of CglD in neonatal meningitis may be associated with its activity of glycerol dehydrogenase. Taken together, our study suggested that CglD is a virulence factor of E. coli K1 contributed to the development of neonatal meningitis.

ZO-1 expression is suppressed by GM-CSF via miR-96/ERG in brain microvascular endothelial cells.

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases in some disorders such as vascular dementia, Alzheimer's disease, and multiple sclerosis. We previously reported that in Alzheimer's disease patients, a high level of GM-CSF in the brain parenchyma downregulated expression of ZO-1, a blood-brain barrier tight junction protein, and facilitated the infiltration of peripheral monocytes across the blood-brain barrier. However, the molecular mechanism underlying regulation of ZO-1 expression by GM-CSF is unclear. Herein, we found that the erythroblast transformation-specific (ETS) transcription factor ERG cooperated with the proto-oncogene protein c-MYC in regulation of ZO-1 transcription in brain microvascular endothelial cells (BMECs). The ERG expression was suppressed by miR-96 which was increased by GM-CSF through the phosphoinositide-3 kinase (PI3K)/Akt pathway. Inhibition of miR-96 prevented ZO-1 down-regulation induced by GM-CSF both in vitro and in vivo. Our results revealed the mechanism of ZO-1 expression reduced by GM-CSF, and provided a potential target, miR-96, which could block ZO-1 down-regulation caused by GM-CSF in BMECs.

Endothelial Atg7 Deficiency Ameliorates Acute Cerebral Injury Induced by Ischemia/Reperfusion.

Ischemic strokes often result in cerebral injury due to ischemia/reperfusion (I/R). Although the local inflammatory responses are known to play a primary role in the brain I/R injury, the underlying mechanism remains unclear. In the current study, we investigated the effect of brain endothelial Atg7 (autophagy related 7) depletion in the acute brain injury induced by ischemia and reperfusion. Endothelial knockout of Atg7 in mice (Atg7 eKO) was found to significantly attenuate both the infarct volume and the neurological defects induced by I/R when compared to the controls. In fact, brain inflammatory responses induced by I/R were alleviated by the Atg7 eKO. Furthermore, an increased expression of pro-inflammatory cytokines, including IL-1ß, IL-6, IL-8, and TNF-α, was observed in brain endothelial cells in response to oxygen/glucose depletion/reoxygenation, which was decreased by the shRNA-mediated Atg7 knockdown. Interestingly, Atg7 knockdown reduced IKKß phosphorylation, leading to NF-κB deactivation and downregulation of the pro-inflammatory cytokines mRNA levels. Further, Atg7 transcriptional regulation function is independent of its role in autophagy. Taken together, our results demonstrated that brain endothelial Atg7 contributes to brain damage during I/R by modulating the expression of pro-inflammatory cytokines. Depletion of Atg7 in brain endothelium has a neuroprotective effect against the ischemia/reperfusion-induced acute cerebral injury during stroke.