Sergio M. Gloor

Molecular and cellular permeability control at the blood-brain barrier

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells. These cells have at least three properties which distinguish them from their peripheral counterparts: (1) tight junctions (TJs) of extremely low permeability; (2) low rates of fluid-phase endocytosis; (3) specific transport and carrier molecules. In combination, these features restrict the nonspecific flux of ions, proteins, and other substances into the central nervous system (CNS) environment. The restriction protects neurons from harmful compositional fluctuations occurring in the blood and allows uptake of essential molecules. Breakdown of the BBB is associated with a variety of CNS disorders and results in aggravation of the condition. Restoration of the BBB is thus one strategy during therapy of CNS diseases. Its success depends on a precise knowledge of the structural and functional principles underlying BBB functionality. In this review we have tried to summarise the current knowledge of TJs, including information gained from non-neuronal systems, and describe selected mechanisms involved in permeability regulation.

High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca - substrate specificity in glycosyl hydrolase family 5.

BACKGROUND . beta-Mannanases hydrolyse the O-glycosidic bonds in mannan, a hemicellulose constituent of plants. These enzymes have potential use in pulp and paper production and are of significant biotechnological interest. Thermostable beta-mannanases would be particularly useful due to their high temperature optimum and broad pH tolerance. The thermophilic actinomycete Thermomonospora fusca secretes at least one beta-mannanase (molecular mass 38 kDa) with a temperature optimum of 80 degreesC. No three-dimensional structure of a mannan-degrading enzyme has been reported until now. RESULTS . The crystal structure of the thermostable beta-mannanase from T. fusca has been determined by the multiple isomorphous replacement method and refined to 1.5 A resolution. In addition to the native enzyme, the structures of the mannotriose- and mannohexaose-bound forms of the enzyme have been determined to resolutions of 1.9 A and 1.6 A, respectively. CONCLUSIONS . Analysis of the -1 subsite of T. fusca mannanase reveals neither a favourable interaction towards the axial HO-C(2) nor a discrimination against the equatorial hydroxyl group of gluco-configurated substrates. We propose that selectivity arises from two possible mechanisms: a hydrophobic interaction of the substrate with Val263, conserved in family 5 bacterial mannanases, which discriminates between the different conformations of the hydroxymethyl group in native mannan and cellulose; and/or a specific interaction between Asp259 and the axial hydroxyl group at the C(2) of the substrate in the -2 subsite. Compared with the catalytic clefts of family 5 cellulases, the groove of T. fusca mannanase has a strongly reduced number of aromatic residues providing platforms for stacking with the substrate. This deletion of every second platform is in good agreement with the orientation of the axial hydroxyl groups in mannan.

Tenascin-R is an intrinsic autocrine factor for oligodendrocyte differentiation and promotes cell adhesion by a sulfatide-mediated mechanism.

O4+ oligodendrocyte (OL) progenitors in the mammalian CNS are committed fully to terminal differentiation into myelin-forming cells. In the absence of other cell types in vitro, OL differentiation reproduces the in vivo development with a correct timing, suggesting the existence of an intrinsic regulatory mechanism that presently is unknown. We have examined the effect of two isoforms of the extracellular matrix (ECM) molecule tenascin-R (TN-R), which is expressed by OLs during the process of myelination, on the adhesion and maturation of OLs in vitro. Here we show that the substrate-bound molecules supported the adhesion of O4+ OLs independently of the CNS region or age from which they were derived. At the molecular level this process was mediated by protein binding to membrane surface sulfatides (Sulf), as indicated by the interference of O4 antibody and Sulf with the attachment of OLs or other Sulf+ cells, erythrocytes, to TN-R substrates and by direct protein–glycolipid binding studies. In the absence of platelet-derived growth factor (PDGF), exogenous TN-R induced myelin gene expression and the upregulation of its own synthesis by cultured cells, resulting in a rapid terminal differentiation of O4+ progenitors. Our findings strongly suggest that TN-R represents an intrinsic regulatory molecule that controls the timed OL differentiation by an autocrine mechanism and imply the relevance of TN-R for CNS myelination and remyelination.

ATP-induced conformational changes of the nucleotide-binding domain of Na,K-ATPase.

The Na,K-ATPase hydrolyzes ATP to drive the coupled extrusion and uptake of Na+ and K+ ions across the plasma membrane. Here, we report two high-resolution NMR structures of the 213-residue nucleotide-binding domain of rat α1 Na,K-ATPase, determined in the absence and the presence of ATP. The nucleotide binds in the anti conformation and shows a relative paucity of interactions with the protein, reflecting the low-affinity ATP-binding state. Binding of ATP induces substantial conformational changes in the binding pocket and in residues located in the hinge region connecting the N- and P-domains. Structural comparison with the Ca-ATPase stabilized by the inhibitor thapsigargin, E2(TG), and the model of the H-ATPase in the E1 form suggests that the observed changes may trigger the series of events necessary for the release of the K+ ions and/or disengagement of the A-domain, leading to the eventual transfer of the γ-phosphate group to the invariant Asp369.

sICAM-1 and TNF-? induce MIP-2 with distinct kinetics in astrocytes and brain microvascular endothelial cells

The dysfunction of the blood‐brain barrier (BBB) occurring after traumatic brain injury (TBI) is mediated by intracerebral neutrophil accumulation, chemokine release (e.g., interleukin (IL)‐8) and upregulation of adhesion molecules (e.g., intercellular adhesion molecule (ICAM)‐1). In patients with severe TBI, we previously found that elevated cerebrospinal fluid (CSF) IL‐8 and soluble (s)ICAM‐1 correlate with BBB dysfunction, and this prompted us to concomitantly monitor IL‐8, sICAM‐1 and their stimulator tumor necrosis factor (TNF)‐α in CSF. Potential mechanisms for upregulation of the IL‐8 analogue, murine macrophage inflammatory protein (MIP)‐2, and sICAM‐1 at the BBB were studied using cultured mouse astrocytes and brain microvascular endothelial cells (MVEC). In CSF of seven patients, IL‐8 and sICAM‐1 were elevated for 19 days after severe TBI, whereas TNF‐α exceeded normal values on 9 days. Stimulation of MVEC and astrocytes with TNF‐α simultaneously induced the release of MIP‐2 reaching saturation by 4–8 hr and of sICAM‐1 increasing continuously from 2–4 hr to 12 hr. Augmented sICAM‐1 production correlated with enhanced membrane‐bound (m)ICAM‐1 expression in both cell types (rs = 0.96 and 0.90, P < 0.0001), but was markedly higher in astrocytes. The release of sICAM‐1 was not influenced by IL‐8 or MIP‐2, although astrocytes and MVEC expressed the IL‐8/MIP‐2 receptor (CXCR‐2) as determined by FACS analysis. Instead, we found that sICAM‐1 strongly induced MIP‐2 secretion by both cell types with kinetics differing from those evoked by TNF‐α. If added together, sICAM‐1 and TNF‐α synergistically induced MIP‐2 production suggesting the involvement of two different pathways for MIP‐2 regulation. J. Neurosci. Res. 60:733–742, 2000.

Down‐regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type‐1 receptor and nuclear factor‐κB activation

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin‐1, ZO‐1 and ZO‐2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO‐1 expression in astrocytes. TNF decreased occludin, but not ZO‐1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down‐regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor‐κB (NF‐κB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type‐1 receptor (TNFR1), TNF type‐2 receptor (TNFR2), or both, respectively, revealed that down‐regulation was mediated entirely by TNFR1. ZO‐1, which can interact with occludin, was found to co‐precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down‐regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF‐κB is involved as a negative regulator.

Relevance of Na,K-ATPase to local extracellular potassium homeostasis and modulation of synaptic transmission

The ion gradients generated by the Na,K‐ATPase are essential for Na+‐coupled transport systems, osmoregulation and restoration of ion concentrations in excitable tissues. Indirectly, the sodium pump controls intracellular Ca2+ concentration through the Na/Ca exchanger. In the nervous system various neurotransmitters can modulate Na,K‐ATPase activity. The great diversity of Na,K‐ATPase subunit isoforms, their complex spatial and temporal regulation of expression and their cellular localisation imply a functional role of the sodium pump in different regulatory pathways. Among these, potassium homeostasis and modulation of synaptic transmission are discussed here.

Endothelial cell barrier impairment induced by glioblastomas and transforming growth factor beta2 involves matrix metalloproteinases and tight junction proteins

Abstract Gliomas, particularly glioblastoma multiforme, perturb the blood-brain barrier and cause brain edema that contributes to morbidity and mortality. The mechanisms underlying this vasogenic edema are poorly understood. We examined the effects of cocultured primary cultured human glioblastoma cells and glioma-derived growth factors on the endothelial cell tight junction proteins claudin 1, claudin 5, occludin, and zonula occludens 1 of brain-derived microvascular endothelial cells and a human umbilical vein endothelial cell line. Cocultured glioblastoma cells and glioma-derived factors (e.g. transforming growth factor &bgr;2) enhanced the paracellular flux of endothelial cell monolayers in conjunction with downregulation of the tight junction proteins. Neutralizing anti-transforming growth factor &bgr;2 antibodies partially restored the barrier properties in this in vitro blood-brain barrier model. The involvement of endothelial cell-derived matrix metalloproteinases (MMPs) was demonstrated by quantitative reverse-transcriptase-polymerase chain reaction analysis and by the determination of MMP activities via zymography and fluorometry in the presence or absence of the MMP inhibitor GM6001. Occludin, claudin 1, and claudin 5 were expressed in microvascular endothelial cells in nonneoplastic brain samples but were significantly reduced in anaplastic astrocytoma and glioblastoma samples. Taken together, these in vitro and in vivo results indicate that glioma-derived factors may induce MMPs and downregulate endothelial tight junction protein and, thus, play a key role in glioma-induced impairment of the blood-brain barrier.

Specific proteolytic cleavage of agrin regulates maturation of the neuromuscular junction

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve–muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and β-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.

A vector for the synthesis of cRNAs encoding Myc epitope-tagged proteins in xenopus laevis oocytes

We describe a plasmid, pNKS2-myc, designed for convenient in-frame fusion of an antibody-specific epitope sequence to the N terminus of a desired cDNA and subsequent synthesis of transcripts that direct the synthesis of the tagged polypeptide in Xenopus laevis (Xl) oocytes. pNKS2-myc contains an SP6 promoter, followed by the translation initiation sequence of the Na,K-pump beta 3 subunit of Xl and the sequence encoding an epitope derived from the human c-myc proto-oncogene product. Appropriate restriction sites allow one to insert virtually any desired cDNA fragment directly behind the epitope-specific sequence and before a long poly(A) tail. After linearization with EcoRI or NotI, polyadenylated cRNA can be synthesized that is efficiently translated in Xl oocytes. The utility of pNKS2-myc is demonstrated by cloning cDNAs coding for Na,K-pump subunits into this vector and injecting the corresponding cRNAs into oocytes. The tagged mouse beta 1 and beta 2 subunit isoforms could be purified from detergent extracts of these cells by immunoprecipitation with a generally available monoclonal antibody (mAb) to the tag, 9E10, as well as with specific mAb that recognize individual beta subunit isoforms. Under native conditions, endogenous and coexpressed exogenous alpha 1 subunits (the catalytic subunit of the Na,K-pump) were co-precipitated, indicating that the N-terminal addition of the decapeptide epitope has no adverse effect on the folding of beta subunits nor on their assembly with alpha subunits. Furthermore, the Myc-specific mAb likewise precipitated a Myc-tagged Na,K-pump alpha 1 subunit together with any of the co-synthesized beta subunits.