Yasuteru Sano

Interleukin-25 expressed by brain capillary endothelial cells maintains blood-brain barrier function in a protein kinase Cepsilon-dependent manner.

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-17, interferon-γ, IL-1β, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-α in BCECs and consequently repairs TNF-α-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cϵ (PKCϵ) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCϵ inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCϵ-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.

Aprataxin, the causative protein for EAOH is a nuclear protein with a potential role as a DNA repair protein

Early‐onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH) is an autosomal recessive neurodegenerative disorder characterized by early‐onset ataxia, ocular motor apraxia, and hypoalbuminemia. Recently, the causative gene for EAOH, APTX, has been identified. Of the two splicing variants of APTX mRNA, the short and the long forms, long‐form APTX mRNA was found to be the major isoform. Aprataxin is mainly located in the nucleus, and, furthermore, the first nuclear localization signal located near the amino terminus of the long‐form aprataxin is essential for its nuclear localization. We found, based on the yeast two‐hybrid and coimmunoprecipitation experiments, that the long‐form but not the short‐form aprataxin interacts with XRCC1 (x‐ray repair cross‐complementing group 1). Interestingly the amino terminus of the long‐form aprataxin is homologous with polynucleotidekinase‐3′‐phosphatase, which has been demonstrated to be involved in base excision repair, a subtype of single‐strand DNA break repair, through interaction with XRCC1, DNA polymerase β, and DNA ligase III. These results strongly support the possibility that aprataxin and XRCC1 constitute a multiprotein complex and are involved in single‐strand DNA break repair, and furthermore, that accumulation of unrepaired damaged DNA underlies the pathophysiological mechanisms of EAOH.

Peripheral nerve pericytes originating from the blood-nerve barrier expresses tight junctional molecules and transporters as barrier-forming cells.

The objective of this study was to establish pure blood–nerve barrier (BNB)‐derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier‐forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature‐sensitive simian virus 40 large T‐antigen gene. These cell lines expressed several pericyte markers such as α‐smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin‐12, ZO‐1, and ZO‐2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO‐1 were localized at the cell–cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans‐pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell–cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood–brain barrier. Furthermore, we also showed that these cell lines expressed many barrier‐related transporters. ABCG2, p‐gp, MRP‐1, and Glut‐1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve‐to‐blood efflux and blood‐to‐peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis. J. Cell. Physiol. 217: 388–399, 2008.

Advanced glycation end-products disrupt the blood–brain barrier by stimulating the release of transforming growth factor–β by pericytes and vascular endothelial growth factor and matrix metalloproteinase–2 by endothelial cells in vitro

Diabetic encephalopathy is now accepted as an important complication of diabetes. The breakdown of the blood-brain barrier (BBB) is associated with dementia in patients with type 2 diabetes mellitus (T2DM). The purpose of this study was to identify the possible mechanisms responsible for the disruption of the BBB after exposure to advanced glycation end-products (AGEs). We investigated the effect of AGEs on the basement membrane and the barrier property of the BBB by Western blot analysis, using our newly established lines of human brain microvascular endothelial cell (BMEC), pericytes, and astrocytes. AGEs reduced the expression of claudin-5 in BMECs by increasing the autocrine signaling through vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) secreted by the BMECs themselves. Furthermore, AGEs increased the amount of fibronectin in the pericytes through a similar up-regulation of the autocrine transforming growth factor (TGF)-β released by pericytes. These results indicated that AGEs induce basement membrane hypertrophy of the BBB by increasing the degree of autocrine TGF-β signaling by pericytes, and thereby disrupt the BBB through the up-regulation of VEGF and MMP-2 in BMECs under diabetic conditions.

Two different clinical phenotypes of Creutzfeldt-Jakob disease with a M232R substitution.

ObjectiveTo describe the clinical features of Creutzfeldt-Jakob disease with a substitution of arginine for methionine (M232R substitution) at codon 232 (CJD232) of the prion protein gene (PRNP).Patients and methodsWe evaluated the clinical and laboratory features of 20 CJD232 patients: age of onset, initial symptoms, duration until becoming akinetic and mute, duration until occurrence of periodic sharp and wave complexes on EEG (PSWC), MRI findings, and the presence of CSF 14-3-3 protein. Immunohistochemically, prion protein (PrP) deposition was studied.ResultsNone of the patients had a family history of CJD. We recognized two clinical phenotypes: a rapidly progressive type (rapidtype) and a slowly progressive type (slow-type). Out of 20 patients, 15 became akinetic and mute, demonstrated myoclonus, and showed PSWC within a mean duration of 3.1, 2.4, and 2.8 months, respectively (rapid-type). Five showed slowly progressive clinical courses (slow-type). Five became akinetic and mute and four demonstrated myoclonus within a mean duration of 20.6 and 15.3 months, respectively, which were significantly longer than those in the rapid-type. Only one demonstrated PSWC 13 months after the onset. Diffuse synaptic-type deposition was demonstrated in four rapidtype patients, and perivacuolar and diffuse synaptic-type deposition in two, and diffuse synaptic-type deposition in one slow-type patient. Three of 50 suspected but non-CJD patients had the M232R substitution.ConclusionsPatients with CJD232 had no family history like patients with sCJD, and showed two different clinical phenotypes in spite of having the same PRNP genotype. More studies are needed to determine whether M232R substitution causes the disease and influences the disease progression.

Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

The ability of the Blood Brain Barrier (BBB) to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS) activity and regulating leukocytes’ access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS): fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF) that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

Effects of neuromyelitis optica–IgG at the blood–brain barrier in vitro

Objective: To address the hypothesis that physiologic interactions between astrocytes and endothelial cells (EC) at the blood–brain barrier (BBB) are afflicted by pathogenic inflammatory signaling when astrocytes are exposed to aquaporin-4 (AQP4) antibodies present in the immunoglobulin G (IgG) fraction of serum from patients with neuromyelitis optica (NMO), referred to as NMO-IgG. Methods: We established static and flow-based in vitro BBB models incorporating co-cultures of conditionally immortalized human brain microvascular endothelial cells and human astrocyte cell lines with or without AQP4 expression. Results: In astrocyte–EC co-cultures, exposure of astrocytes to NMO-IgG decreased barrier function, induced CCL2 and CXCL8 expression by EC, and promoted leukocyte migration under flow, contingent on astrocyte expression of AQP4. NMO-IgG selectively induced interleukin (IL)-6 production by AQP4-positive astrocytes. When EC were exposed to IL-6, we observed decreased barrier function, increased CCL2 and CXCL8 expression, and enhanced leukocyte transmigration under flow. These effects were reversed after application of IL–6 neutralizing antibody. Conclusions: Our results indicate that NMO-IgG induces IL-6 production by AQP4-positive astrocytes and that IL-6 signaling to EC decreases barrier function, increases chemokine production, and enhances leukocyte transmigration under flow.

The Contribution of Suilysin to the Pathogenesis of Streptococcus suis Meningitis

BACKGROUND Streptococcus suis is an emerging zoonotic pathogen, and causes sepsis and meningitis in humans. Although sequence type (ST) 1 and ST104 strains are capable of causing sepsis, ST1 strains commonly cause meningitis. In this study, we investigated the role of suilysin, a member of cholesterol-dependent cytolysins, in differential pathogenicity between ST1 and ST104 strains. METHODS The levels of transcription and translation of the sly gene and messenger RNA of both ST strains were compared by means of quantitative polymerase chain reaction and Western blotting. Survival rates and bacterial densities in brain were compared between mice infected with wild-type and sly-knockout ST1 strain. ST104 infections with or without complementation of suilysin were also assessed. RESULTS The amounts of suilysin produced by ST1 strains were much higher than those produced by ST104 strains. Lower production of suilysin by ST104 strains were attributed to the attenuated sly gene expression, which seemed to be associated with 2 nucleotide insertions in sly promoter region. Furthermore, suilysin contributed to the higher bacterial density and enhanced inflammation in brain and increased mortality. CONCLUSIONS Our data may explain why ST1 strains, but not ST104 strains, commonly cause meningitis and also suggest the contribution of suilysin to the pathogenesis of meningitis in humans.

Sera from patients with multifocal motor neuropathy disrupt the blood-nerve barrier

Objective In multifocal motor neuropathy (MMN), the destruction of the blood-nerve barrier (BNB) has been considered to be the key step in the disease process. The purpose of the present study was to ascertain whether sera from patients with MMN can open the BNB, and which component of patient sera is the most important for this disruption. Methods We evaluated the effects of sera from patients with MMN, patients with amyotrophic lateral sclerosis, and control subjects on the expression of tight junction proteins and vascular cell adhesion molecule-1 (VCAM-1), and on the transendothelial electrical resistance (TEER) in human peripheral nerve microvascular endothelial cells (PnMECs). Results The sera from patients with MMN decreased the claudin-5 protein expression and the TEER in PnMECs. However, this effect was reversed after application of an anti-vascular endothelial growth factor (anti-VEGF) neutralising antibody. The VEGF secreted by PnMECs was significantly increased after exposure to the sera from patients with MMN. The sera from patients with MMN also increased the VCAM-1 protein expression by upregulating the nuclear factor kappa-B (NF-κB) signalling. The immunoglobulin G purified from MMN sera decreased the expression of claudin-5 and increased the VCAM-1 expression in PnMECs. Conclusions The sera from MMN patients may disrupt the BNB function via the autocrine secretion of VEGF in PnMECs, or the exposure to autoantibodies against PnMECs that are contained in the MMN sera. Autoantibodies against PnMECs in MMN sera may activate the BNB by upregulating the VCAM-1 expression, thereby allowing for the entry of a large number of circulating inflammatory cells into the peripheral nervous system.

Autocrine MMP-2/9 secretion increases the BBB permeability in neuromyelitis optica

Objective Pathological breakdown of the blood-brain barrier (BBB) is thought to constitute the beginning of the disease process in neuromyelitis optica (NMO). In the current study, we investigated possible molecular mechanisms responsible for the breakdown of BBB using NMO sera. Methods We analysed the effects of sera obtained from anti-aquaporin 4 (AQP4) antibody-positive NMO spectrum disorder (NMOSD) patients, multiple sclerosis (MS) patients and control subjects on the production of claudin-5, matrix-metalloproteinases (MMPs)-2/9, and vascular cell adhesion protein-1 (VCAM-1) in human brain microvascular endothelial cells (BMECs). We also examined whether immunoglobulin G (IgG) purified from NMOSD sera influences the claudin-5 or VCAM-1 protein expression. Results The disturbance of BBB properties in BMECs following exposure to NMOSD sera was restored after adding the MMP inhibitor, GM6001. The secretion of MMP-2/9 by BMECs significantly increased after applying the NMOSD sera. The sera from NMOSD patients also increased both the MMP-2/9 secretion and the VCAM-1 protein level by BMECs. The IgG purified from NMOSD sera did not influence the BBB properties or the amount of MMP-2/9 proteins, although it did increase the amount of VCAM-1 proteins in BMECs. Reduction in anti-AQP4 antibody titre was not correlated with a reduction in VCAM-1 expression. Conclusions The autocrine secretion of MMP-2/9 by BMECs induced by humoral factors, other than IgG, in sera obtained from NMOSD patients potentially increases BBB permeability. IgG obtained from NMOSD sera, apart from anti-AQP4 antibodies, affect the BBB by upregulating VCAM, thereby facilitating the entry of inflammatory cells into the central nervous system.