Involvement of Claudin-11 in Disruption of Blood-Brain, -Spinal Cord, and -Arachnoid Barriers in Multiple Sclerosis.

It is important to understand the molecular mechanisms of barrier disruption in the central nervous system (CNS) of patients with multiple sclerosis (MS). The purpose of the present study was to clarify whether claudin-11 is involved in the disruption of two endothelial barriers (blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB)) and two epithelial barriers (blood-arachnoid barrier (BAB) and blood-CSF barrier (BCSFB)) in the CNS in MS. Immunohistochemical analysis revealed that, in both normal human and mouse, claudin-11 is co-localized with claudin-5 in the brain and spinal cord capillaries. The absolute protein expression level of claudin-11 was nearly equal to that of claudin-5 in rat brain capillaries, but was 2.81-fold greater in human brain capillaries. The protein expressions of claudin-11 were significantly downregulated in the brain and spinal cord capillaries of an MS patient and experimental autoimmune encephalomyelitis (EAE) mice. Specific downregulation of claudin-11 with siRNA significantly increased the transfer of membrane-impermeable FITC-dextran across human brain capillary endothelial cell (hCMEC/D3) monolayer. As for the epithelial barrier, claudin-11 protein expression was not decreased in choroid plexus epithelial cells forming the BCSFB in EAE mice, whereas it was decreased in brain and spinal cord meninges that form the BAB. Specific downregulation of claudin-11 with siRNA in a rat choroid plexus epithelial cell (TR-CSFB) monolayer significantly increased the permeability of FITC-dextran. In conclusion, our present findings indicate that claudin-11 expression at the BBB, BSCB, and BAB, but not the BCSFB, is downregulated in multiple sclerosis, impairing the functional integrity of these barriers.

Involvement of insulin-degrading enzyme in insulin- and atrial natriuretic peptide-sensitive internalization of amyloid-ß peptide in mouse brain capillary endothelial cells.

Cerebral clearance of amyloid-ß peptide (Aß), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aß1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aß1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAß1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAß1-40, suggesting that hAß1-40 and hANP share a common elimination process. Internalization of [125I]hAß1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAß1-40, suggesting that there is no direct interaction between Npr-C and hAß1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAß1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAß1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.

Screening of PC and PMMA-binding peptides for site-specific immobilization of proteins.

In the present study, we used proteomic research technology to develop a method for the screening and evaluation of material-binding peptides for protein immobilization. Using this screening method, soluble Escherichia coli proteins that preferentially adsorbed onto polycarbonate (PC) and poly(methylmethacrylate) (PMMA) as model plastic materials were first isolated and identified by 2-dimensional electrophoresis (2DE) combined with peptide mass fingerprinting (PMF). The genes of identified protein candidates (ELN, MLT, OMP, and BIF) that exhibited a hexahistidine tag (6×His-tag) were over-expressed by E. coli BL21 (DE3), and the proteins were purified by IMAC affinity chromatography. The candidates for PC and PMMA-binding peptides were isolated from peptide fragments from affinity protein candidates, which were digested with trypsin and chymotrypsin. Consequently, 5 candidates for the PC-binding peptide and 2 candidates for the PMMA-binding peptide were successfully identified by MALDI-TOF MS. All of the peptides identified were introduced to the C-terminus of glutathione S-transferase (GST) as a model protein for immobilization. Adsorption of peptide-fused and wild-type GSTs onto the plastic surfaces was directly monitored using a quartz crystal microbalance (QCM) device. Consequently, genetic fusion of PC-MLT8 and PC-OMP6 as PC-binders and PM-OMP25 as a PMMA-binder significantly enhanced the adsorption rates of GST, achieving an adsorption density that was more than 10 times higher than that of wild-type GST. Furthermore, the residual activity levels of GST-PC-OMP6 and GST-PM-OMP25 in the adsorption state were 2 times higher than that of wild-type GST. Thus, the PC and PMMA-binding peptides identified in this study, namely PC-OMP6 and PM-OMP25, were considerably useful for site-specific immobilization of proteins, while maintaining a higher adsorption density and residual activity levels. The method demonstrated in this study will be applicable to the isolation of a variety of material-binding peptides against the surfaces of unique materials.

1α,25-Dihydroxyvitamin D3 enhances cerebral clearance of human amyloid-ß peptide(1-40) from mouse brain across the blood-brain barrier.

BACKGROUND: Cerebrovascular dysfunction has been considered to cause impairment of cerebral amyloid-ß peptide (Aß) clearance across the blood-brain barrier (BBB). Further, low levels of vitamin D are associated with increased risk of Alzheimer's disease, as well as vascular dysfunction. The purpose of the present study was to investigate the effect of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), an active form of vitamin D, on cerebral Aß clearance from mouse brain. METHODS: The elimination of [125I]hAß(1-40) from mouse brain was examined by using the Brain Efflux Index method to determine the remaining amount of [125I]hAß(1-40) radioactivity after injection into the cerebral cortex. [125I]hAß(1-40) internalization was analyzed using conditionally immortalized mouse brain capillary endothelial cells (TM-BBB4). RESULTS: Twenty-four hours after intraperitoneal injection of 1,25(OH)2D3 (1 µg/mouse), [125I]hAß(1-40) elimination from mouse brain was increased 1.3-fold, and the level of endogenous Aß(1-40) in mouse brain was reduced. These effects were observed at 24 h after i.p. injection of 1,25(OH)2D3, while no significant effect was observed at 48 or 72 h. Vitamin D receptor (VDR) mRNA was detected in mouse brain capillaries, suggesting that 1,25(OH)2D3 has a VDR-mediated genomic action. Furthermore, forskolin, which activates mitogen-activated protein kinase kinase (MEK), enhanced [125I]hAß(1-40) elimination from mouse brain. Forskolin also enhanced [125I]hAß(1-40) internalization in TM-BBB4 cells, and this enhancement was inhibited by a MEK inhibitor, suggesting involvement of non-genomic action. CONCLUSIONS: The active form of vitamin D, 1,25(OH)2D3, appears to enhance brain-to-blood Aß(1-40) efflux transport at the BBB through both genomic and non-genomic actions. Compounds activating these pathways may be candidate agents for modulating Aß(1-40) elimination at the BBB.

Atrial natriuretic peptide is eliminated from the brain by natriuretic peptide receptor-C-mediated brain-to-blood efflux transport at the blood-brain barrier.

Cerebral atrial natriuretic peptide (ANP), which is generated in the brain, has functions in the regulation of brain water and electrolyte balance, blood pressure and local cerebral blood flow, as well as in neuroendocrine functions. However, cerebral ANP clearance is still poorly understood. The purpose of this study was to clarify the mechanism of blood-brain barrier (BBB) efflux transport of ANP in mouse. Western blot analysis showed expression of natriuretic peptide receptor (Npr)-A and Npr-C in mouse brain capillaries. The brain efflux index (BEI) method confirmed elimination of [(125)I]human ANP (hANP) from mouse brain across the BBB. Inhibition studies suggested the involvement of Npr-C in vivo. Furthermore, rapid internalization of [(125)I]hANP by TM-BBB4 cells (an in vitro BBB model) was significantly inhibited by Npr-C inhibitors and by two different Npr-C-targeted short interfering RNAs (siRNAs). Finally, treatment with 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) significantly increased Npr-C expression in TM-BBB4 cells, as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted absolute proteomics. Our results indicate that Npr-C mediates brain-to-blood efflux transport of ANP at the mouse BBB as a pathway of cerebral ANP clearance. It seems likely that levels of natriuretic peptides in the brain are modulated by 1,25(OH)(2)D(3) through upregulation of Npr-C expression at the BBB.

Transmission of circulating cell-free AA amyloid oligomers in exosomes vectors via a prion-like mechanism.

Recent studies clearly demonstrated that several types of pathogenic amyloid proteins acted as agents that could transmit amyloidosis by means of a prion-like mechanism. Systemic AA amyloidosis is one of the most severe complications of chronic inflammatory disorders, particularly rheumatoid arthritis. It is well known that, similar to an infectious prion protein, amyloid-enhancing factor (AEF) acts as a transmissible agent in AA amyloidosis. However, how AEF transmits AA amyloidosis in vivo remained to be fully elucidated. In the present study, we focused on finding cell-free forms of AEF and its carriers in circulation by using the murine transfer model of AA amyloidosis. We first determined that circulating cell-free AEF existed in blood and plasma in mice with systemic AA amyloidosis. Second, we established that plasma exosomes containing AA amyloid oligomers derived from serum amyloid A had AEF activity and could transmit systemic AA amyloidosis via a prion-like mechanism. These novel findings should provide insights into the transmission mechanism of systemic amyloidoses.